Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

PubMed

Witta, Samir E; Gemmill, Robert M; Hirsch, Fred R; Coldren, Christopher D; Hedman, Karla; Ravdel, Larisa; Helfrich, Barbara; Dziadziuszko, Rafal; Chan, Daniel C; Sugita, Michio; Chan, Zeng; Baron, Anna; Franklin, Wilbur; Drabkin, Harry A; Girard, Luc; Gazdar, Adi F; Minna, John D; Bunn, Paul A

2006-01-15

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling.

PubMed

Kaowinn, Sirichat; Jun, Seung Won; Kim, Chang Seok; Shin, Dong-Myeong; Hwang, Yoon-Hwae; Kim, Kyujung; Shin, Bosung; Kaewpiboon, Chutima; Jeong, Hyeon Hee; Koh, Sang Seok; Krämer, Oliver H; Johnston, Randal N; Chung, Young-Hwa

2017-12-01

Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation. We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

Combined inhibition of EMMPRIN and epidermal growth factor receptor prevents the growth and migration of head and neck squamous cell carcinoma cells.

PubMed

Suzuki, Shinsuke; Ishikawa, Kazuo

2014-03-01

It has been reported that the epidermal growth factor receptor (EGFR) expression is associated with the extracellular matrix metalloproteinase inducer (EMMPRIN) in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in head and neck squamous cell carcinoma (HNSCC), HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. EMMPRIN expression in HNSCC cells was upregulated by EGF. In addition, EGF stimulation induced HNSCC cell invasion and MMP-9 expression. This increase in invasion and MMP-9 expression was abrogated by downmodulation of EMMPRIN. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in HNSCC, HNSCC cells were treated with an EMMPRIN function-blocking antibody and the EGFR inhibitor AG1478. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced tumorigenicity and that combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer.

PubMed

Karachaliou, Niki; Codony-Servat, Jordi; Teixidó, Cristina; Pilotto, Sara; Drozdowskyj, Ana; Codony-Servat, Carles; Giménez-Capitán, Ana; Molina-Vila, Miguel Angel; Bertrán-Alamillo, Jordi; Gervais, Radj; Massuti, Bartomeu; Morán, Teresa; Majem, Margarita; Felip, Enriqueta; Carcereny, Enric; García-Campelo, Rosario; Viteri, Santiago; González-Cao, María; Morales-Espinosa, Daniela; Verlicchi, Alberto; Crisetti, Elisabetta; Chaib, Imane; Santarpia, Mariacarmela; Luis Ramírez, José; Bosch-Barrera, Joaquim; Felipe Cardona, Andrés; de Marinis, Filippo; López-Vivanco, Guillermo; Miguel Sánchez, José; Vergnenegre, Alain; Sánchez Hernández, José Javier; Sperduti, Isabella; Bria, Emilio; Rosell, Rafael

2015-12-07

BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.

Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata.

PubMed

Zhu, Wenjie; Fan, Sigang; Huang, Guiju; Zhang, Dongling; Liu, Baosuo; Bi, Xiaomin; Yu, Dahui

2015-07-25

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, proliferation, differentiation and migration. Yet whether it functions in pearl formation or not is not reported. In this study, EGFR was cloned from the pearl oyster Pinctada fucata (named as Pf-EGFR) and its expression profiles were investigated. The cDNA was 2156bp long with an ORF of 1017bp encoding 338 amino acid residues. The deduced polypeptide contained an L domain and a cysteine-rich domain, consistent with the characteristics of ErbB family. The sequence of Pf-EGFR shared 22.78-56.71% identity with other EGFRs. The genomic sequence of Pf-EGFR consisted of six exons and five introns, being 5190bp in total length, and expressed in all the tested tissues with a higher expression level in the pearl sac (P<0.05). In situ hybridization showed that Pf-EGFR was specifically expressed on both the inner side of the outer fold and the outer side of the inner fold of the mantle, as well as on the whole pearl sac including the connective tissues. In addition, Pf-EGFR was markedly increased at larval metamorphosis, significantly higher than other development periods (P<0.05). These results indicated that the Pf-EGFR may facilitate pearl formation as well as larval metamorphosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

PubMed Central

Zhang, Weina; Chen, Lechuang; Ma, Kai; Zhao, Yahui; Liu, Xianghe; Wang, Yu; Liu, Mei; Liang, Shufang; Zhu, Hongxia; Xu, Ningzhi

2016-01-01

Epidermal growth factor receptor (EGFR) is a target of colon cancer therapy, but the effects of this therapy on the tumor microenvironment remain poorly understood. Our in vivo studies showed that cetuximab, an anti-EGFR monoclonal antibody, effectively inhibited AOM/DSS-induced, colitis-associated tumorigenesis, downregulated M2-related markers, and decreased F4/80+/CD206+ macrophage populations. Treatment with conditioned medium of colon cancer cells increased macrophage expression of the M2-related markers arginase-1 (Arg1), CCL17, CCL22, IL-10 and IL-4. By contrast, conditioned medium of EGFR knockout colon cancer cells inhibited expression of these M2-related markers and induced macrophage expression of the M1-related markers inducible nitric oxide synthase (iNOS), IL-12, TNF-α and CCR7. EGFR knockout in colon cancer cells inhibited macrophage-induced promotion of xenograft tumor growth. Moreover, colon cancer-derived insulin-like growth factor-1 (IGF-1) increased Arg1 expression, and treatment with the IGF1R inhibitor AG1024 inhibited that increase. These results suggest that inhibition of EGFR signaling in colon cancer cells modulates cytokine secretion (e.g. IGF-1) and prevents M1-to-M2 macrophage polarization, thereby inhibiting cancer cell growth. PMID:27683110

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas

PubMed Central

Fukuoka, Hidenori; Cooper, Odelia; Ben-Shlomo, Anat; Mamelak, Adam; Ren, Song-Guang; Bruyette, Dave; Melmed, Shlomo

2011-01-01

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease. PMID:22105169

Tendencies for higher co-expression of EGFR and HER2 and downregulation of HER3 in prostate cancer lymph node metastases compared with corresponding primary tumors.

PubMed

Carlsson, J; Shen, L; Xiang, J; Xu, J; Wei, Q

2013-01-01

The epidermal growth factor receptor (EGFR) family members are potential targets for therapy using extra-cellular domain receptor binding agents, such as the antibodies trastuzumab and cetuximab, or antibodies labeled with therapeutically useful radionuclides or toxins. This is especially the case when the tumor cells are resistant to chemotherapy and tyrosine kinase inhibitors. Studies concerning the expression of these receptors in prostate cancer vary in the literature, possibly due to differences in patient inclusion, sample preparations and scoring criteria. In our study, EGFR, HER2 and HER3 expression was analyzed in prostate cancer samples from primary tumors and corresponding lymph node metastases from 12 patients. The expression of HER2 and EGFR was scored from immunohistochemical preparations and the HercepTest criteria (0, 1+, 2+ or 3+), while HER3 expression was scored as no, weak or strong staining. There were 5 EGFR-positive (2+ or 3+) primary tumors and 6 EGFR-positive lymph node metastases, and there was EGFR upregulation in one metastasis. Only 4 of the 12 patients had marked HER2 expression (2+ or 3+) in their primary tumors and there was one downregulation and 5 cases of upregulation in the metastases. Thus, a total of 8 out of 12 analyzed metastases were HER2-positive. Of the 12 primary tumors, 9 expressed HER3 while only 2 of the lymph node metastases expressed recognizable HER3 staining, so 7 metastases appeared to have downregulated HER3 expression. In one of the primary tumors there was positive co-expression of EGFR and HER2, while this co-expression was observed in 4 of the metastases. Thus, there were tendencies for upregulation of HER2, increased co-expression of EGFR and HER2 and downregulation of HER3 in the prostate cancer lymph node metastases in comparison to the primary tumors. The results are encouraging for studies involving more patients. Possible strategies for EGFR- and HER2-targeted therapy are briefly discussed in the present study, especially with regard to the expression and co-expression of EGFR and HER2 in metastases.

Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

PubMed

Jones, Karra A; Gilder, Andrew S; Lam, Michael S; Du, Na; Banki, Michael A; Merati, Aran; Pizzo, Donald P; VandenBerg, Scott R; Gonias, Steven L

2016-05-01

In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phosphorylated Epidermal Growth Factor Receptor Expression Is Associated With Clinicopathologic Parameters and Patient Survival in Mobile Tongue Squamous Cell Carcinoma.

PubMed

Theocharis, Stamatios; Giaginis, Constantinos; Dana, Eugene; Thymara, Irene; Rodriguez, Jose; Patsouris, Efstratios; Klijanienko, Jerzy

2017-03-01

Phosphorylated epidermal growth factor receptor (pEGFR) activates several signaling pathways, resulting in tumor-promoting cellular activities, and has been implicated in malignant transformation and disease progression. The present study evaluated the clinical significance of pEGFR protein expression in mobile tongue squamous cell carcinoma (SCC). The present cohort study included 48 patients with mobile tongue SCC. We evaluated whether pEGFR immunohistochemical protein expression is associated with clinical variables and patient outcome. Of the 48 patients included in the present cohort study, 25 were men and 23 were women. The median patient age was 60 years (interquartile range 53 to 72). pEGFR protein expression was significantly increased in well-differentiated tumors compared with poorly differentiated tumors (P = .001). Elevated pEGFR protein expression was significantly more frequently observed in mobile tongue SCC cases with a well-defined tumor shape and an earlier disease stage (P = .010 and P = .019, respectively). Patients with mobile tongue SCC presenting with elevated pEGFR expression had longer overall and disease-free survival times compared with those with low pEGFR expression (P = .015 and P = .006, respectively; log-rank test). On multivariate analysis, pEGFR expression proved to be an independent prognostic factor of both overall and disease-free survival (P = .008 and P = .044, respectively; Cox regression analysis). The results of the present study support evidence that the pEGFR signaling pathway might be implicated in the malignant transformation of mobile tongue SCC. Additional studies are recommended to validate whether pEGFR could be used as a potential biomarker and therapeutic target in mobile tongue SCC. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Epidermal growth factor receptor gene amplification in surgical resected Japanese lung cancer.

PubMed

Sasaki, Hidefumi; Shimizu, Shigeki; Okuda, Katsuhiro; Kawano, Osamu; Yukiue, Haruhiro; Yano, Motoki; Fujii, Yoshitaka

2009-06-01

To evaluate the epidermal growth factor receptor (EGFR) protein expression and increased copy number as predictors of clinical outcome in patients with non-small-cell lung cancer (NSCLC), we have performed fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). We investigated the EGFR increased copy number and EGFR protein expression statuses in 109 surgically treated NSCLC cases. The presence or absence of EGFR mutations of kinase domains was analyzed by genotyping analysis and sequences, and already reported. EGFR increased copy number was defined as Cappuzzo et al. criteria. FISH positive was found from 36/109 (33.0%) lung cancer patients, including 30 high polysomy cases and 6 gene amplification cases. FISH-positive cases were significantly correlated with worse prognosis (log-rank test p=0.0097). Within EGFR-mutant patients (n=55), FISH-positive cases were also correlated with poor prognosis (p=0.0255). FISH-negative tumors were found to be more frequently well-differentiated histology. Smoking status (never smoker vs. smoker, p=0.1510), and gender (p=0.5248) did not correlated with FISH positive. EGFR IHC results were correlated with FISH results (p=0.004), but not correlated with prognosis (p=0.2815). Although EGFR FISH-positive rate did not correlated with EGFR mutation (p=0.1973), EGFR polysomy or amplification cases were correlated with EGFR mutations (p=0.0023). In conclusion, the EGFR FISH-positive rate in Japanese patients with NSCLC was similar to rates in Western populations, unlike the higher frequencies of EGFR mutation in East Asians. A high EGFR gene copy number might have shorter survival in NSCLC.

HER3 expression is enhanced during progression of lung adenocarcinoma without EGFR mutation from stage 0 to IA1.

PubMed

Kumagai, Toru; Tomita, Yasuhiko; Nakatsuka, Shin-Ichi; Kimura, Madoka; Kunimasa, Kei; Inoue, Takako; Tamiya, Motohiro; Nishino, Kazumi; Susaki, Yoshiyuki; Kusu, Takashi; Tokunaga, Toshiteru; Okami, Jiro; Higashiyama, Masahiko; Imamura, Fumio

2018-04-01

Activating EGFR mutations, HER2, and HER3 are implicated in lung cancer; however, with the exception of EGFR gene amplification in lung adenocarcinoma harboring EGFR mutations, their involvement in disease progression during the early stages is poorly understood. In this paper, we focused on which receptor is correlated with lung adenocarcinoma progression in the presence or absence of EGFR mutation from stage 0 to IA1. HER2 and HER3 expression and activating EGFR mutations in surgically resected lung adenocarcinoma exhibiting ground glass nodules on chest computed tomography and re-classified to stage 0 and IA1 were examined by immunohistochemistry and peptide nucleic acid-locked nucleic acid PCR clamp method, respectively. HER2 and HER3 expression was detected in 22.2% and 86.1% of samples, respectively. The frequency of EGFR mutation was 45.7% and was not significantly different between stage 0 and IA1 (40.0% and 48.0%, respectively), suggesting that EGFR mutation does not correlate with cancer progression from stage 0 to IA1. HER2 expression also did not correlate to progression. However, not only the frequency, but also the intensity of HER3 expression was increased in stage IA1 lung adenocarcinoma, particularly in lung adenocarcinoma without EGFR mutation. HER3 tends to be intensively expressed during the progression of lung adenocarcinoma without EGFR mutation from carcinoma in situ to invasive carcinoma. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Role of GPER1, EGFR and CXCR1 in differentiating between malignant follicular thyroid carcinoma and benign follicular thyroid adenoma

PubMed Central

Zhao, Le; Zhu, Xiao-Yun; Jiang, Rong; Xu, Man; Wang, Ni; Chen, George G; Liu, Zhi-Min

2015-01-01

It is extremely difficult to discriminate between follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA) before surgery, because the morphologies of carcinoma cells and adenoma cells obtained by fine needle aspiration biopsy (FNAB) are similar. Molecular markers may be helpful on this issue. The purpose of this study was to assess the role of GPER1, EGFR and CXCR1 in differential diagnosis between FTC and FTA. GPER1, EGFR and CXCR1 mRNA expression levels were examined in 15 FTCs and 10 FTAs using real-time RT-PCR. FTC showed to have significantly increased mRNA levels of the three molecules compared to FTA (P < 0.001 for all the three molecules). GPER1, EGFR and CXCR1 protein expression in 106 FTCs and 128 FTAs were analyzed using immunohistochemistry. The rates of GPER1, EGFR and CXCR1 high expression were 73.6%, 72.6% and 70.8% in FTC and 30.5%, 28.1% and 27.3% in FTA, respectively. Statistical analysis showed that GPER1, EGFR and CXCR1 protein expression were correlated with one another in FTC and concomitant high expression of the three molecules had stronger correlation with the occurrence of FTC than did each alone. The positive predictive values (PPV) for concomitant high expression of the three molecules for discriminating between FTC and FTA were 91.0% for GPER1/EGFR, 93.8% for GPER1/CXCR1, 92.3% for EGFR/CXCR1 and 98.2% for GPER1/EGFR/CXCR1, respectively. These results indicated that the evaluation of GPER1, EGFR and CXCR1 concomitant high expression may be helpful in differential diagnosis between FTC and FTA. PMID:26617848

Role of GPER1, EGFR and CXCR1 in differentiating between malignant follicular thyroid carcinoma and benign follicular thyroid adenoma.

PubMed

Zhao, Le; Zhu, Xiao-Yun; Jiang, Rong; Xu, Man; Wang, Ni; Chen, George G; Liu, Zhi-Min

2015-01-01

It is extremely difficult to discriminate between follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA) before surgery, because the morphologies of carcinoma cells and adenoma cells obtained by fine needle aspiration biopsy (FNAB) are similar. Molecular markers may be helpful on this issue. The purpose of this study was to assess the role of GPER1, EGFR and CXCR1 in differential diagnosis between FTC and FTA. GPER1, EGFR and CXCR1 mRNA expression levels were examined in 15 FTCs and 10 FTAs using real-time RT-PCR. FTC showed to have significantly increased mRNA levels of the three molecules compared to FTA (P < 0.001 for all the three molecules). GPER1, EGFR and CXCR1 protein expression in 106 FTCs and 128 FTAs were analyzed using immunohistochemistry. The rates of GPER1, EGFR and CXCR1 high expression were 73.6%, 72.6% and 70.8% in FTC and 30.5%, 28.1% and 27.3% in FTA, respectively. Statistical analysis showed that GPER1, EGFR and CXCR1 protein expression were correlated with one another in FTC and concomitant high expression of the three molecules had stronger correlation with the occurrence of FTC than did each alone. The positive predictive values (PPV) for concomitant high expression of the three molecules for discriminating between FTC and FTA were 91.0% for GPER1/EGFR, 93.8% for GPER1/CXCR1, 92.3% for EGFR/CXCR1 and 98.2% for GPER1/EGFR/CXCR1, respectively. These results indicated that the evaluation of GPER1, EGFR and CXCR1 concomitant high expression may be helpful in differential diagnosis between FTC and FTA.

Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer.

PubMed

Bonomi, P D; Gandara, D; Hirsch, F R; Kerr, K M; Obasaju, C; Paz-Ares, L; Bellomo, C; Bradley, J D; Bunn, P A; Culligan, M; Jett, J R; Kim, E S; Langer, C J; Natale, R B; Novello, S; Pérol, M; Ramalingam, S S; Reck, M; Reynolds, C H; Smit, E F; Socinski, M A; Spigel, D R; Vansteenkiste, J F; Wakelee, H; Thatcher, N

2018-06-14

Upregulated expression and aberrant activation of the epidermal growth-factor receptor (EGFR) are found in lung cancer, making EGFR a relevant target for non-small-cell lung cancer (NSCLC). Treatment with anti-EGFR monoclonal antibodies (mAbs) is associated with modest improvement in overall survival in patients with squamous cell lung cancer (SqCLC) who have a significant unmet need for effective treatment options. While there is evidence that using EGFR gene copy number, EGFR mutation, and EGFR protein expression as biomarkers can help select patients who respond to treatment, it is important to consider biomarkers for response in patients treated with combination therapies that include EGFR mAbs. Randomized trials of EGFR-directed mAbs cetuximab and necitumumab in combination with chemotherapy, immunotherapy, or anti-angiogenic therapy in patients with advanced NSCLC, including SqCLC, were searched in the literature. Results of associations of potential biomarkers and outcomes were summarized. Results. Data from phase III clinical trials indicate that patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein (H-score of ≥ 200) and/or gene copy numbers of EGFR (e.g., ≥40% cells with ≥4 EGFR copies as detected by fluorescence in situ hybridization; gene amplification in ≥ 10% of analyzed cells) derive greater therapeutic benefits from EGFR-directed mAbs. Biomarker data are limited for EGFR mAbs used in combination with immunotherapy and are absent when used in combination with anti-angiogenic agents. Therapy with EGFR-directed mAbs in combination with chemotherapy is associated with greater clinical benefits in patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein and/or have increased EGFR gene copy number. These data support validating the role of these as biomarkers to identify those patients who derive the greatest clinical benefit from EGFR mAb therapy. However, data on biomarkers for EGFR-directed mAbs combined with immunotherapy or anti-angiogenic agents remain limited.

Immunohistochemical correlation of epidermal growth factor receptor and c-erbB-2 with histopathologic grading of mucoepidermoid carcinoma.

PubMed

Khiavi, Monir Moradzadeh; Vosoughhosseini, Sepideh; Saravani, Shirin; Halimi, Monireh

2012-01-01

Mucoepidermoid carcinoma is the most common salivary gland malignancy with highly variable biologic potential that correlates with the histopathologic grade of the tumor. Therefore, identification of the histopathologic grade of the mucoepidermoid carcinoma is very important in the treatment and determination of the final prognosis. The present study was performed to survey immunohistochemically Epidermal Growth Factor ReceptorEGFR and c-erbB-2 expression in different grades of mucoepidermoid carcinoma. This retrospective study included 46 formalin-fixed, paraffin-embedded blocks of mucoepidermoid carcinoma. Based on histopathologic parameters, samples were classified into three grades. Then new sections were made and stained by immunohistochemistry (IHC) method for EGFR and c-erbB-2. Finally, EGFR and c-erbB-2 expression and their correlation with histopathologic grading were statistically analyzed by ANOVA. Nineteen samples of normal salivary gland tissue were also chosen as control group. The means of EGFR and c-erbB-2 were 71%, 71%, respectively. Statistically significant correlation was found between EGFR expression and histopathologic grading of mucoepidermoid carcinoma of salivary glands (P < 0.001). There was no statistically significant correlation between histopathologic grading of salivary gland mucoepidermoid carcinoma and c-erbB-2 expression (P = 0.60). There is a parallelism between an increase in EGFR expression and increase in the histopathologic grading of salivary gland mucoepidermoid carcinoma. Therefore, the biologic behavior of salivary gland mucoepidermoid carcinoma can be determined by EGFR expression and it is a useful technique for determination of tumor grades and probably their prognosis.

δ-Catenin Increases the Stability of EGFR by Decreasing c-Cbl Interaction and Enhances EGFR/Erk1/2 Signaling in Prostate Cancer.

PubMed

Shrestha, Nensi; Shrestha, Hridaya; Ryu, Taeyong; Kim, Hangun; Simkhada, Shishli; Cho, Young-Chang; Park, So-Yeon; Cho, Sayeon; Lee, Kwang-Youl; Lee, Jae-Hyuk; Kim, Kwonseop

2018-04-30

δ-Catenin, a member of the p120-catenin subfamily of armadillo proteins, reportedly increases during the late stage of prostate cancer. Our previous study demonstrates that δ-catenin increases the stability of EGFR in prostate cancer cell lines. However, the molecular mechanism behind δ-catenin-mediated enhanced stability of EGFR was not explored. In this study, we hypothesized that δ-catenin enhances the protein stability of EGFR by inhibiting its lysosomal degradation that is mediated by c-casitas b-lineage lymphoma (c-Cbl), a RING domain E3 ligase. c-Cbl monoubiquitinates EGFR and thus facilitates its internalization, followed by lysosomal degradation. We observed that δ-catenin plays a key role in EGFR stability and downstream signaling. δ-Catenin competes with c-Cbl for EGFR binding, which results in a reduction of binding between c-Cbl and EGFR and thus decreases the ubiquitination of EGFR. This in turn increases the expression of membrane bound EGFR and enhances EGFR/Erk1/2 signaling. Our findings add a new perspective on the role of δ-catenin in enhancing EGFR/Erk1/2 signaling-mediated prostate cancer.

Substance-specific importance of EGFR for vascular smooth muscle cells motility in primary culture.

PubMed

Schreier, Barbara; Schwerdt, Gerald; Heise, Christian; Bethmann, Daniel; Rabe, Sindy; Mildenberger, Sigrid; Gekle, Michael

2016-07-01

Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations. In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed. VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Prognostic significance of epidermal growth factor receptor in surgically treated squamous cell lung cancer patients.

PubMed

Niemiec, Joanna; Kołodziejski, Leszek; Dyczek, Sonia; Gasińska, Anna

2004-01-01

Epidermal growth factor receptor (EGFR) is one of signalling pathways activated during premalignant proliferative changes in the airway epithelium. However there is no agreement about prognostic significance of EGFR expression in non-small cell lung cancer (NSCLC). Facts mentioned above prompted us to study EGFR expression in the group of 78 surgically treated squamous cell lung cancer (SqCLC) patients. The EGFR expression was visualized in formalin-fixed, paraffin-embedded sections, using immunohistochemistry. Three methods of assessment of EGFR expression were applied: percentage of cells with membranous EGFR expression--EGFR labellig index (EGFR LI), percentage of fields with membranous EGFR staining (PS%) and staining intensity (absent, weak or strong) in the whole specimen (SI). Mean EGFR LI and PS% values were 30.4 +/- 3.5% and 51.6 +/- 3.9%, respectively. Patients with higher EGFR expression (EGFR LI, PS%, SI) were significantly younger than those with low EGFR expression. EGFR LI was higher in pT3 tumours than in pT1+pT2 tumours, moreover, EGFR expression (EGFR LI, PS%, SI) was significantly higher in G1+G2 tumours than in G3 tumours. There were significant correlations between parameters used for assessment of EGFR expression. PS% < or = 50 indicated shorter disease-specific survival than PS% > 50. However, patients with tumours with both very low and very high EGFR LI (13% > or = EGFR LI > 80%) showed significantly shorter survival than those with medium EGFR LI (13% < GFR LI < or = 80%). Additionally, pTNM and pN significantly influenced patients' survival. In multivariate analysis, EGFR LI and pTNM were independent prognostic parameters influencing disease-specific survival of patients.

SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells.

PubMed

Rybak, Adrian P; Tang, Damu

2013-12-01

SOX2 is an essential transcription factor for stem cells and plays a role in tumorigenesis, however its role in prostate cancer stem cells (PCSCs) remains unclear. We report here a significant upregulation of SOX2 at both mRNA and protein levels in DU145 PCSCs propagated as suspension spheres in vitro. The expression of SOX2 in DU145 PCSCs is positively regulated by epidermal growth factor receptor (EGFR) signaling. Activation of EGFR signaling, following the addition of epidermal growth factor (EGF) or ectopic expression of a constitutively-active EGFR mutant (EGFRvIII), increased SOX2 expression and the self-renewal of DU145 PCSCs. Conversely, a small molecule EGFR inhibitor (AG1478) blocked EGFR activation, reduced SOX2 expression and inhibited PCSC self-renewal activity, implicating SOX2 in mediating EGFR-dependent self-renewal of PCSCs. In line with this notion, ectopic SOX2 expression enhanced EGF-induced self-renewal of DU145 PCSCs, while SOX2 knockdown reduced PCSC self-renewal with EGF treatment no longer capable of enhancing their propagation. Furthermore, SOX2 knockdown reduced the capacity of DU145 PCSCs to grow under anchorage-independent conditions. Finally, DU145 PCSCs generated xenograft tumors more aggressively with elevated levels of SOX2 expression compared to xenograft tumors derived from non-PCSCs. Collectively, we provide evidence that SOX2 plays a critical role in EGFR-mediated self-renewal of DU145 PCSCs. © 2013.

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

PubMed

Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

2017-08-01

Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R (epithelial-like)-derived xenograft tumors but not TE-8 (mesenchymal-like)-derived xenograft tumors. The factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.

Epidermal Growth Factor Receptor Expression Modulates Antitumor Efficacy of Vandetanib or Cediranib Combined With Radiotherapy in Human Glioblastoma Xenografts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wachsberger, Phyllis R., E-mail: Phyllis.wachsberger@jeffersonhospital.org; Lawrence, Yaacov R.; Liu Yi

2012-01-01

Purpose: The purpose of this study was to determine the ability of radiation therapy (RT) combined with the tyrosine kinase inhibitors (TKI) vandetanib (antiepidermal growth factor receptor [EGFR] plus antivascular endothelial growth factor receptor [anti-VEGFR]) and cediranib (anti-VEGFR) to inhibit glioblastoma multiforme (GBM) growth. A secondary aim was to investigate how this regimen is modulated by tumor EGFR expression. Methods and Materials: Radiosensitivity was assessed by clonogenic cell survival assay. VEGF secretion was quantified by enzyme-linked immunosorbent assay. GBM (U87MG wild-type EGFR [wtEGFR] and U87MG EGFR-null) xenografts were treated with vandetanib, cediranib, and RT, alone or in combinations. Excised tumormore » sections were stained for proliferative and survival biomarkers. Results: In vitro, U87MG wtEGFR and U87 EGFR-null cells had similar growth kinetics. Neither TKI affected clonogenic cell survival following RT. However, in vivo, exogenous overexpression of wtEGFR decreased tumor doubling time (T2x) in U87MG xenografts (2.70 vs. 4.41 days for U87MG wtEGFR vs. U87MG vector, respectively). In U87MG EGFR-null cells, TKI combined with radiation was no better than radiation therapy alone. In U87MG wtEGFR, RT in combination with vandetanib (but not with cediranib) significantly increased tumor T2x compared with RT alone (T2x, 10.4 days vs. 4.8 days; p < 0.001). In vivo, growth delay correlated with suppression of pAkt, survivin, and Ki67 expression in tumor samples. The presence of EGFR augmented RT-stimulated VEGF release; this effect was inhibited by vandetanib. Conclusions: EGFR expression promoted tumor growth in vivo but not in vitro, suggesting a microenvironmental effect. GBM xenografts expressing EGFR exhibited greater sensitivity to both cediranib and vandetanib than EGFR-null tumors. Hence EGFR status plays a major role in determining a tumor's in vivo response to radiation combined with TKI, supporting a 'personalized' approach to GBM management.« less

Sensitization of cerebral tissue in nude mice with photodynamic therapy induces ADAM17/TACE and promotes glioma cell invasion

PubMed Central

Zheng, Xuguang; Jiang, Feng; Katakowski, Mark; Zhang, Xuepeng; Jiang, Hao; Zhang, Zheng Gang; Chopp, Michael

2008-01-01

In the present study, we tested the hypothesis that a mild cerebral tissue injury promotes subsequent glioma invasion via activation of the ADAM17-EGFR-PI3K-Akt pathway. Mild injury was induced by Photodynamic therapy (PDT), which employs tissue-penetrating laser light exposure following systemic administration of a tumor-localizing photosensitizer. Athymic nude mice were treated with sublethal PDT (80J/cm2 with 2mg/kg Photofrin). Hypoxic stress and ADAM17-EGFR-PI3K-Akt were measured using Western blot and immunostaining. Additional groups with/without pro-sublethal PDT were subsequently implanted with U87 glioma tumor cell. Tumor invasion and ADAM17-EGFR-PI3K-Akt pathway in tumor area were measured. After a sublethal dose of PDT, HIF-1α expression was increased by a factor of three in PDT-treated normal brain tissue compared to contralateral control brain tissue. PDT-treated brain tissue exhibited a significant increase in ADAM17, p-EGFR, p-Akt expression compared to non-treated tissue. ADAM17 positive area significantly increased from 1.78% to 10.89%. The percentage of p-EGFR and p-Akt positive cells significantly increased from 9.50% and 14.50% to 21.31% and 32.29%,respectively, PDT treatment significantly increased subsequent implanted U87 glioma cell invasion by 3.68-fold and increased ADAM17, EGFR, p-EGFR, Akt, p-Akt expression by 178%, 43.9%,152.7%, 89.6%,and 164.2%, respectively, compared to control group. Our data showed that a sublethal sensitization of cerebral tissue with PDT significantly increased U87 cell invasion in nude mice, and that glioma cell invasion is highly correlated with activation of the ADAM17-EGFR-PI3K-Akt pathway (r=0.928, 0.775, 0.870, 0.872, and 0.883, respectively), most likely via HIF-1α. PMID:18358600

Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas

PubMed Central

Goyal, Amit; Gonda, David; Akers, Johnny; Adhikari, Bandita; Patel, Kunal; Vandenberg, Scott; Yan, Wei; Bao, Zhaoshi; Carter, Bob S.; Wang, Renzhi; Mao, Ying; Jiang, Tao; Chen, Clark C.

2014-01-01

The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients. PMID:25277177

Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

PubMed

Li, Jie; Taich, Zachary J; Goyal, Amit; Gonda, David; Akers, Johnny; Adhikari, Bandita; Patel, Kunal; Vandenberg, Scott; Yan, Wei; Bao, Zhaoshi; Carter, Bob S; Wang, Renzhi; Mao, Ying; Jiang, Tao; Chen, Clark C

2014-09-15

The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

THE SIGNIFICANCE OF EPIDERMAL GROWTH FACTOR RECEPTOR AND SURVIVIN EXPRESSION IN BLADDER CANCER TISSUE AND URINE CYTOLOGY OF PATIENTS WITH TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER.

PubMed

Kehinde, E O; Al-Maghrebi, M; Anim, J T; Kapila, K; George, S S; Al-Juwaiser, A; Memon, A

2013-01-01

To assess whether epidermal growth factor receptor (EGFR) and survivin immunostaining of tumour cells in urinary cytology and tissue of patients with bladder cancer has a prognostic significance. Prospective study Department of Surgery (Division of Urology), Mubarak Al-Kabeer Teaching Hospital and Faculty of Medicine, Kuwait University, Kuwait Urine cytology smears obtainedpriorto cystoscopy in patients with transitional cell carcinoma (TCC) of the bladder were immunostained for EGFR and survivin. Bladder cancer tissue resected at surgery was also immunostained for EGFR and survivin expression. Tissue expression of EGFR and survivin in TCC of the bladder was compared to their expression in urine cytology and relationship to tumour grade and stage. 178 patients were studied (43 newly diagnosed bladder cancer, 58 with recurrent TCC and 77 in disease remission). Twenty five patients with normal urothelium served as controls. The mean sensitivity of urine cytology, tissue survivin immunohistochemistry (IHC) and tissue EGFR IHC was 30.5%, 62% and 59% respectively. The corresponding mean specificity was 95%, 79% and 38% respectively. For grades 1, 2 and 3 bladder tumors, tissue expression positivity for EGFR was 47.8%, 92.9%, 100% and for tissue survivin it was 27.8%, 18.2% and 33.3% respectively. For grades 1, 2 and 3 bladder tumors, urine expression positivity for EGFR was 35.7%, 40% and 67.7% and for urine survivin it was 8.3%, 42.9% and 33.3% respectively. Positive EGFR immunostaining of urine cytology specimen or tumour tissue increases with histological grade of TCC of the bladder. Survivin expression is less consistent in both urine cytology specimen and tissue samples. EGFR immunostaining may provide a useful tool in the grading of bladder TCC and aid in the selection of patients that may benefit from administration of EGFR inhibitors.

Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

PubMed Central

Tseng, Shih-Heng; Chou, Min-Yuan; Chu, I-Ming

2015-01-01

We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24 hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells. PMID:26056447

Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors

PubMed Central

Akbay, Esra A; Koyama, Shohei; Carretero, Julian; Altabef, Abigail; Tchaicha, Jeremy H; Christensen, Camilla L; Mikse, Oliver R; Cherniack, Andrew D; Beauchamp, Ellen M; Pugh, Trevor J; Wilkerson, Matthew D; Fecci, Peter E; Butaney, Mohit; Reibel, Jacob B; Soucheray, Margaret; Cohoon, Travis J; Janne, Pasi A; Meyerson, Matthew; Hayes, D. Neil; Shapiro, Geoffrey I; Shimamura, Takeshi; Sholl, Lynette M; Rodig, Scott J; Freeman, Gordon J; Hammerman, Peter S; Dranoff, Glenn; Wong, Kwok-Kin

2013-01-01

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition. PMID:24078774

Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder.

PubMed

Hashmi, Atif Ali; Hussain, Zubaida Fida; Irfan, Muhammad; Khan, Erum Yousuf; Faridi, Naveen; Naqvi, Hanna; Khan, Amir; Edhi, Muhammad Muzzammil

2018-06-07

Epidermal growth factor receptor (EGFR) has been shown to have abnormal expression in many human cancers and is considered as a marker of poor prognosis. Frequency of over expression in bladder cancer has not been studied in our population; therefore we aimed to evaluate the frequency and prognostic significance of EGFR immunohistochemical expression in locoregional population. We performed EGFR immunohistochemistry on 126 cases of bladder cancer and association of EGFR expression with tumor grade, lamina propria invasion, deep muscle invasion and recurrence of disease was evaluated. High EGFR expression was noted in 26.2% (33 cases), 15.1% (19 cases) and 58.7% (74 cases) revealed low and no EGFR expression respectively. Significant association of EGFR expression was noted with tumor grade, lamina propria invasion, deep muscle invasion and recurrence status while no significant association was seen with age, gender and overall survival. Kaplan- Meier curves revealed significant association of EGFR expression with recurrence while no significant association was seen with overall survival. Significant association of EGFR overexpression with tumor grade, muscularis propria invasion and recurrence signifies its prognostic value; therefore EGFR can be used as a prognostic biomarker in Urothelial bladder carcinoma.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer

PubMed Central

Alvarez-Cubero, Maria J.; Nadal, Rosa; Sanchez-Rovira, Pedro; Salido, Marta; Rodríguez, María; García-Puche, Jose L.; Delgado-Rodriguez, Miguel; Solé, Francisco; García, Maria A.; Perán, Macarena; Rosell, Rafael; Marchal, Juan A.; Lorente, Jose A.

2014-01-01

Circulating tumor cells (CTCs) are frequently associated with epithelialmesenchymal transition (EMT). The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer. PMID:25277187

Differential effect of EGFR inhibitors on tamoxifen-resistant breast cancer cells.

PubMed

Kim, Sangmin; Lee, Jeongmin; Oh, Soo Jin; Nam, Seok Jin; Lee, Jeong Eon

2015-09-01

Although tamoxifen is the most common and effective therapy for treatment of estrogen receptor-α (ER-α) breast cancer patients, resistance of endocrine therapy occurs, either de novo or acquired during therapy. Here, we investigated the clinical value of epidermal growth factor receptor (EGFR) in tamoxifen-resistant (TamR) patients and the differential effect of EGFR inhibitors, neratinib and gefitinib, on TamR breast cancer cell model. The morphology of TamR MCF7 cells showed mesenchymal phenotypes and did not induce cell death by tamoxifen treatment compared with tamoxifen‑sensitive (TamS) MCF7 cells. In addition, mesenchymal marker proteins, including N-cadherin (N-cad), fibronectin (FN), and Slug, significantly increased in TamR cells. In contrast, ER-α and E-cadherin (E-cad) were greatly decreased. We also found that the levels of EGFR and HER2 expression were increased in TamR cells. Furthermore, we observed that EGFR expression was directly involved with poor prognosis of tamoxifen-treated breast cancer patients using the GSE1378 date set. Thus, we treated TamR and TamS cells with EGFR inhibitors, neratinib and gefitinib, respectively. Interestingly, neratinib induced apoptotic cell death of TamR but not gefitinib. Cleaved PARP-1 expression was also increased by neratinib treatment in TamR cells. Therefore, we suggest that neratinib may be a potential therapeutic drug for treating TamR breast cancer.

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

PubMed

Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer

2011-12-01

Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival ofmore » wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.« less

Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells.

PubMed

Chang, Zhi-Gang; Wei, Jun-Min; Qin, Chang-Fu; Hao, Kun; Tian, Xiao-Dong; Xie, Kun; Xie, Xue-Hai; Yang, Yin-Mo

2012-05-01

Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes. After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly. Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

PD-L1 expression according to the EGFR status in primary lung adenocarcinoma.

PubMed

Takada, Kazuki; Toyokawa, Gouji; Tagawa, Tetsuzo; Kohashi, Kenichi; Shimokawa, Mototsugu; Akamine, Takaki; Takamori, Shinkichi; Hirai, Fumihiko; Shoji, Fumihiro; Okamoto, Tatsuro; Oda, Yoshinao; Maehara, Yoshihiko

2018-02-01

It was reported that programmed cell death-ligand 1 (PD-L1) expression is associated with smoking and wild-type epidermal growth factor receptor (EGFR) in lung adenocarcinoma. However, the association between PD-L1 expression and EGFR mutation site in EGFR mutation-positive lung adenocarcinoma is unclear. We retrospectively examined the relationship between PD-L1 expression and EGFR status in 441 surgically resected primary lung adenocarcinomas. Membrane PD-L1 expression on tumor cells was evaluated by immunohistochemical analysis using a PD-L1 antibody (clone SP142) and defined by tumor proportion scores (TPSs) of 0%, 1-4%, 5-49%, and ≥50%, respectively. Two hundred and eighteen (49.4%) patients had wild-type EGFR, and 223 (50.6%) had mutant EGFR-98 (44.0%) with exon 19 deletion, 116 (52.0%) with exon 21 L858R point mutation, and nine (4.0%) with another EGFR mutation. Overall, Fisher's exact test showed that PD-L1 positivity was associated with wild-type EGFR, and there was only one case with PD-L1 TPS ≥50% among the cases with mutant EGFR. The analysis of cases with mutant EGFR indicated no significant association between EGFR mutation site and PD-L1 expression. However, the prevalence of PD-L1 TPS 5-49% was higher among patients with EGFR exon 19 deletion than with EGFR exon 21 L858R point mutation. PD-L1 expression was significantly associated with wild-type EGFR, and PD-L1 TPS ≥50% seldom overlaps with presence of driver oncogene EGFR. There was no significant difference in PD-L1 expression among the EGFR mutation sites. Copyright © 2017 Elsevier B.V. All rights reserved.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

PubMed Central

Wang, Lihong; Cao, Hailong; Lu, Ning; Liu, Liping; Wang, Bangmao; Hu, Tianhui; Israel, Dawn A.; Peek, Richard M.; Polk, D. Brent; Yan, Fang

2013-01-01

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells. PMID:23457600

The importance of immunohistochemical expression of EGFr in squamous cell carcinoma of the oral cavity treated with surgery and postoperative radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smid, Ernst J.; Stoter, T. Rianne; Bloemena, Elisabeth

2006-08-01

Purpose: The aim of this study was to investigate the prognostic significance of epidermal growth factor (EGFr) expression in oral cavity squamous cell carcinoma (OCSCC) treated with curative surgery and postoperative radiotherapy. Methods and Materials: This retrospective study included 165 OCSCC patients. The expression of EGFr was assessed on paraffin-embedded tissue of the primary tumor by immunohistochemistry using a monoclonal antibody directed against EGFr. Intensity of the EGFr expression was scored by two authors blinded for the clinical outcome. Results: In the univariate analysis, locoregional control at 3 years (LRC) in the EGFr-negative cases was 69% compared with 77% inmore » the EGFr-positive cases (p 0.22). In the multivariate analysis for local control, a significant interaction was found between EGFr and overall treatment time of radiation (OTT). After stratification for EGFr expression, the OTT was of no importance in the EGFr-negative cases, whereas a significant difference in LRC was found in the EGFr-positive cases, in which the LRC after 3 years was 69% and 94% in case of an OTT of 0-42 days and >42 days, respectively (p = 0.009; hazard ratio = 3.42; 95% confidence interval, 1.28-8.96). No significant association was found between EGFr expression and overall survival. Conclusions: In the present study, no association was found between EGFr expression and outcome regarding locoregional control and overall survival. However, the results of the present study suggest that patients with squamous cell carcinoma of the oral cavity with high EGFr expression benefit more from a reduction of the overall treatment time of postoperative radiation than those with low EGFr expression.« less

Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme

PubMed Central

Mokri, Poroshista; Lamp, Nora; Linnebacher, Michael; Classen, Carl Friedrich; Erbersdobler, Andreas; Schneider, Björn

2017-01-01

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies. PMID:28934307

A role for the epidermal growth factor receptor signaling in development of intestinal serrated polyps in mice and humans.

PubMed

Bongers, Gerold; Muniz, Luciana R; Pacer, Michelle E; Iuga, Alina C; Thirunarayanan, Nanthakumar; Slinger, Erik; Smit, Martine J; Reddy, E Premkumar; Mayer, Lloyd; Furtado, Glaucia C; Harpaz, Noam; Lira, Sergio A

2012-09-01

Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

ROLE OF GRB2-ASSOCIATED BINDER 1 (GAB1) IN EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)-INDUCED SIGNALING IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

PubMed Central

Hoeben, A.; Martin, D.; Clement, P. M.; Cools, J.; Gutkind, J. S.

2012-01-01

The Epidermal Growth Factor Receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents. PMID:22865653

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as amore » plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.« less

Ventilation-induced increases in EGFR ligand mRNA are not altered by intra-amniotic LPS or ureaplasma in preterm lambs.

PubMed

Hillman, Noah H; Gisslen, Tate; Polglase, Graeme R; Kallapur, Suhas G; Jobe, Alan H

2014-01-01

Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.

The expression of epidermal growth factor (EGF) and its receptor (EGFR) during post-natal testes development in the yak.

PubMed

Pan, Y; Cui, Y; Yu, S; Zhang, Q; Fan, J; Abdul Rasheed, B; Yang, K

2014-12-01

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24 months old. Results of RT-PCR and WB showed that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post-natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6 months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis. © 2014 Blackwell Verlag GmbH.

Balancing Selectivity and Efficacy of Bispecific Epidermal Growth Factor Receptor (EGFR) × c-MET Antibodies and Antibody-Drug Conjugates*

PubMed Central

Sellmann, Carolin; Doerner, Achim; Knuehl, Christine; Rasche, Nicolas; Sood, Vanita; Krah, Simon; Rhiel, Laura; Messemer, Annika; Wesolowski, John; Schuette, Mark; Becker, Stefan; Toleikis, Lars; Kolmar, Harald; Hock, Bjoern

2016-01-01

Bispecific antibodies (bsAbs) and antibody-drug conjugates (ADCs) have already demonstrated benefits for the treatment of cancer in several clinical studies, showing improved drug selectivity and efficacy. In particular, simultaneous targeting of prominent cancer antigens, such as EGF receptor (EGFR) and c-MET, by bsAbs has raised increasing interest for potentially circumventing receptor cross-talk and c-MET-mediated acquired resistance during anti-EGFR monotherapy. In this study, we combined the selectivity of EGFR × c-MET bsAbs with the potency of cytotoxic agents via bispecific antibody-toxin conjugation. Affinity-attenuated bispecific EGFR × c-MET antibody-drug conjugates demonstrated high in vitro selectivity toward tumor cells overexpressing both antigens and potent anti-tumor efficacy. Due to basal EGFR expression in the skin, ADCs targeting EGFR in general warrant early safety assessments. Reduction in EGFR affinity led to decreased toxicity in keratinocytes. Thus, the combination of bsAb affinity engineering with the concept of toxin conjugation may be a viable route to improve the safety profile of ADCs targeting ubiquitously expressed antigens. PMID:27694443

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Overexpression of YAP1 in EGFR mutant lung adenocarcinoma prior to tyrosine kinase inhibitor therapy is associated with poor survival.

PubMed

Hong, Soon Auck; Jang, Si-Hyong; Oh, Mee-Hye; Kim, Sung Joon; Kang, Jin-Hyung; Hong, Sook-Hee

2018-03-01

EGFR tyrosine kinase inhibitor (EGFR TKI) is approved as first-line treatment for advanced-stage EGFR mutant lung adenocarcinoma (LADC). Yes-associated protein 1 (YAP1), a main effector of the Hippo pathway, is associated with adverse prognosis and disruption of EGFR TKI modulation of non-small cell lung cancer. In this study, we demonstrated a prognostic role of YAP1 in EGFR mutant LADC and efficacy of EGFR TKI therapy. A total of 63 patients, including 41 with paired lung cancer specimens before and after EGFR TKI therapy and 22 with non-paired lung cancer specimens prior to EGFR TKI, were enrolled for examination. Expression of YAP1 protein was evaluated using immunohistochemistry. Fifteen paired cases (36.6%) with high nuclear YAP1 expression were detected in the pre-EGFR TKI LADC group and 21 paired cases (52.5%) after treatment with EGFR TKI. Nuclear YAP1 expression was significantly upregulated after EGFR TKI therapy (P = .002). Fifteen paired cases with high nuclear YAP1 expression before EGFR TKI LADCs showed poorer overall survival (OS) (P = .023) and progression-free survival (PFS) (P = .041). Among the 63 patients under study, those with high nuclear YAP1 expression before EGFR TKI showed shorter OS (P = .038) and PFS (P < .001). High nuclear YAP1 expression in cases with acquired EGFR exon 20 T790 M mutant LADCs showed poorer OS (P < .001). We demonstrated that YAP1 burden before clinical application of EGFR TKI plays a crucial role in prognosis of EGFR mutant LADC treated using EGFR TKI. Copyright © 2018 Elsevier GmbH. All rights reserved.

Improved therapeutic effectiveness by combining recombinant p14(ARF) with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma.

PubMed

Liu, Feng; Du, JinTao; Xian, Junming; Liu, Yafeng; Liu, Shixi; Lin, Yan

2015-01-01

The tumor suppressor p14(ARF) and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14(ARF) with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. After human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14(ARF) and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14(ARF) and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. Ad-p14(ARF) combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14(ARF) and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14(ARF) protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14(ARF) was combined with Ad-antisense EGFR. Besides, Ad-p14(ARF) plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14(ARF) or Ad-antisense EGFR alone. Combination Ad-p14(ARF) with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided. Copyright © 2015 Elsevier Inc. All rights reserved.

Trefoil factors: Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

PubMed Central

Kosriwong, Kanuengnuch; Menheniott, Trevelyan R; Giraud, Andrew S; Jearanaikoon, Patcharee; Sripa, Banchob; Limpaiboon, Temduang

2011-01-01

AIM: To investigate trefoil factor (TFF) gene copy number, mRNA and protein expression as potential biomarkers in cholangiocarcinoma (CCA). METHODS: TFF mRNA levels, gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction (PCR), quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA, precancerous bile duct dysplasia and from disease-free controls. The functional impact of recombinant human (rh)TFF2 peptide treatment on proliferation and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling was assessed in the CCA cell line, KMBC, by viable cell counting and immunoblotting, respectively. RESULTS: TFF1, TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls, and was unrelated to gene copy number. TFF1 immunoreactivity was strongly increased in both dysplasia and CCA, whereas TFF2 immunoreactivity was increased only in CCA compared to disease-free controls. By contrast, TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA. Kaplan-Meier analysis found no association of TFF mRNA, protein and copy number with age, gender, histological subtype, and patient survival time. Treatment of KMBC cells with rhTFF2 stimulated proliferation, triggered phosphorylation of EGFR and downstream extracellular signal related kinase (ERK), whereas co-incubation with the EGFR tyrosine kinase inhibitor, PD153035, blocked rhTFF2-dependent proliferation and EGFR/ERK responses. CONCLUSION: TFF mRNA/protein expression is indicative of CCA tumor progression, but not predictive for histological sub-type or survival time. TFF2 is mitogenic in CCA via EGFR/MAPK activation. PMID:21472131

NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobhakumari, Arya; Department of Pathology, The University of Iowa, Iowa City, IA; Schickling, Brandon M.

2013-11-01

Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formationmore » in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy.« less

Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.

PubMed

Yun, Sumi; Kwak, Yoonjin; Nam, Soo Kyung; Seo, An Na; Oh, Heung-Kwon; Kim, Duck-Woo; Kang, Sung-Bum; Lee, Hye Seung

2018-01-17

Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients' survival with CRC. The expression of EGFR ligands, including heparin binding epidermal growth factor like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS, p=0.018) and overall survival (OS, p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

PubMed Central

Zhou, Zhen Ni; Sharma, Ved P.; Beaty, Brian T.; Roh-Johnson, Minna; Peterson, Esther A.; Van Rooijen, Nico; Kenny, Paraic A.; Wiley, H. Steven; Condeelis, John S.; Segall, Jeffrey E.

2013-01-01

Increased expression of HBEGF in ER negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of CSF-1 while the tumor associated macrophages (TAMs) in turn aid in tumor cell invasion by secreting EGF. To determine how the autocrine expression of EGFR ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We find that autocrine HBEGF expression enhanced in vivo intravasation and metastasis, and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of MMP2 and MMP9 expression. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling. PMID:24013225

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo.

PubMed

Zhou, Z N; Sharma, V P; Beaty, B T; Roh-Johnson, M; Peterson, E A; Van Rooijen, N; Kenny, P A; Wiley, H S; Condeelis, J S; Segall, J E

2014-07-17

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

Interferon-γ alters downstream signaling originating from epidermal growth factor receptor in intestinal epithelial cells: functional consequences for ion transport.

PubMed

Paul, Gisela; Marchelletta, Ronald R; McCole, Declan F; Barrett, Kim E

2012-01-13

The epidermal growth factor receptor (EGFr) regulates many cellular functions, such as proliferation, apoptosis, and ion transport. Our aim was to investigate whether long term treatment with interferon-γ (IFN-γ) modulates EGF activation of downstream signaling pathways in intestinal epithelial cells and if this contributes to dysregulation of epithelial ion transport in inflammation. Polarized monolayers of T(84) and HT29/cl.19A colonocytes were preincubated with IFN-γ prior to stimulation with EGF. Basolateral potassium transport was studied in Ussing chambers. We also studied inflamed colonic mucosae from C57BL/6 mice treated with dextran sulfate sodium or mdr1a knock-out mice and controls. IFN-γ increased intestinal epithelial EGFr expression without increasing its phosphorylation. Conversely, IFN-γ caused a significant decrease in EGF-stimulated phosphorylation of specific EGFr tyrosine residues and activation of ERK but not Akt-1. In IFNγ-pretreated cells, the inhibitory effect of EGF on carbachol-stimulated K(+) channel activity was lost. In inflamed colonic tissues, EGFr expression was significantly increased, whereas ERK phosphorylation was reduced. Thus, although it up-regulates EGFr expression, IFN-γ causes defective EGFr activation in colonic epithelial cells via reduced phosphorylation of specific EGFr tyrosine residues. This probably accounts for altered downstream signaling consequences. These observations were corroborated in the setting of colitis. IFN-γ also abrogates the ability of EGF to inhibit carbachol-stimulated basolateral K(+) currents. Our data suggest that, in the setting of inflammation, the biological effect of EGF, including the inhibitory effect of EGF on Ca(2+)-dependent ion transport, is altered, perhaps contributing to diarrheal and other symptoms in vivo.

Comparative prognostic value of epidermal growth factor quantitative protein expression compared with FISH for head and neck squamous cell carcinoma.

PubMed

Pectasides, Eirini; Rampias, Theodore; Kountourakis, Panteleimon; Sasaki, Clarence; Kowalski, Diane; Fountzilas, George; Zaramboukas, Thomas; Rimm, David; Burtness, Barbara; Psyrri, Amanda

2011-05-01

Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome. A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis. Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047). EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information. ©2011 AACR.

MUC4 modulates human glioblastoma cell proliferation and invasion by upregulating EGFR expression.

PubMed

Li, Weihua; Wu, Chunming; Yao, Yiqun; Dong, Bin; Wei, Zhenqing; Lv, Xiupeng; Zhang, Jian; Xu, Yinghui

2014-04-30

Glioblastoma (GBM), the most common primary brain tumor, is the leading cause of deaths related to tumors in the central nervous system. The prognosis of GBM patients is currently poor, and the mechanisms underlying GBM genesis remain unclear. The expression of MUC4, a high-molecular-weight and highly glycosylated protein, has been studied in many cancers. However, information on MUC4 expression in GBM is limited. In this study, we found that MUC4 was overexpressed in GBM cell lines and tissues. The proliferation and invasive potential of GBM cells were significantly increased by the ectopic expression of MUC4. By contrast, RNA interference targeting MUC4 in GBM cells significantly decreased the proliferation and invasive potential of GBM cells. We also found that the expression of epidermal growth factor receptor (EGFR) was modulated by MUC4. EGFR inhibition by siRNA reversed the MUC4-induced proliferation and invasion. These results indicated that MUC4 expression in GBM was important in GBM cell proliferation and invasion, which may be partly associated with EGFR overexpression. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

SIAH and EGFR, Two RAS Pathway Biomarkers, are Highly Prognostic in Locally Advanced and Metastatic Breast Cancer.

PubMed

van Reesema, Lauren L Siewertsz; Zheleva, Vasilena; Winston, Janet S; Jansen, Rick J; O'Connor, Carolyn F; Isbell, Andrew J; Bian, Minglei; Qin, Rui; Bassett, Patricia T; Hinson, Virginia J; Dorsch, Kimberly A; Kirby, Brad W; Van Sciver, Robert E; Tang-Tan, Angela M; Harden, Elizabeth A; Chang, David Z; Allen, Cynthia A; Perry, Roger R; Hoefer, Richard A; Tang, Amy H

2016-09-01

Metastatic breast cancer exhibits diverse and rapidly evolving intra- and inter-tumor heterogeneity. Patients with similar clinical presentations often display distinct tumor responses to standard of care (SOC) therapies. Genome landscape studies indicate that EGFR/HER2/RAS "pathway" activation is highly prevalent in malignant breast cancers. The identification of therapy-responsive and prognostic biomarkers is paramount important to stratify patients and guide therapies in clinical oncology and personalized medicine. In this study, we analyzed matched pairs of tumor specimens collected from 182 patients who received neoadjuvant systemic therapies (NST). Statistical analyses were conducted to determine whether EGFR/HER2/RAS pathway biomarkers and clinicopathological predictors, alone and in combination, are prognostic in breast cancer. SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two logical, sensitive and prognostic biomarkers in metastatic breast cancer. We found that increased SIAH and EGFR expression correlated with advanced pathological stage and aggressive molecular subtypes. Both SIAH expression post-NST and NST-induced changes in EGFR expression in invasive mammary tumors are associated with tumor regression and increased survival, whereas ER, PR, and HER2 were not. These results suggest that SIAH and EGFR are two prognostic biomarkers in breast cancer with lymph node metastases. The discovery of incorporating tumor heterogeneity-independent and growth-sensitive RAS pathway biomarkers, SIAH and EGFR, whose altered expression can be used to estimate therapeutic efficacy, detect emergence of resistant clones, forecast tumor regression, differentiate among partial responders, and predict patient survival in the neoadjuvant setting, has a clear clinical implication in personalizing breast cancer therapy. This work was supported by the Dorothy G. Hoefer Foundation for Breast Cancer Research (A.H. Tang); Center for Innovative Technology (CIT)-Commonwealth Research Commercialization Fund (CRCF) (MF14S-009-LS to A.H. Tang), and National Cancer Institute (CA140550 to A.H. Tang). Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer 89Zr-imgatuzumab.

PubMed

Pool, Martin; Kol, Arjan; Lub-de Hooge, Marjolijn N; Gerdes, Christian A; de Jong, Steven; de Vries, Elisabeth G E; Terwisscha van Scheltinga, Anton G T

2016-10-18

Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89Zr-imgatuzumab, co-injected with equal doses 111In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89Zr-imgatuzumab dose. High 89Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

Erlotinib for Patients with EGFR Wild-Type Metastatic NSCLC: a Retrospective Biomarkers Analysis.

PubMed

Inno, Alessandro; Di Noia, Vincenzo; Martini, Maurizio; D'Argento, Ettore; Di Salvatore, Mariantonietta; Arena, Vincenzo; Schinzari, Giovanni; Orlandi, Armando; Larocca, Luigi Maria; Cassano, Alessandra; Barone, Carlo

2018-03-20

Erlotinib is approved for the treatment of patients with EGFR mutation positive, metastatic NSCLC. It is also approved as second/third line therapy for EGFR mutation negative patients, but in this setting the benefit of erlotinib is modest and there is no validated biomarker for selecting EGFR wild-type patients who may benefit the most from the treatment. We retrospectively assessed EGFR and K-RAS mutational status, and EGFR, c-MET and IGF1-R expression in tumor samples of 72 patients with metastatic NSCLC treated with erlotinib after at least one prior line of chemotherapy, from 2008 to 2012. We analyzed the association between biomarkers and outcome (RR, PFS, and OS). EGFR mutated patients achieved a better RR (56% vs 8%, p = .002), PFS (10 vs 3 months, HR 0.53, p = 0.48) and OS (20 vs 6 months, HR 0.55, p = .07), compared to EGFR wild-type patients. Among 63 EGFR wild-type patients, those with EGFR high-expression had a better outcome in terms of RR (40% vs 2%, p = .002), PFS (7.5 vs 2 months, HR 0.45, p = .007) and OS (30 vs 5 months, HR 0.34, p < .001) compared to patients with EGFR intermediate or low/negative-expression. IGF1-R expression, c-MET expression and K-RAS mutational status did not significantly affect the outcome; however, no patients with K-RAS mutation or c-MET high-expression achieved an objective response. In patients with metastatic, chemo-refractory EGFR wild-type NSCLC, EGFR high-expression may represent a positive predictor of activity for erlotinib, whereas K-RAS mutation and c-MET high-expression may predict lack of activity. These findings deserve further prospective evaluation.

Epiregulin (EREG) is upregulated through an IL-1β autocrine loop in Caco-2 epithelial cells with reduced CFTR function.

PubMed

Massip-Copiz, Macarena; Clauzure, Mariángeles; Valdivieso, Ángel G; Santa-Coloma, Tomás A

2018-03-01

CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl - concentration, which in turn produces an increased interleukin-1β (IL-1β) secretion. The secreted IL-1β then induces an autocrine positive feedback loop, further stimulating IL-1β priming and secretion. Since IL-1β can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1β and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1β loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1β autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl - , and IL-1β. © 2017 Wiley Periodicals, Inc.

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

PubMed

Chandler, L A; Sosnowski, B A; McDonald, J R; Price, J E; Aukerman, S L; Baird, A; Pierce, G F; Houston, L L

1998-09-25

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.

2010-05-15

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK amongmore » these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.« less

High expression of GPER1, EGFR and CXCR1 is associated with lymph node metastasis in papillary thyroid carcinoma.

PubMed

Tang, Cui; Yang, Lei; Wang, Ni; Li, Li; Xu, Man; Chen, George G; Liu, Zhi-Min

2014-01-01

Clinical and epidemiological studies have shown that estrogen may be involved in the development and progression of papillary thyroid carcinoma (PTC). G protein-coupled estrogen receptor 1 (GPER1) is a novel seven-transmembrane estrogen receptor that functions alongside traditional nuclear estrogen receptors (ERs) to regulate the cellular responses to estrogen. The purpose of this study was to examine GPER1, EGFR and CXCR1 expression in PTC and to assess the association of their expression with clinicopathological indicators. GPER1, EGFR and CXCR1 protein expression in 129 PTCs, 61 nodular hyperplasia and 118 normal thyroid tissue specimens were analyzed using immunohistochemistry. The protein expression levels of these three molecules were up-regulated in PTCs. High protein expression of GPER1, EGFR and CXCR1 was significantly correlated with lymph node metastasis (LNM) (P ≤ 0.001). Furthermore, GPER1, EGFR and CXCR1 protein expression were correlated with one another. Concomitant high expression of these molecules had stronger correlation with LNM than did each alone (P = 0.002 for GPER1/EGFR, P = 0.013 for GPER1/CXCR1, P = 0.018 for EGFR/CXCR1 and P < 0.001 for GPER1/EGFR/CXCR1). Additionally, GPER1, EGFR and CXCR1 mRNA expression was assessed in 30 PTCs, 10 nodular hyperplasia and 10 normal thyroid tissue specimens using real-time RT-PCR. GPER1, EGFR and CXCR1 mRNA expression levels were up-regulated in PTCs, and high mRNA expression of GPER1, EGFR and CXCR1 was significantly correlated with LNM (P < 0.001 for all these three molecules). These results demonstrated that the evaluation of GPER1, EGFR and CXCR1 expression in PTC may be useful in predicting the risk of LNM.

COX-2/EGFR expression and survival among women with adenocarcinoma of the lung

PubMed Central

Van Dyke, Alison L.; Cote, Michele L.; Prysak, Geoffrey M.; Claeys, Gina B.; Wenzlaff, Angie S.; Murphy, Valerie C.; Lonardo, Fulvio; Schwartz, Ann G.

2008-01-01

Previous studies suggest that cyclooxygenase-2 (COX-2) expression may predict survival among patients with non-small cell lung cancer. COX-2 may interact with epidermal growth factor receptor (EGFR), suggesting that combined COX-2/EGFR expression may provide predictive value. The extent to which their independent or combined expression is associated with prognosis in women with adenocarcinoma of the lung is unknown. In the present study, we examined relationships between COX-2 expression (n = 238), EGFR expression (n = 158) and dual COX-2/EGFR expression (n = 157) and survival among women with adenocarcinoma of the lung. Overall survival was estimated by constructing Cox proportional hazards models adjusting for other significant variables and stratifying by stage at diagnosis and race. Clinical or demographic parameters were not associated with either COX-2 or EGFR expression. Patients with COX-2-positive tumors tended to have poorer prognosis than did patients with COX-2-negative tumors [hazard ratio (HR) 1.67, 95% confidence interval (CI) 1.01–2.78]. African-Americans with COX-2-positive tumors had a statistically non-significant higher risk of death than African-Americans with COX-2-negative tumors (HR 5.58, 95% CI 0.64–48.37). No association between COX-2 expression and survival was observed among Caucasians (HR 1.29, 95% CI 0.72–2.30). EGFR expression was associated with a 44% reduction in the risk of death (HR 0.56, 95% CI 0.32–0.98). COX-2−/EGFR+ tumor expression, but not COX-2+/EGFR+ tumor expression, was associated with survival when compared with other combined expression results. In conclusion, COX-2 and EGFR expression, but not combined COX-2+/EGFR+ expression, independently predict survival of women with adenocarcinoma of the lung. PMID:18453539

Localization and distribution of neurons that co-express xeroderma pigmentosum-A and epidermal growth factor receptor within Rosenthal's canal.

PubMed

Guthrie, O'neil W

2015-10-01

Xeroderma pigmentosum-A (XPA) is a C4-type zinc-finger scaffolding protein that regulates the removal of bulky-helix distorting DNA damage products from the genome. Phosphorylation of serine residues within the XPA protein is associated with improved protection of genomic DNA and cell death resistance. Therefore, kinase signaling is one important mechanism for regulating the protective function of XPA. Previous experiments have shown that spiral ganglion neurons (SGNs) may mobilize XPA as a general stress response to chemical and physical ototoxicants. Therapeutic optimization of XPA via kinase signaling could serve as a means to improve DNA repair capacity within neurons following injury. The kinase signaling activity of the epidermal growth factor receptor (EGFR) has been shown in tumor cell lines to increase the repair of DNA damage products that are primarily repaired by XPA. Such observations suggest that EGFR may regulate the protective function of XPA. However, it is not known whether SGNs in particular or neurons in general could co-express XPA and EGFR. In the current study gene and protein expression of XPA and EGFR were determined from cochlear homogenates. Immunofluorescence assays were then employed to localize neurons expressing both EGFR and XPA within the ganglion. This work was then confirmed with double-immunohistochemistry. Rosenthal's canal served as the reference space in these experiments and design-based stereology was employed in first-order stereology quantification of immunoreactive neurons. The results confirmed that a population of SGNs that constitutively express XPA may also express the EGFR. These results provide the basis for future experiments designed to therapeutically manipulate the EGFR in order to regulate XPA activity and restore gene function in neurons following DNA damage. Copyright © 2015 Elsevier GmbH. All rights reserved.

Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death.

PubMed

Britain, Colleen M; Holdbrooks, Andrew T; Anderson, Joshua C; Willey, Christopher D; Bellis, Susan L

2018-02-05

The ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated. Using the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays. Results herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance.

A LGG-derived protein promotes IgA production through up-regulation of APRIL expression in intestinal epithelial cells

PubMed Central

Wang, Yang; Liu, Liping; Moore, Daniel J; Shen, Xi; Peek, Richard M.; Acra, Sari A; Li, Hui; Ren, Xiubao; Polk, D Brent; Yan, Fang

2016-01-01

p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting IgA production. p40 up-regulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl , but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice. Thus, p40 up-regulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production. PMID:27353252

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Epidermal growth factor receptor expression in radiation-induced dog lung tumors by immunocytochemical localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, F.L.; Park, J.F.; Dagle, G.E.

1993-06-01

In studies to determine the role of growth factors in radiation-induced lung cancer, epidermal growth factor (EGFR) expression was examined by immunocytochemistry in 51 lung tumors from beagle dogs exposed to inhaled plutonium; 21 of 51 (41%) tumors were positive for EGFR. The traction of tumors positive for EGFR and the histological type of EGFR-positive tumors in the plutonium-exposed dogs were not different from spontaneous dog lung tumors, In which 36% were positive for EGFR. EGFR involvement in Pu-induced lung tumors appeared to be similar to that in spontaneous lung tumors. However, EGFR-positive staining was observed in only 1 ofmore » 16 tumors at the three lowest Pu exposure levels, compared to 20 of 35 tumors staining positive at the two highest Pu exposure levels. The results in dogs were in good agreement with the expression of EGFR reported in human non-small cell carcinoma of the lung, suggesting that Pu-induced lung tumors in the dog may be a suitable animal model to investigate the role of EGFR expression in lung carcinogenesis. In humans, EGFR expression in lung tumors has been primarily related to histological tumor types. In individual dogs with multiple primary lung tumors, the tumors were either all EGFR positive or EGFR negative, suggesting that EGFR expression may be related to the response of the individual dog as well as to the histological type of tumor.« less

Autocrine-Derived Epidermal Growth Factor Receptor Ligands Contribute to Recruitment of Tumor-Associated Macrophage and Growth of Basal Breast Cancer Cells In Vivo

PubMed Central

Nickerson, Nicole K.; Mill, Christopher P.; Wu, Hsin-Jung; Riese, David J.; Foley, John

2014-01-01

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-α, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-α in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-α increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-α increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-α on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-α were somewhat interchangeable in their effects on EGFR signaling; however, TGF-α had a greater effect in vitro and AREG had a greater effect in vivo. PMID:23879171

Autocrine-derived epidermal growth factor receptor ligands contribute to recruitment of tumor-associated macrophage and growth of basal breast cancer cells in vivo.

PubMed

Nickerson, Nicole K; Mill, Christopher P; Wu, Hsin-Jung; Riese, David J; Foley, John

2013-01-01

Epidermal growth factor receptor (EGFR) expression has been linked to progression of basal breast cancers. Many breast cancer cells harbor the EGFR and produce its family of ligands, suggesting they may participate in autocrine and paracrine signaling with cells of the tumor microenvironment. EGFR ligand expression was profiled in the basal breast cancer cell line MDA-231 where AREG, TGF-alpha, and HBEGF were the three ligands most highly expressed. Autocrine signaling was modulated through silencing or overexpression of these three ligands using lentiviral constructs and the impact measured using motility, proliferation, and cytokine expression assays. Changes in receptor phosphorylation and receptor turnover were examined. Knockdown of AREG or TGF-alpha in vitro resulted in decreased motility (p < 0.05) and decreased expression of macrophage chemoattractants. Overexpression of TGF-alpha increased motility and chemoattractant expression, whereas AREG did not. HBEGF modulation had no effect on any cellular behaviors. All the cells with altered ligand production were inoculated into female athymic nude mice to form mammary fat pad tumors, followed by immunohistochemical analysis for necrosis, angiogenesis, and macrophage recruitment. In vivo, knockdown of AREG or TGF-alpha increased survival (p < 0.001) while decreasing angiogenesis (p < 0.001), tumor growth (p < 0.001), and macrophage attraction (p < 0.001). Overexpression of AREG appeared to elicit a greater effect than TGF-alpha on mammary fat pad tumor growth by increasing angiogenesis (p < 0.001) and macrophage attraction to the tumor (p < 0.01). We propose these changes in mammary tumor growth were the result of increased recruitment of macrophages to the tumor by cells with altered autocrine EGFR signaling. We conclude that AREG and TGF-alpha were somewhat interchangeable in their effects on EGFR signaling; however, TGF-alpha had a greater effect in vitro and AREG had a greater effect in vivo.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

PubMed Central

Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

2016-01-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

Genetic deletion of the EGFR ligand epigen does not affect mouse embryonic development and tissue homeostasis.

PubMed

Dahlhoff, Maik; Schäfer, Matthias; Wolf, Eckhard; Schneider, Marlon R

2013-02-15

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor with manifold functions during development, tissue homeostasis and disease. EGFR activation, the formation of homodimers or heterodimers (with the related ERBB2-4 receptors) and downstream signaling is initiated by the binding of a family of structurally related growth factors, the EGFR ligands. Genetic deletion experiments clarified the biological function of all family members except for the last characterized ligand, epigen. We employed gene targeting in mouse embryonic stem cells to generate mice lacking epigen expression. Loss of epigen did not affect mouse development, fertility, or organ physiology. Quantitative RT-PCR analysis revealed increased expression of betacellulin and EGF in a few organs of epigen-deficient mice, suggesting a functional compensation by these ligands. In conclusion, we completed the genetic analysis of EGFR ligands and show that epigen has non-essential functions or functions that can be compensated by other EGFR ligands during growth and tissue homeostasis. Copyright © 2012 Elsevier Inc. All rights reserved.

Ultrasound-Targeted Microbubble Destruction to Deliver siRNA Cancer Therapy

PubMed Central

Carson, Andrew R; McTiernan, Charles F; Lavery, Linda; Grata, Michelle; Leng, Xiaoping; Wang, Jianjun; Chen, Xucai; Villanueva, Flordeliza S

2012-01-01

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound targeted microbubble destruction (UTMD) could be used to enhance delivery of EGFR-directed small inhibitory RNA (siRNA) to murine squamous cell carcinomas. Custom designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling times, relative to controls receiving EGFR siRNA loaded microbubbles but not ultrasound or control siRNA loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof of concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy. PMID:23010078

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours.

PubMed

Shiomitsu, K; Johnson, C L; Malarkey, D E; Pruitt, A F; Thrall, D E

2009-06-01

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.

Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bian, Yong, E-mail: drbiany@126.com; Yu, Yun; Wang, Shanshan

2015-08-07

Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDACmore » patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.« less

Predictive value of EGFR overexpression and gene amplification on icotinib efficacy in patients with advanced esophageal squamous cell carcinoma

PubMed Central

Fan, Qingxia; Lu, Ping; Ma, Changwu; Liu, Wei; Liu, Ying; Li, Weiwei; Hu, Shaoxuan; Ling, Yun; Guo, Lei; Ying, Jianming; Huang, Jing

2016-01-01

This study aimed to search for a molecular marker for targeted epithelial growth factor receptor (EGFR) inhibitor Icotinib by analyzing protein expression and amplification of EGFR proto-oncogene in esophageal squamous cell carcinoma (ESCC) patients. Immunohistochemistry and fluorescence in situ hybridization (FISH) was used to assess EGFR expression and gene amplification status in 193 patients with ESCC. We also examined the association between EGFR overexpression and the efficacy of a novel EGFR TKI, icotinib, in 62 ESCC patients. Of the 193 patients, 95 (49.2%) patients showed EGFR overexpression (3+), and 47(24.4%) patients harbored EGFR FISH positivity. EGFR overexpression was significantly correlated with clinical stage and lymph node metastasis (p<0.05). In addition, EGFR overexpression was significantly correlated with EGFR FISH positivity (p<0.001). Among the 62 patients who received icotinib, the response rate was 17.6% for patients with high EGFR-expressing tumors, which was markedly higher than the rate (0%) for patients with low to moderate EGFR-expressing tumors (p=0.341). Furthermore, all cases responded to icotinib showed EGFR overexpression. In conclusion, our study suggests that EGFR overexpression might potentially be used in predicting the efficacy in patients treated with Icotinib. These data have implications for both clinical trial design and therapeutic strategies. PMID:27013591

Predictive value of EGFR overexpression and gene amplification on icotinib efficacy in patients with advanced esophageal squamous cell carcinoma.

PubMed

Wang, Xi; Niu, Haitao; Fan, Qingxia; Lu, Ping; Ma, Changwu; Liu, Wei; Liu, Ying; Li, Weiwei; Hu, Shaoxuan; Ling, Yun; Guo, Lei; Ying, Jianming; Huang, Jing

2016-04-26

This study aimed to search for a molecular marker for targeted epithelial growth factor receptor (EGFR) inhibitor Icotinib by analyzing protein expression and amplification of EGFR proto-oncogene in esophageal squamous cell carcinoma (ESCC) patients.Immunohistochemistry and fluorescence in situ hybridization (FISH) was used to assess EGFR expression and gene amplification status in 193 patients with ESCC. We also examined the association between EGFR overexpression and the efficacy of a novel EGFR TKI, icotinib, in 62 ESCC patients.Of the 193 patients, 95 (49.2%) patients showed EGFR overexpression (3+), and 47(24.4%) patients harbored EGFR FISH positivity. EGFR overexpression was significantly correlated with clinical stage and lymph node metastasis (p<0.05). In addition, EGFR overexpression was significantly correlated with EGFR FISH positivity (p<0.001). Among the 62 patients who received icotinib, the response rate was 17.6% for patients with high EGFR-expressing tumors, which was markedly higher than the rate (0%) for patients with low to moderate EGFR-expressing tumors (p=0.341). Furthermore, all cases responded to icotinib showed EGFR overexpression.In conclusion, our study suggests that EGFR overexpression might potentially be used in predicting the efficacy in patients treated with Icotinib. These data have implications for both clinical trial design and therapeutic strategies.

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation.

PubMed

Cairns, Junmei; Fridley, Brooke L; Jenkins, Gregory D; Zhuang, Yongxian; Yu, Jia; Wang, Liewei

2018-03-01

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1-dependent inhibition of EGFR In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context-dependent manner. EGFR inhibition can only sensitize EGFR-high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR-low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR-low cancer as a promising therapeutic approach. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

PubMed

Pagano, Eleonora; Calvo, Juan Carlos

2003-10-15

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors. Copyright 2003 Wiley-Liss, Inc.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos

PubMed Central

Dackor, J.; Strunk, K. E.; Wehmeyer, M. M.; Threadgill, D. W.

2007-01-01

Homozygosity for the Egfrtm1Mag null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta was quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfrtm1Mag homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation. PMID:17822758

Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Z. N.; Sharma, V. P.; Beaty, B. T.

2014-10-13

Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and inmore » particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.« less

Sex difference in EGFR pathways in mouse kidney-potential impact on the immune system.

PubMed

Liu, Fengxia; Jiao, Yan; Jiao, Yun; Garcia-Godoy, Franklin; Gu, Weikuan; Liu, Qingyi

2016-11-24

Epidermal growth factor receptor (Egfr) has been the target of several drugs for cancers. The potential gender differences in genes in the Egfr axis have been suggested in humans and in animal models. Female and male mice from the same recombinant inbred (RI) strain have the same genomic components except the sex difference. A population of different RI mouse strains allows to conduct precise analysis of molecular pathways and regulation of Egfr between female and male mice. The whole genome expression profiles of 70 genetically diverse RI strains of mice were used to compare three major molecular aspects of Egfr gene: the relative expression levels, gene network and expression quantitative trait loci (eQTL) that regulate the expression of Egfr between female and male mice. Our data showed that there is a significant sex difference in the expression levels in kidney. A considerable number of genes in the gene network of Egfr are sex differentially expressed. The expression levels of Egfr in mice are statistical significant different between C57BL/6 J (B6) and DBA/2 J (D2) genotypes in male while no difference in female mice. The eQTLs that regulate the expression levels of Egfr between female and male mice are also different. Furthermore, the differential expression levels of Egfr showed significantly different correlations with two known biological traits between male and female mice. Overall there is a substantial sex difference in the Egfr pathways in mice. These data may have significant impact on drug target design, development, formulation, and dosage determinant for women and men in clinical trials.

Phospholipase D2 Enhances Epidermal Growth Factor-Induced Akt Activation in EL4 Lymphoma Cells.

PubMed

Chahal, Manpreet S; Brauner, Daniel J; Meier, Kathryn E

2010-07-02

Phospholipase D2 (PLD2) generates phosphatidic acid through hydrolysis of phosphatidylcholine. PLD2 has been shown to play a role in enhancing tumorigenesis. The epidermal growth factor receptor (EGFR) can both activate and interact with PLD2. Murine lymphoma EL4 cells lacking endogenous PLD2 present a unique model to elucidate the role of PLD2 in signal transduction. In the current study, we investigated effects of PLD2 on EGF response. Western blotting and RT-PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not parental cells or cells transfected with inactive PLD2. EGF-mediated proliferation in cells expressing active PLD2 is dependent on the activities of both the EGFR and the PI3K/Akt pathway, as demonstrated by studies using protein kinase inhibitors. EGF-induced invasion through a synthetic extracellular matrix is enhanced in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 acts in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells.

Phospholipase D2 Enhances Epidermal Growth Factor-Induced Akt Activation in EL4 Lymphoma Cells

PubMed Central

Chahal, Manpreet S.; Brauner, Daniel J.; Meier, Kathryn E.

2010-01-01

Phospholipase D2 (PLD2) generates phosphatidic acid through hydrolysis of phosphatidylcholine. PLD2 has been shown to play a role in enhancing tumorigenesis. The epidermal growth factor receptor (EGFR) can both activate and interact with PLD2. Murine lymphoma EL4 cells lacking endogenous PLD2 present a unique model to elucidate the role of PLD2 in signal transduction. In the current study, we investigated effects of PLD2 on EGF response. Western blotting and RT-PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not parental cells or cells transfected with inactive PLD2. EGF-mediated proliferation in cells expressing active PLD2 is dependent on the activities of both the EGFR and the PI3K/Akt pathway, as demonstrated by studies using protein kinase inhibitors. EGF-induced invasion through a synthetic extracellular matrix is enhanced in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 acts in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells. PMID:27713341

NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest.

PubMed

Diluvio, Giulia; Del Gaudio, Francesca; Giuli, Maria Valeria; Franciosa, Giulia; Giuliani, Eugenia; Palermo, Rocco; Besharat, Zein Mersini; Pignataro, Maria Gemma; Vacca, Alessandra; d'Amati, Giulia; Maroder, Marella; Talora, Claudio; Capalbo, Carlo; Bellavia, Diana; Checquolo, Saula

2018-05-25

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells.

PubMed

Xie, Guofeng; Cheng, Kunrong; Shant, Jasleen; Raufman, Jean-Pierre

2009-04-01

Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-kappaB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation.

Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells

PubMed Central

Xie, Guofeng; Cheng, Kunrong; Shant, Jasleen; Raufman, Jean-Pierre

2009-01-01

Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-κB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation. PMID:19221016

PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

PubMed Central

Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

2015-01-01

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

Huntingtin interacting protein 1 is a novel brain tumor marker that associates with epidermal growth factor receptor.

PubMed

Bradley, Sarah V; Holland, Eric C; Liu, Grace Y; Thomas, Dafydd; Hyun, Teresa S; Ross, Theodora S

2007-04-15

Huntingtin interacting protein 1 (HIP1) is a multidomain oncoprotein whose expression correlates with increased epidermal growth factor receptor (EGFR) levels in certain tumors. For example, HIP1-transformed fibroblasts and HIP1-positive breast cancers have elevated EGFR protein levels. The combined association of HIP1 with huntingtin, the protein that is mutated in Huntington's disease, and the known overexpression of EGFR in glial brain tumors prompted us to explore HIP1 expression in a group of patients with different types of brain cancer. We report here that HIP1 is overexpressed with high frequency in brain cancers and that this overexpression correlates with EGFR and platelet-derived growth factor beta receptor expression. Furthermore, serum samples from patients with brain cancer contained anti-HIP1 antibodies more frequently than age-matched brain cancer-free controls. Finally, we report that HIP1 physically associates with EGFR and that this association is independent of the lipid, clathrin, and actin interacting domains of HIP1. These findings suggest that HIP1 may up-regulate or maintain EGFR overexpression in primary brain tumors by directly interacting with the receptor. This novel HIP1-EGFR interaction may work with or independent of HIP1 modulation of EGFR degradation via clathrin-mediated membrane trafficking pathways. Further investigation of HIP1 function in brain cancer biology and validation of its use as a prognostic or predictive brain tumor marker are now warranted.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine.

PubMed

Muta, Yu; Fujita, Yoshihisa; Sumiyama, Kenta; Sakurai, Atsuro; Taketo, M Mark; Chiba, Tsutomu; Seno, Hiroshi; Aoki, Kazuhiro; Matsuda, Michiyuki; Imajo, Masamichi

2018-06-05

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR.

PubMed

Ingthorsson, S; Andersen, K; Hilmarsdottir, B; Maelandsmo, G M; Magnusson, M K; Gudjonsson, T

2016-08-11

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation.

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

PubMed Central

Ingthorsson, S; Andersen, K; Hilmarsdottir, B; Maelandsmo, G M; Magnusson, M K; Gudjonsson, T

2016-01-01

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492HER2) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492HER2 (D492HER2/EGFR) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492HER2/EGFR xenografts grow slower than the D492HER2 tumors, while overexpression of EGFR alone (D492EGFR) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492HER2 xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation. PMID:26686087

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells.

PubMed

Piyush, Tushar; Chacko, Anisha R; Sindrewicz, Paulina; Hilkens, John; Rhodes, Jonathan M; Yu, Lu-Gang

2017-11-01

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1-EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

Mucinous Colorectal Adenocarcinoma: Influence of EGFR and E-Cadherin Expression on Clinicopathologic Features and Prognosis.

PubMed

Foda, Abd AlRahman M; AbdelAziz, Azza; El-Hawary, Amira K; Hosni, Ali; Zalata, Khalid R; Gado, Asmaa I

2015-08-01

Previous studies have shown conflicting results on epidermal growth factor receptor (EGFR) and E-cadherin expression in colorectal carcinoma and their prognostic significance. To the best of our knowledge, this study is the first to investigate EGFR and E-cadherin expression, interrelation and relation to clinicopathologic, histologic parameters, and survival in rare colorectal mucinous adenocarcinoma (MA). In this study, we studied tumor tissue specimens from 150 patients with colorectal MA and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tips technique, and immunohistochemistry for EGFR and E-cadherin was performed. All relations were analyzed using established statistical methodologies. NMA expressed EGFR and E-cadherin in significantly higher rates with significant heterogenous pattern than MA. EGFR and E-cadherin positivity rates were significantly interrelated in both NMA and MA groups. In the NMA group, high EGFR expression was associated with old age, male sex, multiplicity of tumors, lack of mucinous component, and association with schistosomiasis. However, in the MA group, high EGFR expression was associated only with old age and MA subtype rather than signet ring carcinoma subtype. Conversely, high E-cadherin expression in MA cases was associated with old age, fungating tumor configuration, MA subtype, and negative intratumoral lymphocytic response. However, in the NMA cases, none of these factors was statistically significant. In a univariate analysis, neither EGFR nor E-cadherin expression showed a significant impact on disease-free or overall survival. Targeted therapy against EGFR and E-cadherin may not be useful in patients with MA. Neither EGFR nor E-cadherin is an independent prognostic factor in NMA or MA.

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

PubMed Central

Alagappan, Dhivyaa; Lazzarino, Deborah A; Felling, Ryan J; Balan, Murugabaskar; Kotenko, Sergei V; Levison, Steven W

2009-01-01

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries. PMID:19570028

MUC5AC, a Gel-Forming Mucin Accumulating in Gallstone Disease, Is Overproduced via an Epidermal Growth Factor Receptor Pathway in the Human Gallbladder

PubMed Central

Finzi, Laetitia; Barbu, Véronique; Burgel, Pierre-Regis; Mergey, Martine; Kirkwood, Kimberly S.; Wick, Elizabeth C.; Scoazec, Jean-Yves; Peschaud, Frédérique; Paye, François; Nadel, Jay A.; Housset, Chantal

2006-01-01

Despite evidence that mucin overproduction is critical in the pathogenesis of gallstones, the mechanisms triggering mucin production in gallstone disease are unknown. Here, we tested the potential implication of an inflammation-dependent epidermal growth factor receptor (EGF-R) pathway in the regulation of gallbladder mucin synthesis. In gallbladder tissue sections from subjects with cholesterol gallstones, mucus accumulation was associated with neutrophil infiltration and with increased expressions of EGF-R and of tumor necrosis factor-α (TNF-α). In primary cultures of human gallbladder epithelial cells, TNF-α induced EGF-R overexpression. In the presence of TNF-α, EGF-R ligands (either EGF or transforming growth factor-α) caused significant increases in MUC5AC mRNA and protein production, whereas expression of the other gallbladder mucins MUC1, MUC3, and MUC5B was unchanged. In addition, on gallbladder tissue sections from subjects with gallstones, increased MUC5AC immunoreactivity was detected in the epithelium and within mucus gel in the lumen. Studies in primary cultures demonstrated that MUC5AC up-regulation induced by the combination of TNF-α with EGF-R ligands was completely blunted by inhibitors of EGF-R tyrosine kinase and mitogen-activated protein/extracellular signal-related kinase kinase. In conclusion, an inflammation-dependent EGF-R cascade causes overproduction of the gel-forming mucin MUC5AC, which accumulates in cholesterol gallstone disease. The ability to interrupt this cascade is of potential interest in the prevention of cholesterol gallstones. PMID:17148666

Expression of EGFR Under Tumor Hypoxia: Identification of a Subpopulation of Tumor Cells Responsible for Aggressiveness and Treatment Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoogsteen, Ilse J., E-mail: i.hoogsteen@rther.umcn.nl; Marres, Henri A.M.; Hoogen, Franciscus J.A. van den

2012-11-01

Purpose: Overexpression of epidermal growth factor receptor (EGFR) and tumor hypoxia have been shown to correlate with worse outcome in several types of cancer including head-and-neck squamous cell carcinoma. Little is known about the combination and possible interactions between the two phenomena. Methods and Materials: In this study, 45 cases of histologically confirmed squamous cell carcinomas of the head and neck were analyzed. All patients received intravenous infusions of the exogenous hypoxia marker pimonidazole prior to biopsy. Presence of EGFR, pimonidazole binding, and colocalization between EGFR and tumor hypoxia were examined using immunohistochemistry. Results: Of all biopsies examined, respectively, 91%more » and 60% demonstrated EGFR- and pimonidazole-positive areas. A weak but significant association was found between the hypoxic fractions of pimonidazole (HFpimo) and EGFR fractions (F-EGFR) and between F-EGFR and relative vascular area. Various degrees of colocalization between hypoxia and EGFR were found, increasing with distance from the vasculature. A high fraction of EGFR was correlated with better disease-free and metastasis-free survival, whereas a high degree of colocalization correlated with poor outcome. Conclusions: Colocalization of hypoxia and EGFR was demonstrated in head-and-neck squamous cell carcinomas, predominantly at longer distances from vessels. A large amount of colocalization was associated with poor outcome, which points to a survival advantage of hypoxic cells that are also able to express EGFR. This subpopulation of tumor cells might be indicative of tumor aggressiveness and be partly responsible for treatment resistance.« less

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

PubMed

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase-dependent and kinase-independent functions, both potentially involved in CCRCC progression. These results might have important implications on therapeutic approaches to CCRCC, since the disruption of the interaction between EGFR/SGLT1, mediated by anti-EGFR antibodies and/or SGLT1 inhibitors, might constitute a novel therapeutic target for CCRCC treatment, and new clinical trials should be evaluated on the basis of this therapeutic proposal.

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

PubMed Central

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase-dependent and kinase-independent functions, both potentially involved in CCRCC progression. These results might have important implications on therapeutic approaches to CCRCC, since the disruption of the interaction between EGFR/SGLT1, mediated by anti-EGFR antibodies and/or SGLT1 inhibitors, might constitute a novel therapeutic target for CCRCC treatment, and new clinical trials should be evaluated on the basis of this therapeutic proposal. PMID:27073724

Regulation of miR-21 expression in human melanoma via UV-ray-induced melanin pigmentation.

PubMed

Lin, Kuan-Yu; Chen, Chien-Min; Lu, Cheng-You; Cheng, Chun-Yuan; Wu, Yu-Hsin

2017-08-01

Excessive environmental ultraviolet (UV) radiation produces genetic mutations that can lead to skin cancer. This study was designed to assess the potential inhibitory activity of microRNA-21 (miR-21) on the UV irradiation-stimulated melanogenesis signal pathway in melanoma cells. The molecular mechanism of miR-21-induced inhibitory activity on UV-ray-stimulated melanogenesis-regulating proteins was examined in A375.S2 human melanoma and B16F10 mouse melanoma cells. UV irradiation for 30 min induced melanogenesis signal pathway by increasing melanin production and the number of A375.S2 cells. Similarly, UV radiation increased the expression of α-melanocyte-stimulating hormone (α-MSH) protein and decreased the melanogenesis-regulating signal, such as EGFR and Akt phosphorylation. Notably, miR-21 overexpression in UV-ray-stimulated A375.S2 cells decreased α-MSH expression and increased EGFR and Akt phosphorylation levels. Furthermore, miR-21 on UV-ray- induced melanogenesis was down-regulated by the Akt inhibitor and the EGFR inhibitor (Gefitinib). Results suggest that the suppressive activity of miR-21 on UV-ray-stimulated melanogenesis may involve the down-regulation of α-MSH and the activation in both of EGFR and Akt. © 2017 Wiley Periodicals, Inc.

Sym004, a Novel EGFR Antibody Mixture, Can Overcome Acquired Resistance to Cetuximab1

PubMed Central

Iida, Mari; Brand, Toni M; Starr, Megan M; Li, Chunrong; Huppert, Evan J; Luthar, Neha; Pedersen, Mikkel W; Horak, Ivan D; Kragh, Michael; Wheeler, Deric L

2013-01-01

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (CtxR) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for CtxR tumor cells. Sym004 treatment of CtxR clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo CtxR NCI-H226 mouse xenografts and subsequently treated CtxR tumors with Sym004. Sym004 treatment of mice harboring CtxR tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in CtxR tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for CtxR tumors. PMID:24204198

Induction of anaplastic lymphoma kinase (ALK) as a novel mechanism of EGFR inhibitor resistance in head and neck squamous cell carcinoma patient-derived models.

PubMed

Ouyang, Xiaoming; Barling, Ashley; Lesch, Aletha; Tyner, Jeffrey W; Choonoo, Gabrielle; Zheng, Christina; Jeng, Sophia; West, Toni M; Clayburgh, Daniel; Courtneidge, Sara A; McWeeney, Shannon K; Kulesz-Martin, Molly

2018-06-01

Head and neck squamous cell carcinoma (HNSCC) currently only has one FDA-approved cancer intrinsic targeted therapy, the epidermal growth factor receptor (EGFR) inhibitor cetuximab, to which only approximately 10% of tumors are sensitive. In order to extend therapy options, we subjected patient-derived HNSCC cells to small-molecule inhibitor and siRNA screens, first, to find effective combination therapies with an EGFR inhibitor, and second, to determine a potential mechanistic basis for repurposing the FDA approved agents for HNSCC. The combinations of EGFR inhibitor with anaplastic lymphoma kinase (ALK) inhibitors demonstrated synergy at the highest ratio in our cohort, 4/8 HNSCC patients' derived tumor cells, and this corresponded with an effectiveness of siRNA targeting ALK combined with the EGFR inhibitor gefitinib. Co-targeting EGFR and ALK decreased HNSCC cell number and colony formation ability and increased annexin V staining. Because ALK expression is low and ALK fusions are infrequent in HNSCC, we hypothesized that gefitinib treatment could induce ALK expression. We show that ALK expression was induced in HNSCC patient-derived cells both in 2D and 3D patient-derived cell culture models, and in patient-derived xenografts in mice. Four different ALK inhibitors, including two (ceritinib and brigatinib) FDA approved for lung cancer, were effective in combination with gefitinib. Together, we identified induction of ALK by EGFR inhibitor as a novel mechanism potentially relevant to resistance to EGFR inhibitor, a high ratio of response of HNSCC patient-derived tumor cells to a combination of ALK and EGFR inhibitors, and applicability of repurposing ALK inhibitors to HNSCC that lack ALK aberrations.

Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer

PubMed Central

Herrmann, Amanda C.; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J.; Hong, Waun Ki; Perez-Soler, Roman

2016-01-01

Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

PubMed

Regales, Lucia; Balak, Marissa N; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A; Solit, David B; Rosen, Neal; Zakowski, Maureen F; Pao, William

2007-08-29

The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

Targeting Nuclear EGFR: Strategies for Improving Cetuximab Therapy in Lung Cancer

DTIC Science & Technology

2015-12-01

xenograft models to determine if, in vivo, SFK or AKT inhibition can block EGFR translocation to the nucleus leading to increased membrane expression...resistant NSCLC tumors using mouse xenografts . Mice bearing resistant tumors will be treated with 1) vehicle, 2) cetuximab, 3) dasatinib, 4) MK2206...phosphorylation of Y1101 and EGFR dimerization with HER3 leading to nuclear translocation. Key Research Accomplishments for Aim 2: • In vivo xenograft tumors

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice1

PubMed Central

Sasaki, Takamitsu; Kitadai, Yasuhiko; Nakamura, Toru; Kim, Jang-Seong; Tsan, Rachel Z; Kuwai, Toshio; Langley, Robert R; Fan, Dominic; Kim, Sun-Jin; Fidler, Isaiah J

2007-01-01

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer. PMID:18084614

Activation of EGFR and ERBB2 by Helicobacter pylori results in survival of gastric epithelial cells with DNA damage.

PubMed

Chaturvedi, Rupesh; Asim, Mohammad; Piazuelo, M Blanca; Yan, Fang; Barry, Daniel P; Sierra, Johanna Carolina; Delgado, Alberto G; Hill, Salisha; Casero, Robert A; Bravo, Luis E; Dominguez, Ricardo L; Correa, Pelayo; Polk, D Brent; Washington, M Kay; Rose, Kristie L; Schey, Kevin L; Morgan, Douglas R; Peek, Richard M; Wilson, Keith T

2014-06-01

The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Modification of cytokine-induced killer cells with chimeric antigen receptors (CARs) enhances antitumor immunity to epidermal growth factor receptor (EGFR)-positive malignancies.

PubMed

Ren, Xuequn; Ma, Wanli; Lu, Hong; Yuan, Lei; An, Lei; Wang, Xicai; Cheng, Guanchang; Zuo, Shuguang

2015-12-01

Epidermal growth factor receptor (EGFR, ErbB1, Her-1) is a cell surface molecule overexpressing in a variety of human malignancies and, thus, is an excellent target for immunotherapy. Immunotherapy targeting EGFR-overexpressing malignancies using genetically modified immune effector cells is a novel and promising approach. In the present study, we have developed an adoptive cellular immunotherapy strategy based on the chimeric antigen receptor (CAR)-modified cytokine-induced killer (CAR-CIK) cells specific for the tumor cells expressing EGFR. To generate CAR-CIK cells, a lentiviral vector coding the EGFR-specific CAR was constructed and transduced into the CIK cells. The CAR-CIK cells showed significantly enhanced cytotoxicity and increased production of cytokines IFN-γ and IL-2 when co-cultured with EGFR-positive cancer cells. In tumor xenografts, adoptive immunotherapy of CAR-CIK cells could inhibit tumor growth and prolong the survival of EGFR-overexpressing human tumor xenografts. Moreover, tumor growth inhibition and prolonged survival in mice with EGFR(+) human cancer were associated with the increased persistence of CAR-CIK cells in vivo. Our study indicates that modification with EGFR-specific CAR strongly enhances the antitumor activity of the CIK cells against EGFR-positive malignancies.

Epidermal Growth Factor Receptor Is Related to Poor Survival in Glioblastomas: Single-Institution Experience

PubMed Central

Choi, Youngmin; Lee, Hyung-Sik; Hur, Won-Joo; Sung, Ki-Han; Kim, Ki-Uk; Choi, Sun-Seob; Kim, Su-Jin; Kim, Dae-Cheol

2013-01-01

Purpose There are conflicting results surrounding the prognostic significance of epidermal growth factor receptor (EGFR) status in glioblastoma (GBM) patients. Accordingly, we attempted to assess the influence of EGFR expression on the survival of GBM patients receiving postoperative radiotherapy. Materials and Methods Thirty three GBM patients who had received surgery and postoperative radiotherapy at our institute, between March 1997 and February 2006, were included. The evaluation of EGFR expression with immunohistochemistry was available for 30 patients. Kaplan-Meier survival analysis and Cox regression were used for statistical analysis. Results EGFR was expressed in 23 patients (76.7%), and not expressed in seven (23.3%). Survival in EGFR expressing GBM patients was significantly less than that in non-expressing patients (median survival: 12.5 versus 17.5 months, p=0.013). Patients who received more than 60 Gy showed improved survival over those who received up to 60 Gy (median survival: 17.0 versus 9.0 months, p=0.000). Negative EGFR expression and a higher radiation dose were significantly correlated with improved survival on multivariate analysis. Survival rates showed no differences according to age, sex, and surgical extent. Conclusion The expression of EGFR demonstrated a significantly deleterious effect on the survival of GBM patients. Therefore, approaches targeting EGFR should be considered in potential treatment methods for GBM patients, in addition to current management strategies. PMID:23225805

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent anti-tumor activity

PubMed Central

Caruso, Hillary G.; Hurton, Lenka V.; Najjar, Amer; Rushworth, David; Ang, Sonny; Olivares, Simon; Mi, Tiejuan; Switzer, Kirsten; Singh, Harjeet; Huls, Helen; Lee, Dean A.; Heimberger, Amy B.; Champlin, Richard E.; Cooper, Laurence J. N.

2015-01-01

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells. PMID:26330164

Epidermal Growth Factor-Dependent Transformation by a Human EGF Receptor Proto-Oncogene

NASA Astrophysics Data System (ADS)

Velu, Thierry J.; Beguinot, Laura; Vass, William C.; Willingham, Mark C.; Merlino, Glenn T.; Pastan, Ira; Lowy, Douglas R.

1987-12-01

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 × 105 EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 107 focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

EGFR-L858R mutant enhances lung adenocarcinoma cell invasive ability and promotes malignant pleural effusion formation through activation of the CXCL12-CXCR4 pathway

PubMed Central

Tsai, Meng-Feng; Chang, Tzu-Hua; Wu, Shang-Gin; Yang, Hsiao-Yin; Hsu, Yi-Chiung; Yang, Pan-Chyr; Shih, Jin-Yuan

2015-01-01

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R–dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation. PMID:26338423

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

PubMed

Garousi, Javad; Andersson, Ken G; Dam, Johan H; Olsen, Birgitte B; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Ståhl, Stefan; Löfblom, John; Thisgaard, Helge; Tolmachev, Vladimir

2017-07-20

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111 In-labelled DOTA-conjugated Z EGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z EGFR:2377 . DOTA-Z EGFR:2377 was labelled with 57 Co (T 1/2  = 271.8 d), 55 Co (T 1/2  = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68 Ga (T 1/2  = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57 Co was used in animal studies. Both 57 Co-DOTA-Z EGFR:2377 and 68 Ga-DOTA-Z EGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z EGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57 Co-DOTA-Z EGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68 Ga-DOTA-Z EGFR:2377 . The results of this study suggest that the positron-emitting cobalt isotope 55 Co would be an optimal label for DOTA-Z EGFR:2377 and further development should concentrate on this radionuclide as a label.

Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLC.

PubMed

Gaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M; Schnabel, Philipp A; Vollmer, Ekkehard; Reck, Martin; Goldmann, Torsten

2014-09-17

Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. EGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p=0.001). Overall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p=0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_165.

MiR-615 inhibits cell proliferation, migration and invasion by targeting EGFR in human glioblastoma.

PubMed

Ji, Yanwei; Sun, Qingshan; Zhang, Jianbin; Hu, Haoran

2018-05-15

MiR-615 and epidermal growth factor receptor (EGFR) are associated with a number of disease processes and pathogenesis. However, little is known about the mechanisms of miR-615 and EGFR in human glioblastoma multiforme (GBM). Here, we found that down-regulation of miR-615 expression occurred in GBM tissues and cells, and was inversely correlated with overall survival, relapse-free survival, WHO grade as well as EGFR expression. We further determined that miR-615 functions as a tumor suppressor by inhibiting GBM cell proliferation, cell cycle, migration and invasion, and promoting cell apoptosis. In-vivo assay validated the inhibition effect of miR-615 on tumor growth and EGFR expression. Luciferase reporter assays demonstrated that miR-615 targeted the 3'-untranslated region (3'-UTR) of EGFR. Besides, over-expression of EGFR reversed the inhibition effects of miR-615, while silencing of EGFR aggravated these inhibition effects. In conclusions, we identified that miR-615 plays a tumor suppressor role in GBM cell proliferation, migration and invasion by targeting EGFR expression, and miR-615 may act as a novel biomarker for early diagnosis or therapeutic targets of GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.

PubMed

Girgert, Rainer; Emons, Günter; Gründker, Carsten

2017-02-01

Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptor α expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclin D1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑ and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200 nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7 days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200 nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70, 147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.

Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor

PubMed Central

Cohen, Gadi; Lecht, Shimon; Arien-Zakay, Hadar; Ettinger, Keren; Amsalem, Orit; Oron-Herman, Mor; Yavin, Eylon; Prus, Diana; Benita, Simon; Nissan, Aviram; Lazarovici, Philip

2012-01-01

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues. PMID:23144978

Bio-imaging of colorectal cancer models using near infrared labeled epidermal growth factor.

PubMed

Cohen, Gadi; Lecht, Shimon; Arien-Zakay, Hadar; Ettinger, Keren; Amsalem, Orit; Oron-Herman, Mor; Yavin, Eylon; Prus, Diana; Benita, Simon; Nissan, Aviram; Lazarovici, Philip

2012-01-01

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

Conditional internalization of PEGylated nanomedicines by PEG engagers for triple negative breast cancer therapy

NASA Astrophysics Data System (ADS)

Su, Yu-Cheng; Burnouf, Pierre-Alain; Chuang, Kuo-Hsiang; Chen, Bing-Mae; Cheng, Tian-Lu; Roffler, Steve R.

2017-06-01

Triple-negative breast cancer (TNBC) lacks effective treatment options due to the absence of traditional therapeutic targets. The epidermal growth factor receptor (EGFR) has emerged as a promising target for TNBC therapy because it is overexpressed in about 50% of TNBC patients. Here we describe a PEG engager that simultaneously binds polyethylene glycol and EGFR to deliver PEGylated nanomedicines to EGFR+ TNBC. The PEG engager displays conditional internalization by remaining on the surface of TNBC cells until contact with PEGylated nanocarriers triggers rapid engulfment of nanocargos. PEG engager enhances the anti-proliferative activity of PEG-liposomal doxorubicin to EGFR+ TNBC cells by up to 100-fold with potency dependent on EGFR expression levels. The PEG engager significantly increases retention of fluorescent PEG probes and enhances the antitumour activity of PEGylated liposomal doxorubicin in human TNBC xenografts. PEG engagers with specificity for EGFR are promising for improved treatment of EGFR+ TNBC patients.

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering overall survival.

Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Guodong; Peng, Tao; Zhou, Xuhong

2013-11-01

Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messengermore » RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and senescence and, therefore, the tumor cells regression.« less

Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways

PubMed Central

Saxena, Ruchi; Chandra, Vishal; Manohar, Murli; Hajela, Kanchan; Debnath, Utsab; Prabhakar, Yenamandra S.; Saini, Karan Singh; Konwar, Rituraj; Kumar, Sandeep; Megu, Kaling; Roy, Bal Gangadhar; Dwivedi, Anila

2013-01-01

Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors. PMID:23840429

Combined caveolin-1 and epidermal growth factor receptor expression as a prognostic marker for breast cancer.

PubMed

Liang, Ya-Nan; Liu, Yu; Wang, Letian; Yao, Guodong; Li, Xiaobo; Meng, Xiangning; Wang, Fan; Li, Ming; Tong, Dandan; Geng, Jingshu

2018-06-01

Previous studies have indicated that caveolin-1 (Cav-1) is able to bind the signal transduction factor epidermal growth factor receptor (EGFR) to regulate its tyrosine kinase activity. The aim of the present study was to evaluate the clinical significance of Cav-1 gene expression in association with the expression of EGFR in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Cav-1 and EGFR expression using immunohistochemistry, and clinical significance was assessed using multivariate Cox regression analysis, Kaplan-Meier estimator curves and the log-rank test. Stromal Cav-1 was downregulated in 38.56% (118/306) of tumor tissues, whereas cytoplasmic EGFR and Cav-1 were overexpressed in 53.92% (165/306) and 44.12% (135/306) of breast cancer tissues, respectively. EGFR expression was positively associated with cytoplasmic Cav-1 and not associated with stromal Cav-1 expression in breast cancer samples; however, low expression of stromal Cav-1 was negatively associated with cytoplasmic Cav-1 expression in total tumor tissues, and analogous results were identified in the chemotherapy group. Multivariate Cox's proportional hazards model analysis revealed that, for patients in the estrogen receptor (ER)(+) group, the expression of stromal Cav-1 alone was a significant prognostic marker of breast cancer. However, in the chemotherapy, human epidermal growth factor receptor 2 (HER-2)(-), HER-2(+) and ER(-) groups, the use of combined markers was more effective prognostic marker. Stromal Cav-1 has a tumor suppressor function, and the combined marker stromal Cav-1/EGFR expression was identified as an improved prognostic marker in the diagnosis of breast cancer. Parenchymal expression of Cav-1 is able to promote EGFR signaling in breast cancer, potentially being required for EGFR-mediated initiation of mitosis.

Mutational status of EGFR and KIT in thymoma and thymic carcinoma.

PubMed

Yoh, Kiyotaka; Nishiwaki, Yutaka; Ishii, Genichiro; Goto, Koichi; Kubota, Kaoru; Ohmatsu, Hironobu; Niho, Seiji; Nagai, Kanji; Saijo, Nagahiro

2008-12-01

This study was conducted to evaluate the prevalence of EGFR and KIT mutations in thymomas and thymic carcinomas as a means of exploring the potential for molecularly targeted therapy with tyrosine kinase inhibitors. Genomic DNA was isolated from 41 paraffin-embedded tumor samples obtained from 24 thymomas and 17 thymic carcinomas. EGFR exons 18, 19, and 21, and KIT exons 9, 11, 13, and 17, were analyzed for mutations by PCR and direct sequencing. Protein expression of EGFR and KIT was evaluated immunohistochemically. EGFR mutations were detected in 2 of 20 thymomas, but not in any of the thymic carcinomas. All of the EGFR mutations detected were missense mutations (L858R and G863D) in exon 21. EGFR protein was expressed in 71% of the thymomas and 53% of the thymic carcinomas. The mutational analysis of KIT revealed only a missense mutation (L576P) in exon 11 of one thymic carcinoma. KIT protein was expressed in 88% of the thymic carcinomas and 0% of the thymomas. The results of this study indicate that EGFR and KIT mutations in thymomas and thymic carcinomas are rare, but that many of the tumors express EGFR or KIT protein.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells*

PubMed Central

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T.; Friedman, Henry; Bigner, Darell D.; Ali-Osman, Francis

2015-01-01

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. PMID:26429914

[Mechanisms for effect of osthole on inhibiting the growth and invasion of bladder cancer cells].

PubMed

Liu, Jun; Xu, Ran; Zhao, Xiaokun

2016-04-01

To investigate the effect of osthole on epidermal growth factor receptor tyrosine kinase (EGFR-TPK), matrix-metalloproteinase-2 (MMP-2), aminopeptidase N (APN) in bladder cancer cell and the underlying mechanism.
 The T24 cell lines were cultured. The inhibitory effects of osthole on EGFR-TPK, APN and MMP-2 were evaluated by spectrophotometric and MTT assay. The caspase-3 activity and the expression COX-2 and VEGF in T24 were examined. The activity of NF-κB was determined by electrophoretic mobility shift assay.
 The half inhibition concentrations (IC50) of osthole on EGFR-TPK, APN and MMP-2 were (45.33±3.98), (28.21±3.23) and (8.11±0.54) µmol/L, respectively. The growth inhibitory rates for T24 cells were increased in a dose-dependent manner (P<0.05). The caspase-3 activities were significantly increased in T24 cells in the osthole group compared with control group, while the expression of angiogenesis related-protein COX-2, VEGF, and NF-κB in T24 cells were decreased.
 Through the inhibitory effect on EGFR-TPK, APN and MMP-2, osthole can decrease COX-2, VEGF and NF-κB expression while increase the activity of caspase-3, eventually blocking the growth and invasion of bladder cancer cell.

Epidermal growth factor expression in esophageal adenocarcinoma: a clinically relevant target?

PubMed

Harper, Nicholas; Li, Yan; Farmer, Russell; Martin, Robert C G

2012-05-01

There has been recent widespread enthusiasm in epidermal growth factor (EGFR) as a molecularly active target in esophageal adenocarcinoma (EAC). However, there is limited data on the extent of EGFR expression in EAC. Thus, the aim of this study was to evaluated EGFR, pErk1/2, and total Erk1/2 expression in malignant and benign specimens. Baseline expression of EGFR in the human normal squamous, Barrett's, and EAC cell lines were determined as well as after bile acid treatment and curcumin pretreatment. In addition, EGFR expression was also evaluated in 60 matched normal and malignant EAC resected specimens. The in vitro studies in the Het-1a, BarT, and OE19 cell lines failed to show any measurable expression of EGFR via Western blot technique. The marker serving as the positive control for the study, MnSOD, showed expression in each cell line for all three treatment regimens at approximately 24 kDa EGFR, showing moderate staining in the malignant tumor specimens and low staining in the benign tissue specimens. pErk1/2 showed low staining in the malignant tumor specimens and no staining in the benign tissue specimens. Total Erk1/2 showed high staining in both the malignant tumor specimens and benign tissue specimens. The differences in the mean staining scores for the malignant versus benign tissue specimens for pErk1/2 and total Erk1/2 are not statistically significant (p = 0.0726 and p = 0.7054, respectively). Thus, in conclusion, EGFR expression has been confirmed to be limited to non-existent in EAC and thus its use as a clinically active target is limited at best. Prior to the use of these expensive anti-EGFR therapies, confirmation of overexpression should be verified.

In situ analysis of integrin and growth factor receptor signaling pathways in human glioblastomas suggests overlapping relationships with focal adhesion kinase activation.

PubMed

Riemenschneider, Markus J; Mueller, Wolf; Betensky, Rebecca A; Mohapatra, Gayatry; Louis, David N

2005-11-01

Deregulated integrin signaling is common in cancers, including glioblastoma. Integrin binding and growth factor receptor signaling activate focal adhesion kinase (FAK) and subsequently up-regulate extracellular regulated kinases (ERK-1/2), leading to cell-cycle progression and cell migration. Most studies of this pathway have used in vitro systems or tumor lysate-based approaches. We examined these pathways primarily in situ using a panel of 30 glioblastomas and gene expression arrays, immunohistochemistry, and fluorescence in situ hybridization, emphasizing the histological distribution of molecular changes. Within individual tumors, increased expression of FAK, p-FAK, paxillin, ERK-1/2, and p-ERK-1/2 occurred in regions of elevated EGFR and/or PDGFRA expression. Moreover, FAK activation levels correlated with EGFR and PDGFRA expression, and p-FAK and EGFR expression co-localized at the single-cell level. In addition, integrin expression was enriched in EGFR/PDGFRA-overexpressing areas but was more regionally confined than FAK, p-FAK, and paxillin. Integrins beta8 and alpha5beta1 were most commonly expressed, often in a perinecrotic or perivascular pattern. Taken together, our data suggest that growth factor receptor overexpression facilitates alterations in the integrin signaling pathway. Thus, FAK may act in glioblastoma as a downstream target of growth factor signaling, with integrins enhancing the impact of such signaling in the tumor microenvironment.

An EGFR inhibitor enhances the efficacy of SN38, an active metabolite of irinotecan, in SN38-refractory gastric carcinoma cells

PubMed Central

Yashiro, M; Qiu, H; Hasegawa, T; Zhang, X; Matsuzaki, T; Hirakawa, K

2011-01-01

Background: Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer. Methods: Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38 were, respectively, established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell cycle on gastric cancer cells were examined. Results: Gefitinib or lapatinib showed synergistic anti-tumour effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumour growth by OCUM-2M/SN38 cells than either alone group. Conclusion: The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumour effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism. PMID:21997136

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

PubMed Central

Lara-Guerra, Humberto; Kawashima, Hiroyuki; Sakai, Ryo; Jayachandran, Gitanjali; Majidi, Mourad; Mehran, Reza; Wang, Jing; Bekele, B. Nebiyou; Baladandayuthapani, Veerabhadran; Yoo, Suk-Young; Wang, Ying; Ying, Jun; Meng, Feng; Ji, Lin; Roth, Jack A.

2015-01-01

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR. PMID:26053020

Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease

PubMed Central

Cortés-Ramírez, Dionisio A.; Rodríguez-Tojo, María J.; Coca-Meneses, Juan C.; Marichalar-Mendia, Xabier

2014-01-01

The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. Objectives: To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Material and Methods: Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Results: Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk. Key words:Oral lichenoid disease, oral lichen planus, oral lichenoid lesion, oral carcinogenesis, EGFR. PMID:24880441

Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin.

PubMed

Stache, Christina; Bils, Christiane; Fahlbusch, Rudolf; Flitsch, Jörg; Buchfelder, Michael; Stefanits, Harald; Czech, Thomas; Gaipl, Udo; Frey, Benjamin; Buslei, Rolf; Hölsken, Annett

2016-12-01

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

Immuno-PET Imaging and Radioimmunotherapy of 64Cu-/177Lu-Labeled Anti-EGFR Antibody in Esophageal Squamous Cell Carcinoma Model.

PubMed

Song, In Ho; Lee, Tae Sup; Park, Yong Serk; Lee, Jin Sook; Lee, Byung Chul; Moon, Byung Seok; An, Gwang Il; Lee, Hae Won; Kim, Kwang Il; Lee, Yong Jin; Kang, Joo Hyun; Lim, Sang Moo

2016-07-01

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with (64)Cu or (177)Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model. Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with (64)Cu or (177)Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of (64)Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of (177)Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using (18)F-FDG PET and immunohistochemical staining. (64)Cu- or (177)Lu-labeled antibodies showed high radiolabeling yield (>98%), stability (>90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of (64)Cu- and (177)Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with (177)Lu-cetuximab showed significant inhibition of tumor growth (P < 0.01) and marked reduction of (18)F-FDG SUV compared with that of control (P < 0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P < 0.01). (64)Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and (177)Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical (64)Cu-/(177)Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Alterations in epidermal growth factor receptors 1 and 2 in esophageal squamous cell carcinomas

PubMed Central

2012-01-01

Background Esophageal squamous cell carcinoma (ESCC) shows a 5-year survival rate below 10%, demonstrating the urgency in improving its treatment. Alterations in epidermal growth factor receptors are closely related to malignancy transformation in a number of tumors and recent successful targeted therapies have been directed to these molecules. Therefore, in this study, we analyzed the expression of EGFR and HER2 and evaluated EGFR mutation profile as well as the presence of mutations in hotspots of KRAS and BRAF in ESCC patients. Methods We performed RT-qPCR, immunohistochemistry and Fluorescent in situ hybridization to determine EGFR and HER2 expression in ESCC patients, and direct sequencing and PCR-RFLP for mutations and polymorphism analysis. Results Our results showed an increased EGFR mRNA expression in tumors compared to surrounding tissue (p <0.05), with 11% of the cases presenting at least a four-fold difference between tumor and paired adjacent mucosa. EGFR protein overexpression was present only in 4% of the cases. The median expression of HER2 mRNA was not different between tumors and adjacent mucosa. Still, 7% of the tumors presented at least a 25-fold higher expression of this gene when compared to its paired counterpart. Immunohistochemical analysis revealed that 21% of the tumors were positive for HER2 (scores 2+ and 3+), although only 3+ tumors presented amplification of this gene. Mutation analysis for EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (V600E) showed no mutations in any of the hotspots of these genes in almost 100 patients analyzed. EGFR presented synonymous polymorphisms at codon 836 (C>T) in 2.1% of the patients, and at codon 787 (G>A) in 79.2% of the cases. This last polymorphism was also evaluated in 304 healthy controls, which presented a similar frequency (73.7%) in comparison with ESCC patients. The absence of mutations of EGFR, KRAS and BRAF as well as the overexpression of EGFR and HER2 in less than 10% of the patients suggest that this signaling pathway is altered in only a small proportion of patients with ESCC. Conclusion HER receptors target therapies may have the potential to be effective in only a minor fraction of patients with ESCC. PMID:23207070

EGFR transactivation is involved in TNF-α-induced expression of thymic stromal lymphopoietin in human keratinocyte cell line.

PubMed

Segawa, Ryosuke; Shigeeda, Kenichi; Hatayama, Takahiro; Dong, Jiangxu; Mizuno, Natsumi; Moriya, Takahiro; Hiratsuka, Masahiro; Hirasawa, Noriyasu

2018-03-01

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine involved in the pathology of inflammatory skin diseases, such as atopic dermatitis and psoriasis. Tumor necrosis factor (TNF)-α, a key cytokine in inflammatory skin diseases, is a known TSLP inducer. TNF-α activates NF-κB and induces transactivation of epidermal growth factor receptor (EGFR) in epithelial cells. However, the detailed mechanism of TSLP induction by TNF-α has remained unclear. We investigated the involvement of TNF-α-induced EGFR transactivation in TSLP expression. HaCaT cells were stimulated with TNF-α or EGF in the presence or absence of an EGFR kinase inhibitor or other signaling inhibitors. The expression of TSLP mRNA was analyzed by RT-PCR and the phosphorylation level of signal proteins was analyzed by western blot. TSLP promoter and NF-κB transcription activities were analyzed by luciferase assay. TNF-α-induced TSLP expression was inhibited by the EGFR kinase inhibitor AG1478. While TSLP expression was induced by EGF, it was inhibited by the MEK inhibitor, U0126. Inhibitors of p38 and ADAM proteases suppressed the TNF-α-induced TSLP expression and EGFR phosphorylation, but not the EGF-induced expression. TNF-α-induced EGFR transactivation results in TSLP induction through ERK activation. The activation of p38 and ADAM proteases mediates TNF-α-induced EGFR phosphorylation. These findings suggested that the TNF-α-induced EGFR transactivation pathway could be a target for the treatment of inflammatory skin diseases. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

PubMed

Cheng, Qing; Wållberg, Helena; Grafström, Jonas; Lu, Li; Thorell, Jan-Olov; Hägg Olofsson, Maria; Linder, Stig; Johansson, Katarina; Tegnebratt, Tetyana; Arnér, Elias S J; Stone-Elander, Sharon; Ahlzén, Hanna-Stina Martinsson; Ståhl, Stefan

2016-12-01

Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression.

PubMed

Dai, Yumin; Hogan, Shelly; Schmelz, Eva M; Ju, Young H; Canning, Corene; Zhou, Kequan

2011-01-01

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration

PubMed Central

Wodziak, Dariusz; Dong, Aiwen; Basin, Michael F.; Lowe, Anson W.

2016-01-01

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis. PMID:27764193

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of epidermal growth factor receptor protein and gene alteration on Taiwanese hepatocellular carcinomas.

PubMed

Su, Yu-Hung; Ng, Kwai-Fong; Yu, Ming-Chin; Wu, Ting-Jung; Yeh, Ta-Sen; Lee, Wei-Chen; Lin, Yong-Shiang; Hsieh, Tsung-Han; Lin, Chun-Yen; Yeh, Chau-Ting; Chen, Tse-Ching

2015-09-01

Epidermal growth factor receptor (EGFR) overexpression is associated with disease progression and poor survival in a variety of solid tumors. The role of EGFR in hepatocellular carcinoma (HCC) remains controversial. One hundred thirty-eight HCCs were analyzed for total EGFR (t-EGFR) and phospho-EGFR (p-EGFR) expression and gene amplification using immunohistochemistry and fluorescence in situ hybridization. The role of EGFR was analyzed in relation to the clinicopathological features. Weak to strong p-EGFR immunostaining was noted in 42 of the 138 HCCs. p-EGFR expression correlated with alcoholism (P = 0.03) and chronic hepatitis B infection (P = 0.041). There was no correlation between t-EGFR expression and any of the clinicopathological features. Amplification of the EGFR gene was not identified in the 138 HCCs, but 39.1% of the HCCs showed balanced polysomy of both the EGFR gene and centromere 7. Moreover, 65 tumors showed > 2.2 copies per tumor cell. EGFR copy number gain (CNG) was significantly correlated with gender (P = 0.0491), tumor grade (P = 0.006), and vascular invasion (P = 0.005). HCCs with EGFR CNG also had a poor recurrence-free survival (RFS), as compared with HCCs without EGFR CNG (P = 0.031). When exploring the impact of gender, a significant association of EGFR CNG was found with tumor grade (P = 0.044) and cirrhosis (P = 0.015) exclusively in the male group only; however, the OS and RFS analysis show no significant difference between male and female groups. EGFR CNG was related to crucial clinicopathological features and early recurrence, indicating that EGFR CNG might be a poor prognosis factor for Taiwanese HCC. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Renal effects of metallothionein induction by zinc in vitro and in vivo.

PubMed

Schanz, Moritz; Schaaf, Lea; Dippon, Juergen; Biegger, Dagmar; Fritz, Peter; Alscher, Mark Dominik; Kimmel, Martin

2017-03-16

Metallothionein (MTT) is an endogenous antioxidant that can be induced by both zinc (Zn) and ischemia. In kidneys, increased MTT expression exerts a putative protective role in diabetes and hypoxia. Our goal was to further investigate the behavior of MTT under the influence of Zn and hypoxia in vitro and in vivo. MTT expression was measured in vitro in cell cultures of proximal tubular cells (LCC-PK1) by immune-histochemistry and real-time PCR after incubation with increasing concentrations of Zn under hypoxic and non-hypoxic conditions. In addition, in vivo studies were carried out in 54 patients to study MTT induction through Zn. This is a sub-study of a prospective, randomized, double-blind trial on prevention of contrast-media-induced nephropathy using Placebo, Zn and N-Acetylcysteine. Blood samples were obtained before and after 2 days p.o. treatment with or without Zn (60 mg). ELISA-based MTT level measurements were done to evaluate the effects of Zn administration. For in vivo analysis, we considered the ratio of MTT to baseline MTT (MTT 1 /MTT 0 ) and the ratio of eGFR (eGFR 1 /eGFR 0 ), correspondingly. In vitro quantitative immuno-histochemical analysis (IHC) and real-time PCR showed that at increasing levels of Zn (5, 10, and 15 μg/ml) led to a progressive increase of MTTs: Median (IQR) expression of IHC also increased progressively from 0.10 (0.09-0.12), 0.15 (0.12-0.18), 0.25 (0.25-0.27), 0.59 (0.48-0.70) (p < 0.0001). Median (IQR) expression of PCR: 0.59 (0.51-1.72), 1.62 (1.38-4.70), 3.58 (3.06-10.42) and 10.81 (9.24-31.47) (p < 0.0001). In contrast, hypoxia did not change MTT-levels in vitro (p > 0.05). In vivo no significant differences (p = 0.96) occurred in MTT-levels after 2 days of Zn administration compared with no Zn intake. Nevertheless, there was a significant correlation between MTT (MTT 1 /MTT 0 ) and eGFR (eGFR 1 /eGFR 0 ) in case of Zn administration (rho = -0.49; 95%-CI: -0.78 to -0.03; p = 0.04). We found that Zn did induce MTTs in vitro, whereas hypoxia had no significant impact. In contrast, no significant increase of MTTs was detected after in vivo administration of Zn. However, there was a significant negative correlation between MTT and eGFR in vivo in case of Zn administration, this could indicate a protective role of MTTs in a setting of reduced kidney function, which is possibly influenced by Zn. ClinicalTrials.gov Identifier: NCT00399256 . Retrospectively registered 11/13/2006.

EGFR Is Regulated by TFAP2C in Luminal Breast Cancer and Is a Target for Vandetanib.

PubMed

De Andrade, James P; Park, Jung M; Gu, Vivian W; Woodfield, George W; Kulak, Mikhail V; Lorenzen, Allison W; Wu, Vincent T; Van Dorin, Sarah E; Spanheimer, Philip M; Weigel, Ronald J

2016-03-01

Expression of TFAP2C in luminal breast cancer is associated with reduced survival and hormone resistance, partially explained through regulation of RET. TFAP2C also regulates EGFR in HER2 breast cancer. We sought to elucidate the regulation and functional role of EGFR in luminal breast cancer. We used gene knockdown (KD) and treatment with a tyrosine kinase inhibitor (TKI) in cell lines and primary cancer isolates to determine the role of RET and EGFR in regulation of p-ERK and tumorigenesis. KD of TFAP2C decreased expression of EGFR in a panel of luminal breast cancers, and chromatin immunoprecipitation sequencing (ChIP-seq) confirmed that TFAP2C targets the EGFR gene. Stable KD of TFAP2C significantly decreased cell proliferation and tumor growth, mediated in part through EGFR. While KD of RET or EGFR reduced proliferation (31% and 34%, P < 0.01), combined KD reduced proliferation greater than either alone (52% reduction, P < 0.01). The effect of the TKI vandetanib on proliferation and tumor growth response of MCF-7 cells was dependent upon expression of TFAP2C, and dual KD of RET and EGFR eliminated the effects of vandetanib. The response of primary luminal breast cancers to TKIs assessed by ERK activation established a correlation with expression of RET and EGFR. We conclude that TFAP2C regulates EGFR in luminal breast cancer. Response to vandetanib was mediated through the TFAP2C target genes EGFR and RET. Vandetanib may provide a therapeutic effect in luminal breast cancer, and RET and EGFR can serve as molecular markers for response. ©2016 American Association for Cancer Research.

EGFR Is Regulated by TFAP2C in Luminal Breast Cancer and Is a Target for Vandetanib

PubMed Central

De Andrade, James P.; Park, Jung M.; Gu, Vivian W.; Woodfield, George W.; Kulak, Mikhail V.; Lorenzen, Allison W.; Wu, Vincent T.; Van Dorin, Sarah E.; Spanheimer, Philip M.; Weigel, Ronald J.

2016-01-01

Expression of TFAP2C in luminal breast cancer is associated with reduced survival and hormone resistance, partially explained through regulation of RET. TFAP2C also regulates EGFR in HER2 breast cancer. We sought to elucidate the regulation and functional role of EGFR in luminal breast cancer. We used gene knockdown (KD) and treatment with a tyrosine kinase inhibitor (TKI) in cell lines and primary cancer isolates to determine the role of RET and EGFR in regulation of p-ERK and tumorigenesis. KD of TFAP2C decreased expression of EGFR in a panel of luminal breast cancers and ChIP-seq confirmed that TFAP2C targets the EGFR gene. Stable KD of TFAP2C significantly decreased cell proliferation and tumor growth, mediated in part through EGFR. While KD of RET or EGFR reduced proliferation (31% and 34%, p < 0.01), combined KD reduced proliferation greater than either alone (52% reduction, p < 0.01). The effect of the TKI vandetanib on proliferation and tumor growth response of MCF-7 cells was dependent upon expression of TFAP2C and dual KD of RET and EGFR eliminated the effects of vandetanib. The response of primary luminal breast cancers to TKIs assessed by ERK activation established a correlation with expression of RET and EGFR. We conclude that TFAP2C regulates EGFR in luminal breast cancer. Response to vandetanib was mediated though the TFAP2C target genes EGFR and RET. Vandetanib may provide a therapeutic effect in luminal breast cancer, and RET and EGFR can serve as molecular markers for response. PMID:26832794

Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease.

PubMed

Cortés-Ramírez, Dionisio-Alejandro; Rodríguez-Tojo, María-Jose; Coca-Meneses, Juan-Carlos; Marichalar-Mendia, Xabier; Aguirre-Urizar, José-Manuel

2014-09-01

The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk.

[Study on the correlation between EGFR-STAT3 signal pathway and laryngeal papilloma].

PubMed

Wang, Xinhua; Sun, Jingwu

2009-09-01

To explore the relationship between the expression of EGFR and STAT3 in human laryngeal papilloma and its biological behavior. Reverse transcription polymerase chain reaction(RT-PCR), immunohistochemical staining and Western blot were used to evaluate the mRNA and protein expression of EGFR and STAT3 (p-STAT3) in 42 laryngeal papilloma tissues and 15 samples of normal laryngeal tissue, and the relationship between the protein expression of them and clinic pathological parameters was also analyzed. The mRNA expression levels of EGFR and STAT3 in laryngeal papilloma tissue were significantly higher than that in normal laryngeal tissue (P < 0.05, P < 0.01). Protein positive expression of EGFR and p-STAT3 were also detected in a significantly greater proportion of laryngeal papilloma than normal laryngeal tissue by immunohistochemistry and western blot (P < 0.01, P < 0.05). There was relationship between EGFR and p-STAT3 overexpression in laryngeal papilloma (P < 0.05). The expression p-STAT3 was correlated with the recurrence and canceration of laryngeal papilloma (P < 0.05). The EGFR-STAT3 signal transduction pathway may be involved in the pathogenesis of laryngeal papilloma,, and the persistent activation of STAT3 gene plays an important role in the recurrence and canceration of laryngeal papilloma.

Decreased glutathione biosynthesis contributes to EGFR T790M-driven erlotinib resistance in non-small cell lung cancer.

PubMed

Li, Hongde; Stokes, William; Chater, Emily; Roy, Rajat; de Bruin, Elza; Hu, Yili; Liu, Zhigang; Smit, Egbert F; Heynen, Guus Jje; Downward, Julian; Seckl, Michael J; Wang, Yulan; Tang, Huiru; Pardo, Olivier E

2016-01-01

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib are novel effective agents in the treatment of EGFR-driven lung cancer, but their clinical impact is often impaired by acquired drug resistance through the secondary T790M EGFR mutation. To overcome this problem, we analysed the metabonomic differences between two independent pairs of erlotinib-sensitive/resistant cells and discovered that glutathione (GSH) levels were significantly reduced in T790M EGFR cells. We also found that increasing GSH levels in erlotinib-resistant cells re-sensitised them, whereas reducing GSH levels in erlotinib-sensitive cells made them resistant. Decreased transcription of the GSH-synthesising enzymes (GCLC and GSS) due to the inhibition of NRF2 was responsible for low GSH levels in resistant cells that was directly linked to the T790M mutation. T790M EGFR clinical samples also showed decreased expression of these key enzymes; increasing intra-tumoural GSH levels with a small-molecule GST inhibitor re-sensitised resistant tumours to erlotinib in mice. Thus, we identified a new resistance pathway controlled by EGFR T790M and a therapeutic strategy to tackle this problem in the clinic.

Expression of EGF and EGFR strongly correlates with metastasis of pancreatic ductal carcinoma.

PubMed

Pryczynicz, Anna; Guzińska-Ustymowicz, Katarzyna; Kemona, Andrzej; Czyzewska, Jolanta

2008-01-01

The epidermal growth factor family members: EGF, EGFR and the c-erbB-2(HER-2/neu) gene product have been found to play a role in carcinomas of the stomach, liver, breast, ovary and lungs. Recent reports have indicated that they are also involved in the growth of pancreatic ductal carcinoma, its invasiveness and metastasis. Thirty-six patients with pancreatic ductal carcinoma were analysed with respect to sex, age, histological type, malignancy grade (G), pTN status (pTN), local lymph node involvement and distant metastasis. The tumor levels of EGF, EGFR and c-erbB-2 expression were determined immunohistochemically. Expression of c-erbB-2 was observed in 24/36 cases, EGF in 13/36 cases and EGFR in 18/36 cases. Overexpression of EGF and EGFR was associated with metastasis to lymph nodes and other organs. A correlation was also found between EGF expression and the presence of EGFR in the tumour. The expression of c-erbB-2 protein was not found to correlate with any parameters. EGF and EGFR play a key role in neoplastic spread through lymph node involvement and metastasis to other organs.

Knockdown of HIP1 expression promotes ligand‑induced endocytosis of EGFR in HeLa cells.

PubMed

Li, Dan; Chen, Fenglin; Ding, Jian; Lin, Na; Li, Zonghai; Wang, Xiaozhong

2017-12-01

Huntington-interacting protein 1 (HIP1) is associated with various tumor types; however, its precise functions in tumor cells are unclear. In this study, the effects of HIP1 on the degradation of EGFR, which have important roles in carcinogenesis after EGF stimulation, were examined. After screening 17 cell lines, the coexpression of HIP1 and EGFR was detected in HeLa cells. Accordingly, the expression of HIP1 was knocked down in HeLa cells using various HIP1 siRNA sequences. The endocytosis of EGFR and localization of clathrin in HeLa cells were examined after stimulation by EGF at various concentrations (i.e., 1.5 and 100 ng/ml). After HIP1 expression was blocked by siRNAs, EGFR endocytosis was accelerated and this effect was dependent on the EGF concentration. This endocytosis was colocalized with clathrin expression. These findings indicate that the inhibition of HIP1 can accelerate the endocytosis and degradation of EGFR. Furthermore, they suggest that HIP1 is a potential therapeutic target for various cancer types, particularly those with high EGFR expression, but further research is needed to examine this hypothesis.

Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

PubMed Central

Li, Dan; Chen, Fenglin; Ding, Jian; Lin, Na; Li, Zonghai; Wang, Xiaozhong

2017-01-01

Huntington-interacting protein 1 (HIP1) is associated with various tumor types; however, its precise functions in tumor cells are unclear. In this study, the effects of HIP1 on the degradation of EGFR, which have important roles in carcinogenesis after EGF stimulation, were examined. After screening 17 cell lines, the coexpression of HIP1 and EGFR was detected in HeLa cells. Accordingly, the expression of HIP1 was knocked down in HeLa cells using various HIP1 siRNA sequences. The endocytosis of EGFR and localization of clathrin in HeLa cells were examined after stimulation by EGF at various concentrations (i.e., 1.5 and 100 ng/ml). After HIP1 expression was blocked by siRNAs, EGFR endocytosis was accelerated and this effect was dependent on the EGF concentration. This endocytosis was colocalized with clathrin expression. These findings indicate that the inhibition of HIP1 can accelerate the endocytosis and degradation of EGFR. Furthermore, they suggest that HIP1 is a potential therapeutic target for various cancer types, particularly those with high EGFR expression, but further research is needed to examine this hypothesis. PMID:29039605

Dual-modality wide-field photothermal quantitative phase microscopy and depletion of cell populations

NASA Astrophysics Data System (ADS)

Turko, Nir A.; Barnea, Itay; Blum, Omry; Korenstein, Rafi; Shaked, Natan T.

2015-03-01

We review our dual-modality technique for quantitative imaging and selective depletion of populations of cells based on wide-field photothermal (PT) quantitative phase imaging and simultaneous PT cell extermination. The cells are first labeled by plasmonic gold nanoparticles, which evoke local plasmonic resonance when illuminated by light in a wavelength corresponding to their specific plasmonic resonance peak. This reaction creates changes of temperature, resulting in changes of phase. This phase changes are recorded by a quantitative phase microscope (QPM), producing specific imaging contrast, and enabling bio-labeling in phase microscopy. Using this technique, we have shown discrimination of EGFR over-expressing (EGFR+) cancer cells from EGFR under-expressing (EGFR-) cancer cells. Then, we have increased the excitation power in order to evoke greater temperatures, which caused specific cell death, all under real-time phase acquisition using QPM. Close to 100% of all EGFR+ cells were immediately exterminated when illuminated with the strong excitation beam, while all EGFR- cells survived. For the second experiment, in order to simulate a condition where circulating tumor cells (CTCs) are present in blood, we have mixed the EGFR+ cancer cells with white blood cells (WBCs) from a healthy donor. Here too, we have used QPM to observe and record the phase of the cells as they were excited for selective visualization and then exterminated. The WBCs survival rate was over 95%, while the EGFR+ survival rate was under 5%. The technique may be the basis for real-time detection and controlled treatment of CTCs.

AXL mediates resistance to cetuximab therapy

PubMed Central

Brand, Toni M.; Iida, Mari; Stein, Andrew P.; Corrigan, Kelsey L.; Braverman, Cara; Luthar, Neha; Toulany, Mahmoud; Gill, Parkash S.; Salgia, Ravi; Kimple, Randall J.; Wheeler, Deric L.

2014-01-01

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers. PMID:25136066

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer

PubMed Central

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo, erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity. PMID:29312548

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

PubMed

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-12-12

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo , erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity.

Propofol enhances the cisplatin-induced apoptosis on cervical cancer cells via EGFR/JAK2/STAT3 pathway.

PubMed

Li, Haoran; Lu, Yan; Pang, Yangyang; Li, Mengjiao; Cheng, Xi; Chen, Jiawei

2017-02-01

The main purpose of this study was to evaluate propofol and its combined effect with cisplatin on apoptosis of cervical cancer cells and molecular mechanisms of this phenomenon. The effects of propofol and cisplatin on cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. Besides, protein expression of EGFR/JAK2/STAT3 pathway was determined by western blot. STAT3 was over-expressed in cervical cancer cells by STAT3 cDNA. Expression of EGFR and STAT3 protein of human tissues was evaluated by immunohistochemistry (IHC) assay. In this study, we found that not only propofol alone could inhibit cervical cancer cells viability but also could increase the inhibitory effect of cisplatin on cervical cancer cells growth. Meanwhile, propofol sensitized cervical cancer cells to cisplatin-induced apoptosis but not affected normal cervical cells. In genetic level, propofol could enhance the anti-tumor effect of cisplatin through EGFR/JAK2/STAT3 pathway. Further studies indicated that overexpression of EGFR and STAT3 is related to poor prognoses in cervical cancer patients, which contributed to confirm the clinical role of combined application of propofol and cisplatin. Propofol enhances the cisplatin-induced cell apoptosis cervical cancer cells via EGFR/JAK2/STAT3 pathway and may be developed as a potential therapeutic agent to treat cervical cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Histone Deacetylase 3 Inhibition Overcomes BIM Deletion Polymorphism-Mediated Osimertinib Resistance in EGFR-Mutant Lung Cancer.

PubMed

Tanimoto, Azusa; Takeuchi, Shinji; Arai, Sachiko; Fukuda, Koji; Yamada, Tadaaki; Roca, Xavier; Ong, S Tiong; Yano, Seiji

2017-06-15

Purpose: The BIM deletion polymorphism is associated with apoptosis resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, in non-small cell lung cancer (NSCLC) harboring EGFR mutations. Here, we investigated whether the BIM deletion polymorphism contributes to resistance against osimertinib, a third-generation EGFR-TKI. In addition, we determined the efficacy of a histone deacetylase (HDAC) inhibitor, vorinostat, against this form of resistance and elucidated the underlying mechanism. Experimental Design: We used EGFR -mutated NSCLC cell lines, which were either heterozygous or homozygous for the BIM deletion polymorphism, to evaluate the effect of osimertinib in vitro and in vivo Protein expression was examined by Western blotting. Alternative splicing of BIM mRNA was analyzed by RT-PCR. Results: EGFR -mutated NSCLC cell lines with the BIM deletion polymorphism exhibited apoptosis resistance to osimertinib in a polymorphism dosage-dependent manner, and this resistance was overcome by combined use with vorinostat. Experiments with homozygous BIM deletion-positive cells revealed that vorinostat affected the alternative splicing of BIM mRNA in the deletion allele, increased the expression of active BIM protein, and thereby induced apoptosis in osimertinib-treated cells. These effects were mediated predominantly by HDAC3 inhibition. In xenograft models, combined use of vorinostat with osimertinib could regress tumors in EGFR -mutated NSCLC cells homozygous for the BIM deletion polymorphism. Moreover, this combination could induce apoptosis even when tumor cells acquired EGFR -T790M mutations. Conclusions: These findings indicate the importance of developing HDAC3-selective inhibitors, and their combined use with osimertinib, for treating EGFR -mutated lung cancers carrying the BIM deletion polymorphism. Clin Cancer Res; 23(12); 3139-49. ©2016 AACR . ©2016 American Association for Cancer Research.

EGFR is involved in dermatofibrosarcoma protuberans progression to high grade sarcoma.

PubMed

Osio, Amélie; Xu, Shuo; El Bouchtaoui, Morad; Leboeuf, Christophe; Gapihan, Guillaume; Lemaignan, Christine; Bousquet, Guilhem; Lebbé, Céleste; Janin, Anne; Battistella, Maxime

2018-02-02

Dermatofibrosarcoma protuberans (DFSP), amounting to 6% of all soft tissue sarcomas, has a slow growth rate, contrasting with a likelihood for local recurrence and a 10-20% evolution to higher-grade sarcoma, or "transformed DFSP" (DFSP-T). At molecular level, the characteristic COL1A1-PDGFB rearrangement, leading to sustained PDGFR signaling, is not linked to the evolutive potential. Here, we studied EGFR, another tyrosine kinase receptor, using laser-microdissection to select the different histologic components of DFSP (DFSP center, DFSP infiltrative periphery, DFSP-T higher-grade sarcoma), in 22 patients followed over 3 to 156 months. EGFR protein and mRNA were expressed in 13/22 patients with DFSP or DFSP-T, and increased with tumor progression, both in microdissected areas of higher-grade sarcomas and in microdissected areas of local extension. No cancer-associated EGFR gene mutation or copy-number variation, nor any KRAS, BRAF, NRAS hotspot mutations were found in any microdissected area. Among epithelial-mesenchymal transition factors tested, SNAIL 1/2 had the same expression pattern as EGFR while ZEB1/2 or TWIST1/2 did not. Using a proteome profiler phospho-kinase array on 3 DFSP and 3 DFSP-T cryopreserved tissue samples, EGFR phosphorylation was detected in each case. Among EGFR downstream pathways, we found positive correlations between phosphorylation levels of EGFR and STAT5a/b (r = 0.87, p < 0.05) and TOR (r = 0.95, p < 0.01), but not ERK in the MAPK pathway (r = -0.18, p > 0.70). We thus demonstrated that in DFSP evolution to high grade sarcoma, EGFR and SNAIL were involved, with EGFR activation and signaling through TOR and STAT5a/b downstream effectors, which could lead on to new therapies for advanced DFSP.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

PubMed

Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

2016-06-01

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

Epidermal Growth Factor Receptor Expression As Prognostic Marker in Patients With Anal Carcinoma Treated With Concurrent Chemoradiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraunholz, Ingeborg, E-mail: inge.fraunholz@kgu.de; Rödel, Franz; Kohler, Daniela

Purpose: To investigate the prognostic value of epidermal growth factor receptor (EGFR) expression in pretreatment tumor biopsy specimens of patients with anal cancer treated with concurrent 5-fluorouracil and mitomycin C-based chemoradiation therapy (CRT). Methods and Materials: Immunohistochemical staining for EGFR was performed in pretreatment biopsy specimens of 103 patients with anal carcinoma. EGFR expression was correlated with clinical and histopathologic characteristics and with clinical endpoints, including local failure-free survival (LFFS), colostomy-free survival (CFS), distant metastases-free survival (DMFS), cancer-specific survival (CSS), and overall survival (OS). Results: EGFR staining intensity was absent in 3%, weak in 23%, intermediate in 36% and intensemore » in 38% of the patients. In univariate analysis, the level of EGFR staining was significantly correlated with CSS (absent/weak vs intermediate/intense expression: 5-year CSS, 70% vs 86%, P=.03). As a trend, this was also observed for DMFS (70% vs 86%, P=.06) and LFFS (70% vs 87%, P=.16). In multivariate analysis, N stage, tumor differentiation, and patients’ sex were independent prognostic factors for CSS, whereas EGFR expression only reached borderline significance (hazard ratio 2.75; P=.08). Conclusion: Our results suggest that elevated levels of pretreatment EGFR expression could be correlated with favorable clinical outcome in anal cancer patients treated with CRT. Further studies are warranted to elucidate how EGFR is involved in the response to CRT.« less

Down-regulation of microRNA-146a is associated with high-risk human papillomavirus infection and epidermal growth factor receptor overexpression in penile squamous cell carcinoma.

PubMed

Peta, Elektra; Cappellesso, Rocco; Masi, Giulia; Sinigaglia, Alessandro; Trevisan, Marta; Grassi, Angela; Di Camillo, Barbara; Vassarotto, Elisa; Fassina, Ambrogio; Palù, Giorgio; Barzon, Luisa

2017-03-01

Dysregulation of host microRNA expression has been involved in the development and progression of human papillomavirus (HPV)-related tumors. Analysis of miR-146a expression in a series of 59 penile squamous cell carcinomas (PSCCs) showed that its levels were lower in high-risk HPV-positive than in HPV-negative PSCCs and inversely correlated with expression of epidermal growth factor receptor (EGFR), a known target for miR-146a. Analysis of genotype distribution for rs2910164, a common functional polymorphism of miR-146a, did not identify correlations with miR-146a levels and EGFR expression in PSCCs. In vitro experiments demonstrated that E6 of HPV type 16, but not low-risk HPV-6, down-regulated miR-146a in human foreskin keratinocytes and up-regulated EGFR. Ectopic expression of miR-146a decreased expression of EGFR and inhibited proliferation of keratinocytes and cervical carcinoma cells. EGFR is commonly overexpressed in penile cancer and in other squamous cell carcinomas. Molecular mechanisms leading to EGFR overexpression and activation are known for HPV-negative cancers and include amplification or mutations of the EGFR gene. The results of this study indicate that down-regulation of miR-146a may represent another mechanism of EGFR overexpression in PSCCs, which can be mediated by high-risk HPV E6 in HPV-related tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

EGFR expression in circulating tumor cells from high-grade metastatic soft tissue sarcomas.

PubMed

Braun, Alexcia Camila; de Mello, Celso Abdon Lopes; Corassa, Marcelo; Abdallah, Emne Ali; Urvanegia, Ana Cláudia; Alves, Vanessa Silva; Flores, Bianca C T C P; Díaz, Mônica; Nicolau, Ulisses Ribaldo; Silva, Virgilio Souza E; Calsavara, Vinicius; Paterlini-Brechót, Patrizia; Chinen, Ludmilla Thomé Domingos

2018-06-03

Soft tissue Sarcomas (STS) are rare malignances, with high mortality rates. Half of patients develop metastasis. The presence of isolated Circulating Tumor Cells (CTCs) and Circulating Tumor Microemboli (CTM) in the blood may be early markers of tumor invasion. Epidermal Growth Factor (EGF) family receptors can also influence this process. to quantify CTCs and identify CTM as well as the EGF Receptor (EGFR) protein expression in these cells and correlate with clinical outcome in metastatic STS. Approximately 8mL of blood was prospectively collected from patients with different types of high-grade STS, before the beginning of chemotherapy. The samples were processed and filtered by ISET (Rarecells, France) for the isolation and quantification of CTCs and CTMs. EGFR expression was analyzed by immunocytochemistry (ICC) on CTCs/ CTMs. We analyzed 18 patients with median age of 49 years (18-77 y). The positivity for EGFR protein expression in CTCs was observed in 93.75% of the patients. This result shows that targeting EGFR positive CTCs from STS origen can be translated in clinical benefit for some patients. In addition, if target therapy is chosen, the EGFR expression in CTCs can be used in follow-up to measure treatment effectiveness. This is the first study to demonstrate the expression of EGFR protein in CTCs from sarcoma patients. It may open an area for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of EGFR in sustaining tumor metastasis in sarcomas.

MicroRNA-142 Inhibits Proliferation and Promotes Apoptosis in Airway Smooth Muscle Cells During Airway Remodeling in Asthmatic Rats via the Inhibition of TGF-β -Dependent EGFR Signaling Pathway.

PubMed

Wang, Jing; Wang, Hu-Shan; Su, Zhen-Bo

2018-06-27

Asthma is a heterogeneous disease characterized by chronic airway inflammation resulting from airway hyper-responsiveness to diverse stimuli. In this study, we investigated whether microRNA-142 (miR-142) expression affects proliferation and apoptosis in airway smooth muscle cells (ASMCs) during airway remodeling in asthmatic rats. Thirty six Wistar rats were randomly classified into a control group and an model group. miR-142 mimics and inhibitors were constructed, and ASMCs were transfected using liposomes according to the following groups: blank, negative control (NC), miR-142 mimics, miR-142 inhibitors, si-TGF-β and miR-142 inhibitors + si-TGF-β. We verified that miR-142 targets TGF-β using a dual-luciferase reporter assay. The expression levels of miR-142, TGF-β, EGFR and apoptosis signaling pathway-related genes were determined using RT-qPCR and western blotting. Changes in cell proliferation, cell cycle progression and apoptosis were analyzed using MTT assays and flow cytometry. Rats with asthma had higher expression levels of EGFR and Akt and lower miR-142 levels. miR-142 was negatively correlated with TGF-β expression. In ASMCs, the expression of TGF-β, EGFR, Akt, phosphorylated-Akt (p-Akt), Bcl-2 and Bcl-xl and the rate of early apoptosis were decreased while expression of Bax and p21 and the proliferation rate were elevated with the upregulation of miR-142. The opposite results were observed with the downregulation of miR-142. Finally, the proliferative rate was decreased while the apoptosis rate was increased and expression levels of EGFR, Akt, p-Akt, Bcl-2 and Bcl-xl were reduced while Bax and p21 were elevated in the ASMCs transfected with miR-142 inhibitors and si-TGF-β. The results of our study suggest that miR-142 inhibits proliferation and promotes apoptosis in ASMCs during airway remodeling in asthmatic rats by inhibiting TGF-β expression via a mechanism involving the EGFR signaling pathway. © 2018 The Author(s). Published by S. Karger AG, Basel.

Up-regulation of miR-146a contributes to the inhibition of invasion of pancreatic cancer cells

PubMed Central

Li, Yiwei; VandenBoom, Timothy G.; Wang, Zhiwei; Kong, Dejuan; Ali, Shadan; Philip, Philip A.; Sarkar, Fazlul H.

2009-01-01

Pancreatic cancer (PC) is an aggressive malignancy with high mortality and is believed to be in part due to its highly invasive and metastatic behavior, which is associated with over-expression of EGFR and activation of NF-κB. Emerging evidence also suggest critical roles of microRNAs (miRNAs) in the regulation of various pathobiological processes including metastasis in PC and in other human malignancies. In the present study, we found lower expression of miR-146a in PC cells compared to normal human pancreatic duct epithelial (HPDE) cells. Interestingly, re-expression of miR-146a inhibited the invasive capacity of Colo357 and Panc-1 PC cells with concomitant down-regulation of EGFR and IRAK-1. Mechanistic studies including miR-146a re-expression, anti-miR-146 transfection, and EGFR knock-down experiment showed that there was a crosstalk between EGFR, MTA-2, IRAK-1, IκBα and NF-κB. Most importantly, we found that the treatment of PC cells with “natural agents” [3,3′-diinodolylmethane (DIM) or isoflavone] led to an increase in the expression of miR-146a and consequently down-regulated the expression of EGFR, MTA-2, IRAK-1 and NF-κB, resulting in the inhibition of invasion of Colo357 and Panc-1 cells. These results provide experimental evidence in support of the role of DIM and isoflavone as potential non-toxic agents as regulators of miRNA, which could be useful for the inhibition of cancer cell invasion and metastasis, and further suggesting that these agents could be important for designing novel targeted strategy for the treatment of PC. PMID:25242818

Epidermal growth factor receptor expression is related to post-mitotic events in cerebellar development: regulation by thyroid hormone.

PubMed

Carrasco, Emilce; Blum, Mariann; Weickert, Cynthia Shannon; Casper, Diana

2003-01-10

It has been established that thyroid hormone and neurotrophic factors both orchestrate developmental events in the brain. However, it is not clear how these two influences are related. In this study, we investigated the effects of thyroid hormone on cerebellar development and the coincident expression of transforming growth factor-alpha (TGF-alpha), a ligand in the epidermal growth factor (EGF) family, and the epidermal growth factor receptor (EGFR). Profiles of thyroid hormone expression were measured in postnatal animals and were found to peak at postnatal day 15 (P15). These levels dropped below detectable levels when mice were made hypothyroid with propylthiouracil (PTU). TGF-alpha and EGFR expression, as determined by RNAse protection assay, was maximal at P6 in normal animals, but remained low in hypothyroid animals, suggesting that thyroid hormone was responsible for their induction. In situ hybridization and immunohistochemical analysis of EGFR expression revealed that this receptor was present on granule cells within the inner zone of the external granule cell layer (EGL), suggesting that EGFR-ligands were not inducing granule cell proliferation. The persistence of EGFR expression on migrating granule cells and subsequent down-regulation of expression in the internal granule cell layer (IGL) implicates a role for EGFR-ligands in differentiation and/or migration. In hypothyroid animals, we observed a delayed progression of granule cell migration, consistent with the persistence of EGFR labeling in the EGL, and in the 'pile-up' of labeled cells at the interface between the molecular layer and the Purkinje cell layer. Taken together, these results implicate thyroid hormone in the coordinated expression of TGF-alpha and EGFR, which are positioned to play a role in post-mitotic developmental events in the cerebellum.

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization.

PubMed

Aifa, Sami; Aydin, Jan; Nordvall, Gunnar; Lundström, Ingemar; Svensson, Samuel P S; Hermanson, Ola

2005-01-01

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.

ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion.

PubMed

Xiao, Li-Jie; Lin, Ping; Lin, Feng; Liu, Xin; Qin, Wei; Zou, Hai-Feng; Guo, Liang; Liu, Wei; Wang, Shu-Juan; Yu, Xiao-Guang

2012-05-01

ADAM17, also known as tumor necrosis factor-α converting enzyme (TACE), is involved in proteolytic ectodomain shedding of cell surface molecules and cytokines. Although aberrant expression of ADAM17 has been shown in various malignancies, the function of ADAM17 in prostate cancer has not been clarified. In the present study, we sought to elucidate whether ADAM17 contributes to prostate cancer cell invasion, as well as the mechanism involved in the process. The expression pattern of ADAM17 was investigated in human prostate cancer cells. The results showed that ADAM17 expression levels are correlated with the invasive ability of androgen-independent prostate cancer cell lines. Further, ADAM17 was overexpressed in cells showing high invasion characteristics, activation of the EGFR-MEK-ERK pathway, up-regulation of MMP-2, MMP-9, and an increased TGF-α release into the supernatant. However, AG1478, PD98059 and antibody against TGF-α deactivating the EGFR-MEK-ERK signaling pathway, abolished up-regulation of MMP-2, MMP-9 and prevented cell invasion. In addition, cells with knockdown of ADAM17 by siRNA exhibited low invasive ability, deactivated EGFR-MEK-ERK signaling pathway, reduced TGF-α released and down-regulation of MMP-2, MMP-9. However, these effects could be reversed by simultaneous addition of TGF-α. These data demonstrated that ADAM17 contributes to androgen-independent prostate cancer cell invasion by shedding of EGFR ligand TGF-α, which subsequently activates the EGFR-MEK-ERK signaling pathway, leading finally to overexpression of MMP-2 and MMP-9. This study suggests that the ADAM17 expression level may be a new predictive biomarker of invasion and metastasis of prostate cancer, and ADAM17 could provide a target for treating metastatic PCa.

Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations.

PubMed

Masuzawa, Keita; Yasuda, Hiroyuki; Hamamoto, Junko; Nukaga, Shigenari; Hirano, Toshiyuki; Kawada, Ichiro; Naoki, Katsuhiko; Soejima, Kenzo; Betsuyaku, Tomoko

2017-12-01

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations (exon 19 deletion and L858R, with or without T790M), osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations.

Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations

PubMed Central

Masuzawa, Keita; Yasuda, Hiroyuki; Hamamoto, Junko; Nukaga, Shigenari; Hirano, Toshiyuki; Kawada, Ichiro; Naoki, Katsuhiko; Soejima, Kenzo; Betsuyaku, Tomoko

2017-01-01

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations (exon 19 deletion and L858R, with or without T790M), osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations. PMID:29285266

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR

DOE PAGES

Wang, Yunshan; Zhang, Pengju; Liu, Ziming; ...

2014-11-21

CUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear. Expression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively. We found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpressionmore » of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities. In conclusion, our results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.« less

Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis.

PubMed

Karachaliou, Niki; Chaib, Imane; Cardona, Andres Felipe; Berenguer, Jordi; Bracht, Jillian Wilhelmina Paulina; Yang, Jie; Cai, Xueting; Wang, Zhigang; Hu, Chunping; Drozdowskyj, Ana; Servat, Carles Codony; Servat, Jordi Codony; Ito, Masaoki; Attili, Ilaria; Aldeguer, Erika; Capitan, Ana Gimenez; Rodriguez, July; Rojas, Leonardo; Viteri, Santiago; Molina-Vila, Miguel Angel; Ou, Sai-Hong Ignatius; Okada, Morihito; Mok, Tony S; Bivona, Trever G; Ono, Mayumi; Cui, Jean; Cajal, Santiago Ramón Y; Frias, Alex; Cao, Peng; Rosell, Rafael

2018-03-01

Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells

PubMed Central

Bazzani, Lorenzo; Donnini, Sandra; Giachetti, Antonio; Christofori, Gerhard; Ziche, Marina

2018-01-01

Prostaglandin E2 (PGE2) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1, PTGS2, MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE2-induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression. PMID:29599917

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells.

PubMed

Bazzani, Lorenzo; Donnini, Sandra; Giachetti, Antonio; Christofori, Gerhard; Ziche, Marina

2018-03-13

Prostaglandin E 2 (PGE 2 ) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE 2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE 2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE 2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE 2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1 , PTGS2 , MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE 2 -induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling.

PubMed

Sun, Xiang; Xu, Yang; Wang, Yi; Chen, Qian; Liu, Liu; Bao, Yangyi

2018-05-15

BACKGROUND Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the treatment of non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. However, the survival of patients with EGFR-TKI administration is limited by the inevitable development of acquired drug resistance. Recently, multi-targeted drugs combination has been shown to be a promising strategy to improve the efficacy of EGFR-TKI treatment and enable the reduction of drug resistance in NSCLC. MATERIAL AND METHODS Humanized NSCLC cell lines PC9 and A549 were co-cultured with thalidomide and/or icotinib to test for anti-tumor efficiency. Cell proliferation was measured by MTT assay, cell apoptosis by flow cytometry and cell migration by wound healing assay. Western blot was performed to determine the expression of caspase-3, -8, -9, Bax, EGFR, VEGF-R, AKT, ERK, MMP2, MMP9, and NF-κB. The xenograft mouse model was used to explore the effects of thalidomide and icotinib in vivo. Immunohistochemical testing was used to determine the expression of Ki-67 and TUNEL staining in tumor tissues. RESULTS Treatments of thalidomide and/or icotinib reduced cell viability, induced apoptosis, and suppressed migration. Attenuation of pEGFR and pVEGF-R resulted in deactivation of ERK and AKT pathways, which eventually increased the anti-proliferative response. In PC9 xenograft model, combined administration of thalidomide and icotinib restrained tumor growth with remarkable reduced Ki-67 index and increased TUNEL positive cells. CONCLUSIONS Thalidomide sensitizes icotinib to increase apoptosis and prevent migration, and it may be a potentially promising anti-tumor drug in lung cancer multi-modality therapy.

Gender-related survival differences associated with EGFR polymorphisms in metastatic colon cancer.

PubMed

Press, Oliver A; Zhang, Wu; Gordon, Michael A; Yang, Dongyun; Lurje, Georg; Iqbal, Syma; El-Khoueiry, Anthony; Lenz, Heinz-Josef

2008-04-15

Evidence is accumulating supporting gender-related differences in the development of colonic carcinomas. Sex steroid hormone receptors are expressed in the colon and interact with epidermal growth factor receptor (EGFR), a gene widely expressed in colonic tissue. Increased EGFR expression is linked with poor prognosis in colon cancer. Within the EGFR gene there are two functional polymorphisms of interest: a polymorphism located at codon 497 (HER-1 R497K) and a dinucleotide (CA)(n) repeat polymorphism located within intron 1. These germ-line polymorphisms of EGFR were analyzed in genomic DNA from 318 metastatic colon cancer patients, 177 males and 141 females, collected from 1992 to 2003. Gender-related survival differences were associated with the HER-1 R497K polymorphism (P(interaction) = 0.003). Females with the HER-1 497 Arg/Arg variant had better overall survival (OS) when compared with the Lys/Lys and/or Lys/Arg variants. In males the opposite was true. The EGFR dinucleotide (CA)(n) repeat also trended with a gender-related OS difference (P(interaction) = 0.11). Females with both short <20 (CA)(n) repeat alleles had better OS than those with any long >or=20 (CA)(n) repeats. In males the opposite was true. Combination analysis of the two polymorphisms taken together also revealed the same gender-related survival difference (P(interaction) = 0.002). These associations were observed using multivariable analysis. The two polymorphisms were not in linkage disequilibrium and are independent of one another. This study supports the role of functional EGFR polymorphisms as independent prognostic markers in metastatic colon cancer. As a prognostic factor, these variants had opposite prognostic implications based on gender.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

The induction of C/EBPβ contributes to vitamin D inhibition of ADAM17 expression and parathyroid hyperplasia in kidney disease.

PubMed

Arcidiacono, Maria Vittoria; Yang, Jing; Fernandez, Elvira; Dusso, Adriana

2015-03-01

In secondary hyperparathyroidism (SHPT), enhanced parathyroid levels of transforming growth factor-α (TGFα) increase EGF receptor (EGFR) activation causing parathyroid hyperplasia, high parathyroid hormone (PTH) and also reductions in vitamin D receptor (VDR) that limit vitamin D suppression of SHPT. Since anti-EGFR therapy is not an option in human SHPT, we evaluated ADAM17 as a therapeutic target to suppress parathyroid hyperplasia because ADAM17 is required to release mature TGFα, the most potent EGFR-activating ligand. Computer analysis of the ADAM17 promoter identified TGFα and C/EBPβ as potential regulators of the ADAM17 gene. Their regulation of ADAM17 expression, TGFα/EGFR-driven growth and parathyroid gland (PTG) enlargement were assessed in promoter-reporter assays in A431 cells and corroborated in rat and human SHPT, using erlotinib as anti-EGFR therapy to suppress TGFα signals, active vitamin D to induce C/EBPβ or the combination. While TGFα induced ADAM17-promoter activity by 2.2-fold exacerbating TGFα/EGFR-driven growth, ectopic C/EBPβ expression completely prevented this vicious synergy. Accordingly, in advanced human SHPT, parathyroid ADAM17 levels correlated directly with TGFα and inversely with C/EBPβ. Furthermore, combined erlotinib + calcitriol treatment suppressed TGFα/EGFR-cell growth and PTG enlargement more potently than erlotinib in part through calcitriol induction of C/EBPβ to inhibit ADAM17-promoter activity, mRNA and protein. Importantly, in rat SHPT, the correction of vitamin D deficiency effectively reversed the resistance to paricalcitol induction of C/EBPβ to suppress ADAM17 expression and PTG enlargement, reducing PTH by 50%. In SHPT, correction of vitamin D and calcitriol deficiency induces parathyroid C/EBPβ to efficaciously attenuate the severe ADAM17/TGFα synergy, which drives PTG enlargement and high PTH. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Sulforaphane attenuates EGFR signaling in NSCLC cells.

PubMed

Chen, Chi-Yuan; Yu, Zhu-Yun; Chuang, Yen-Shu; Huang, Rui-Mei; Wang, Tzu-Chien V

2015-06-03

EGFR, a receptor tyrosine kinase (RTK), is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of many cancers, including NSCLC. However, intrinsic and acquired resistance to TKI remains a common obstacle. One strategy that may help overcome EGFR-TKI resistance is to target EGFR for degradation. As EGFR is a client protein of heat-shock protein 90 (HSP90) and sulforaphane is known to functionally regulate HSP90, we hypothesized that sulforaphane could attenuate EGFR-related signaling and potentially be used to treat NSCLC. Our study revealed that sulforaphane displayed antitumor activity against NSCLC cells both in vitro and in vivo. The sensitivity of NSCLC cells to sulforaphane appeared to positively correlate with the inhibition of EGFR-related signaling, which was attributed to the increased proteasomal degradation of EGFR. Combined treatment of NSCLC cells with sulforaphane plus another HSP90 inhibitor (17-AAG) enhanced the inhibition of EGFR-related signaling both in vitro and in vivo. We have shown that sulforaphane is a novel inhibitory modulator of EGFR expression and is effective in inhibiting the tumor growth of EGFR-TKI-resistant NSCLC cells. Our findings suggest that sulforaphane should be further explored for its potential clinical applications against NSCLC.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

Alternative HER/PTEN/Akt Pathway Activation in HPV Positive and Negative Penile Carcinomas

PubMed Central

Stankiewicz, Elzbieta; Prowse, David M.; Ng, Mansum; Cuzick, Jack; Mesher, David; Hiscock, Frances; Lu, Yong-Jie; Watkin, Nicholas; Corbishley, Catherine; Lam, Wayne; Berney, Daniel M.

2011-01-01

Background The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). Methodology/Principal Findings 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. Conclusions/Significance EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors. PMID:21407808

Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas.

PubMed

Stankiewicz, Elzbieta; Prowse, David M; Ng, Mansum; Cuzick, Jack; Mesher, David; Hiscock, Frances; Lu, Yong-Jie; Watkin, Nicholas; Corbishley, Catherine; Lam, Wayne; Berney, Daniel M

2011-03-02

The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors.

[99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation.

PubMed

Babaei, Mohammad Hossein; Almqvist, Ylva; Orlova, Anna; Shafii, Mohammad; Kairemo, Kalevi; Tolmachev, Vladimir

2005-06-01

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.

Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

PubMed

Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

2016-05-31

Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

Endophilin-1 regulates blood-brain barrier permeability via EGFR-JNK signaling pathway.

PubMed

Chen, Lin; Liu, Wenjing; Wang, Ping; Xue, Yixue; Su, Qingjie; Zeng, Chaosheng; Shang, Xiuli

2015-05-05

Endophilin-1 (Endo1), a multifunctional protein, is essential for synaptic vesicle endocytosis. However, the role and mechanism of endophilin-1 in blood-brain barrier (BBB) function are still unclear. This study was performed to determine whether endophilin-1 regulated BBB permeability via the EGFR-JNK signaling pathway. In the present study, we found that endophilin-1 over-expression in human cerebral microvascular endothelial cell (hCMEC/D3) increased BBB permeability and meanwhile reduced the expression levels of epidermal growth factor receptor (EGFR), phosphorylated c-Jun N-terminal kinase (p-JNK). While endophilin-1 knockdown led to the contrary results. After JNK inhibitor SP600125 was administered to the endophilin-1 silenced hCMEC/D3 cells, the transendothelial electrical resistance (TEER) value was decreased and the permeability coefficient values to 4kDa and 40kDa FITC-dextran were increased. Results observed by Transmission electron microscopy (TEM) showed that tight junctions (TJs) were opened. Moreover, immunofluorescence and Western blot assays revealed the discontinuous distribution of TJ-associated proteins ZO-1, occludin on cell-cell boundaries and a significant decrease in protein expressing levels. Therefore, these results indicated that endophilin-1 positively regulated BBB permeability via the EGFR-JNK signaling pathway in hCMEC/D3 cells, which would provide an experimental basis for further research on endophilin-1 mediated the opening of BBB. Copyright © 2015 Elsevier B.V. All rights reserved.

S-nitrosylation of EGFR and Src activates an oncogenic signaling network in human basal-like breast cancer.

PubMed

Switzer, Christopher H; Glynn, Sharon A; Cheng, Robert Y-S; Ridnour, Lisa A; Green, Jeffrey E; Ambs, Stefan; Wink, David A

2012-09-01

Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor negative (ER-) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in epidermal growth factor receptor (EGFR) tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER- breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER- breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt, and β-catenin) and the loss of PP2A tumor suppressor activity. In addition, NO signaling increased cellular EMT, expression and activity of COX-2, and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell-like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients.

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer.

PubMed

Shields, Sarah; Conroy, Emer; O'Grady, Tony; McGoldrick, Alo; Connor, Kate; Ward, Mark P; Useckaite, Zivile; Dempsey, Eugene; Reilly, Rebecca; Fan, Yue; Chubb, Anthony; Matallanas, David Gomez; Kay, Elaine W; O'Connor, Darran; McCann, Amanda; Gallagher, William M; Coppinger, Judith A

2018-03-20

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

PubMed Central

Shields, Sarah; Conroy, Emer; O’Grady, Tony; McGoldrick, Alo; Connor, Kate; Ward, Mark P.; Useckaite, Zivile; Dempsey, Eugene; Reilly, Rebecca; Fan, Yue; Chubb, Anthony; Matallanas, David Gomez; Kay, Elaine W.; O’Connor, Darran; McCann, Amanda; Gallagher, William M.; Coppinger, Judith A.

2018-01-01

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC. PMID:29644001

Synthesis and biological evaluation of new water-soluble photoactive chlorin conjugate for targeted delivery.

PubMed

Otvagin, Vasilii F; Nyuchev, Alexander V; Kuzmina, Natalia S; Grishin, Ivan D; Gavryushin, Andrei E; Romanenko, Yuliya V; Koifman, Oscar I; Belykh, Dmitrii V; Peskova, Nina N; Shilyagina, Natalia Yu; Balalaeva, Irina V; Fedorov, Alexey Yu

2018-01-20

A new water-soluble conjugate, consisting of a chlorin-based photosensitizing part, and a 4-arylaminoquinazoline moiety with high potential affinity to an epidermal growth factor receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), suitable for photodynamic therapy (PDT), was synthesized starting from methylpheophorbide-a in seven steps. An increased accumulation of this compound in A431 cells with high level of EGFR expression, in comparison with CHO and HeLa cells with low EGFR expression was observed. The prepared conjugate exhibits dark and photoinduced cytotoxicity at micromolar concentrations with IC 50dark /IC 50light ratio of 11-18. In tumor-bearing mice, the conjugate preferentially accumulates in the tumor tissue. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Rutin inhibits B[a]PDE-induced cyclooxygenase-2 expression by targeting EGFR kinase activity.

PubMed

Choi, Seunghwan; Lim, Tae-Gyu; Hwang, Mun Kyung; Kim, Yoon-A; Kim, Jiyoung; Kang, Nam Joo; Jang, Tae Su; Park, Jun-Seong; Yeom, Myeong Hun; Lee, Ki Won

2013-11-15

Rutin is a well-known flavonoid that exists in various natural sources. Accumulative studies have represented the biological effects of rutin, such as anti-oxidative and anti-inflammatory effects. However, the underlying mechanisms of rutin and its direct targets are not understood. We investigated whether rutin reduced B[a]PDE-induced-COX-2 expression. The transactivation of AP-1 and NF-κB were inhibited by rutin. Rutin also attenuated B[a]PDE-induced Raf/MEK/ERK and Akt activation, but had no effect on the phosphorylation of EGFR. An in vitro kinase assay revealed rutin suppressed EGFR kinase activity. We also confirmed direct binding between rutin and EGFR, and found that the binding was regressed by ATP. The EGFR inhibitor also inhibited the B[a]PDE-induced MEK/ERK and Akt signaling pathways and subsequently, suppressed COX-2 expression and promoter activity, in addition to suppressing the transactivation of AP-1 and NF-κB. In EGFR(-/-)mouse embryonic fibroblast cells, B[a]PDE-induced COX-2 expression was also diminished. Collectively, rutin inhibits B[a]PDE-induced COX-2 expression by suppressing the Raf/MEK/ERK and Akt signaling pathways. EGFR appeared to be the direct target of rutin. Copyright © 2013 Elsevier Inc. All rights reserved.

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter

PubMed Central

Qu, X; Sandmann, T; Frierson, H; Fu, L; Fuentes, E; Walter, K; Okrah, K; Rumpel, C; Moskaluk, C; Lu, S; Wang, Y; Bourgon, R; Penuel, E; Pirzkall, A; Amler, L; Lackner, M R; Tabernero, J; Hampton, G M; Kabbarah, O

2016-01-01

Key molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) are well described. However, the mechanisms through which clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we used an integrative genomics approach to examine CRC progression. We used laser capture microdissection to isolate colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas and metastases, and used gene expression profiling to identify pathways that were differentially expressed between the different cell types. We identified a number of potentially important transcriptional changes in developmental and oncogenic pathways, and noted a marked upregulation of EREG in primary and metastatic cancer cells. We confirmed this pattern of gene expression by in situ hybridization and observed staining consistent with autocrine expression in the tumor cells. Upregulation of EREG during the adenoma–carcinoma transition was associated with demethylation of two key sites within its promoter, and this was accompanied by an increase in the levels of epidermal growth factor receptor (EGFR) phosphorylation, as assessed by reverse-phase protein analysis. In CRC cell lines, we demonstrated that EREG demethylation led to its transcriptional upregulation, higher levels of EGFR phosphorylation, and sensitization to EGFR inhibitors. Low levels of EREG methylation in patients who received cetuximab as part of a phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers, including those of the head and neck, lung and bladder. Therefore, we propose that upregulation of EREG expression through promoter demethylation might be an important means of activating the EGFR pathway during the genesis of CRC and potentially other cancers. PMID:27270421

The expression of SALL4 is significantly associated with EGFR, but not KRAS or EML4-ALK mutations in lung cancer.

PubMed

Jia, Xiangbo; Qian, Rulin; Zhang, Binbin; Zhao, Song

2016-10-01

Lung cancer is the leading cause of cancer-related deaths worldwide; unfortunately, its prognosis is still very poor. Therefore, developing the target molecular is very important for lung cancer diagnosis and treatment, especially in the early stage. With this in view, spalt-like transcription factor 4 ( SALL4 ) is considered a potential biomarker for diagnosis and prognosis in cancers, including lung cancer. In order to better investigate the association between the expression of SALL4 and driver genes mutation, 450 histopathologically diagnosed patients with lung cancer and 11 non-cancer patients were enrolled to test the expression of SALL4 and the status of driver genes mutation. This investigation included epidermal growth factor receptor ( EGFR ), kirsten rat sarcoma viral oncogene homolog ( KRAS ), and a fusion gene of the echinoderm microtubule-associated protein-like 4 ( EML4 ) and the anaplastic lymphoma kinase ( ALK ). The results of the study showed that females harbored more EGFR mutation in adenocarcinoma (ADC). The mutation rate of KRAS and EML4-ALK was about 5%, and the double mutations of EGFR/EML4-ALK were higher than EGFR/KRAS . In the expression analysis, the expression of SALL4 was much higher in cancer tissues than normally expected, especially in tissues that carried EGFR mutation (P<0.05), however, there were no significant differences between different mutation types. Likewise, there were no significant differences between expression of SALL4 and KRAS and EML4-ALK mutations. SALL4 is up regulated in lung cancer specimens and harbors EGFR mutation; this finding indicates that SALL4 expression may be relevant with EGFR , which could provide a new insight to lung cancer therapy. The mechanism needs further investigation and analysis.

Targeting the Golgi apparatus to overcome acquired resistance of non-small cell lung cancer cells to EGFR tyrosine kinase inhibitors

PubMed Central

Katayama, Ryohei; Fang, Siyang; Tsutsui, Saki; Akatsuka, Akinobu; Shan, Mingde; Choi, Hyeong-Wook; Fujita, Naoya; Yoshimatsu, Kentaro; Shiina, Isamu; Yamori, Takao; Dan, Shingo

2018-01-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) were demonstrated to provide survival benefit in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR; however, emergence of acquired resistance to EGFR-TKIs has been shown to cause poor outcome. To overcome the TKI resistance, drugs with different mode of action are required. We previously reported that M-COPA (2-methylcoprophilinamide), a Golgi disruptor, suppressed the growth of gastric cancers overexpressing receptor tyrosine kinases (RTKs) such as hepatocyte growth factor receptor (MET) via downregulating their cell surface expression. In this study, we examined the antitumor effect of M-COPA on NSCLC cells with TKI resistance. As a result, M-COPA effectively downregulated cell surface EGFR and its downstream signals, and finally exerted in vivo antitumor effect in NSCLC cells harboring secondary (T790M/del19) and tertiary (C797S/T790M/del19) mutated EGFR, which exhibit acquired resistance to first- and third generation EGFR-TKIs, respectively. M-COPA also downregulated MET expression potentially involved in the acquired resistance to EGFR-TKIs via bypassing the EGFR pathway blockade. These results provide the first evidence that targeting the Golgi apparatus might be a promising therapeutic strategy to overcome the vicious cycle of TKI resistance in EGFR-mutated NSCLC cells via downregulating cell surface RTK expression. PMID:29416720

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor α protein

PubMed Central

Fiori, JL; Sanghvi, M; O'Connell, MP; Krzysik-Walker, SM; Moaddel, R; Bernier, M

2011-01-01

BACKGROUND AND PURPOSE AM251 is an inverse agonist of the cannabinoid 1 receptor (CB1R) that can exert ‘off-target’ effects in vitro and in CB1R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function. EXPERIMENTAL APPROACH The various biological functions of AM251 were measured in CB1R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. KEY RESULTS The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα. CONCLUSIONS AND IMPLICATIONS AM251 up-regulates EGFR expression and signalling via a novel non-CB1R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines. PMID:21449913

Elevated Src family kinase activity stabilizes E-cadherin-based junctions and collective movement of head and neck squamous cell carcinomas

PubMed Central

Veracini, Laurence; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Etienne-Grimaldi, Marie-Christine; Milano, Gérard; Bozec, Alexandre; Babin, Emmanuel; Sudaka, Anne; Thariat, Juliette; Van Obberghen-Schilling, Ellen

2015-01-01

EGF receptor (EGFR) overexpression is thought to drive head and neck carcinogenesis however clinical responses to EGFR-targeting agents have been modest and alternate targets are actively sought to improve results. Src family kinases (SFKs), reported to act downstream of EGFR are among the alternative targets for which increased expression or activity in epithelial tumors is commonly associated to the dissolution of E-cadherin-based junctions and acquisition of a mesenchymal-like phenotype. Robust expression of total and activated Src was observed in advanced stage head and neck tumors (N=60) and in head and neck squamous cell carcinoma lines. In cultured cancer cells Src co-localized with E-cadherin in cell-cell junctions and its phosphorylation on Y419 was both constitutive and independent of EGFR activation. Selective inhibition of SFKs with SU6656 delocalized E-cadherin and disrupted cellular junctions without affecting E-cadherin expression and this effect was phenocopied by knockdown of Src or Yes. These findings reveal an EGFR-independent role for SFKs in the maintenance of intercellular junctions, which likely contributes to the cohesive invasion E-cadherin-positive cells in advanced tumors. Further, they highlight the need for a deeper comprehension of molecular pathways that drive collective cell invasion, in absence of mesenchymal transition, in order to combat tumor spread. PMID:25779657

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR.

PubMed

Wang, Zheng; Wu, Xue; Liang, Yan-Ni; Wang, Li; Song, Zhong-Xing; Liu, Jian-Li; Tang, Zhi-Shu

2016-09-27

Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G₀/G₁ phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

Intrinsic and Extrinsic Regulation of PD-L2 Expression in Oncogene-Driven Non-Small Cell Lung Cancer.

PubMed

Shibahara, Daisuke; Tanaka, Kentaro; Iwama, Eiji; Kubo, Naoki; Ota, Keiichi; Azuma, Koichi; Harada, Taishi; Fujita, Jiro; Nakanishi, Yoichi; Okamoto, Isamu

2018-03-27

The interaction of programmed cell death ligand 2 (PD-L2) with programmed cell death 1 is implicated in tumor immune escape. The regulation of PD-L2 expression in tumor cells has remained unclear, however. We here examined intrinsic and extrinsic regulation of PD-L2 expression in NSCLC. PD-L2 expression was evaluated by reverse transcription and real-time polymerase chain reaction analysis and by flow cytometry. BEAS-2B cells stably expressing an activated mutant form of EGFR or the echinoderm microtubule associated protein like 4 (EML4)-ALK receptor tyrosine kinase fusion oncoprotein manifested increased expression of PD-L2 at both the mRNA and protein levels. Furthermore, treatment of NSCLC cell lines that harbor such driver oncogenes with corresponding EGFR or ALK tyrosine kinase inhibitors or depletion of EGFR or ALK by small interfering RNA transfection suppressed expression of PD-L2, demonstrating that activating EGFR mutations or echinoderm microtubule associated protein like 4 gene (EML4)-ALK receptor tyrosine kinase gene (ALK) fusion intrinsically induce PD-L2 expression. We also found that interferon gamma (IFN-γ) extrinsically induced expression of PD-L2 through signal transducer and activator of transcription 1 signaling in NSCLC cells. Oncogene-driven expression of PD-L2 in NSCLC cells was inhibited by knockdown of the transcription factors signal transducer and activator of transcription 3 (STAT3) or c-FOS. IFN-γ also activated STAT3 and c-FOS, suggesting that these proteins may also contribute to the extrinsic induction of PD-L2 expression. Expression of PD-L2 is induced intrinsically by activating EGFR mutations or EML4-ALK fusion and extrinsically by IFN-γ, with STAT3 and c-FOS possibly contributing to both intrinsic and extrinsic pathways. Our results thus provide insight into the complexity of tumor immune escape in NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Relation of epidermal growth factor receptor and estrogen receptor-independent pS2 protein to the malignant transformation of mucinous cystic neoplasms of the pancreas.

PubMed

Kirby, R E; Lewandrowski, K B; Southern, J F; Compton, C C; Warshaw, A L

1995-01-01

To evaluate the role of epidermal growth factor receptor (EGF-R) and pS2 protein in the evolution of malignancy in mucinous cystic tumors of the pancreas. Mucinous cystic tumors of the pancreas include histologically benign but premalignant mucinous cystic neoplasms and mucinous cystadenocarcinoma. The molecular events leading to transformation from a benign to a malignant mucinous tumor are not known. Overexpression of EGF-R and detection of an estrogen-induced protein (pS2) has been demonstrated in ductal adenocarcinomas of the pancreas, but these factors have not been evaluated in mucinous cystic tumors. Twenty-six mucinous tumors were examined for EGF-R, pS2 protein, and estrogen and progesterone receptors. Eight (61.2%) of 13 malignant tumors exhibited increased expression of EGF-R, whereas EGF-R was not detected in any of the 13 benign tumors (P = .002). The pS2 protein was detected in nine of 11 malignant and 11 of 11 benign tumors (P = .480). Estrogen and progesterone receptors were not detected in the epithelium of either tumor type. The median survival time of the patients with EGF-R-negative tumors was 29.0 months compared with 14.5 months for those with EGF-R-positive tumors, but this difference did not reach significance owing to the small population size. Overexpression of EGF-R in mucinous cystic tumors, as in ductal adenocarcinomas, may be an important feature associated with malignancy and may have prognostic significance. Failure to detect EGF-R in histologically benign epithelium suggests that the upregulation of EGF-R may be important in the evolution of aggressive behavior. The expression of pS2 protein appears to be independent of estrogen and may play a role in the proliferative activity of mucinous tumors. However, pS2 expression is not a feature associated exclusively with malignancy.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells.

PubMed

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T; Friedman, Henry; Bigner, Darell D; Ali-Osman, Francis

2015-12-25

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

AXL mediates resistance to cetuximab therapy.

PubMed

Brand, Toni M; Iida, Mari; Stein, Andrew P; Corrigan, Kelsey L; Braverman, Cara M; Luthar, Neha; Toulany, Mahmoud; Gill, Parkash S; Salgia, Ravi; Kimple, Randall J; Wheeler, Deric L

2014-09-15

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR. ©2014 American Association for Cancer Research.

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

PubMed Central

Bessette, Darrell C.; Tilch, Erik; Seidens, Tatjana; Quinn, Michael C. J.; Wiegmans, Adrian P.; Shi, Wei; Cocciardi, Sibylle; McCart-Reed, Amy; Saunus, Jodi M.; Simpson, Peter T.; Grimmond, Sean M.; Lakhani, Sunil R.; Khanna, Kum Kum; Waddell, Nic; Al-Ejeh, Fares; Chenevix-Trench, Georgia

2015-01-01

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance. PMID:25969993

Activation of EGFR and ERBB2 by Helicobacter pylori Results in Survival of Gastric Epithelial Cells with DNA Damage

PubMed Central

Chaturvedi, Rupesh; Asim, Mohammad; Piazuelo, M. Blanca; Yan, Fang; Barry, Daniel P.; Sierra, Johanna Carolina; Delgado, Alberto G.; Hill, Salisha; Casero, Robert A.; Bravo, Luis E.; Dominguez, Ricardo L.; Correa, Pelayo; Polk, D. Brent; Washington, M. Kay; Rose, Kristie L.; Schey, Kevin L.; Morgan, Douglas R.; Peek, Richard M.; Wilson, Keith T.

2014-01-01

BACKGROUND & AIMS The gastric cancer-causing pathogen Helicobacter pylori upregulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOXhigh cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H pylori-infected Egfrwa5 mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. Phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsies from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS SMOX expression and DNA damage were decreased, and apoptosis increased in H pylori-infected Egfrwa5 mice. H pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damagehigh apoptosislow cells. Phosphoproteomic analysis revealed increased EGFR and ERBB2 signaling. Immunoblot analysis demonstrated the presence of a phosphorylated (p)EGFR–ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damagehigh apoptosislow cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR–ERBB2, and pERBB2 were increased predominantly in tissues demonstrating gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR–ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis. PMID:24530706

In vivo imaging of the dynamics of different variants of EGFR in glioblastomas.

PubMed

Shah, Khalid

2011-01-01

A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression.

Characterization of the expression and clinical features of epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in esophageal carcinoma

PubMed Central

NIYAZ, MADINIYAT; ANWER, JURAT; LIU, HUI; ZHANG, LIWEI; SHAYHEDIN, ILYAR; AWUT, IDIRIS

2015-01-01

The present study aimed to understand the expression characteristics of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) in individuals of Uygur, Han and Kazak ethnicity with esophageal carcinoma in Xinjiang (China) and their interrelation analysis, and to investigate the expression differences in these genes between esophageal carcinoma and pericarcinoma tissue samples, and between the three ethnic groups. The expression levels of EGFR and VEGFR-2 from 119 pairs of esophageal carcinoma tissue and corresponding pericarcinoma tissue from Uygur, Han and Kazak patients with esophageal carcinoma were detected by immunohistochemistry following surgical resection, and an additional five carcinoma in situ specimens were also tested. The relative expression was analyzed among the ethnic groups and clinicopathological parameters. The positive rate of EGFR in esophageal carcinoma tissue from patients of Uygur, Han and Kazak heritage was 70.73, 68.42 and 67.5%, respectively. For VEGFR-2 the positive rate was 73.17, 68.42 and 67.5%, respectively. No significant difference was detected in their expression between the three ethnic groups (P>0.05); however, EGFR and VEGFR-2 overexpression were correlated with lymph node metastasis (P<0.05). VEGF expression was also correlated with the expression of VEGFR-2 in esophageal carcinoma tissues. EGFR was positive in carcinoma in situ samples, while VEGFR-2 was negative. The overexpression of EGFR is therefore an early event and may have a significant role in the progression of esophageal carcinoma pathogenesis. EGFR overexpression may correlate with the expression of VEGFR-2 in esophageal cancer. These results may aid the early diagnosis of esophageal cancer, and the development of individual target treatment in the future. PMID:26788193

Sulindac metabolites inhibit epidermal growth factor receptor activation and expression.

PubMed

Pangburn, Heather A; Kraus, Hanna; Ahnen, Dennis J; Rice, Pamela L

2005-09-02

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased mortality from colorectal cancer (CRC). NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2) signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF) receptor (EGFR). HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068), total EGFR, phosphorylated ERK1/2 (pERK1/2), total ERK1/2, activated caspase-3, and alpha-tubulin. EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. These results suggest that downregulation of EGFR signaling by sulindac metabolites may occur, at least in part, by inhibiting activation and expression of EGFR. Inhibition of EGFR signaling may account for part of the growth inhibitory and chemopreventive effects of these compounds.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors.

PubMed

Felsberg, Jörg; Hentschel, Bettina; Kaulich, Kerstin; Gramatzki, Dorothee; Zacher, Angela; Malzkorn, Bastian; Kamp, Marcel; Sabel, Michael; Simon, Matthias; Westphal, Manfred; Schackert, Gabriele; Tonn, Jörg C; Pietsch, Torsten; von Deimling, Andreas; Loeffler, Markus; Reifenberger, Guido; Weller, Michael

2017-11-15

Purpose: Approximately 40% of all glioblastomas have amplified the EGFR gene, and about half of these tumors express the EGFRvIII variant. The prognostic role of EGFRvIII in EGFR -amplified glioblastoma patients and changes in EGFRvIII expression in recurrent versus primary glioblastomas remain controversial, but such data are highly relevant for EGFRvIII-targeted therapies. Experimental Design: EGFR -amplified glioblastomas from 106 patients were assessed for EGFRvIII positivity. Changes in EGFR amplification and EGFRvIII status from primary to recurrent glioblastomas were evaluated in 40 patients with EGFR -amplified tumors and 33 patients with EGFR -nonamplified tumors. EGFR single-nucleotide variants (SNV) were assessed in 27 patients. Data were correlated with outcome and validated in 150 glioblastoma patients from The Cancer Genome Atlas (TCGA) consortium. Results: Sixty of 106 EGFR -amplified glioblastomas were EGFRvIII-positive (56.6%). EGFRvIII positivity was not associated with different progression-free or overall survival. EGFRvIII status was unchanged at recurrence in 35 of 40 patients with EGFR -amplified primary tumors (87.5%). Four patients lost and one patient gained EGFRvIII positivity at recurrence. None of 33 EGFR- nonamplified glioblastomas acquired EGFR amplification or EGFRvIII at recurrence. EGFR SNVs were frequent in EGFR -amplified tumors, but were not linked to survival. Conclusions: EGFRvIII and EGFR SNVs are not prognostic in EGFR -amplified glioblastoma patients. EGFR amplification is retained in recurrent glioblastomas. Most EGFRvIII-positive glioblastomas maintain EGFRvIII positivity at recurrence. However, EGFRvIII expression may change in a subset of patients at recurrence, thus repeated biopsy with reassessment of EGFRvIII status is recommended for patients with recurrent glioblastoma to receive EGFRvIII-targeting agents. Clin Cancer Res; 23(22); 6846-55. ©2017 AACR . ©2017 American Association for Cancer Research.

Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

PubMed Central

Harris, Deshea L.

2007-01-01

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that EGFR is internalized in response to EGF stimulation suggests that it could interact with and be regulated by PTP1B. The ability of PTP1B inhibitor to sustain EGFR Tyr992 phosphorylation and increase the number of Ki67-positive cells indicates that PTP1B plays a role in the negative regulation of EGF-induced signaling and helps suppress cell cycle entry. PMID:17563729

EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible factor (HIF)-1α and STAT3.

PubMed

Koh, Jaemoon; Jang, Ji-Young; Keam, Bhumsuk; Kim, Sehui; Kim, Moon-Young; Go, Heounjeong; Kim, Tae Min; Kim, Dong-Wan; Kim, Chul-Woo; Jeon, Yoon Kyung; Chung, Doo Hyun

2016-03-01

Programmed cell death (PD)-1/PD-1 ligand-1 (PD-L1)-targeted therapy has emerged as a promising therapeutic strategy for lung cancer. However, whether EML4-ALK regulates PD-L1 expression in lung cancer remains unknown. A total of 532 pulmonary adenocarcinomas (pADCs), including 58 ALK -translocated tumors, were immunohistochemically evaluated for PD-L1 and PD-1. H23 ( EGFR Wild-type EML4-ALK - PD-L1 Low ) and H2228 ( EGFR Wild-type EML4-ALK + PD-L1 High ) cells were transfected with EML4-ALK or ALK short interfering RNAs and used to investigate the alterations in PD-L1 expression. PD-L1 expression was detected in 81% of ALK -translocated pADCs; this value was significantly higher than those of pADCs with EGFR mutation, KRAS mutation or lacking ALK, EGFR or KRAS mutation ( p <0.005 for all). Moreover, ALK -translocated pADC with PD-L1 expression showed significantly higher numbers of tumor-infiltrating PD-1 + cells. ALK knockdown or inhibition (crizotinib treatment) in H2228 cells downregulated PD-L1 expression. Transfection of H23 cells with EML4-ALK enhanced PD-L1 expression, which was compromised by crizotinib treatment. This ALK-dependent upregulation of PD-L1 expression was mediated by STAT3 and hypoxia-inducible factor (HIF)-1α under normoxia and hypoxia. Furthermore, EML4-ALK enhanced HIF-1α expression through increasing transcription and decreasing ubiquitination of HIF-1α. In ALK -translocated pADC tissues, significant positive correlations between PD-L1 and nuclear HIF-1α ( p < 0.05) or pSTAT3 expression levels ( p <0.005) were observed. Among patients with ALK -translocated pADC, strong PD-L1 expression was significantly associated with shorter progression-free ( p = 0.001) and overall survival ( p = 0.002) after crizotinib treatment. Collectively, our findings demonstrate that ALK- derived pADCs increase PD-L1 expression via HIF-1α and/or STAT3, thus providing a rationale for PD-1/PD-L1 pathway-targeted therapy in ALK -translocated lung cancer.

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

DOE PAGES

Su, Xinhui; Cheng, Kai; Jeon, Jongho; ...

2014-06-27

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-Z EGFR:1907 and 18F-FBEM-Z EGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-Z EGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al 18F-NOTA-Zmore » EGFR:1907 and 18F-CBT-Z EGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z EGFR:1907 with NOTA and radiolabeling with Al 18F to produce Al 18F-NOTA-Z EGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole ( 18F-CBT) was conjugated to Cys-Z EGFR:1907 to produce 18F-CBT-Z EGFR:1907. Binding affinity and specificity of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al 18F-NOTA-Z EGFR:1907 or 18F-CBT-Z EGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-Z EGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al 18F-NOTA-Z EGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for 18F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al 18F-NOTA-Z EGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.« less

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Xinhui; Cheng, Kai; Jeon, Jongho

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-Z EGFR:1907 and 18F-FBEM-Z EGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-Z EGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al 18F-NOTA-Zmore » EGFR:1907 and 18F-CBT-Z EGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z EGFR:1907 with NOTA and radiolabeling with Al 18F to produce Al 18F-NOTA-Z EGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole ( 18F-CBT) was conjugated to Cys-Z EGFR:1907 to produce 18F-CBT-Z EGFR:1907. Binding affinity and specificity of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al 18F-NOTA-Z EGFR:1907 or 18F-CBT-Z EGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-Z EGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al 18F-NOTA-Z EGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for 18F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al 18F-NOTA-Z EGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.« less

Cancer-associated fibroblasts promote epithelial-mesenchymal transition and EGFR-TKI resistance of non-small cell lung cancers via HGF/IGF-1/ANXA2 signaling.

PubMed

Yi, Yanmei; Zeng, Shanshan; Wang, Zhaotong; Wu, Minhua; Ma, Yuanhuan; Ye, Xiaoxia; Zhang, Biao; Liu, Hao

2018-03-01

The involvement of the tumor stromal cells in acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism remains unclear. In the present study, we investigated the role and mechanism underlying Cancer-associated fibroblasts (CAFs) in TKI resistance of NSCLCs. In vitro and in vivo experiments showed that HCC827 and PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR-TKI gefitinib when cultured with CAFs isolated from NSCLC tissues. Moreover, we showed that CAFs could induce epithelial-mesenchymal transition (EMT) phenotype of HCC827 and PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition markers. Using proteomics-based method, we identified that CAFs significantly increased the expression of the Annexin A2 (ANXA2). More importantly, knockdown of ANXA2 completely reversed EMT phenotype and gefitinib resistance induced by CAFs. Furthermore, we found that CAFs increased the expression and phosphorylation of ANXA2 by secretion of growth factors HGF and IGF-1 and by activation of the corresponding receptors c-met and IGF-1R. Dual inhibition of HGF/c-met and IGF-1/IGF-1R pathways could significantly suppress ANXA2, and markedly reduced CAFs-induced EMT and gefitinib resistance. Taken together, these findings indicate that CAFs promote EGFR-TKIs resistance through HGF/IGF-1/ANXA2/EMT signaling and may be an ideal therapeutic target in NSCLCs with EGFR-activating mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted Doxorubicin-Loaded Bacterially Derived Nano-Cells for the Treatment of Neuroblastoma.

PubMed

Sagnella, Sharon M; Trieu, Jennifer; Brahmbhatt, Himanshu; MacDiarmid, Jennifer A; MacMillan, Alex; Whan, Renee M; Fife, Christopher M; McCarroll, Joshua A; Gifford, Andrew J; Ziegler, David S; Kavallaris, Maria

2018-05-01

Advanced stage neuroblastoma is an aggressive disease with limited treatment options for patients with drug-resistant tumors. Targeted delivery of chemotherapy for pediatric cancers offers promise to improve treatment efficacy and reduce toxicity associated with systemic chemotherapy. The EnGeneIC Dream Vector (EDV TM ) is a nanocell, which can package chemotherapeutic drugs and target tumors via attachment of bispecific proteins to the surface of the nanocell. Phase I trials in adults with refractory tumors have shown an acceptable safety profile. Herein we investigated the activity of EGFR-targeted and doxorubicin-loaded EDV TM ( EGFR EDV TM Dox ) for the treatment of neuroblastoma. Two independent neuroblastoma cell lines with variable expression of EGFR protein [SK-N-BE(2), high; SH-SY-5Y, low] were used. EGFR EDV TM Dox induced apoptosis in these cells compared to control, doxorubicin, or non-doxorubicin loaded EGFR EDV TM In three-dimensional tumor spheroids, imaging and fluorescence life-time microscopy revealed that EGFR EDV TM Dox had a marked enhancement of doxorubicin penetration compared to doxorubicin alone, and improved penetration compared to non-EGFR-targeted EDV TM Dox , with enhanced spheroid penetration leading to increased apoptosis. In two independent orthotopic human neuroblastoma xenograft models, short-term studies (28 days) of tumor-bearing mice led to a significant decrease in tumor size in EGFR EDV TM Dox -treated animals compared to control, doxorubicin, or non-EGFR EDV TM Dox There was increased TUNEL staining of tumors at day 28 compared to control, doxorubicin, or non-EGFR EDV TM Dox Moreover, overall survival was increased in neuroblastoma mice treated with EGFR EDV TM Dox ( P < 0007) compared to control. Drug-loaded bispecific-antibody targeted EDVs TM offer a highly promising approach for the treatment of aggressive pediatric malignancies such as neuroblastoma. Mol Cancer Ther; 17(5); 1012-23. ©2018 AACR . ©2018 American Association for Cancer Research.

Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang

2011-07-15

Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blotmore » and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.« less

Cetuximab Resistance in Head and Neck Cancer Is Mediated by EGFR-K521 Polymorphism.

PubMed

Braig, Friederike; Kriegs, Malte; Voigtlaender, Minna; Habel, Beate; Grob, Tobias; Biskup, Karina; Blanchard, Veronique; Sack, Markus; Thalhammer, Anja; Ben Batalla, Isabel; Braren, Ingke; Laban, Simon; Danielczyk, Antje; Goletz, Steffen; Jakubowicz, Elzbieta; Märkl, Bruno; Trepel, Martin; Knecht, Rainald; Riecken, Kristoffer; Fehse, Boris; Loges, Sonja; Bokemeyer, Carsten; Binder, Mascha

2017-03-01

Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K 521 (K-allele), which is expressed in >40% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progression-free survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K 521 was reduced slightly, but ligand-mediated EGFR activation was intact. We found a lack of glycan sialyation on EGFR-K 521 that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K 521 expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform. Cancer Res; 77(5); 1188-99. ©2016 AACR . ©2016 American Association for Cancer Research.

Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation.

PubMed

Tomas, Alejandra; Jones, Sylwia; Vaughan, Simon O; Hochhauser, Daniel; Futter, Clare E

2017-08-01

EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome. © 2017. Published by The Company of Biologists Ltd.

An Immunohistochemical Study of Anaplastic Lymphoma Kinase and Epidermal Growth Factor Receptor Mutation in Non-Small Cell Lung Carcinoma.

PubMed

Verma, Sonal; Kumar, Madhu; Kumari, Malti; Mehrotra, Raj; Kushwaha, R A S; Goel, Madhumati; Kumar, Ashutosh; Kant, Surya

2017-07-01

Lung cancer is one of the leading causes of cancer related death. Targeted treatment for specific markers may help in reducing the cancer related morbidity and mortality. To study expression of Anaplastic Lymphoma Kinase (ALK)and Epidermal Growth Factor Receptor (EGFR) mutations in patients of Non-Small Cell Lung Cancer NSCLC, that are the targets for specific ALK inhibitors and EGFR tyrosine kinase inhibitors. Total 69 cases of histologically diagnosed NSCLC were examined retrospectively for immunohistochemical expression of EGFR and ALK, along with positive control of normal placental tissue and anaplastic large cell lymphoma respectively. Of the NSCLC, Squamous Cell Carcinoma (SCC) accounted for 71.0% and adenocarcinoma was 26.1%. ALK expression was seen in single case of 60-year-old female, non-smoker with adenocarcinoma histology. EGFR expression was seen in both SCC (59.18%) and adenocarcinoma in (77.78%) accounting for 63.77% of all cases. Both ALK and EGFR mutation were mutually exclusive. EGFR expression was seen in 63.77% of cases, highlighting the importance of its use in routine analysis, for targeted therapy and better treatment results. Although, ALK expression was seen in 1.45% of all cases, it is an important biomarker in targeted cancer therapy. Also, the mutually exclusive expression of these two markers need further studies to develop a diagnostic algorithm for NSCLC patients.

The apoptotic effect of simvastatin via the upregulation of BIM in nonsmall cell lung cancer cells.

PubMed

Lee, Hwa Young; Kim, In Kyoung; Lee, Hye In; Mo, Jin Young; Yeo, Chang Dong; Kang, Hyeon Hui; Moon, Hwa Sik; Lee, Sang Haak

2016-01-01

Statins are known to have pleiotropic effects that induce cell death in certain cancer cells. BIM is a member of the bcl-2 gene family, which promotes apoptotic cell death. This study investigated the hypothesis that simvastatin has pro-apoptotic effects in epidermal growth factor receptor (EGFR)-mutated lung cancer cell lines via the upregulation of the expression of the BIM protein. The cytotoxic effects of simvastatin on gefitinib-sensitive (HCC827, E716-A750del) and -resistant (H1975, T790M + L858R) nonsmall cell lung cancer (NSCLC) cells were compared. Cell proliferation and expression of apoptosis-related and EGFR downstream signaling proteins were evaluated. Expression of BIM was compared in H1975 cells after treatment with simvastatin or gefitinib. SiRNA-mediated BIM depletion was performed to confirm whether the cytotoxicity of simvastatin was mediated by the expression of BIM. H1975 cells showed significantly reduced viability compared with HCC827 cells after treatment with simvastatin (2 μM) for 48 hours. In simvastatin-treated H1975 cells, expression of pro-apoptotic proteins was increased and the phosphorylation of ERK 1/2 (p-ERK 1/2) was reduced. Expression of BIM was suppressed by gefitinib (1 μM) treatment in H1975 cells, but it was significantly increased by treatment with simvastatin. BIM depletion by siRNA transfection enhanced the viability of H1975 cells that received simvastatin treatment and increased their expression of anti-apoptotic proteins. Simvastatin restored the expression of BIM to induce apoptotic cell death in NSCLC cells harboring an EGFR-resistant mutation. Our study suggests the potential utility of simvastatin as a BIM-targeted treatment for NSCLC.

LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kou, Changhua, E-mail: chkoukou@hotmail.com; Zhou, Tian; Han, Xilin

2015-08-21

Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cellmore » proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling. - Highlights: • Promoter methylation of LRIG1 occurred in colorectal cancer cells and tumors. • Restoration of LRIG1 inhibits tumor growth in vitro and in vivo. • Overexpression or knockdown of LRIG1 regulates EGFR/AKT and downstream apoptosis. • Methylation of LRIG1 correlates with its mRNA and protein downregulation. • LRIG1 was firstly identified as an epigenetic target in cancer.« less

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

PubMed

Lee, Ming-Fen; Pan, Min-Hsiung; Chiou, Yi-Siou; Cheng, An-Chin; Huang, Han

2011-11-09

Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT

PubMed Central

Choudhary, Kumari Sonal; Rohatgi, Neha; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

2016-01-01

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend. PMID:27253373

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT.

PubMed

Choudhary, Kumari Sonal; Rohatgi, Neha; Halldorsson, Skarphedinn; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

2016-06-01

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

PubMed

Shao, Genbao; Lai, Wensheng; Wan, Xiaolei; Xue, Jing; Wei, Ye; Jin, Jie; Zhang, Liuping; Lin, Qiong; Shao, Qixiang; Zou, Shengqiang

2017-05-01

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclin D1. Significantly, blockage of epidermal growth factor receptor (EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

PubMed Central

Hilmarsdottir, Bylgja; Gustafsdottir, Sigrun M.; Franzdottir, Sigridur Rut; Arason, Ari Jon; Steingrimsson, Eirikur; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2013-01-01

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland. PMID:23573284

Expression and functional role of sprouty-2 in breast morphogenesis.

PubMed

Sigurdsson, Valgardur; Ingthorsson, Saevar; Hilmarsdottir, Bylgja; Gustafsdottir, Sigrun M; Franzdottir, Sigridur Rut; Arason, Ari Jon; Steingrimsson, Eirikur; Magnusson, Magnus K; Gudjonsson, Thorarinn

2013-01-01

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Role of Pgrmc1 in estrogen maintenance of meiotic arrest in zebrafish oocytes through Gper/Egfr.

PubMed

Aizen, Joseph; Thomas, Peter

2015-04-01

The regulation of receptor trafficking to the cell surface and its effect on responses of target cells to growth factors and hormones remain poorly understood. Initial evidence has been recently obtained using cancer cells that surface expression of the epidermal growth factor receptor (EGFR) is dependent on its association with progesterone receptor membrane component 1 (PGRMC1). Estrogen inhibition of oocyte maturation (OM) in zebrafish is mediated through G-protein-coupled estrogen membrane receptor 1 (Gper1) and involves activation of Egfr. Therefore, in this study, the potential roles of Pgrmc1 in the cell surface expression and functions of Egfr in normal cells were investigated in this in vitro OM model of Egfr action using an inhibitor of PGMRC1 signaling, AG205. A single ∼60 kDa protein band, which corresponds to the size of the Pgrmc1 dimer, was detected on plasma membranes of fully grown oocytes by western blotting. Co-treatment with the PGRMC1 inhibitor AG205 (20 μM) blocked the inhibitory effects of 100 nM estradiol-17β and the GPER agonist, G-1, on spontaneous maturation of denuded zebrafish oocytes. Moreover, reversal of these estrogen effects on OM by the EGFR inhibitors AG1478 and AG825 (50 μM) was prevented by co-incubation with the PGRMC1 inhibitor. Inhibition of Pgrmc1 signaling with AG205 also caused a decrease in Egfr-dependent signaling and Egfr expression on oocyte cell membranes. These results indicate that maintenance of Pgrmc1 signaling is required for Egfr expression on zebrafish oocyte cell membranes and for conserving the functions of Egfr in maintaining meiotic arrest through estrogen activation of Gper. © 2015 Society for Endocrinology.

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

PubMed

Wechsler, Erin Isaacson; Tugizov, Sharof; Herrera, Rossana; Da Costa, Maria; Palefsky, Joel M

2018-05-01

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

Preselection of EGFR mutations in non-small-cell lung cancer patients by immunohistochemistry: comparison with DNA-sequencing, EGFR wild-type expression, gene copy number gain and clinicopathological data.

PubMed

Gaber, Rania; Watermann, Iris; Kugler, Christian; Vollmer, Ekkehard; Perner, Sven; Reck, Martin; Goldmann, Torsten

2017-01-01

Targeting epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC) having EGFR mutations is associated with an improved overall survival. The aim of this study is to verify, if EGFR mutations detected by immunohistochemistry (IHC) is a convincing way to preselect patients for DNA-sequencing and to figure out, the statistical association between EGFR mutation, wild-type EGFR overexpression, gene copy number gain, which are the main factors inducing EGFR tumorigenic activity and the clinicopathological data. Two hundred sixteen tumor tissue samples of primarily chemotherapeutic naïve NSCLC patients were analyzed for EGFR mutations E746-A750del and L858R and correlated with DNA-sequencing. Two hundred six of which were assessed by IHC, using 6B6 and 43B2 specific antibodies followed by DNA-sequencing of positive cases and 10 already genotyped tumor tissues were also included to investigate debugging accuracy of IHC. In addition, EGFR wild-type overexpression was IHC evaluated and EGFR gene copy number determination was performed by fluorescence in situ hybridization (FISH). Forty-one÷206 (19.9%) cases were positive for mutated EGFR by IHC. Eight of them had EGFR mutations of exons 18-21 by DNA-sequencing. Hit rate of 10 already genotyped NSCLC mutated cases was 90% by IHC. Positive association was found between EGFR mutations determined by IHC and both EGFR overexpression and increased gene copy number (p=0.002 and p<0.001, respectively). Additionally, positive association was detected between EGFR mutations, high tumor grade and clinical stage (p<0.001). IHC staining with mutation specific antibodies was demonstrated as a possible useful screening test to preselect patients for DNA-sequencing.

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Zhi-Dong; Xu, Liang; Tang, Kan-Kai

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβmore » phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF-κB-dependent manner. • NF-κB-dependent cyclin D1 upregulation is required for the EGFR-mediated cytoprotection against hypoxia-induced injury. • Endogenous EGFR activity antagonizes hypoxia-induced PC12 cells injury.« less

c-Met Expression Is a Marker of Poor Prognosis in Patients With Locally Advanced Head and Neck Squamous Cell Carcinoma Treated With Chemoradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baschnagel, Andrew M.; Williams, Lindsay; Hanna, Alaa

2014-03-01

Purpose: To examine the prognostic significance of c-Met expression in relation to p16 and epidermal growth factor receptor (EGFR) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with definitive concurrent chemoradiation. Methods and Materials: Archival tissue from 107 HNSCC patients treated with chemoradiation was retrieved, and a tissue microarray was assembled. Immunohistochemical staining of c-Met, p16, and EGFR was performed. c-Met expression was correlated with p16, EGFR, clinical characteristics, and clinical endpoints including locoregional control (LRC), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS). Results: Fifty-one percent of patients were positive for p16,more » and 53% were positive for EGFR. Both p16-negative (P≤.001) and EGFR-positive (P=.019) status predicted for worse DFS. Ninety-three percent of patients stained positive for c-Met. Patients were divided into low (0, 1, or 2+ intensity) or high (3+ intensity) c-Met expression. On univariate analysis, high c-Met expression predicted for worse LRC (hazard ratio [HR] 2.27; 95% CI, 1.08-4.77; P=.031), DM (HR 4.41; 95% CI, 1.56-12.45; P=.005), DFS (HR 3.00; 95% CI, 1.68-5.38; P<.001), and OS (HR 4.35; 95% CI, 2.13-8.88; P<.001). On multivariate analysis, after adjustment for site, T stage, smoking history, and EGFR status, only high c-Met expression (P=.011) and negative p16 status (P=.003) predicted for worse DFS. High c-Met expression was predictive of worse DFS in both EGFR-positive (P=.032) and -negative (P=.008) patients. In the p16-negative patients, those with high c-Met expression had worse DFS (P=.036) than did those with low c-Met expression. c-Met expression was not associated with any outcome in the p16-positive patients. Conclusions: c-Met is expressed in the majority of locally advanced HNSCC cases, and high c-Met expression predicts for worse clinical outcomes. High c-Met expression predicted for worse DFS in p16-negative patients but not in p16-positive patients. c-Met predicted for worse outcome regardless of EGFR status.« less

EGF receptor uses SOS1 to drive constitutive activation of NFκB in cancer cells

PubMed Central

De, Sarmishtha; Dermawan, Josephine Kam Tai; Stark, George R.

2014-01-01

Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation. PMID:25071181

Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling

PubMed Central

Lin, Meei-Hua; Chang, Kuo-Wei; Lin, Shu-Chun; Miner, Jeffrey H.

2010-01-01

Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family (Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations. PMID:20513444

Long-term treatment with EGFR inhibitor erlotinib attenuates renal inflammatory cytokines but not nephropathy in Alport syndrome mouse model.

PubMed

Omachi, Kohei; Miyakita, Rui; Fukuda, Ryosuke; Kai, Yukari; Suico, Mary Ann; Yokota, Tsubasa; Kamura, Misato; Shuto, Tsuyoshi; Kai, Hirofumi

2017-12-01

Alport syndrome (AS) is a hereditary kidney disease caused by mutation of type IV collagen. Loss of collagen network induces collapse of glomerular basement membrane (GBM) structure. The previous studies showed that upregulation of some tyrosine kinase receptors signaling accompanied GBM disorder in AS mouse model. EGFR signaling is one of the well-known receptor kinase signaling that is involved in glomerular diseases. However, whether EGFR signaling is relevant to AS progression is still uninvestigated. Here, we determined the involvement of EGFR in AS and the effect of suppressing EGFR signaling by erlotinib treatment on AS progression. Phosphorylated EGFR expression was investigated by Western blotting analysis and immunostaining of kidney tissues of Col4a5 mutant mice (a mouse model of X-linked AS). To check the effect of blocking EGFR signaling in AS, we administered erlotinib to AS mice once a day (10 mg/kg/day) orally for 18 weeks. Renal function parameters (proteinuria, serum creatinine, and BUN) and renal histology were assessed, and the gene expressions of inflammatory cytokines were analyzed in renal tissues. Phosphorylated EGFR expression was upregulated in AS mice kidney tissues. Erlotinib slightly reduced the urinary protein and suppressed the expression of renal injury markers (Lcn2, Lysozyme) and inflammatory cytokines (Il-6, Il-1β and KC). Erlotinib did not improve renal pathology, such as glomerular sclerosis and fibrosis. These findings suggest that EGFR signaling is upregulated in kidney, but although inhibiting this signaling pathway suppressed renal inflammatory cytokines, it did not ameliorate renal dysfunction in AS mouse model.

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres as a therapeutic target against EGFR-positive non-small cell lung cancer.

PubMed

Cheng, Chun-Chia; Chou, Kuei-Fang; Wu, Cheng-Wen; Su, Nai-Wen; Peng, Cheng-Liang; Su, Ying-Wen; Chang, Jungshan; Ho, Ai-Sheng; Lin, Huan-Chau; Chen, Caleb Gon-Shen; Yang, Bi-Ling; Chang, Yu-Cheng; Chiang, Ya-Wen; Lim, Ken-Hong; Chang, Yi-Fang

2018-02-01

YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell context-dependent dual effects of EFEMP1 stabilizes subpopulation equilibrium in responding to changes of in vivo growth environment.

PubMed

Hu, Yuanjie; Ke, Chao; Ru, Ning; Chen, Yumay; Yu, Liping; Siegel, Eric R; Linskey, Mark E; Wang, Ping; Zhou, Yi-Hong

2015-10-13

Conflicting functions of EFEMP1 in cancer have been reported. Using two syngeneic glioma cell lines (U251 and U251-NS) carrying two different principal cell subpopulations that express high or low EGFR, and that are able to interconvert via mis-segregation of chromosome 7 (Chr7), we studied EFEMP1's cell-context-dependent functions in regulating subpopulation equilibrium, here defined by the percentage of cells carrying different copies of Chr7. We found that EFEMP1 attenuated levels of EGFR and cellular respiration in high-EGFR-expressing cells, but increased levels of NOTCH1, MMP2, cell invasiveness, and both oxidative phosphorylation and glycolytic respiration in low-EGFR-expressing cells. Consistently, EFEMP1 suppressed intracranial xenograft formation in U251 and promoted its formation in U251-NS. Interestingly, subpopulation equilibria in xenografts of U251-NS without EFEMP1 overexpression were responsive to inoculum size (1, 10 and 100 thousand cells), which may change the tumor-onset environment. It was not observed in xenografts of U251-NS with EFEMP1 overexpression. The anti-EGFR function of EFEMP1 suppressed acceleration of growth of U251-NS, but not the subpopulation equilibrium, when serially passed under a different (serum-containing adherent) culture condition. Overall, the data suggest that the orthotopic environment of the brain tumor supports EFEMP1 in carrying out both its anti-EGFR and pro-invasive/cancer stem cell-transforming functions in the two glioma cell subpopulations during formation of a single tumor, where EFEMP1 stabilizes the subpopulation equilibrium in response to alterations of the growth environment. This finding implies that EFEMP1 may restrain cancer plasticity in coping with ever-changing tumor microenvironments and/or therapeutic-intervention stresses.

Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy.

PubMed

Musiani, Daniele; Konda, John David; Pavan, Simona; Torchiaro, Erica; Sassi, Francesco; Noghero, Alessio; Erriquez, Jessica; Perera, Timothy; Olivero, Martina; Di Renzo, Maria Flavia

2014-09-01

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments. © FASEB.

Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling

PubMed Central

Dasgupta, Santanu; Menezes, Mitchell E.; Das, Swadesh K.; Emdad, Luni; Janjic, Aleksandar; Bhatia, Shilpa; Mukhopadhyay, Nitai D; Shao, Chunbo; Sarkar, Devanand; Fisher, Paul B.

2013-01-01

Purpose Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Experimental Design Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors. Results Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident. Conclusions Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC. PMID:23873690

Colorectal adenocarcinoma with mucinous component: relation of MMP-13, EGFR, and E-cadherin expressions to clinicopathological features and prognosis.

PubMed

Foda, Abd Al-Rahman Mohammad; El-Hawary, Amira Kamal; Aziz, Azza Abdel

2015-06-01

The aim of this study was to compare colorectal adenocarcinoma with mucinous component, ordinary adenocarcinoma (OA) and mucinous adenocarcinoma (MA) regarding clinicopathological parameters, survival, EGFR, MMP-13, and E-cadherin. We studied tumor tissue specimens from 28 patients with adenocarcinoma with mucinous component, 47 with OA, and 56 with MA, who underwent radical surgery from January 2007 to January 2012 at the Gastroenterology Centre, Mansoura University, Egypt. High density manual tissue microarrays were constructed and immunohistochemistry for EGFR, MMP-13, and E-cadherin was done. Colorectal adenocarcinoma with mucinous component (AWMC) was significantly associated with more perineural invasion, lower EGFR, and MMP-13 expressions than OA, with no difference in E-cadherin expression. Conversely, only microscopic abscess formation was significantly more with colorectal AWMC than MC with no difference in EGFR, MMP-13 and E-cadherin expression between both groups. Colorectal AWMC showed a better survival than MA with no difference with OA. In a univariate analysis, EGFR, MMP-13, and E-cadherin expressions did not show a significant impact on disease-free or overall survival in patients with colorectal AWMC. Colorectal AWMC remains a vague entity that resembles OA in some clinicopathological and molecular respects as well as MA. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Combined targeting of PDK1 and EGFR triggers regression of glioblastoma by reversing the Warburg effect.

PubMed

Velpula, Kiran Kumar; Bhasin, Arnima; Asuthkar, Swapna; Tsung, Andrew J

2013-12-15

Glioblastoma multiforme is the most aggressive primary brain tumor in adults. Overexpression of the EGF receptor (EGFR) is recognized as a widespread oncogenic signature in glioblastoma multiforme, but the complexity of its contributions is not fully understood, nor the most effective ways to leverage anti-EGFR therapy in this setting. Hypoxia is known to drive the aggressive character of glioblastoma multiforme by promoting aerobic glycolysis rather than pyruvate oxidation carried out in mitochondria (OXPHOS), a phenomenon termed the Warburg effect, which is a general feature of oncogenesis. In this study, we report that hypoxia drives expression of the pyruvate dehydrogenase kinase (PDK1) and EGFR along with the hypoxia-inducing factor (HIF)-1α in human glioblastoma multiforme cells. PDK1 is a HIF-1-regulated gene and our findings indicated that hypoxia-induced PDK1 expression may promote EGFR activation, initiating a feed-forward loop that can sustain malignant progression. RNAi-mediated attenuation of PDK1 and EGFR lowered PDK1-EGFR activation and decreased HIF-1α expression, shifting the Warburg phenotype to OXPHOS and inhibiting glioblastoma multiforme growth and proliferation. In clinical specimens of glioblastoma multiforme, we found that immunohistochemical expression of PDK1, EGFR, and HIF-1α were elevated in glioblastoma multiforme specimens when compared with normal brain tissues. Collectively, our studies establish PDK1 as a key driver and candidate therapeutic target in glioblastoma multiforme. ©2013 AACR.

An Immunohistochemical Study of Anaplastic Lymphoma Kinase and Epidermal Growth Factor Receptor Mutation in Non-Small Cell Lung Carcinoma

PubMed Central

Verma, Sonal; Kumari, Malti; Mehrotra, Raj; Kushwaha, R A S; Goel, Madhumati; Kumar, Ashutosh; Kant, Surya

2017-01-01

Introduction Lung cancer is one of the leading causes of cancer related death. Targeted treatment for specific markers may help in reducing the cancer related morbidity and mortality. Aim To study expression of Anaplastic Lymphoma Kinase (ALK)and Epidermal Growth Factor Receptor (EGFR) mutations in patients of Non-Small Cell Lung Cancer NSCLC, that are the targets for specific ALK inhibitors and EGFR tyrosine kinase inhibitors. Materials and Methods Total 69 cases of histologically diagnosed NSCLC were examined retrospectively for immunohistochemical expression of EGFR and ALK, along with positive control of normal placental tissue and anaplastic large cell lymphoma respectively. Results Of the NSCLC, Squamous Cell Carcinoma (SCC) accounted for 71.0% and adenocarcinoma was 26.1%. ALK expression was seen in single case of 60-year-old female, non-smoker with adenocarcinoma histology. EGFR expression was seen in both SCC (59.18%) and adenocarcinoma in (77.78%) accounting for 63.77% of all cases. Both ALK and EGFR mutation were mutually exclusive. Conclusion EGFR expression was seen in 63.77% of cases, highlighting the importance of its use in routine analysis, for targeted therapy and better treatment results. Although, ALK expression was seen in 1.45% of all cases, it is an important biomarker in targeted cancer therapy. Also, the mutually exclusive expression of these two markers need further studies to develop a diagnostic algorithm for NSCLC patients. PMID:28892905

Differential Efficacy of Combined Therapy With Radiation and AEE788 in High and Low EGFR-Expressing Androgen-Independent Prostate Tumor Models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huamani, Jessica; Willey, Christopher; Thotala, Dinesh

2008-05-01

Purpose: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT {+-} AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flowmore » and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusions: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.« less

EGFR and Ras regulate DDX59 during lung cancer development.

PubMed

Yang, Lin; Zhang, Hanyin; Chen, Dan; Ding, Peikun; Yuan, Yunchang; Zhang, Yandong

2018-02-05

Oncogenes EGFR and ras are frequently mutated and activated in human lung cancers. In this report, we found that both EGFR and Ras signaling can upregulate RNA helicase DDX59 in lung cancer cells. DDX59 can be induced through the mitogen activated protein kinase (MAPK) pathway after EGFR or Ras activation. Inhibitors for Ras/Raf/MAP pathway significantly decreased DDX59 expression at both protein and mRNA levels. Through immunohistochemistry, we found that DDX59 protein expression correlated with Ras and EGFR mutation status in human lung adenocarcinoma. Finally, through a xenograft nude mice model, we demonstrated that DDX59 is pivotal for EGFR mutated lung cancer cell growth in vivo. Our study identified a novel protein downstream of Ras and EGFR, which may serve as a potential therapeutic drug target for lung cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model.

PubMed

Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

2016-09-01

Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo , and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

Erlotinib is a viable treatment for tumors with acquired resistance to cetuximab

PubMed Central

Brand, Toni M; Dunn, Emily F; Iida, Mari; Myers, Rebecca A; Kostopoulos, Kellie T; Li, Chunrong; Peet, Chimera R

2011-01-01

The epidermal growth factor receptor (EGFR) is an ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of tumorigenesis in many human tumors. Currently there are five EGFR inhibitors used in oncology, two monoclonal antibodies (panitumumab and cetuximab) and three tyrosine kinase inhibitors (erlotinib, gefitinib and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical success; however, many tumors remain non-responsive or acquire resistance during therapy. To explore potential molecular mechanisms of acquired resistance to cetuximab we previously established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to escalating doses of cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in the activation of EGFR, HER2 and HER3 receptors as well as increased signaling through the MAP K and AKT pathways. RNAi studies demonstrated dependence of cetuximab-resistant clones on the EGFR signaling network. These findings prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to the EGFR targeted TKI erlotinib. In vitro, erlotinib was able to decrease signaling through the EGFR axis, decrease cellular proliferation and induce apoptosis. To determine if erlotinib could have therapeutic benefit in vivo, we established cetuximab-resistant NCI-H226 mouse xenografts, and subsequently treated them with erlotinib. Mice harboring cetuximab-resistant tumors treated with erlotinib exhibited either a tumor regression or growth delay as compared with vehicle controls. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation and increased rates of apoptosis. The work presented herein suggests that (1) cells with acquired resistance to cetuximab maintain their dependence on EGFR and (2) tumors developing resistance to cetuximab can benefit from subsequent treatment with erlotinib, providing rationale for its use in the setting of cetuximab resistance. PMID:21725209

Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.

PubMed

Omidfar, Kobra; Rasaee, Mohhamad Javad; Kashanian, Soheila; Paknejad, Malieheh; Bathaie, Zahra

2007-01-01

Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

Prognostic Significance of NSCLC and Response to EGFR-TKIs of EGFR-Mutated NSCLC Based on PD-L1 Expression.

PubMed

Kobayashi, Kenichi; Seike, Masahiro; Zou, Fenfei; Noro, Rintaro; Chiba, Mika; Ishikawa, Arimi; Kunugi, Shinobu; Kubota, Kaoru; Gemma, Akihiko

2018-02-01

Recent clinical trials have shown that immune checkpoint blockades that target either PD-1 or PD-L1 yield remarkable responses in a subgroup of patients with non-small cell lung cancer (NSCLC). We retrospectively examined, by immunohistochemical analysis, 211 NSCLC samples. Using 32 independent samples, we also evaluated PD-L1 expression in NSCLC patients with EGFR gene mutations treated by EGFR-TKIs. Overall survival of PD-L1-positive stages I-III NSCLC and stage I NSCLC and stages I-III squamous cell carcinoma (SQ) were significantly shorter than those of PD-L1-negative NSCLC (p<0.01 and p=0.02 and p=0.01, respectively). In stage I NSCLC and stages I-III SQ, PD-L1 expression was found to be independent predictor of death after multivariate analysis. Response to EGFR-TKIs was not significantly different between PD-L1-positive and PD-L1-negative NSCLC patients with EGFR mutations. PD-L1 expression was a significant independent predictor of poor outcome in NSCLC patients. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Marshall, Marianne E.; Hinz, Trista K.; Kono, Scott A.; Singleton, Katherine R.; Bichon, Brady; Ware, Kathryn E.; Marek, Lindsay; Frederick, Barbara A.; Raben, David; Heasley, Lynn E.

2011-01-01

Purpose We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and EGFR-dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed while only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and ERK signaling in three HNSCC cell lines while three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR TKIs with no contribution from FGFR pathways. Conclusions FGFR signaling was dominant or co-dominant with EGFR in six HNSCC lines while three lines exhibited little or no role for FGFRs and were highly EGFR-dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. PMID:21673064

Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis.

PubMed

Taute, Stefanie; Pfister, Herbert J; Steger, Gertrud

2017-01-01

Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor formation in cooperation with UV-light.

Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis

PubMed Central

Taute, Stefanie; Pfister, Herbert J.; Steger, Gertrud

2017-01-01

Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor formation in cooperation with UV-light. PMID:29176966

Differential Receptor Tyrosine Kinase PET Imaging for Therapeutic Guidance.

PubMed

Wehrenberg-Klee, Eric; Turker, N Selcan; Heidari, Pedram; Larimer, Benjamin; Juric, Dejan; Baselga, José; Scaltriti, Maurizio; Mahmood, Umar

2016-09-01

Inhibitors of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway hold promise for the treatment of breast cancer, but resistance to these treatments can arise via feedback loops that increase surface expression of the receptor tyrosine kinases (RTK) epidermal growth factor receptor 1 (EGFR) and human epidermal growth factor receptor 3 (HER3), leading to persistent growth pathway signaling. We developed PET probes that provide a method of imaging this response in vivo, determining which tumors may use this escape pathway while avoiding the need for repeated biopsies. Anti-EGFR-F(ab')2 and anti-HER3-F(ab')2 were generated from monoclonal antibodies by enzymatic digestion, conjugated to DOTA, and labeled with (64)Cu. A panel of breast cancer cell lines was treated with increasing concentrations of the AKT inhibitor GDC-0068 or the PI3K inhibitor GDC-0941. Pre- and posttreatment expression of EGFR and HER3 was compared using Western blot and correlated to probe accumulation with binding studies. Nude mice xenografts of HCC-70 or MDA-MB-468 were treated with either AKT inhibitor or PI3K inhibitor and imaged with either EGFR or HER3 PET probe. Changes in HER3 and EGFR PET probe accumulation correlate to RTK expression change as assessed by Western blot (R(2) of 0.85-0.98). EGFR PET probe PET/CT imaging of HCC70 tumors shows an SUV of 0.32 ± 0.03 for vehicle-, 0.50 ± 0.01 for GDC-0941-, and 0.62 ± 0.01 for GDC-0068-treated tumors, respectively (P < 0.01 for both comparisons to vehicle). HER3 PET probe PET/CT imaging of MDAMB468 tumors shows an SUV of 0.35 ± 0.02 for vehicle- and 0.73 ± 0.05 for GDC-0068-treated tumors (P < 0.01). Our imaging studies, using PET probes specific to EGFR and HER3, show that changes in RTK expression indicative of resistance to PI3K and AKT inhibitors can be seen within days of therapy initiation and are of sufficient magnitude as to allow reliable clinical interpretation. Noninvasive PET monitoring of these RTK feedback loops should help to rapidly assess resistance to PI3K and AKT inhibitors and guide selection of an appropriate combinatorial therapeutic regimen on an individual patient basis. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.

PubMed

Kim, Sun-Jin; Uehara, Hisanori; Karashima, Takashi; Shepherd, David L; Killion, Jerald J; Fidler, Isaiah J

2003-03-01

We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice. Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry. Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions. These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.

S100A9 and EGFR gene signatures predict disease progression in muscle invasive bladder cancer patients after chemotherapy.

PubMed

Kim, W T; Kim, J; Yan, C; Jeong, P; Choi, S Y; Lee, O J; Chae, Y B; Yun, S J; Lee, S C; Kim, W J

2014-05-01

In our previous gene expression profile analysis, IL1B, S100A8, S100A9, and EGFR were shown to be important mediators of muscle invasive bladder cancer (MIBC) progression. The aim of the present study was to investigate the ability of these gene signatures to predict disease progression after chemotherapy in patients with locally recurrent or metastatic MIBC. Patients with locally advanced MIBC who received chemotherapy were enrolled. The expression signatures of four genes were measured and carried out further functional analysis to confirm our findings. Two of the four genes, S100A9 and EGFR, were determined to significantly influence disease progression (P = 0.023, 0.045, respectively). Based on a receiver operating characteristic curve, a cut-off value for disease progression was determined. Patients with the good-prognostic signature group had a significantly longer time to progression and cancer-specific survival time than those with the poor-prognostic signature group (P < 0.001, 0.042, respectively). In the multivariate Cox regression analysis, gene signature was the only factor that significantly influenced disease progression [hazard ratio: 4.726, confidence interval: 1.623-13.763, P = 0.004]. In immunohistochemical analysis, S100A9 and EGFR positivity were associated with disease progression after chemotherapy. Protein expression of S100A9/EGFR showed modest correlation with gene expression of S100A9/EGFR (r = 0.395, P = 0.014 and r = 0.453, P = 0.004). Our functional analysis provided the evidence demonstrating that expression of S100A9 and EGFR closely associated chemoresistance, and that inhibition of S100A9 and EGFR may sensitize bladder tumor cells to the cisplatin-based chemotherapy. The S100A9/EGFR level is a novel prognostic marker to predict the chemoresponsiveness of patients with locally recurrent or metastatic MIBC.

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

PubMed

Figueroa, Javier M; Skog, Johan; Akers, Johnny; Li, Hongying; Komotar, Ricardo; Jensen, Randy; Ringel, Florian; Yang, Isaac; Kalkanis, Steven; Thompson, Reid; LoGuidice, Lori; Berghoff, Emily; Parsa, Andrew; Liau, Linda; Curry, William; Cahill, Daniel; Bettegowda, Chetan; Lang, Frederick F; Chiocca, E Antonio; Henson, John; Kim, Ryan; Breakefield, Xandra; Chen, Clark; Messer, Karen; Hochberg, Fred; Carter, Bob S

2017-10-19

RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Development of New Mouse Lung Tumor Models Expressing EGFR T790M Mutants Associated with Clinical Resistance to Kinase Inhibitors

PubMed Central

Regales, Lucia; Balak, Marissa N.; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A.; Solit, David B.; Rosen, Neal; Zakowski, Maureen F.; Pao, William

2007-01-01

Background The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. Methodology/Principal Findings To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFRT790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFRL858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFRT790M-expressing animals develop tumors with longer latency than EGFRL858R+T790M-bearing mice and in the absence of additional kinase domain mutations. Conclusions/Significance These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFRT790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations. PMID:17726540

Rab11a differentially modulates epidermal growth factor-induced proliferation and motility in immortal breast cells.

PubMed

Palmieri, Diane; Bouadis, Amina; Ronchetti, Ruban; Merino, Maria J; Steeg, Patricia S

2006-11-01

The development of cancer prevention strategies depends on the elucidation of molecular pathways underlying oncogenesis. In a previous proteomic study of matched normal breast ducts and Ductal Carcinoma in Situ (DCIS), we identified overexpression of Rab11a in DCIS. Rab11a is not well studied in cancer, but is known to regulate the recycling of internalized cell surface proteins and receptors from the early endosome through the trans-Golgi network. Using immunohistochemistry, we confirmed our observation, noting increased Rab11a expression in 19 of 22 (86%) DCIS cases compared to matched normal breast epithelium. To study the function of Rab11a, immortal, nontumorigenic MCF10A breast cells were stimulated with ligands to the EGF receptor (EGFR) after transfection with empty vector (control), Rab11a, or a S25N dominant-negative (DN) Rab11a. Using an iodinated ligand:receptor recycling assay, transfection of Rab11a accelerated, while DN-Rab11a postponed EGFR recycling in vitro. The signaling and in vitro phenotypic consequences of Rab11a expression and function were studied. Transfection of DN-Rab11a increased Erk1/2 activation downstream of EGF, but exerted no effect on the Akt pathway. Expression of DN-Rab11a inhibited MCF10A proliferation by 50-60%, and also inhibited anchorage-dependent colonization. Notably, DN-Rab11a transfection increased motility toward EGFR ligands. The data provide a first demonstration that Rab11a modulates EGFR recycling, and promotes the proliferation but inhibits the motility of an immortal breast line, consistent with the DCIS phenotype.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

PubMed

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

Involvement of Epidermal Growth Factor Receptor Signaling in Estrogen Inhibition of Oocyte Maturation Mediated Through the G Protein-Coupled Estrogen Receptor (Gper) in Zebrafish (Danio rerio)1

PubMed Central

Peyton, Candace; Thomas, Peter

2011-01-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression. PMID:21349822

Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor (Gper) in zebrafish (Danio rerio).

PubMed

Peyton, Candace; Thomas, Peter

2011-07-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression.

Autophagosome-mediated EGFR down-regulation induced by the CK2 inhibitor enhances the efficacy of EGFR-TKI on EGFR-mutant lung cancer cells with resistance by T790M.

PubMed

So, Kwang Sup; Kim, Cheol Hyeon; Rho, Jin Kyung; Kim, Sun Ye; Choi, Yun Jung; Song, Joon Seon; Kim, Woo Sung; Choi, Chang Min; Chun, Young Jin; Lee, Jae Cheol

2014-01-01

Protein kinase CK2 has diverse functions promoting and maintaining cancer phenotypes. We investigated the effect of CK2 inhibition in lung cancer cells with T790M-mediated resistance to the EGFR-TK inhibitor. Resistant sublines of PC-9 to gefitinib (PC-9/GR) and erlotinib (PC-9/ER) were established by previous study, and T790M secondary mutation was found in both resistant sublines. A decrease of EGFR by siRNA treatment effectively controlled the growth of resistant cells, thus suggesting that they still have EGFR-dependency. CX-4945, a potent and selective CK2 inhibitor, induced autophagy in PC-9/GR and PC-9/ER, and which was supported by the induction of autophagic vacuoles and microtubule-associated protein 1 light chain 3 (LC3) expression, and the increase of punctate fluorescent signals in resistant cells pre-transfected with green fluorescent protein (GFP)-tagged LC3. However, the withdrawal of CX-4945 led to the recovery of cancer cells with autophagy. We found that the induction of autophagy by CX-4945 in both resistant cells was CK2 dependent by using small interfering RNA against CK2. The treatment with CX-4945 alone induced a minimal growth inhibition in resistant cells. However, combined treatment of CX-4945 and EGFR-TKI effectively inhibited cancer-cell proliferation and induced apoptosis. CX-4945 increased the translocation of EGFR from the cell surface into the autophagosome, subsequently leading to the decrease of EGFR while inhibition of autophagy by 3MA or Atg7-targeted siRNA pretreatment reduced the decrease of EGFR by CX-4945. Accordingly, apoptosis by a combination of CX-4945 and EGFR-TKI was suppressed by 3MA or Atg7-targeted siRNA pretreatment, thus suggesting that autophagosome-mediated EGFR down-regulation would have an important role regarding apoptotic cell death by EGFR-TKI. Combined treatment of the CK2 inhibitor and EGFR-TKI may be a promising strategy for overcoming T790M-mediated resistance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achanzar, William E.; Moyer, Carolyn F.; Marthaler, Laura T.

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR){alpha}/{gamma} agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPAR{alpha}, PPAR{delta}, PPAR{gamma}, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPAR{gamma} agonist pioglitazone daily for themore » final 3 days to directly assess the effects of diet acidification on responsiveness to PPAR{gamma} agonism. Urothelial cell PPAR{alpha} and {gamma} expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPAR{alpha}, {delta}, or {gamma} mRNA or protein expression, PPAR{alpha}- or {gamma}-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPAR{gamma}-regulated gene expression. These results support the contention that urine acidification does not prevent PPAR{gamma} agonist-induced bladder tumors by altering PPAR{alpha}, {gamma}, or EGFR expression or PPAR signaling in rat bladder urothelium.« less

Anti-EGFR-Conjugated Hollow Gold Nanospheres Enhance Radiocytotoxic Targeting of Cervical Cancer at Megavoltage Radiation Energies

NASA Astrophysics Data System (ADS)

Liu, Jiao; Liang, Ying; Liu, Ting; Li, Dengke; Yang, Xingsheng

2015-05-01

The study aimed to confirm that anti-epidermal growth factor receptor (EGFR) monoclonal antibody-conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaken by cervical cancer cells and induce its apoptosis when combined with radiotherapy, as a result enhancing radiosensitivity of cervical cancer cells. HGNs with a mean diameter of 54.6 ± 7.11 nm and wall thickness of 5.01 ± 2.23 nm were viewed by transmission electron microscopy (TEM). Cell uptake was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The cytotoxicity on HeLa cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry (FCM). The expression of several critical apoptosis-related proteins, including Bcl-2, Bax, Bad, and active caspase 3, was tested by western blot analysis. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticle uptake compared to naked HGNs. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of HeLa cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax, Bad, and caspase 3 when combined with radiation. Therefore, anti-EGFR/HGNs can increase the targeted uptake of HGNs by HeLa cells and enhance radiocytotoxic targeting of cervical cancer at megavoltage radiation energies.

MicroRNA-137 inhibits growth of glioblastoma through EGFR suppression

PubMed Central

Zhang, Zhenxing; Song, Xiaofeng; Tian, He; Miao, Ye; Feng, Xu; Li, Yang; Wang, Honglei

2017-01-01

Aberrant expression of certain microRNAs (miRNAs) has been shown to contribute to the development of Glioblastoma multiforme (GBM). However, the involvement of miR-137 in the carcinogenesis of GBM has not been reported. Here, we showed that miR-137 levels in GBM tissues were significantly lower than the paired normal brain tissue in patients’ specimens. Moreover, low miR-137 levels in GBM tissue were associated with poor prognosis. In vitro, overexpression of miR-137 decreased GBM cell growth and increased cell apoptosis, while depletion of miR-137 enhanced cell growth and decreased cell apoptosis. Combined bioinformatics analysis and dual luciferase reporter assay showed that miR-137 may target the 3’-UTR of the epidermal growth factor receptor (EGFR) to reduce its protein translation, resulting in suppression of EGFR signaling in GBM cells. Together, our data suggest that reduction in miR-137 levels in GBM tissues may increase cell growth and decrease cell apoptosis, possibly through suppression of EGFR. PMID:28386374

Berberine inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors in gastric cancer.

PubMed

Wang, Junxiong; Yang, Shuo; Cai, Xiqiang; Dong, Jiaqiang; Chen, Zhangqian; Wang, Rui; Zhang, Song; Cao, Haichao; Lu, Di; Jin, Tong; Nie, Yongzhan; Hao, Jianyu; Fan, Daiming

2016-11-15

Cetuximab plus chemotherapy for advanced gastric cancer (GC) shows an active result in phase 2 trials. Unfortunately, Combination of cetuximab does not provide enough benefit to chemotherapy alone in phase 3 trials. Studies have demonstrated that berberine can suppress the activation of EGFR in tumors. In this study, we evaluated whether berberine could enhance the effects of EGFR-TKIs in GC cell lines and xenograft models. Our data suggest that berberine could effectively enhance the activity of erlotinib and cetuximab in vitro and in vivo. Berberine was found to inhibit growth in GC cell lines and to induce apoptosis. These effects were linked to inhibition of EGFR signaling activation, including the phosphorylation of STAT3. The expressions of Bcl-xL and Cyclind1 proteins were decreased, whereas the levels of cleavage of poly-ADP ribose polymerase (PARP) were considerably increased in the cell lines in response to berberine treatment. These results suggest a potential role for berberine in the treatment of GC, particularly in combination with EGFR-TKIs therapy. Berberine may be a competent therapeutic agent in GC where it can enhance the effects of EGFR inhibitors.

Berberine inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors in gastric cancer

PubMed Central

Wang, Junxiong; Yang, Shuo; Cai, Xiqiang; Dong, Jiaqiang; Chen, Zhangqian; Wang, Rui; Zhang, Song; Cao, Haichao; Lu, Di; Jin, Tong; Nie, Yongzhan; Hao, Jianyu; Fan, Daiming

2016-01-01

Cetuximab plus chemotherapy for advanced gastric cancer (GC) shows an active result in phase 2 trials. Unfortunately, Combination of cetuximab does not provide enough benefit to chemotherapy alone in phase 3 trials. Studies have demonstrated that berberine can suppress the activation of EGFR in tumors. In this study, we evaluated whether berberine could enhance the effects of EGFR-TKIs in GC cell lines and xenograft models. Our data suggest that berberine could effectively enhance the activity of erlotinib and cetuximab in vitro and in vivo. Berberine was found to inhibit growth in GC cell lines and to induce apoptosis. These effects were linked to inhibition of EGFR signaling activation, including the phosphorylation of STAT3. The expressions of Bcl-xL and Cyclind1 proteins were decreased, whereas the levels of cleavage of poly-ADP ribose polymerase (PARP) were considerably increased in the cell lines in response to berberine treatment. These results suggest a potential role for berberine in the treatment of GC, particularly in combination with EGFR-TKIs therapy. Berberine may be a competent therapeutic agent in GC where it can enhance the effects of EGFR inhibitors. PMID:27738318

Association of EGFR mutation or ALK rearrangement with expression of DNA repair and synthesis genes in never-smoker women with pulmonary adenocarcinoma.

PubMed

Ren, Shengxiang; Chen, Xiaoxia; Kuang, Peng; Zheng, Limou; Su, Chunxia; Li, Jiayu; Li, Bing; Wang, Yongshen; Liu, Lu; Hu, Qiong; Zhang, Jie; Tang, Liang; Li, Xuefei; Zhou, Caicun; Schmid-Bindert, Gerald

2012-11-15

Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement may predict the outcome of targeted drug therapy and also are associated with the efficacy of chemotherapy in patients with nonsmall cell lung cancer (NSCLC). The authors of this report investigated the relation of EGFR mutation or ALK rearrangement status and the expression of DNA repair or synthesis genes, including excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), thymidylate synthetase (TS), and breast cancer-early onset (BRCA1), as a potential explanation for these observations. In total, 104 resected lung adenocarcinomas from women who were nonsmokers were analyzed concurrently for EGFR mutations, ALK rearrangements, and mRNA expression of the ERCC1, RRM1, TS, and BRCA1 genes. EGFR mutations were detected with a proprietary detection kit, ALK rearrangements were detected by polymerase chain reaction analysis, and genetic mRNA expression was detected by real-time polymerase chain reaction analysis. Of 104 patients, 73 (70.2%) had EGFR mutations, and 10 (9.6%) had ALK rearrangements. ERCC1 mRNA levels in patients who had EGFR mutations were 3.44 ± 1.94 × 10(-3) , which were significantly lower than the levels in patients who were positive for ALK rearrangements and in patients who were negative for both biomarkers (4.60 ± 1.95 × 10(-3) and 4.95 ± 2.33 × 10(-3) , respectively; P = .010). However, TS mRNA levels were significantly lower in patients who had EGFR mutations (1.15 ± 1.38 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .006) or ALK rearrangements (1.21 ± 0.78 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .020) than in patients who were negative for both biomarkers. NSCLC specimens that harbored activating EGFR mutations were more likely to express low ERCC1 and TS mRNA levels, whereas patients with NSCLC who had ALK rearrangement were more likely to express low TS mRNA levels. Copyright © 2012 American Cancer Society.

Chronic activation of wild-type epidermal growth factor receptor and loss of Cdkn2a cause mouse glioblastoma formation.

PubMed

Acquaviva, Jaime; Jun, Hyun Jung; Lessard, Julie; Ruiz, Rolando; Zhu, Haihao; Donovan, Melissa; Woolfenden, Steve; Boskovitz, Abraham; Raval, Ami; Bronson, Roderick T; Pfannl, Rolf; Whittaker, Charles A; Housman, David E; Charest, Al

2011-12-01

Glioblastoma multiforme (GBM) is characterized by overexpression of epidermal growth factor receptor (EGFR) and loss of the tumor suppressors Ink4a/Arf. Efforts at modeling GBM using wild-type EGFR in mice have proven unsuccessful. Here, we present a unique mouse model of wild-type EGFR-driven gliomagenesis. We used a combination of somatic conditional overexpression and ligand-mediated chronic activation of EGFR in cooperation with Ink4a/Arf loss in the central nervous system of adult mice to generate tumors with the histopathologic and molecular characteristics of human GBMs. Sustained, ligand-mediated activation of EGFR was necessary for gliomagenesis, functionally substantiating the clinical observation that EGFR-positive GBMs from patients express EGFR ligands. To gain a better understanding of the clinically disappointing EGFR-targeted therapies for GBM, we investigated the molecular responses to EGFR tyrosine kinase inhibitor (TKI) treatment in this model. Gefitinib treatment of primary GBM cells resulted in a robust apoptotic response, partially conveyed by mitogen-activated protein kinase (MAPK) signaling attenuation and accompanied by BIM(EL) expression. In human GBMs, loss-of-function mutations in the tumor suppressor PTEN are a common occurrence. Elimination of PTEN expression in GBM cells posttumor formation did not confer resistance to TKI treatment, showing that PTEN status in our model is not predictive. Together, these findings offer important mechanistic insights into the genetic determinants of EGFR gliomagenesis and sensitivity to TKIs and provide a robust discovery platform to better understand the molecular events that are associated with predictive markers of TKI therapy.

Dependence of Relative Expression of NTR1 and EGFR on Cell Density and Extracellular pH in Human Pancreatic Cancer Cell Lines

PubMed Central

Olszewski-Hamilton, Ulrike; Hamilton, Gerhard

2011-01-01

Pancreatic adenocarcinoma is a devastating disease characterized by early dissemination and poor prognosis. These solid tumors express receptors for neuropeptides like neurotensin (NT) or epidermal growth factor (EGF) and exhibit acidic regions when grown beyond a certain size. We previously demonstrated increases in intracellular Ca2+ levels, intracellular pH and interleukin-8 (IL-8) secretion in BxPC-3 and PANC-1 pancreatic cancer cells in response to a stable NT analog. The present study aimed at investigation of the dependence of the relative expression of NT receptor 1 (NTR1) and EGFR in BxPC-3 and MIA PaCa-2 cells on cell density and extracellular pH (pHe). MTT assays revealed the NTR1 inhibitor SR 142948-sensitive Lys8-ψ-Lys9NT (8–13)-induced proliferation in BxPC-3 and PANC-1 cells. Confluent cultures of BxPC3 and HT-29 lines exhibited highest expression of NTR1 and lowest of EGFR and expression of NTR1 was maximal at slightly acidic pHe. IL-8 production was stimulated by Lys8-ψ-Lys9NT (8–13) and even enhanced at both acidic and alkaline pHe in BxPC-3 and PANC-1 cells. In conclusion, our in vitro study suggests that one contributing factor to the minor responses obtained with EGFR-directed therapy may be downregulation of this receptor in tumor cell aggregates, possibly resulting in acquisition of a more aggressive phenotype via other growth factor receptors like NTR1. PMID:24212612

EGFR molecular profiling in advanced NSCLC: a prospective phase II study in molecularly/clinically selected patients pretreated with chemotherapy.

PubMed

Milella, Michele; Nuzzo, Carmen; Bria, Emilio; Sperduti, Isabella; Visca, Paolo; Buttitta, Fiamma; Antoniani, Barbara; Merola, Roberta; Gelibter, Alain; Cuppone, Federica; D'Alicandro, Valerio; Ceribelli, Anna; Rinaldi, Massimo; Cianciulli, Anna; Felicioni, Lara; Malatesta, Sara; Marchetti, Antonio; Mottolese, Marcella; Cognetti, Francesco

2012-04-01

The optimal use of epidermal growth factor receptor (EGFR)-related molecular markers to prospectively identify tyrosine kinase inhibitor (TKI)-sensitive patients, particularly after a previous chemotherapy treatment, is currently under debate. We designed a prospective phase II study to evaluate the activity of EGFR-TKI in four different patient groups, according to the combination of molecular (EGFR gene mutations, EGFR gene copy number and protein expression, and phosphorylated AKT expression, pAKT) and clinicopathological (histology and smoking habits) factors. Correlations between molecular alterations and clinical outcome were also explored retrospectively for first-line chemotherapy and EGFR-TKI treatment. Patients who had progressed during or after first-line chemotherapy were prospectively assigned to EGFR-TKI treatment as follows: (G1) EGFR mutation (n = 12); (G2) highly polysomic/amplified EGFR (n = 18); (G3) EGFR and/or pAKT positive (n = 41); (G4) adenocarcinoma/bronchoalveolar carcinoma and no smoking history (n = 15). G1 and G4 had the best and second-best overall response rate (25% and 20%, respectively), whereas the worst outcome was observed in G2 (ORR, 6%; p = 0.05). Disease control was highest in G1 and G4 (>50%) and lowest in G3 (<20%) (p = 0.02). Patients selected by EGFR mutation or clinical parameters (G1 and G4) also had significantly better progression-free survival and overall survival (p = 0.02 and p = 0.01, respectively). Multivariate analysis confirmed the impact of sex, smoking history, EGFR/KRAS mutation, and pAKT on outcomes and allowed us to derive an efficient predictive model. Histology, EGFR mutations, and pAKT were independent predictors of response to first-line chemotherapy at retrospective analysis, whereas pAKT and human epidermal growth factor receptor 2 expression were the only independent predictors of progression-free survival and overall survival. Selection of patients based on either EGFR mutation or clinical characteristics seems an effective approach to optimize EGFR-TKI treatment in chemotherapy-pretreated non-small-cell lung cancer patients.

Dysregulation of EGFR Pathway in EphA2 Cell Subpopulation Significantly Associates with Poor Prognosis in Colorectal Cancer.

PubMed

De Robertis, Mariangela; Loiacono, Luisa; Fusilli, Caterina; Poeta, Maria Luana; Mazza, Tommaso; Sanchez, Massimo; Marchionni, Luigi; Signori, Emanuela; Lamorte, Giuseppe; Vescovi, Angelo Luigi; Garcia-Foncillas, Jesus; Fazio, Vito Michele

2017-01-01

EphA2 receptor is involved in multiple cross-talks with other cellular networks, including EGFR, FAK, and VEGF pathways, with which it collaborates to stimulate cell migration, invasion, and metastasis. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC. Gene expression analysis was performed in EphA2 high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I-III CRC, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159-70. ©2016 AACR. ©2016 American Association for Cancer Research.

Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

PubMed

Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

2016-06-01

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

Expression of Epidermal Growth Factor Receptor and Transforming Growth Factor Alpha in Cancer Bladder: Schistosomal and Non-Schistosomal

PubMed Central

Badawy, Afkar A.; El-Hindawi, Ali; Hammam, Olfat; Moussa, Mona; Helal, Noha S.; Kamel, Amira

2017-01-01

Introduction Overexpression of epidermal growth factor receptor (EGFR) has been described in several solid tumors including bladder cancer. Transforming growth factor alpha (TGFα) is frequently deregulated in neoplastic cells and plays a role in the development of bladder cancer. TGFα-EGFR ligand-receptor combination constitutes an important event in multistep tumorigenesis. Methods This study was done on 30 bladder biopsies from patients with urothelial carcinoma, 15 with squamous cell carcinoma, 10 with cystitis and 5 normal control bladder specimens. All were immuohistochemically stained with EGFR and TGFα antibodies. Results EGFR and TGFα were over-expressed in higher grades and late stages of bladder cancer. Moreover, they show higher expression in squamous cell carcinoma compared to urothelial carcinoma and in schistosomal associated lesions than in non-schistosomal associated lesions. Conclusion EGFR and TGFα could be used as prognostic predictors in early stage and grade of bladder cancer cases, especially those with schistosomal association. In addition they can help in selecting patients who can get benefit from anti-EGFR molecular targeted therapy. PMID:28413380

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

PubMed

Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

2010-08-01

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

Novel role of MDA-9/syntenin in regulating urothelial cell proliferation by modulating EGFR signaling.

PubMed

Dasgupta, Santanu; Menezes, Mitchell E; Das, Swadesh K; Emdad, Luni; Janjic, Aleksandar; Bhatia, Shilpa; Mukhopadhyay, Nitai D; Shao, Chunbo; Sarkar, Devanand; Fisher, Paul B

2013-09-01

Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors. Significantly higher (P=0.002-0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P=0.01), grade (P=0.03), and invasion status (P=0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P=0.0012), invasion (P=0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P=0.04, EGFR; P=0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P=0.03) and invasion (P=0.04) was also evident. Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC. ©2013 AACR.

Phenytoin enhances the phosphorylation of epidermal growth factor receptor and fibroblast growth factor receptor in the subventricular zone and promotes the proliferation of neural precursor cells and oligodendrocyte differentiation.

PubMed

Galvez-Contreras, Alma Y; Gonzalez-Castaneda, Rocio E; Campos-Ordonez, Tania; Luquin, Sonia; Gonzalez-Perez, Oscar

2016-01-01

Phenytoin is a widely used antiepileptic drug that induces cell proliferation in several tissues, such as heart, bone, skin, oral mucosa and neural precursors. Some of these effects are mediated via fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR). These receptors are strongly expressed in the adult ventricular-subventricular zone (V-SVZ), the main neurogenic niche in the adult brain. The aim of this study was to determine the cell lineage and cell fate of V-SVZ neural progenitors expanded by phenytoin, as well as the effects of this drug on EGFR/FGFR phosphorylation. Male BALB/C mice received 10 mg/kg phenytoin by oral cannula for 30 days. We analysed the proliferation of V-SVZ neural progenitors by immunohistochemistry and western blot. Our findings indicate that phenytoin enhanced twofold the phosphorylation of EGFR and FGFR in the V-SVZ, increased the number of bromodeoxyuridine (BrdU)+/Sox2+ and BrdU+/doublecortin+ cells in the V-SVZ, and expanded the population of Olig2-expressing cells around the lateral ventricles. After phenytoin removal, a large number of BrdU+/Receptor interacting protein (RIP)+ cells were observed in the olfactory bulb. In conclusion, phenytoin enhanced the phosphorylation of FGFR and EGFR, and promoted the expression of neural precursor markers in the V-SVZ. In parallel, the number of oligodendrocytes increased significantly after phenytoin removal. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

PubMed

Yamazaki, Shota; Higuchi, Youichi; Ishibashi, Masayuki; Hashimoto, Hiroko; Yasunaga, Masahiro; Matsumura, Yasuhiro; Tsuchihara, Katsuya; Tsuboi, Masahiro; Goto, Koichi; Ochiai, Atsushi; Ishii, Genichiro

2018-06-01

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

PubMed Central

Bazzani, Lorenzo; Donnini, Sandra; Finetti, Federica; Christofori, Gerhard; Ziche, Marina

2017-01-01

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression. PMID:28415726

Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

PubMed

Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

2005-11-21

It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

Anti-EGFR Peptide-Conjugated Triangular Gold Nanoplates for Computed Tomography/Photoacoustic Imaging-Guided Photothermal Therapy of Non-Small Cell Lung Cancer.

PubMed

Zhao, Ying; Liu, Wenfei; Tian, Ying; Yang, Zhenlu; Wang, Xiaofen; Zhang, Yunlei; Tang, Yuxia; Zhao, Shuang; Wang, Chunyan; Liu, Ying; Sun, Jing; Teng, Zhaogang; Wang, Shouju; Lu, Guangming

2018-05-23

Non-small cell lung cancer (NSCLC) is difficult to cure because of the high recurrence rate and the side effects of current treatments. It is urgent to develop a new treatment that is safer and more effective than current treatments against NSCLC. Herein, we constructed anti-epidermal growth factor receptor (EGFR) peptide-conjugated PEGylated triangular gold nanoplates (TGN-PEG-P75) as a targeting photothermal therapy (PTT) agent to treat NSCLC under the guidance of computed tomography (CT) and photoacoustic (PA) imaging. The surface of TGNs is successfully conjugated with a novel peptide P75 that has the specific affinity to epidermal growth factor receptor (EGFR). It is found that the EGFR is overexpressed in NSCLC cells. The TGN-PEG-P75 has uniform edge length (77.9 ± 7.0 nm) and neutrally charged surface. The cell uptake experiments demonstrate remarkable affinity of the TGN-PEG-P75 to high EGFR expression cells than low EGFR expression cells (5.1-fold). Thanks to the strong near-infrared absorbance, high photothermal conversion efficiency, and the increased accumulation in tumor cells via the interaction of P75 and EGFR, TGN-PEG-P75 exhibits 3.8-fold superior therapeutic efficacy on HCC827 cells than TGN-PEG. The in vivo CT/PA dual-modal imaging of the TGN-PEG-P75 is helpful in selecting the optimal treatment time and providing real-time visual guidance of PTT. Furthermore, treatments on HCC827 tumor-bearing mouse model demonstrate that the growth of NSCLC cells can be effectively inhibited by the TGN-PEG-P75 through PTT, indicating the great promise of the nanoplatform for treating NSCLC in vivo.

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities.

PubMed

Niesen, Judith; Stein, Christoph; Brehm, Hannes; Hehmann-Titt, Grit; Fendel, Rolf; Melmer, Georg; Fischer, Rainer; Barth, Stefan

2015-12-01

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA'). The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA' (from cetuximab) and scFv1711-ETA' (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA'-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC.

PubMed

Che, Ting-Fang; Lin, Ching-Wen; Wu, Yi-Ying; Chen, Yu-Ju; Han, Chia-Li; Chang, Yih-leong; Wu, Chen-Tu; Hsiao, Tzu-Hung; Hong, Tse-Ming; Yang, Pan-Chyr

2015-11-10

Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo. Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

NASA Astrophysics Data System (ADS)

Lin, Wen Jen; Chien, Wei Hsuan

2015-09-01

The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly( d,l-lactide- co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brunetto de Farias, Caroline; Children's Cancer Institute, 90420-140 Porto Alegre, RS; Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS

2012-08-24

Highlights: Black-Right-Pointing-Pointer BDNF protected HT-29 colorectal cancer cells from the antitumor effect of cetuximab. Black-Right-Pointing-Pointer TrkB inhibition potentiated the antitumor effect of cetuximab. Black-Right-Pointing-Pointer BDNF/TrkB signaling might be involved in resistance to anti-EGFR therapy. -- Abstract: The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling canmore » protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.« less

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

RAB-7 Antagonizes LET-23 EGFR Signaling during Vulva Development in Caenorhabditis elegans

PubMed Central

Skorobogata, Olga; Rocheleau, Christian E.

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(−) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans. PMID:22558469

RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans.

PubMed

Skorobogata, Olga; Rocheleau, Christian E

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

PubMed

Li, He; Huang, Yao; Jiang, Du-Qing; Cui, Lian-Zhen; He, Zhou; Wang, Chao; Zhang, Zhi-Wei; Zhu, Hai-Li; Ding, Yong-Mei; Li, Lin-Fang; Li, Qiang; Jin, Hua-Jun; Qian, Qi-Jun

2018-02-07

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

Inhibition of DYRK1A destabilizes EGFR and reduces EGFR-dependent glioblastoma growth

PubMed Central

Pozo, Natividad; Zahonero, Cristina; Fernández, Paloma; Liñares, Jose M.; Ayuso, Angel; Hagiwara, Masatoshi; Pérez, Angel; Ricoy, Jose R.; Hernández-Laín, Aurelio; Sepúlveda, Juan M.; Sánchez-Gómez, Pilar

2013-01-01

Glioblastomas (GBMs) are very aggressive tumors that are resistant to conventional chemo- and radiotherapy. New molecular therapeutic strategies are required to effectively eliminate the subpopulation of GBM tumor–initiating cells that are responsible for relapse. Since EGFR is altered in 50% of GBMs, it represents one of the most promising targets; however, EGFR kinase inhibitors have produced poor results in clinical assays, with no clear explanation for the observed resistance. We uncovered a fundamental role for the dual-specificity tyrosine phosphorylation–regulated kinase, DYRK1A, in regulating EGFR in GBMs. We found that DYRK1A was highly expressed in these tumors and that its expression was correlated with that of EGFR. Moreover, DYRK1A inhibition promoted EGFR degradation in primary GBM cell lines and neural progenitor cells, sharply reducing the self-renewal capacity of normal and tumorigenic cells. Most importantly, our data suggest that a subset of GBMs depends on high surface EGFR levels, as DYRK1A inhibition compromised their survival and produced a profound decrease in tumor burden. We propose that the recovery of EGFR stability is a key oncogenic event in a large proportion of gliomas and that pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for EGFR-dependent GBMs. PMID:23635774

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

PubMed Central

Hughes, Shannon K.; Oudin, Madeleine J.; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A.; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S.; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A.; Gertler, Frank B.

2015-01-01

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express MenaINV, which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5′ inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When MenaINV is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor–induced signaling. Disruption of this attenuation by MenaINV sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. PMID:26337385

Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Ping; Zhang Qing; Torossian, Artour

2012-07-01

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used tomore » investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.« less

High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.

PubMed

Kumar, S S; Tomita, Y; Wrin, J; Bruhn, M; Swalling, A; Mohammed, M; Price, T J; Hardingham, J E

2017-06-01

Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

Gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells.

PubMed

Chen, Ying-Jung; Lin, Ku-Nan; Jhang, Li-Mei; Huang, Chia-Hui; Lee, Yuan-Chin; Chang, Long-Sen

2016-05-25

Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, W.-N.; Luo, S.-F.; Wu, C.-B.

2008-04-15

In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-{kappa}B in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2,more » or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.« less

GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer.

PubMed

Mo, Zhiqiang; Liu, Manran; Yang, Fangfang; Luo, Haojun; Li, Zhenhua; Tu, Gang; Yang, Guanglun

2013-11-29

Tamoxifen is widely used to treat hormone-dependent breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Here, we investigated the role of estrogen G-protein coupled receptor 30 (GPR30) on Tamoxifen resistance in breast cancer. Primary tumors (PTs) of breast cancer and corresponding metastases (MTs) were used to evaluate the expression of GPR30 and epidermal growth factor receptor (EGFR) immunohistochemically. Tamoxifen-resistant (TAM-R) subclones derived from parent MCF-7 cells were used to investigate the role of GPR30 in the development of tamoxifen resistance, using MTT assay, western blot, RT-PCR, immunofluorescence, ELISA and flow cytometry. TAM-R xenografts were established to assess anti-tumor effects of combination therapy with GPR30 antagonist G15 plus 4-hydroxytamoxifen (Tam), using tumor volume measurement and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In 53 human breast cancer specimens, GPR30 expression in MTs increased compared to matched PTs; in MTs, the expression patterns of GPR30 and EGFR were closely related. Compared to parent MCF-7 cells, TAM-R cells had greater growth responses to 17β-estradiol (E2), GPR30 agonist G1 and Tam, and significantly higher activation of Mitogen-activated protein (MAP) kinases; but this increased activity was abolished by G15 or AG1478. In TAM-R cells, GPR30 cell-surface translocation facilitated crosstalk with EGFR, and reduced cAMP generation, attenuating inhibition of EGFR signaling. Combination therapy both promoted apoptosis in TAM-R cells and decreased drug-resistant tumor progression. Long-term endocrine treatment facilitates the translocation of GPR30 to cell surfaces, which interferes with the EGFR signaling pathway; GPR30 also attenuates the inhibition of MAP kinases. These factors contribute to tamoxifen resistance development in breast cancer. Combination therapy with GPR30 inhibitors and tamoxifen may provide a new therapeutic option for drug-resistant breast cancer.

Identification and suppression of epidermal growth factor receptor variant III signaling in fibroblast-like synoviocytes from aggressive rheumatoid arthritis by the mimotope.

PubMed

Niu, Jianying; Li, Changhong; Jin, Yinji; Xing, Rui; Sun, Lin; Yu, Ruohan; Jian, Leilei; Liu, Xiangyuan; Yang, Lin

2018-06-01

Epidermal growth factor receptor (EGFR) signaling has been reported to play a vital role in the pathogenesis of rheumatoid arthritis (RA). In current study, we sought to observe whether the active immunization induced by the mimotope could recognize EGFR, inhibit their signaling and disrupt the pathogenic behavior of fibroblast-like synoviocytes (FLS) from RA patients. We prepared a linked EGFR mimotope and performed series of experiments to detect whether the mimotope could induce the desired immune responses. To our surprises, we detected the expression of EGFR variant III (EGFRvIII), but not EGFR in the synovial tissues and FLS from patients with aggressive RA by the linked EGFR mimotope-induced antibodies (LEMIA). Meanwhile, LEMIA could inhibit the signaling caused by the autophosphorylation of EGFRvIII in the FLS. The proliferation, migration, invasion and anti-apoptosis capabilities of the EGFRvIII-expressed FLS were disrupted by LEMIA. These results suggest that EGFRvIII signaling may participate in the malignant behaviors of FLS from aggressive RA. Meanwhile, the linked EGFR mimotope could be used to detect the expression of EGFRvIII and developed to be a potential therapy agent against the aggressive FLS. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Loss of EGFR-ASAP1 signaling in metastatic and unresectable hepatoblastoma.

PubMed

Ranganathan, Sarangarajan; Ningappa, Mylarappa; Ashokkumar, Chethan; Higgs, Brandon W; Min, Jun; Sun, Qing; Schmitt, Lori; Subramaniam, Shankar; Hakonarson, Hakon; Sindhi, Rakesh

2016-12-02

Hepatoblastoma (HBL), the most common childhood liver cancer is cured with surgical resection after chemotherapy or with liver transplantation if local invasion and multifocality preclude resection. However, variable survival rates of 60-80% and debilitating chemotherapy sequelae argue for more informed treatment selection, which is not possible by grading the Wnt-β-catenin over activity present in most HBL tumors. A hypothesis-generating whole transcriptome analysis shows that HBL tumors removed at transplantation are enriched most for cancer signaling pathways which depend predominantly on epidermal growth factor (EGF) signaling, and to a lesser extent, on aberrant Wnt-β-catenin signaling. We therefore evaluated whether EGFR, ASAP1, ERBB2 and ERBB4, which signal downstream after ligation of EGF, and which show aberrant expression in several other invasive cancers, would also predict HBL tumor invasiveness. Immunohistochemistry of HBL tumors (n = 60), which are histologically heterogeneous, shows that compared with well-differentiated fetal cells, less differentiated embryonal and undifferentiated small cells (SCU) progressively lose EGFR and ASAP1 expression. This trend is exaggerated in unresectable, locally invasive or metastatic tumors, in which embryonal tumor cells are EGFR-negative, while SCU cells are EGFR-negative and ASAP1-negative. Loss of EGFR-ASAP1 signaling characterizes undifferentiated and invasive HBL. EGFR-expressing HBL tumors present novel therapeutic targeting opportunities.

PANC-1 pancreatic cancer cell growth inhibited by cucurmosin alone and in combination with an epidermal growth factor receptor-targeted drug.

PubMed

Wang, Congfei; Yang, Aiqin; Zhang, Baoming; Yin, Qiang; Huang, Heguang; Chen, Minghuang; Xie, Jieming

2014-03-01

To investigate the inhibition of PANC-1 pancreatic cancer cell growth by cucurmosin (CUS) and its possible mechanism. We observed the inhibition of PANC-1 cell growth by sulforhodamine B and colony-forming experiments in vitro and established nonobese diabetic/severe combined immunodeficiency mouse subcutaneous tumor models in vivo. We used Western blot to analyze protein levels related to apoptosis and epidermal growth factor receptor (EGFR) signaling pathways after drug intervention, whereas the messenger RNA expression of EGFR was analyzed by quantitative real-time polymerase chain reaction. Sulforhodamine B and colony-forming experiments indicated that CUS inhibited PANC-1 cell proliferation in a dose- and time-dependent manner. A stronger inhibitory effect was observed when CUS was combined with gefitinib. The subcutaneous tumor growth was also inhibited. Western blot showed that all the examined proteins decreased, except for 4E-BP1 and the active fragments of caspase 3 and caspase 9 increased. Epidermal growth factor receptor expression did not change significantly in quantitative real-time polymerase chain reaction. Cucurmosin can strongly inhibit the growth of PANC-1 cells in vitro and in vivo. Cucurmosin can down-regulate EGFR protein expression, but not at the messenger RNA level. Cucurmosin can also inhibit the ras/raf and phosphatidylinositol 3-kinase/Akt downstream signaling pathways and enhance the sensitivity of the EGFR-targeted drug gefitinib.

Src mediates cigarette smoke-induced resistance to tyrosine kinase inhibitors in NSCLC cells

PubMed Central

Filosto, Simone; Baston, David S.; Chung, Samuel; Becker, Cathleen R.; Goldkorn, Tzipora

2015-01-01

The EGF Receptor (EGFR) is a proto-oncogene commonly dysregulated in several cancers including non-small cell lung cancer (NSCLC) and, thus, is targeted for treatment using tyrosine kinase inhibitors (TKIs) such as Erlotinib. However, despite the efficacy observed in NSCLC patients harboring oncogenic variants of the EGFR, general ineffectiveness of TKIs in NSCLC patients who are current and former smokers necessitates identification of novel mechanisms to overcome this phenomenon. Previously, we showed that NSCLC cells harboring either wild-type (WT) EGFR or oncogenic mutant (MT) L858R EGFR become resistant to the effects of TKIs when exposed to cigarette smoke (CS), evidenced by their auto-phosphorylation and prolonged downstream signaling. Here, we present Src as a target mediating CS-induced resistance to TKIs in both WT EGFR and L858R MT EGFR expressing NSCLC cells. First, we show that CS exposure of A549 cells leads to time-dependent activation of Src which then abnormally binds to the WT EGFR causing TKI resistance, contrasting previous observations of constitutive binding between inactive Src and TKI-sensitive L858R MT EGFR. Next, we demonstrate that Src inhibition restores TKI sensitivity in CS-exposed NSCLC cells, preventing EGFR auto-phosphorylation in the presence of Erlotinib. Furthermore, we show that over-expression of a dominant-negative Src (Y527F/K295R) restores TKI sensitivity to A549 exposed to CS. Importantly, the TKI resistance that emerges even in CS-exposed L858R EGFR expressing NSCLC cells could be eliminated with Src inhibition. Together, these findings offer new rationale for using Src inhibitors for treating TKI-resistant NSCLC commonly observed in smokers. PMID:23686837

Epidermal growth factor receptor (EGFR) is overexpressed in high-grade dysplasia and adenocarcinoma of the esophagus and may represent a biomarker of histological progression in Barrett's esophagus (BE).

PubMed

Cronin, James; McAdam, Elizabeth; Danikas, Antonios; Tselepis, Chris; Griffiths, Paul; Baxter, John; Thomas, Linzi; Manson, James; Jenkins, Gareth

2011-01-01

The assessment of cancer risk in patients with Barrett's esophagus (BE) is currently fraught with difficulty. The current gold standard method of assessing cancer risk is histological assessment, with the appearance of high-grade dysplasia (HGD) as the key event monitored. Sampling error during endoscopy limits the usefulness of this approach, and there has been much recent interest in supplementing histological assessment with molecular markers, which may aid in patient stratification. No molecular marker has been yet validated to accurately correlate with esophageal histological progression. Here, we assessed the suitability of several membranous proteins as biomarkers by correlating their abundance with histological progression. In all, 107 patient samples, from 100 patients, were arranged on a tissue microarray (TMA) and represented the various stages of histological progression in BE. This TMA was probed with antibodies for eight receptor proteins (mostly membranous). Epidermal growth factor receptor (EGFR) staining was found to be the most promising biomarker identified with clear increases in staining accompanying histological progression. Further, immunohistochemistry was performed using the full-tissue sections from BE, HGD, and adenocarcinoma tissues, which confirmed the stepwise increase in EGFR abundance. Using a robust H-score analysis, EGFR abundance was shown to increase 13-fold in the adenocarcinoma tissues compared to the BE tissues. EGFR was "overexpressed" in 35% of HGD specimens and 80% of adenocarcinoma specimens when using the H-score of the BE patients (plus 3 s.d.) as the threshold to define overexpression. EGFR staining was also noted to be higher in BE tissues adjacent to HGD/adenocarcinoma. Western blotting, although showing more EGFR protein in the adenocarcinomas compared to the BE tissue, was highly variable. EGFR overexpression was accompanied by aneuploidy (gain) of chromosome 7, plus amplification of the EGFR locus. Finally, the bile acid deoxycholic acid (DCA) (at neutral and acidic pH) and acid alone was capable of upregulating EGFR mRNA in vitro, and in the case of neutral pH DCA, this was NF-κB dependent. EGFR is overexpressed during the histological progression in BE tissues and hence may be useful as a biomarker of histological progression. Furthermore, as EGFR is a membranous protein expressed on the luminal surface of the esophageal mucosa, it may also be a useful target for biopsy guidance during endoscopy.

Effects of miR‑138‑5p and miR‑204‑5p on the migration and proliferation of gastric cancer cells by targeting EGFR.

PubMed

Wang, Yi; Zhang, Haiyang; Ge, Shaohua; Fan, Qian; Zhou, Likun; Li, Hongli; Bai, Ming; Ning, Tao; Liu, Rui; Wang, Xia; Deng, Ting; Zhang, Le; Ying, Guoguang; Ba, Yi

2018-06-01

GC (gastric cancer) remains one of the most lethal malignancies worldwide. EGFR (epidermal growth factor receptor) plays an important role in the malignant process of GC, therefore, the present study addressed the relationship between EGFR and its potential regulators and examined their regulatory mechanisms in GC. We examined differences in the expression levels of EGFR in GC and adjacent non‑cancerous tissues. Bioinformatics analyses and dual luciferase reporter assays were used to confirm the putative relationship between miR‑138 or miR‑204 and EGFR, and their relationship was further detected using western blotting, RT‑PCR, and a series of cell studies. EGFR proteins were abundantly expressed in GC tissues, however EGFR mRNA levels remained indistinctive. Consequently, EGFR was revealed as a putative target of miR‑138 and miR‑204 which bound to the 3'UTR of EGFR mRNA. Further analysis revealed that miR‑138 and miR‑204 were significantly downregulated in GC tissues and the overexpression of miR‑138 and miR‑204 in GC cell lines resulted in the significant inhibition of EGFR protein levels and GC cell proliferation and metastasis. Rescue experiments confirmed that the roles of the two microRNAs were specific to EGFR. EGFR is a pivotal oncogene in GC progression that may be regulated by miR‑138 and miR‑204.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma

NASA Astrophysics Data System (ADS)

Sun, Chong; Wang, Liqin; Huang, Sidong; Heynen, Guus J. J. E.; Prahallad, Anirudh; Robert, Caroline; Haanen, John; Blank, Christian; Wesseling, Jelle; Willems, Stefan M.; Zecchin, Davide; Hobor, Sebastijan; Bajpe, Prashanth K.; Lieftink, Cor; Mateus, Christina; Vagner, Stephan; Grernrum, Wipawadee; Hofland, Ingrid; Schlicker, Andreas; Wessels, Lodewyk F. A.; Beijersbergen, Roderick L.; Bardelli, Alberto; di Nicolantonio, Federica; Eggermont, Alexander M. M.; Bernards, Rene

2014-04-01

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a `drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Randomized, placebo-controlled window trial of EGFR, Src, or combined blockade in head and neck cancer.

PubMed

Bauman, Julie E; Duvvuri, Umamaheswar; Gooding, William E; Rath, Tanya J; Gross, Neil D; Song, John; Jimeno, Antonio; Yarbrough, Wendell G; Johnson, Faye M; Wang, Lin; Chiosea, Simion; Sen, Malabika; Kass, Jason; Johnson, Jonas T; Ferris, Robert L; Kim, Seungwon; Hirsch, Fred R; Ellison, Kimberly; Flaherty, John T; Mills, Gordon B; Grandis, Jennifer R

2017-03-23

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II-IVa HNSCC were randomized to 7-21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms ( P = 0.0014); however, no effect was seen with dasatinib alone ( P = 0.24). High baseline pMAPK expression was associated with response to erlotinib ( P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib ( P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

Randomized, placebo-controlled window trial of EGFR, Src, or combined blockade in head and neck cancer

PubMed Central

Bauman, Julie E.; Duvvuri, Umamaheswar; Gooding, William E.; Rath, Tanya J.; Gross, Neil D.; Song, John; Jimeno, Antonio; Yarbrough, Wendell G.; Johnson, Faye M.; Wang, Lin; Chiosea, Simion; Sen, Malabika; Kass, Jason; Johnson, Jonas T.; Ferris, Robert L.; Kim, Seungwon; Hirsch, Fred R.; Ellison, Kimberly; Flaherty, John T.; Mills, Gordon B.

2017-01-01

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II–IVa HNSCC were randomized to 7–21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms (P = 0.0014); however, no effect was seen with dasatinib alone (P = 0.24). High baseline pMAPK expression was associated with response to erlotinib (P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib (P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma. PMID:28352657

STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients

PubMed Central

Srivastava, Raghvendra M.; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-bae, Jie; Wang, Lin; Seethala, Raja R.; Branstetter, Barton F.; Ferrone, Soldano; Ferris, Robert L.

2015-01-01

The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was utilized to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. PMID:25972070

The Epidermal Growth Factor Receptor Critically Regulates Endometrial Function during Early Pregnancy

PubMed Central

Large, Michael J.; Wetendorf, Margeaux; Lanz, Rainer B.; Hartig, Sean M.; Creighton, Chad J.; Mancini, Michael A.; Kovanci, Ertug; Lee, Kuo-Fen; Threadgill, David W.; Lydon, John P.; Jeong, Jae-Wook; DeMayo, Francesco J.

2014-01-01

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets. PMID:24945252

EGFR-Targeted Adenovirus Dendrimer Coating for Improved Systemic Delivery of the Theranostic NIS Gene

PubMed Central

Grünwald, Geoffrey K; Vetter, Alexandra; Klutz, Kathrin; Willhauck, Michael J; Schwenk, Nathalie; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Zach, Christian; Wagner, Ernst; Göke, Burkhard; Holm, Per S; Ogris, Manfred; Spitzweg, Christine

2013-01-01

We recently demonstrated tumor-selective iodide uptake and therapeutic efficacy of combined radiovirotherapy after systemic delivery of the theranostic sodium iodide symporter (NIS) gene using a dendrimer-coated adenovirus. To further improve shielding and targeting we physically coated replication-selective adenoviruses carrying the hNIS gene with a conjugate consisting of cationic poly(amidoamine) (PAMAM) dendrimer linked to the peptidic, epidermal growth factor receptor (EGFR)-specific ligand GE11. In vitro experiments demonstrated coxsackie-adenovirus receptor-independent but EGFR-specific transduction efficiency. Systemic injection of the uncoated adenovirus in a liver cancer xenograft mouse model led to high levels of NIS expression in the liver due to hepatic sequestration, which were significantly reduced after coating as demonstrated by 123I-scintigraphy. Reduction of adenovirus liver pooling resulted in decreased hepatotoxicity and increased transduction efficiency in peripheral xenograft tumors. 124I-PET-imaging confirmed EGFR-specificity by significantly lower tumoral radioiodine accumulation after pretreatment with the EGFR-specific antibody cetuximab. A significantly enhanced oncolytic effect was observed following systemic application of dendrimer-coated adenovirus that was further increased by additional treatment with a therapeutic dose of 131I. These results demonstrate restricted virus tropism and tumor-selective retargeting after systemic application of coated, EGFR-targeted adenoviruses therefore representing a promising strategy for improved systemic adenoviral NIS gene therapy. PMID:24193032

Cadmium promotes the proliferation of triple-negative breast cancer cells through EGFR-mediated cell cycle regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Zhengxi, E-mail: weizhengxi@gmail.com; Song, Xiulong; Shaikh, Zahir A., E-mail: zshaikh@uri.edu

Cadmium (Cd) is a carcinogenic metal which is implicated in breast cancer by epidemiological studies. It is reported to promote breast cancer cell growth in vitro through membrane receptors. The study described here examined Cd-mediated growth of non-metastatic human breast cancer derived cells that lack receptors for estrogen, progesterone, and HER2. Treatment of triple-negative HCC 1937 cells with 0.1–0.5 μM Cd increased cell growth by activation of AKT and ERK. Accelerated cell cycle progression was achieved by increasing the levels of cyclins A, B, and E, as well as those of CDKs 1 and 2. Although triple negative cells lackmore » estrogen receptor, they express high levels of EGFR. Therefore, further studies on HCC 1937 and another triple-negative cell line, HCC 38, were conducted using specific siRNA and an inhibitor of EGFR to determine whether EGFR was responsible for mediating the effect of Cd. The results revealed that in both cell types EGFR was not only activated upon Cd treatment, but was also essential for the downstream activation of AKT and ERK. Based on these observations, it is concluded that, in breast cancer cells lacking estrogen receptor, sub-micromolar concentration of Cd can promote cell proliferation. Furthermore, that EGFR plays a critical role in this process. - Highlights: • Sub-micromolar concentrations of Cd promote cell growth in breast cancer cells that lack ER, PR, and HER2. • The increase in cell number is not due to reduction in apoptosis. • Growth promotion involves AKT and ERK signaling and downstream stimulation of cell cycle progression. • Initiation of cell growth by Cd occurs at the cell membrane and requires the activation of EGFR.« less

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers

PubMed Central

Essenburg, Curt J.; Turner, Lisa; Madaj, Zachary; Winn, Mary E.; Melnik, Marianne K.; Korkaya, Hasan; Maroun, Christiane R.; Christensen, James G.; Steensma, Matthew R.; Boerner, Julie L.; Graveel, Carrie R.

2016-01-01

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients. PMID:27655711

Spatial localization of EGF family ligands and receptors in the zebrafish ovarian follicle and their expression profiles during folliculogenesis.

PubMed

Tse, Anna Chung-Kwan; Ge, Wei

2010-07-01

The roles of epidermal growth factor (EGF) family in the ovary have received increasing attention recently. Despite this, the production sites of EGF family members in the ovarian follicle still remain controversial. Using zebrafish as the model, the present study investigated spatial distribution of several EGF family ligands and receptors in the follicle as well as their temporal expression profiles during folliculogenesis. RT-PCR analysis on the somatic follicle layer and oocyte revealed that all EGF family ligands examined (egf, tgfa, btc and hbegf) were mostly or exclusively expressed in the oocyte. In contrast, their common receptor (egfr) was expressed exclusively in the follicle layer. By comparison, members of activin family showed an opposite pattern of distribution. Activin subunits (inhbaa and inhbb) were both expressed exclusively in the follicle layer whereas activin receptors and follistatin were abundantly present in the oocyte. During folliculogenesis, egf, tgfa and hbegf increased their expression together with egfr in the fast secondary growth phase. The developmental profiles of EGF family during embryogenesis appeared to argue for an important role for EGF family in folliculogenesis rather than embryogenesis as maternal molecules. The present study provided clear evidence for the existence of two paracrine pathways in the follicle, the oocyte-derived EGF family ligands and follicle cell-derived activins, which may mediate oocyte-to-follicle cell and follicle cell-to-oocyte communications, respectively. The functional relationship between these two signaling systems in the follicle is suggested by the observation that all four EGFR ligands examined significantly stimulated activin subunit expression in cultured follicle cells. (c) 2009 Elsevier Inc. All rights reserved.

G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells.

PubMed

Vivacqua, Adele; Lappano, Rosamaria; De Marco, Paola; Sisci, Diego; Aquila, Saveria; De Amicis, Francesca; Fuqua, Suzanne A W; Andò, Sebastiano; Maggiolini, Marcello

2009-11-01

In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer

PubMed Central

Albitar, Maher; Sudarsanam, Sucha; Ma, Wanlong; Jiang, Shiping; Chen, Wayne; Funari, Vincent; Blocker, Forrest; Agersborg, Sally

2018-01-01

Background The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer. Methods In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing. Results Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. Conclusions This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification. PMID:29568386

Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism

PubMed Central

Hamdi, M M; Mutungi, G

2011-01-01

Abstract Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression. PMID:21606113

Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rastogi, Ichwaku; Rajanna, Supriya; Webb, Andrew

According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) inmore » the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, β-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of β-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or β-Catenin siRNA can increase drug sensitivity of TKI-resistant cells. - Highlights: • Resistance to TKIs in NSCLC cells is mediated via modulation in EMT related proteins. • EMT may induce c-Met mediated TKI resistance, similar to EGFR TKI resistance. • Role of β-catenin and cadherins in TKI resistance was validated by FACS and qPCR. • Knockdown of β-catenin or Zeb-1 can increase TKI sensitivity in TKI-resistant cells. • Targeting key EMT related proteins may overcome TKI resistance in NSCLC.« less

Effect of non-thermal air atmospheric pressure plasma jet treatment on gingival wound healing

NASA Astrophysics Data System (ADS)

Lee, Jung-Hwan; Choi, Eun-Ha; Kim, Kwang-Mahn; Kim, Kyoung-Nam

2016-02-01

Non-thermal atmospheric pressure plasmas have been applied in the biomedical field for the improvement of various cellular activities. In dentistry, the healing of gingival soft tissue plays an important role in health and aesthetic outcomes. While the biomedical application of plasma has been thoroughly studied in dentistry, a detailed investigation of plasma-mediated human gingival fibroblast (HGF) migration for wound healing and its underlying biological mechanism is still pending. Therefore, the aim of this study is to apply a non-thermal air atmospheric pressure plasma jet (NTAAPPJ) to HGF to measure the migration and to reveal the underlying biological mechanisms involved in the migration. After the characterization of NTAAPPJ by optical emission spectroscopy, the adherent HGF was treated with NTAAPPJ or air with a different flow rate. Cell viability, lipid peroxidation, migration, intracellular reactive oxygen species (ROS), and the expression of migration-related genes (EGFR, PAK1, and MAPK3) were investigated. The level of statistical significance was set at 0.05. NTAAPPJ and air treatment with a flow rate of 250-1000 standard cubic centimetres per minute (sccm) for up to 30 s did not induce significant decreases in cell viability or membrane damage. A significant increase in the migration of mitomycin C-treated HGF was observed after 30 s of NTAAPPJ treatment compared to 30 s air-only treatment, which was induced by high levels of intracellular reactive oxygen species (ROS). An increase in migration-related gene expression and EGFR activation was observed following NTAAPPJ treatment in an air flow rate-dependent manner. This is the first report that NTAAPPJ treatment induces an increase in HGF migration without changing cell viability or causing membrane damage. HGF migration was related to an increase in intracellular ROS, changes in the expression of three of the migration-related genes (EGFR, PAK1, and MAPK1), and EGFR activation. Therefore, NTAAPPJ for gingival tissue healing is a promising method for health and aesthetic outcomes.

EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis.

PubMed

Gainor, Justin F; Shaw, Alice T; Sequist, Lecia V; Fu, Xiujun; Azzoli, Christopher G; Piotrowska, Zofia; Huynh, Tiffany G; Zhao, Ling; Fulton, Linnea; Schultz, Katherine R; Howe, Emily; Farago, Anna F; Sullivan, Ryan J; Stone, James R; Digumarthy, Subba; Moran, Teresa; Hata, Aaron N; Yagi, Yukako; Yeap, Beow Y; Engelman, Jeffrey A; Mino-Kenudson, Mari

2016-09-15

PD-1 inhibitors are established agents in the management of non-small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, anaplastic lymphoma kinase (ALK)-positive, and EGFR wild-type/ALK-negative patients. We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORR) were assessed using RECIST v1.1. PD-L1 expression and CD8(+) tumor-infiltrating lymphocytes (TIL) were evaluated by IHC. Objective responses were observed in 1 of 28 (3.6%) EGFR-mutant or ALK-positive patients versus 7 of 30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced EGFR-mutant (N = 68) and ALK-positive (N = 27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre-tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5%, and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI-resistant biopsies (N = 57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8(+) TILs (grade ≥2) were observed in only 1 pretreatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens and was not observed in any ALK-positive, pre- or post-TKI specimens. NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8(+) TILs within the tumor microenvironment may underlie these clinical observations. Clin Cancer Res; 22(18); 4585-93. ©2016 AACRSee related commentary by Gettinger and Politi, p. 4539. ©2016 American Association for Cancer Research.

Decreased GFR estimated by MDRD or Cockcroft-Gault equation predicts incident CVD: the strong heart study.

PubMed

Shara, Nawar M; Resnick, Helaine E; Lu, Li; Xu, Jiaqiong; Vupputuri, Suma; Howard, Barbara V; Umans, Jason G

2009-01-01

Kidney function, expressed as glomerular filtration rate (GFR), is commonly estimated from serum creatinine (Scr) and, when decreased, may serve as a nonclassical risk factor for incident cardiovascular disease (CVD). The ability of estimated GFR (eGFR) to predict CVD events during 5-10 years of follow-up is assessed using data from the Strong Heart Study (SHS), a large cohort with a high prevalence of diabetes. eGFRs were calculated with the abbreviated Modification of Diet in Renal Disease study (MDRD) and the Cockcroft-Gault (CG) equations. These estimates were compared in participants with normal and abnormal Scr. The association between eGFR and incident CVD was assessed. More subjects were labeled as having low eGFR (<60 ml/min per 1.73 m2) by the MDRD or CG equation, than by Scr alone. When Scr was in the normal range, both equations labeled similar numbers of participants as having low eGFRs, although concordance between the equations was poor. However, when Scr was elevated, the MDRD equation labeled more subjects as having low eGFR. Persons with low eGFR had increased risk of CVD. The MDRD and CG equations labeled more participants as having decreased GFR than did Scr alone. Decreased eGFR was predictive of CVD in this American Indian population with a high prevalence of obesity and type 2 diabetes mellitus.

Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

PubMed Central

Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

2008-01-01

Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B expression, but not EGFR expression, was elevated in HCEC from older donors, suggesting that the reduced proliferative activity of these cells in response to EGF is due, at least in part, to increased PTP1B activity. The fact that inhibition of PTP1B increased the relative number of cells entering S-phase strongly suggests that PTP1B helps negatively regulate EGF-stimulated cell cycle entry in HCEC. These results also suggest that it may be possible to increase the proliferative activity of HCEC, particularly in cells from older donors, by inhibiting the activity of this important protein tyrosine phosphatase. PMID:18253097

Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

PubMed

Wei, Hong; Zhu, Zijie; Lu, Longtao

2017-01-01

Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Hyeon-Ok; Hong, Sung-Eun; Kim, Chang Soon

2015-08-15

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drugmore » alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.« less

Laminin-5γ-2 (LAMC2) Is Highly Expressed in Anaplastic Thyroid Carcinoma and Is Associated With Tumor Progression, Migration, and Invasion by Modulating Signaling of EGFR

PubMed Central

Kanojia, Deepika; Okamoto, Ryoko; Jain, Saket; Madan, Vikas; Chien, Wenwen; Sampath, Abhishek; Ding, Ling-Wen; Xuan, Meng; Said, Jonathan W.; Doan, Ngan B.; Liu, Li-Zhen; Yang, Henry; Gery, Sigal; Braunstein, Glenn D.; Koeffler, H. Phillip

2014-01-01

Context: Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy having no effective treatment. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein involved in cell migration and tumor invasion and might represent an ideal target for the development of novel therapeutic approaches for ATC. Objective: The objective of the investigation was to study the role of LAMC2 in ATC tumorigenesis. Design: LAMC2 expression was evaluated by RT-PCR, Western blotting, and immunohistochemistry in tumor specimens, adjacent noncancerous tissues, and cell lines. The short hairpin RNA (shRNA) approach was used to investigate the effect of LAMC2 knockdown on the tumorigenesis of ATC. Results: LAMC2 was highly expressed in ATC samples and cell lines compared with normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed the migration, invasion, and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered the expression of genes associated with migration, invasion, proliferation, and survival. Immunoprecipitation studies showed that LAMC2 bound to epidermal growth factor receptor (EGFR) in the ATC cells. Silencing LAMC2 partially blocked epidermal growth factor-mediated activation of EGFR and its downstream pathway. Interestingly, cetuximab (an EGFR blocking antibody) or EGFR small interfering RNA additively enhanced the antiproliferative activity of the LAMC2 knockdown ATC cells compared with the control cells. Conclusions: To our knowledge, this is the first report investigating the effect of LAMC2 on cell growth, cell cycle, migration, invasion, and EGFR signaling in ATC cells, suggesting that LAMC2 may be a potential therapeutic target for the treatment of ATC. PMID:24170107

Spectrum of somatic EGFR, KRAS, BRAF, PTEN mutations and TTF-1 expression in Brazilian lung cancer patients.

PubMed

Carneiro, Juliana G; Couto, Patricia G; Bastos-Rodrigues, Luciana; Bicalho, Maria Aparecida C; Vidigal, Paula V; Vilhena, Alyne; Amaral, Nilson F; Bale, Allen E; Friedman, Eitan; De Marco, Luiz

2014-01-01

Lung cancer is the leading global cause of cancer-related mortality. Inter-individual variability in treatment response and prognosis has been associated with genetic polymorphisms in specific genes: EGFR, KRAS, BRAF, PTEN and TTF-1. Somatic mutations in EGFR and KRAS genes are reported at rates of 15-40% in non-small cell lung cancer (NSCLC) in ethnically diverse populations. BRAF and PTEN are commonly mutated genes in various cancer types, including NSCLC, with PTEN mutations exerting an effect on the therapeutic response of EGFR/AKT/PI3K pathway inhibitors. TTF-1 is expressed in approximately 80% of lung adenocarcinomas and its positivity correlates with higher prevalence of EGFR mutation in this cancer type. To determine molecular markers for lung cancer in Brazilian patients, the rate of the predominant EGFR, KRAS, BRAF and PTEN mutations, as well as TTF-1 expression, was assessed in 88 Brazilian NSCLC patients. EGFR exon 19 deletions (del746-750) were detected in 3/88 (3·4%) patients. Activating KRAS mutations in codons 12 and 61 were noted in five (5·7%) and two (2·3%) patients, respectively. None of the common somatic mutations were detected in either the BRAF or PTEN genes. TTF-1 was overexpressed in 40·7% of squamous-cell carcinoma (SCC). Our findings add to a growing body of data that highlights the genetic heterogeneity of the abnormal EGFR pathway in lung cancer among ethnically diverse populations.

Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

PubMed

Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

2014-07-01

The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma

PubMed Central

Ma, Yufang; Tang, Nan; Thompson, Reid; Mobley, Bret C.; Clark, Steven W.; Sarkaria, Jann N.; Wang, Jialiang

2015-01-01

Purpose Aberrant activation of epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR inhibitors exhibit at best modest efficacy in glioblastoma. This is in sharp contrast to the observations in EGFR-mutant lung cancer. We examined whether activation of functionally redundant receptor tyrosine kinases (RTKs) conferred resistance to EGFR inhibitors in glioblastoma. Experimental Design We collected a panel of patient-derived glioblastoma xenograft (PDX) lines that maintained expression of wild type or mutant EGFR in serial xenotransplantation and tissue cultures. Using this physiologically relevant platform, we tested the abilities of several RTK ligands to protect glioblastoma cells against an EGFR inhibitor, gefitinib. Based on the screening results, we further developed a combination therapy co-targeting EGFR and insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF1R). Results Insulin and IGF1 induced significant protection against gefitinib in the majority of EGFR-dependent PDX lines with one exception that did not expression InsR or IGF1R. Blockade of the InsR/IGF1R pathway synergistically improved sensitivity to gefitinib or dacomitinib. Gefitinib alone effectively attenuated EGFR activities and the downstream MEK/ERK pathway. However, repression of AKT and induction of apoptosis required concurrent inhibition of both EGFR and InsR/IGF1R. A combination of gefitinib and OSI-906, a dual InsR/IGF1R inhibitor, was more effective than either agent alone to treat subcutaneous glioblastoma xenograft tumors. Conclusions Our results suggest that activation of the InsR/IGF1R pathway confers resistance to EGFR inhibitors in EGFR-dependent glioblastoma through AKT regulation. Concurrent blockade of these two pathways holds promise to treat EGFR-dependent glioblastoma. PMID:26561558

Effect of verteporfin-PDT on epithelial growth factor receptor (EGFR) signaling pathway in cholangiocarcinoma cell lines

NASA Astrophysics Data System (ADS)

Andreola, Fausto; Cerec, Virginie; Pereira, Stephen P.

2009-06-01

EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional chemo/radiotherapy have been reported in preclinical studies for different cancers but not cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii) response to combination of PDT and EGFR-targeting agents. We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic, respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor (Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA damage.

Intracellular Insulin-like Growth Factor-I Induces Bcl-2 Expression in Airway Epithelial Cells 1

PubMed Central

Chand, Hitendra S.; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S.; Randell, Scott H.; Tesfaigzi, Yohannes

2012-01-01

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and IGF-1 coincided with induced Bcl-2 expression compared to controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using shRNA showed that intracellular (IC)-IGF-1 was increasing Bcl-2 expression. Blocking EGFR or IGF-1R activation also suppressed IC-IGF-1, and abolished the Bcl-2 induction. Induced expression and co-localization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and EGFR pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

Mitogen-Inducible Gene-6 Mediates Feedback Inhibition from Mutated BRAF towards the Epidermal Growth Factor Receptor and Thereby Limits Malignant Transformation

PubMed Central

Milewska, Malgorzata; Romano, David; Herrero, Ana; Guerriero, Maria Luisa; Birtwistle, Marc; Quehenberger, Franz; Hatzl, Stefan; Kholodenko, Boris N.; Segatto, Oreste; Kolch, Walter; Zebisch, Armin

2015-01-01

BRAF functions in the RAS-extracellular signal-regulated kinase (ERK) signaling cascade. Activation of this pathway is necessary to mediate the transforming potential of oncogenic BRAF, however, it may also cause a negative feedback that inhibits the epidermal growth factor receptor (EGFR). Mitogen-inducible gene-6 (MIG-6) is a potent inhibitor of the EGFR and has been demonstrated to function as a tumor suppressor. As MIG-6 can be induced via RAS-ERK signaling, we investigated its potential involvement in this negative regulatory loop. Focus formation assays were performed and demonstrated that MIG-6 significantly reduces malignant transformation induced by oncogenic BRAF. Although this genetic interaction was mirrored by a physical interaction between MIG-6 and BRAF, we did not observe a direct regulation of BRAF kinase activity by MIG-6. Interestingly, a selective chemical EGFR inhibitor suppressed transformation to a similar degree as MIG-6, whereas combining these approaches had no synergistic effect. By analyzing a range of BRAF mutated and wildtype cell line models, we could show that BRAF V600E causes a strong upregulation of MIG-6, which was mediated at the transcriptional level via the RAS-ERK pathway and resulted in downregulation of EGFR activation. This feedback loop is operational in tumors, as shown by the analysis of almost 400 patients with papillary thyroid cancer (PTC). Presence of BRAF V600E correlated with increased MIG-6 expression on the one hand, and with inactivation of the EGFR and of PI3K/AKT signaling on the other hand. Importantly, we also observed a more aggressive disease phenotype when BRAF V600E coexisted with low MIG-6 expression. Finally, analysis of methylation data was performed and revealed that higher methylation of MIG-6 correlated to its decreased expression. Taken together, we demonstrate that MIG-6 efficiently reduces cellular transformation driven by oncogenic BRAF by orchestrating a negative feedback circuit directed towards the EGFR. PMID:26065894

EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus.

PubMed

Birkman, Eva-Maria; Ålgars, Annika; Lintunen, Minnamaija; Ristamäki, Raija; Sundström, Jari; Carpén, Olli

2016-07-07

Approximately 50 % of gastric adenocarcinomas belong to a molecular subgroup characterised by chromosomal instability and a strong association with the intestinal histological subtype. This subgroup typically contains alterations in the receptor tyrosine kinase-RAS pathway, for example EGFR or HER2 gene amplifications leading to protein overexpression. In clinical practice, HER2 overexpressing metastatic gastric cancer is known to respond to treatment with anti-HER2 antibodies. By contrast, anti-EGFR antibodies have not been able to provide survival benefit in clinical trials, which, however, have not included patient selection based on the histological subtype or EGFR gene copy number analysis of the tumours. To examine the role of EGFR as a potential biomarker, we studied the prevalence, clinicopathological associations as well as prognostic role of EGFR and HER2 expression and gene amplification in intestinal adenocarcinomas of the stomach, gastro-oesophageal junction and distal oesophagus. Tissue samples from 220 patients were analysed with EGFR and HER2 immunohistochemistry. Those samples with moderate/strong staining intensity were further analysed with silver in situ hybridization to quantify gene copy numbers. The results were associated with clinical patient characteristics and survival. Moderate/strong EGFR protein expression was found in 72/220 (32.7 %) and EGFR gene amplification in 31/220 (14.1 %) of the tumours, while moderate/strong HER2 protein expression was detected in 31/220 (14.1 %) and HER2 gene amplification in 29/220 (13.2 %) of the tumours. EGFR and HER2 genes were co-amplified in eight tumours (3.6 %). EGFR gene amplification was more common in tumours of distal oesophagus/gastro-oesophageal junction/cardia than in those of gastric corpus (p = 0.013). It was associated with shortened time to cancer recurrence (p = 0.026) and cancer specific survival (p = 0.033). EGFR gene amplification is relatively common in intestinal adenocarcinomas and associates with decreased survival. It is rarely concurrent with HER2 gene amplification, suggesting that anti-EGFR therapies might be applicable to some patients not eligible for anti-HER2 treatment. Analogous to HER2 testing, determination of EGFR gene amplification status in concert with immunohistochemistry could improve the specificity of patient selection when investigating the possible benefits of anti-EGFR therapies in the treatment of gastric adenocarcinomas.

Gefitinib targets EGFR dimerization and ERK1/2 phosphorylation to inhibit pleural mesothelioma cell proliferation.

PubMed

Favoni, Roberto E; Pattarozzi, Alessandra; Lo Casto, Michele; Barbieri, Federica; Gatti, Monica; Paleari, Laura; Bajetto, Adriana; Porcile, Carola; Gaudino, Giovanni; Mutti, Luciano; Corte, Giorgio; Florio, Tullio

2010-03-01

Altered EGFR activity is a causal factor for human tumor development, including malignant pleural mesotheliomas. The aim of the present study was the evaluation of the effects of Gefitinib on EGF-induced mesothelioma cell proliferation and the intracellular mechanisms involved. Cell proliferation, DNA synthesis and apoptosis were measured by MTT, thymidine incorporation and FACS analysis; EGFR, ERK1/2 and Akt expression and phosphorylation by Western blot, whereas receptor sites were analyzed by binding studies. Gefitinib inhibited EGF-induced proliferation in two mesothelioma cell lines, derived from pleural effusion (IST-Mes2) or tumor biopsy (ZL55). The treatment with Gefitinib induced cell cycle arrest in both cell lines, while apoptosis was observed only for high concentrations and prolonged drug exposure. EGF-dependent mesothelioma cell proliferation was mediated by EGFR and ERK1/2 phosphorylation, while Akt was not affected. Gefitinib inhibited both EGFR and ERK1/2 activation, being maximal at drug concentrations that induce cytostatic effects, suggesting that the proapoptotic activity of Gefitinib is independent from EGFR inhibition. Gefitinib treatment increased EGFR Bmax, possibly through membrane stabilization of inactive receptor dimers that we show to be induced by the drug also in the absence of EGF. EGFR activation of ERK1/2 represents a key pathway for pleural mesothelioma cell proliferation. Low concentrations of Gefitinib cause mesothelioma cell cycle arrest through the blockade of EGFR activity while high concentrations induce apoptosis. Finally, we propose that the formation of inactive EGFR dimers may contribute to the antitumoral activity of Gefitinib.

Tumour gene expression predicts response to cetuximab in patients with KRAS wild-type metastatic colorectal cancer.

PubMed

Baker, J B; Dutta, D; Watson, D; Maddala, T; Munneke, B M; Shak, S; Rowinsky, E K; Xu, L-A; Harbison, C T; Clark, E A; Mauro, D J; Khambata-Ford, S

2011-02-01

Although it is accepted that metastatic colorectal cancers (mCRCs) that carry activating mutations in KRAS are unresponsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, a significant fraction of KRAS wild-type (wt) mCRCs are also unresponsive to anti-EGFR therapy. Genes encoding EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are promising gene expression-based markers but have not been incorporated into a test to dichotomise KRAS wt mCRC patients with respect to sensitivity to anti-EGFR treatment. We used RT-PCR to test 110 candidate gene expression markers in primary tumours from 144 KRAS wt mCRC patients who received monotherapy with the anti-EGFR antibody cetuximab. Results were correlated with multiple clinical endpoints: disease control, objective response, and progression-free survival (PFS). Expression of many of the tested candidate genes, including EREG and AREG, strongly associate with all clinical endpoints. Using multivariate analysis with two-layer five-fold cross-validation, we constructed a four-gene predictive classifier. Strikingly, patients below the classifier cutpoint had PFS and disease control rates similar to those of patients with KRAS mutant mCRC. Gene expression appears to identify KRAS wt mCRC patients who receive little benefit from cetuximab. It will be important to test this model in an independent validation study.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weng, M.-S.; Ho, C.-T.; Ho, Y.-S.

Fatty acid synthase (FAS) is a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids. Most breast cancers require lipogenesis for growth. Here, we demonstrated the effects of theanaphthoquinone (TNQ), a member of the thearubigins generated by the oxidation of theaflavin (TF-1), on the expression of FAS in human breast cancer cells. TNQ was found to suppress the EGF-induced expression of FAS mRNA and FAS protein in MDA-MB-231 cells. Expression of FAS has previously been shown to be regulated by the SREBP family of transcription factors. In this study, we demonstrated that the EGF-induced nuclear translocation of SREBP-1more » was blocked by TNQ. Moreover, TNQ also modulated EGF-induced ERK1/2 and Akt phosphorylation. Treatment of MDA-MB-231 cells with PI 3-kinase inhibitors, LY294002 and Wortmannin, inhibited the EGF-induced expression of FAS and nuclear translocation of SREBP-1. Treatment with TNQ inhibited EGF-induced EGFR/ErbB-2 phosphorylation and dimerization. Furthermore, treatment with kinase inhibitors of EGFR and ErbB-2 suggested that EGFR/ErbB-2 activation was involved in EGF-induced FAS expression. In constitutive FAS expression, TNQ inhibited FAS expression and Akt autophosphorylation in BT-474 cells. The PI 3-kinase inhibitors and tyrosine kinase inhibitors of EGFR and ErbB-2 also reduced constitutive FAS expression. In addition, pharmacological blockade of FAS by TNQ decreased cell viability and induced cell death in BT-474 cells. In summary, our findings suggest that TNQ modulates FAS expression by the regulation of EGFR/ErbB-2 pathways and induces cell death in breast cancer cells.« less

Structural and energetic basis for the molecular recognition of dual synthetic vs. natural inhibitors of EGFR/HER2.

PubMed

Bello, Martiniano; Saldaña-Rivero, Lucia; Correa-Basurto, José; García, Benjamín; Sánchez-Espinosa, Victor Armando

2018-05-01

Activation of EGFR starts by ligand binding at the extracellular domain which results in homo and heterodimerization, leading to phosphorylation, activation of downstream signaling pathways which upregulate expression of genes, proliferation and angiogenesis. Abnormalities in the expression of EGFR play a critical role in the development of different types of cancer. HER2 is the preferred heterodimerization partner for EGFR; this biological characteristic together with the high percentage of structural homology has been exploited in the design of dual synthetic inhibitors against EGFR/HER2. Herein we combined structural data and molecular dynamics (MD) simulations coupled to an MMGBSA approach to provide insight into the binding mechanism between two dual synthetics (lapatinib and TAK-285) and one dual natural inhibitor (EGCG) which target EGFR/HER2. In addition, we proposed some EGCG derivatives which were filtered through in silico screening. Structural analysis demonstrated that the coupling of synthetic, natural or newly designed compounds impacts the conformational space of EGFR and HER2 differently. Energetic analysis points out that lapatinib and TAK-285 have better affinity for inactive EGFR than the active EGFR state or HER2, whereas some EGCG derivatives seem to form binding affinities similar to those observed for lapatinib or TAK-285. Copyright © 2018 Elsevier B.V. All rights reserved.

EZH2 expression is a prognostic biomarker in patients with colorectal cancer treated with anti-EGFR therapeutics.

PubMed

Yamamoto, Itaru; Nosho, Katsuhiko; Kanno, Shinichi; Igarashi, Hisayoshi; Kurihara, Hiroyoshi; Ishigami, Keisuke; Ishiguro, Kazuya; Mitsuhashi, Kei; Maruyama, Reo; Koide, Hideyuki; Okuda, Hiroyuki; Hasegawa, Tadashi; Sukawa, Yasutaka; Okita, Kenji; Takemasa, Ichiro; Yamamoto, Hiroyuki; Shinomura, Yasuhisa; Nakase, Hiroshi

2017-03-14

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that suppresses microRNA-31 (miR-31) in various human malignancies including colorectal cancer. We recently suggested that miR-31 regulates the signaling pathway downstream of epidermal growth factor receptor (EGFR) in colorectal cancer. Therefore, we conducted this study for assessing the relationship between EZH2 expression and clinical outcomes in patients with colorectal cancer treated with anti-EGFR therapeutics. We immunohistochemically evaluated EZH2 expression and assessed miR-31 and gene mutations [KRAS (codon 61/146), NRAS (codon 12/13/61), and BRAF (codon 600)] in 109 patients with colorectal cancer harboring KRAS (codon 12/13) wild-type. We also evaluated the progression-free survival (PFS) and overall survival (OS). In the result, low EZH2 expression was significantly associated with shorter PFS (log-rank test: P = 0.023) and OS (P = 0.036) in patients with colorectal cancer. In the low-miR-31-expression group and the KRAS (codon 61/146), NRAS, and BRAF wild-type groups, a significantly shorter PFS (P = 0.022, P = 0.039, P = 0.021, and P = 0.036, respectively) was observed in the EZH2 low-expression groups than in the high-expression groups. In the multivariate analysis, low EZH2 expression was associated with a shorter PFS (P = 0.046), independent of the mutational status and miR-31. In conclusion, EZH2 expression was associated with survival in patients with colorectal cancer who were treated with anti-EGFR therapeutics. Moreover, low EZH2 expression was independently associated with shorter PFS in patients with cancer, suggesting that EZH2 expression is a useful additional prognostic biomarker for anti-EGFR therapy.

EGFR LI and Ki-67 LI are independent prognostic parameters influencing survivals of surgically treated squamous cell lung cancer patients.

PubMed

Niemiec, J; Kolodziejski, L; Dyczek, S

2005-01-01

In literature there are still opinion differences concerning the prognostic significance of epidermal growth factor receptor (EGFR) expression and proliferative potential in patients with non small cell lung cancer (NSCLC). This prompted us to study those parameters. The Ki-67 labeling index (Ki-67 LI), EGFR labeling index (EGFR LI), and mitotic index (MI) were analyzed in the group of 78 consecutive, surgically treated squamous cell lung cancer (SqCLC) patients. The expression of Ki-67 and EGFR protein was visualized on formalin fixed, paraffin embedded sections using immunohistochemistry (IHC). Mitotic index was assessed on formalin fixed, paraffin embedded sections, stained with hematoxylin and eosin using morphological criteria. Mean values of Ki-67 LI and MI were higher for G2+G3 tumors than for G1 tumors. EGFR LI was higher for G1+G2 than for G3 tumors, and for pT3 than for pT1+pT2 tumors. Patients having tumors with Ki-67 < or =28% or (EGFR LI < or =13% or EGFR LI >80%) survived significantly shorter than those having tumors with Ki-67 LI >28% or 13%< EGFR LI < or =80%. In multivariate analysis, 13%> or = EGFR LI <80% and Ki-67 LI < or =28% were independent negative prognostic parameters influencing survivals of SqCLC patients.

Optimization of genetics to create therapies for metastatic (stage IV) non-small-cell lung cancer.

PubMed

Rosell, Rafael; Moran, Teresa; Viteri, Santiago; Carcereny, Enric; Gasco, Amaya; Quiroga, Vanessa; Wei, Jia; Camps, Carlos; Massuti, Bartomeu

2010-07-01

Non-small-cell lung cancer (NSCLC) is a disseminated disease in 50% of cases, with a gloomy prognosis and median survivals of < 1 year. Based on substantial advances, cancer biology insights and novel biotechnology tools, customized treatment provides hints that cisplatin-based treatment can be optimized in favorable subgroups of patients according to gene expression DNA repair profiles. In 2004, it was discovered that 10-15% of NSCLC can harbor a new class of EGFR mutation conferring specific sensitivity to EGFR tyrosine kinase inhibitors. The homologous recombination pathway provides information for customizing cisplatin-based chemotherapy. BRCA1 plays a central role in this pathway that can be used in tailoring chemotherapy. Patient subgroups can obtain significant increases in progression-free survival. For EGFR lung-addicted cancers, treatment with EGFR tyrosine kinase inhibitors like erlotinib provide impressive improvement in progression-free survival--up to 14 months with significant enhanced survival. Customized chemotherapy based on BRCA1 models can contribute to demonstrating this approach's clinical relevance, and the implementation of EGFR mutation assessment is warranted to identify EGFR-addicted lung cancers with a different prognosis that could benefit from a specifically targeted therapy approach.

LRIG1 modulates aggressiveness of head and neck cancers by regulating EGFR-MAPK-SPHK1 signaling and extracellular matrix remodeling.

PubMed

Sheu, J J-C; Lee, C-C; Hua, C-H; Li, C-I; Lai, M-T; Lee, S-C; Cheng, J; Chen, C-M; Chan, C; Chao, S C-C; Chen, J-Y; Chang, J-Y; Lee, C-H

2014-03-13

EGFR overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found at 3p14, and its copy-number loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and western blot analysis demonstrated that LRIG1 modulated extracellular matrix (ECM) remodeling and EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs and SPHK1. In addition, LRIG1 induction triggered cell morphology changes and integrin inactivation, which coupled with reduced SNAI2 expression. By contrast, knockdown of endogenous LRIG1 in TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration and invasiveness. Such tumor-promoting effects could be abolished by specific MAPK or SPHK1 inhibitors. Our data suggest LRIG1 as a tumor suppressor for head and neck cancers; LRIG1 downregulation in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.

Effect of topical rebamipide on goblet cells in the lid wiper of human conjunctiva

PubMed Central

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu; Ishida, Susumu

2017-01-01

It has been demonstrated that topical administration of rebamipide, which is an antiulcer agent, increases the mucin level of the tear film and ameliorates ocular surface conditions such as lid wiper epitheliopathy. The aim of the present study was to analyze the changes in goblet cell number, cell proliferation, and epidermal growth factor receptor (EGFR) induced by topical rebamipide addition to the lid wiper of humans. A total of 30 eyelid tissue samples were obtained during involutional entropion surgeries, fixed in paraformaldehyde, embedded in paraffin and divided into two groups: Rebamipide or non-rebamipide. The tissues in the rebamipide group were obtained from patients who had a medical history of topical rebamipide use prior to surgery. The number of goblet cells was counted under light microscopy. A total of 22 eyelid tissue samples were further examined using immunohistochemistry with anti-Ki-67 and anti-EGFR antibodies to evaluate cell proliferation and EGFR expression, respectively. Histologically, the lid wiper and palpebral conjunctiva were clearly identified in the tissues. The number of goblet cells was significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0367). There was no significant difference in lid wiper cell proliferation between the rebamipide and non-rebamipide groups. Immunohistochemistry revealed that EGFR levels in the lid wiper epithelial cells were significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0237). These results suggest that topical rebamipide application increases the number of goblet cells in the lid wiper, which in turn upregulates the expression of EGFR. These findings may be clinically relevant and provide a therapeutic basis for the treatment of ocular disease such as dry eye and lid wiper epitheliopathy. PMID:28587435

Effect of topical rebamipide on goblet cells in the lid wiper of human conjunctiva.

PubMed

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu; Ishida, Susumu

2017-06-01

It has been demonstrated that topical administration of rebamipide, which is an antiulcer agent, increases the mucin level of the tear film and ameliorates ocular surface conditions such as lid wiper epitheliopathy. The aim of the present study was to analyze the changes in goblet cell number, cell proliferation, and epidermal growth factor receptor (EGFR) induced by topical rebamipide addition to the lid wiper of humans. A total of 30 eyelid tissue samples were obtained during involutional entropion surgeries, fixed in paraformaldehyde, embedded in paraffin and divided into two groups: Rebamipide or non-rebamipide. The tissues in the rebamipide group were obtained from patients who had a medical history of topical rebamipide use prior to surgery. The number of goblet cells was counted under light microscopy. A total of 22 eyelid tissue samples were further examined using immunohistochemistry with anti-Ki-67 and anti-EGFR antibodies to evaluate cell proliferation and EGFR expression, respectively. Histologically, the lid wiper and palpebral conjunctiva were clearly identified in the tissues. The number of goblet cells was significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0367). There was no significant difference in lid wiper cell proliferation between the rebamipide and non-rebamipide groups. Immunohistochemistry revealed that EGFR levels in the lid wiper epithelial cells were significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0237). These results suggest that topical rebamipide application increases the number of goblet cells in the lid wiper, which in turn upregulates the expression of EGFR. These findings may be clinically relevant and provide a therapeutic basis for the treatment of ocular disease such as dry eye and lid wiper epitheliopathy.

The USP8 mutational status may predict drug susceptibility in corticotroph adenomas of Cushing's disease.

PubMed

Hayashi, Kyohei; Inoshita, Naoko; Kawaguchi, Kohei; Ibrahim Ardisasmita, Arif; Suzuki, Hisanori; Fukuhara, Noriaki; Okada, Mitsuo; Nishioka, Hiroshi; Takeuchi, Yasuhiro; Komada, Masayuki; Takeshita, Akira; Yamada, Shozo

2016-02-01

Somatic mutations in the ubiquitin-specific peptidase USP8 gene were recently detected in one- to two-third(s) of corticotroph adenomas of Cushing's disease (CD). These mutations may lead to the deubiquitination of EGFR, thereby increasing EGFR signaling, which has been implicated in ACTH hypersecretion. Our objective was to determine the impact of USP8 mutations on the clinicopathological features of CD. USP8 mutations as well as clinicopathological characteristics were examined in 60 corticotroph adenomas including 15 Crooke's cell adenomas (CCAs), a rare histological variant presenting with generally aggressive behavior, using qRT-PCR and/or immunohistochemistry. USP8 mutations were exclusively detected in women, except for one case, with a prevalence of 42.2% in non-CCA and 13.3% in CCA (overall 35%). Clinically well-behaved presentations including microadenoma and curative resection were more common in mutated cases. The expression of EGFR was not associated with the mutation status. In contrast, mutated tumors expressed significantly higher levels of POMC, SSTR5, and MGMT. Microadenomas that strongly express POMC were common among mutated tumors, which may lead to the mechanisms by which very small adenomas secrete excess ACTH to present overt CD. While USP8 mutations were less likely to enhance tumorous ACTH hypersecretion via EGFR-mediated activation, the presence of USP8 mutations may predict favorable responses to the somatostatin analog pasireotide, which exhibits high affinity for SSTR5. In contrast, non-mutated aggressive tumors such as CCA may respond better to the alkylating agent temozolomide because of their significantly weak expression of MGMT. © 2016 European Society of Endocrinology.

Photothermal optical coherence tomography of epidermal growth factor receptor in live cells using immunotargeted gold nanospheres

NASA Astrophysics Data System (ADS)

Skala, Melissa C.; Crow, Matthew J.; Wax, Adam; Izatt, Joseph A.

2009-02-01

Molecular imaging is a powerful tool for investigating disease processes and potential therapies in both in vivo and in vitro systems. However, high resolution molecular imaging has been limited to relatively shallow penetration depths that can be accessed with microscopy. Optical coherence tomography (OCT) is an optical analogue to ultrasound with relatively good penetration depth (1-2 mm) and resolution (~1-10 μm). We have developed and characterized photothermal OCT as a molecular contrast mechanism that allows for high resolution molecular imaging at deeper penetration depths than microscopy. Our photothermal system consists of an amplitude-modulated heating beam that spatially overlaps with the focused spot of the sample arm of a spectral-domain OCT microscope. Validation experiments in tissue-like phantoms containing gold nanospheres that absorb at 532 nm revealed a sensitivity of 14 parts per million nanospheres (weight/weight) in a tissue-like environment. The nanospheres were then conjugated to anti-EGFR, and molecular targeting was confirmed in cells that over-express EGFR (MDA-MB-468) and cells that express low levels of EGFR (MDA-MB-435). Molecular imaging in three-dimensional tissue constructs was confirmed with a significantly lower photothermal signal (p<0.0001) from the constructs composed of cells that express low levels of EGFR compared to the over-expressing cell constructs (300% signal increase). This technique could potentially augment confocal and multiphoton microscopy as a method for deep-tissue, depth-resolved molecular imaging with relatively high resolution and target sensitivity, without photobleaching or cytotoxicity.

Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation

PubMed Central

Ymer, Susie I.; Greenall, Sameer A.; Cvrljevic, Anna; Cao, Diana X.; Donoghue, Jacqui F.; Epa, V. Chandana; Scott, Andrew M.; Adams, Timothy E.; Johns, Terrance G.

2011-01-01

The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation. PMID:24212795

Epidermal growth factor receptor signaling mediates aldosterone-induced profibrotic responses in kidney

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Lili; Yang, Min; Ding, Wei

Aldosterone has been recognized as a risk factor for the development of chronic kidney disease (CKD). Studies have indicated that enhanced activation of epidermal growth factor receptor (EGFR) is associated with the development and progression of renal fibrosis. But if EGFR is involved in aldosterone-induced renal fibrosis is less investigated. In the present study, we examined the effect of erlotinib, an inhibitor of EGFR tyrosine kinase activity, on the progression of aldosterone-induced renal profibrotic responses in a murine model underwent uninephrectomy. Erlotinib-treated rats exhibited relieved structural lesion comparing with rats treated with aldosterone alone, as characterized by glomerular hypertrophy, mesangialmore » cell proliferation and expansion. Also, erlotinib inhibited the expression of TGF-β, α-SMA and mesangial matrix proteins such as collagen Ⅳ and fibronectin. In cultured mesangial cells, inhibition of EGFR also abrogated aldosterone-induced expression of extracellular matrix proteins, cell proliferation and migration. We also demonstrated that aldosterone induced the phosphorylation of EGFR through generation of ROS. And the activation of EGFR resulted in the phosphorylation of ERK1/2, leading to the activation of profibrotic pathways. Taken together, we concluded that aldosterone-mediated tissue fibrosis relies on ROS induced EGFR/ERK activation, highlighting EGFR as a potential therapeutic target for modulating renal fibrosis. - Highlights: • EGFR was involved in aldosterone-induced renal profibrotic responses. • Aldosterone-induced EGFR activation was mediated by MR-dependent ROS generation. • EGFR activated the MAPK/ERK1/2 signaling to promote renal fibrosis.« less

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells.

PubMed

Han, Jianfeng; Chu, Jianhong; Keung Chan, Wing; Zhang, Jianying; Wang, Youwei; Cohen, Justus B; Victor, Aaron; Meisen, Walter H; Kim, Sung-hak; Grandi, Paola; Wang, Qi-En; He, Xiaoming; Nakano, Ichiro; Chiocca, E Antonio; Glorioso, Joseph C; Kaur, Balveen; Caligiuri, Michael A; Yu, Jianhua

2015-07-09

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

KRAS, EGFR, PDGFR-α, KIT and COX-2 status in carcinoma showing thymus-like elements (CASTLE)

PubMed Central

2014-01-01

Background CASTLE (Carcinoma showing thymus-like elements) is a rare malignant neoplasm of the thyroid resembling lymphoepithelioma-like and squamous cell carcinoma of the thymus with different biological behaviour and a better prognosis than anaplastic carcinoma of the thyroid. Methods We retrospectively investigated 6 cases of this very rare neoplasm in order to investigate the mutational status of KRAS, EGFR, PDGFR-α and KIT, as well as the immunohistochemical expression pattern of CD117, EGFR and COX-2, and possibly find new therapeutic targets. Results Diagnosis was confirmed by a moderate to strong expression of CD5, CD117 and CK5/6, whereas thyroglobulin, calcitonin and TTF-1 were negative in all cases. Tumors were also positive for COX-2 and in nearly all cases for EGFR. In four cases single nucleotide polymorphisms (SNPs) could be detected in exon 12 of the PDGFR-α gene (rs1873778), in three cases SNPs were found in exon 20 of the EGFR gene (rs1050171). No mutations were found in the KIT and KRAS gene. Conclusions All tumors showed a COX-2 expression as well as an EGFR expression except for one case and a wild-type KRAS status. No activating mutations in the EGFR, KIT and PDGFR-α gene could be detected. Our data may indicate a potential for targeted therapies, but if these therapeutic strategies are of benefit in CASTLE remains to be determined. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1658499296115016 PMID:24934485

Brk/PTK6 Sustains Activated EGFR Signaling through Inhibiting EGFR Degradation and Transactivating EGFR

PubMed Central

Li, X; Lu, Y; Liang, K; Hsu, J -M.; Albarracin, C; Mills, G B; Hung, M-C; Fan, Z

2011-01-01

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from Cbl-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy. PMID:22231447

Changes in the EGFR amplification and EGFRvIII expression between paired primary and recurrent glioblastomas

PubMed Central

Gao, Ya; Kerkhof, Melissa; Kros, Johan M.; Gorlia, Thierry; van Zwieten, Kitty; Prince, Jory; van Duinen, Sjoerd; Sillevis Smitt, Peter A.; Taphoorn, Martin; French, Pim J.

2015-01-01

Abstract Background The efficacy of novel targeted therapies is often tested at the time of tumor recurrence. However, for glioblastoma (GBM) patients, surgical resections at recurrence are performed only in a minority of patients; therefore, molecular data are predominantly derived from the initial tumor. Molecular data of the initial tumor for patient selection into personalized medicine trials can therefore be used only when the specific genetic change is retained in the recurrent tumor. Methods In this study we determined whether EGFR amplification and expression of the most common mutation in GBMs (EGFRvIII) is retained at tumor recurrence. Because retention of genetic changes may be dependent on the initial treatment, we only used a cohort of GBM samples that were uniformly treated according to the current standard of care (ie, chemo-irradiation with temozolomide). Results Our data show that, in spite of some quantitative differences, the EGFR amplification status remains stable in the majority (84%) of tumors evaluated. EGFRvIII expression remained similar in 79% of GBMs. However, within the tumors expressing EGFRvIII at initial diagnosis, approximately one-half lose their EGFRvIII expression at tumor recurrence. Conclusions The relative stability of EGFR amplification indicates that molecular data obtained in the primary tumor can be used to predict the EGFR status of the recurrent tumor, but care should be taken in extrapolating EGFRvIII expression from the primary tumor, particularly when expressed at first diagnosis. PMID:25691693

Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity.

PubMed

Bao, Bin; Mitrea, Cristina; Wijesinghe, Priyanga; Marchetti, Luca; Girsch, Emily; Farr, Rebecca L; Boerner, Julie L; Mohammad, Ramzi; Dyson, Greg; Terlecky, Stanley R; Bollig-Fischer, Aliccia

2017-03-10

Among breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prog-nosis. TNBC does not express estrogen receptor-alpha, progesterone receptor, or the HER2 oncogene; therefore, TNBC lacks targets for molecularly-guided therapies. The concept that EGFR oncogene inhibitor drugs could be used as targeted treatment against TNBC has been put forth based on estimates that 30-60% of TNBC express high levels of EGFR. However, results from clinical trials testing EGFR inhibitors, alone or in combination with cytotoxic chemotherapy, did not improve patient outcomes. Results herein offer an explanation as to why EGFR inhibitors failed TNBC patients and support how combining a select antioxidant and an EGFR-specific small molecule kinase inhibitor (SMKI) could be an effective, novel therapeutic strategy. Treatment with CAT-SKL-a re-engineered protein form of the antioxidant enzyme catalase-inhibited cancer stem-like cells (CSCs), and treatment with the EGFR-specific SMKI erlotinib inhibited non-CSCs. Thus, combining the antioxidant CAT-SKL with erlotinib targeted both CSCs and bulk cancer cells in cultures of EGFR-expressing TNBC-derived cells. We also report evidence that the mechanism for CAT-SKL inhibition of CSCs may depend on antioxidant-induced downregulation of a short alternative mRNA splicing variant of the methyl-CpG binding domain 2 gene, isoform MBD2c.

Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy

PubMed Central

Musiani, Daniele; Konda, John David; Pavan, Simona; Torchiaro, Erica; Sassi, Francesco; Noghero, Alessio; Erriquez, Jessica; Perera, Timothy; Olivero, Martina; Di Renzo, Maria Flavia

2014-01-01

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.—Musiani, D., Konda, J. D., Pavan, S., Torchiaro, E., Sassi, F., Noghero, A., Erriquez, J., Perera, T., Olivero, M., Di Renzo, M. F. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy. PMID:24903273

Monoclonal antibody-tagged receptor-targeted contrast agents for detection of cancers

NASA Astrophysics Data System (ADS)

Soukos, N. S.; Hamblin, Michael R.; Deutsch, Thomas F.; Hasan, Tayyaba

2001-07-01

Oral cancer and precancer overexpress the epidermal growth factor receptor (EGFR) and monoclonal antibodies against EGFR coupled to photoactive dyes may have a potential both as a diagnostic and treatment modalities for oral premalignancy. We asked whether an anti-EGFR mab (C225) conjugated with the fluorescence dye indocyanine Cy5.5 could detect dysplastic changes in the hamster cheek pouch carcinogenesis model. Secondly, we tested whether the same antibody conjugated with the photosensitizer chlorin (e6) could be used together with illumination to reduce levels of expression of EGFR as evaluated by the immunophotodetection procedure. Increased fluorescence appeared to correlate with development of premalignancy when the C225-Cy5.5 conjugate was used. Areas with increased fluorescence signal were found in carcinogen-treated but clinically normal cheek pouches, that revealed dysplastsic changes by histology. The immunophotodetection procedure was carried out after photoummunotherapy with the C225-ce6 conjugate, and showed a significant reduction in fluorescence in the illuminated compared to the non-illuminated areas in the carcinogen- treated but not the normal cheek pouch. The results demonstrate that the use of anti-EGFR Mab targeted photoactive dyes may serve as a feedback controlled optical diagnosis and therapy procedure for oral premalignant lesions.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

PubMed

Yamaura, Takumi; Ezaki, Junji; Okabe, Naoyuki; Takagi, Hironori; Ozaki, Yuki; Inoue, Takuya; Watanabe, Yuzuru; Fukuhara, Mitsuro; Muto, Satoshi; Matsumura, Yuki; Hasegawa, Takeo; Hoshino, Mika; Osugi, Jun; Shio, Yutaka; Waguri, Satoshi; Tamura, Hirosumi; Imai, Jun-Ichi; Ito, Emi; Yanagisawa, Yuka; Honma, Reiko; Watanabe, Shinya; Suzuki, Hiroyuki

2018-02-01

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor ( EGFR ) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B ( FAM83B ) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR . Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR . Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR .

Type I γ Phosphatidylinositol Phosphate 5-Kinase i5 Controls the Ubiquitination and Degradation of the Tumor Suppressor Mitogen-inducible Gene 6*

PubMed Central

Sun, Ming; Cai, Jinyang; Anderson, Richard A.; Sun, Yue

2016-01-01

Mitogen-inducible gene 6 (Mig6) is a tumor suppressor, and the disruption of Mig6 expression is associated with cancer development. Mig6 directly interacts with epidermal growth factor receptor (EGFR) to suppress the activation and downstream signaling of EGFR. Therefore, loss of Mig6 enhances EGFR-mediated signaling and promotes EGFR-dependent carcinogenesis. The molecular mechanism modulating Mig6 expression in cancer remains unclear. Here we demonstrate that type I γ phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), stabilizes Mig6 expression. Knockdown of PIPKIγi5 leads to the loss of Mig6 expression, which dramatically enhances and prolongs EGFR-mediated cell signaling. Loss of PIPKIγi5 significantly promotes Mig6 protein degradation via proteasomes, but it does not affect the Mig6 mRNA level. PIPKIγi5 directly interacts with the E3 ubiquitin ligase neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1). The C-terminal domain of PIPKIγi5 and the WW1 and WW2 domains of NEDD4-1 are required for their interaction. The C2 domain of NEDD4-1 is required for its interaction with PtdIns(4,5)P2. By binding with NEDD4-1 and producing PtdIns(4,5)P2, PIPKIγi5 perturbs NEDD4-1-mediated Mig6 ubiquitination and the subsequent proteasomal degradation. Thus, loss of NEDD4-1 can rescue Mig6 expression in PIPKIγi5 knockdown cells. In this way, PIPKIγi5, NEDD4-1, and Mig6 form a novel molecular nexus that controls EGFR activation and downstream signaling. PMID:27557663

EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis.

PubMed

Wu, Nan-Lin; Huang, Duen-Yi; Hsieh, Shie-Liang; Hsiao, Cheng-Hsiang; Lee, Te-An; Lin, Wan-Wan

2013-10-01

Decoy receptor 3 (DcR3) is a soluble receptor of Fas ligand (FasL), LIGHT (TNFSF14) and TNF-like molecule 1A (TL1A) and plays pleiotropic roles in many inflammatory and autoimmune disorders and malignant diseases. In cutaneous biology, DcR3 is expressed in primary human epidermal keratinocytes and is upregulated in skin lesions in psoriasis, which is characterized by chronic inflammation and angiogenesis. However, the regulatory mechanisms of DcR3 over-expression in skin lesions of psoriasis are unknown. Here, we demonstrate that DcR3 can be detected in both dermal blood vessels and epidermal layers of psoriatic skin lesions. Analysis of serum samples showed that DcR3 was elevated, but FasL was downregulated in psoriatic patients compared with normal individuals. Additional cell studies revealed a central role of epidermal growth factor receptor (EGFR) in controlling the basal expression of DcR3 in keratinocytes. Activation of EGFR by epidermal growth factor (EGF) and transforming growth factor (TGF)-α strikingly upregulated DcR3 production. TNF-αenhanced DcR3 expression in both keratinocytes and endothelial cells compared with various inflammatory cytokines involved in psoriasis. Additionally, TNF-α-enhanced DcR3 expression in keratinocytes was inhibited when EGFR was knocked down or EGFR inhibitor was used. The NF-κB pathway was critically involved in the molecular mechanisms underlying the action of EGFR and inflammatory cytokines. Collectively, the novel regulatory mechanisms of DcR3 expression in psoriasis, particularly in keratinocytes and endothelial cells, provides new insight into the pathogenesis of psoriasis and may also contribute to the understanding of other diseases that involve DcR3 overexpression. Copyright © 2013 Elsevier B.V. All rights reserved.

[Cetuximab in combination with icotinib overcomes the acquired resistance caused by EGFR T790M mutation in non-small cell lung cancer].

PubMed

Wang, Meng; Zhang, Lianmin; Zhao, Xiaoliang; Liu, Jun; Chen, Yulong; Wang, Changli

2014-09-01

The aim of this study was to investigate the effects of combination of icotinib and cetuximab on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC, and provide experimental evidence for rational treatment of NSCLC. The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4, 5-dimethylthiazol-2-yl)- 5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. The expression of molecular markers of tumor proliferation PCNA and Ki-67 protein was further examined by immunohistochemistry, and the expression of EGFR-signaling-related proteins in tissue sections taken from H1975 tumor xenografts was assessed by Western blot assay. Sensitivity to EGFR inhibitors was detected in human H1975 tumor xenograft in nude mice. The in vitro experiment showed that the proliferative ability of H1975 cells was inhibited in a dose-dependent manner, along with the increasing doses of cetuximab and icotinib, and the combination of cetuximab with icotinib resulted in a more pronounced growth inhibition of the H1975 cells. The apoptosis rate of H1975 cells after treatment with 0.5 µmol/L icotinib and 1 µg/ml cetuximab was (22.03 ± 2.41)% and that after treatment with 5 µmol/L icotinib and 10 µg/ml cetuximab was (42.75 ± 2.49)%, both were significantly higher than that after treatment with the same dose of icotinib or cetuximab alone (P < 0.05). The nude mouse experiment showed that the transplanted tumor was growing to (614.5 ± 10.8) mm(3) in the blank control group and to (611.2 ± 8.7) mm(3) at 28 days after icotinib treatment, but (30.8 ± 2.0) mm(3) in the cetuximab treatment group and 0 mm(3) in the cetuximab combined with icotinib group. There was a significantly decreased expression of Ki-67 and PCNA proteins and down-regulation of phosphorylation of EGFR signaling-related proteins in the cetuximab combined with icotinib group. The combination of icotinib with cetuximab can exert synergistic inhibitory effect on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC H1975 cells, interrupts the EGFR-downstream signaling pathway, and enhances the anticancer activity of chemotherapeutic drugs. Our results provide further experimental evidence for the clinical studies of combination of icotinib with cetuximab in the treatment of NSCLC patients associated with secondary drug resistance caused by T790M mutation of EGFR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penrose, Harrison; Heller, Sandra; Cable, Chloe

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTORmore » and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.« less

G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells.

PubMed

Albanito, Lidia; Madeo, Antonio; Lappano, Rosamaria; Vivacqua, Adele; Rago, Vittoria; Carpino, Amalia; Oprea, Tudor I; Prossnitz, Eric R; Musti, Anna Maria; Andò, Sebastiano; Maggiolini, Marcello

2007-02-15

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib

PubMed Central

Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

2014-01-01

Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF “like” ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation. PMID:25249545

Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib.

PubMed

Younes, Mohamad; Wu, Zherui; Dupouy, Sandra; Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

2014-09-30

Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF "like" ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation.

Systemic analysis of different colorectal cancer cell lines and TCGA datasets identified IGF-1R/EGFR-PPAR-CASPASE axis as important indicator for radiotherapy sensitivity.

PubMed

Chen, Lin; Zhu, Zhe; Gao, Wei; Jiang, Qixin; Yu, Jiangming; Fu, Chuangang

2017-09-05

Insulin-like growth factor 1 receptor (IGF-1R) is proved to contribute the development of many types of cancers. But, little is known about its roles in radio-resistance of colorectal cancer (CRC). Here, we demonstrated that low IGF-1R expression value was associated with the better radiotherapy sensitivity of CRC. Besides, through Quantitative Real-time PCR (qRT-PCR), the elevated expression value of epidermal growth factor receptor (EGFR) was observed in CRC cell lines (HT29, RKO) with high radio-sensitivity compared with those with low sensitivity (SW480, LOVO). The irradiation induced apoptosis rates of wild type and EGFR agonist (EGF) or IGF-1R inhibitor (NVP-ADW742) treated HT29 and SW480 cells were quantified by flow cytometry. As a result, the apoptosis rate of EGF and NVP-ADW742 treated HT29 cells was significantly higher than that of those wild type ones, which indicated that high EGFR and low IGF-1R expression level in CRC was associated with the high sensitivity to radiotherapy. We next conducted systemic bioinformatics analysis of genome-wide expression profiles of CRC samples from the Cancer Genome Atlas (TCGA). Differential expression analysis between IGF-1R and EGFR abnormal CRC samples, i.e. CRC samples with higher IGF-1R and lower EGFR expression levels based on their median expression values, and the rest of CRC samples identified potential genes contribute to radiotherapy sensitivity. Functional enrichment of analysis of those differential expression genes (DEGs) in the Database for Annotation, Visualization and Integrated Discovery (DAVID) indicated PPAR signaling pathway as an important pathway for the radio-resistance of CRC. Our study identified the potential biomarkers for the rational selection of radiotherapy for CRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Acquired Resistance Mechanisms to Combination Met-TKI/EGFR-TKI Exposure in Met-Amplified EGFR-TKI-Resistant Lung Adenocarcinoma Harboring an Activating EGFR Mutation.

PubMed

Yamaoka, Toshimitsu; Ohmori, Tohru; Ohba, Motoi; Arata, Satoru; Kishino, Yasunari; Murata, Yasunori; Kusumoto, Sojiro; Ishida, Hiroo; Shirai, Takao; Hirose, Takashi; Ohnishi, Tsukasa; Sasaki, Yasutsuna

2016-12-01

Met-amplified EGFR-tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) harboring an activating EGFR mutation is responsive to concurrent EGFR-TKI and Met-TKI treatment in a preclinical model. Here, we determined that Met-amplified gefitinib-resistant cells acquire dual resistance to inhibition of EGFR and Met tyrosine kinase activities. PC-9 lung adenocarcinoma cells harboring 15-bp deletions (Del E746_A750) in EGFR exon 19 were treated with increasing concentrations of the Met-TKI PHA665752 and 1 μmol/L gefitinib for 1 year; three resistant clones were established via Met amplification. The three dual-resistance cell lines (PC-9DR2, PC-9DR4, and PC-9DR6, designated as DR2, DR4, and DR6, respectively) exhibited different mechanisms for evading both EGFR and Met inhibition. None of the clones harbored a secondary mutation of EGFR T790M or a Met mutation. Insulin-like growth factor (IGF)/IGF1 receptor activation in DR2 and DR4 cells acted as a bypass signaling pathway. Met expression was attenuated to a greater extent in DR2 than in PC-9 cells, but was maintained in DR4 cells by overexpression of IGF-binding protein 3. In DR6 cells, Met was further amplified by association with HSP90, which protected Met from degradation and induced SET and MYND domain-containing 3 (SMYD3)-mediated Met transcription. This is the first report describing the acquisition of dual resistance mechanisms in NSCLC harboring an activating EGFR mutation to Met-TKI and EGFR-TKI following previous EGFR-TKI treatment. These results might inform the development of more effective therapeutic strategies for NSCLC treatment. Mol Cancer Ther; 15(12); 3040-54. ©2016 AACR. ©2016 American Association for Cancer Research.

Epidermal growth factor receptor signaling pathway is frequently altered in ampullary carcinoma at protein and genetic levels.

PubMed

Mikhitarian, Kaidi; Pollen, Maressa; Zhao, Zhiguo; Shyr, Yu; Merchant, Nipun B; Parikh, Alexander; Revetta, Frank; Washington, M Kay; Vnencak-Jones, Cindy; Shi, Chanjuan

2014-05-01

Our objective was to explore alteration of the epidermal growth factor receptor (EGFR) signaling pathway in ampullary carcinoma. Immunohistochemical studies were employed to evaluate expression of amphiregulin as well as expression and activation of EGFR. A lab-developed assay was used to identify mutations in the EGFR pathway genes, including KRAS, BRAF, PIK3CA, PTEN, and AKT1. A total of 52 ampullary carcinomas were identified, including 25 intestinal-type and 24 pancreatobiliary-type tumors, with the intestinal type being associated with a younger age at diagnosis (P=0.03) and a better prognosis (P<0.01). Expression of amphiregulin correlated with better differentiation (P<0.01), but no difference was observed between two major histologic types. Expression and activation of EGFR was more commonly seen in the pancreatobiliary type (P<0.01). Mutations were detected in 50% of the pancreatobiliary type and 60% of the intestinal type. KRAS was the most common gene mutated in the pancreatobiliary type (42%) as well as the intestinal type (52%). Other mutations detected included PIK3CA, SMAD4 and BRAF. KRAS mutations at codons 12 and 13 did not adversely affect overall survival. In conclusion, EGFR expression and activation were different between intestinal- and pancreatobiliary-type ampullary carcinoma. KRAS mutation was common in both histologic types; however, the incidence appeared to be lower in the pancreatobiliary type compared with its pancreatic counterpart, pancreatic ductal adenocarcinoma. Mutational analysis of the EGFR pathway genes may provide important insights into personalized treatment for patients with ampullary carcinoma.

The Epidermal Growth Factor Receptor Promotes Glomerular Injury and Renal Failure in Rapidly Progressive Crescentic Glomerulonephritis; the Identification of Possible Therapy

PubMed Central

Bollée, Guillaume; Flamant, Martin; Schordan, Sandra; Fligny, Cécile; Rumpel, Elisabeth; Milon, Marine; Schordan, Eric; Sabaa, Nathalie; Vandermeersch, Sophie; Galaup, Ariane; Rodenas, Anita; Casal, Ibrahim; Sunnarborg, Susan W; Salant, David J; Kopp, Jeffrey B.; Threadgill, David W; Quaggin, Susan E; Dussaule, Jean-Claude; Germain, Stéphane; Mesnard, Laurent; Endlich, Karlhans; Boucheix, Claude; Belenfant, Xavier; Callard, Patrice; Endlich, Nicole; Tharaux, Pierre-Louis

2011-01-01

Rapidly progressive glomerulonephritis (RPGN) is a clinical a morphological expression of severe glomerular injury. Glomerular injury manifests as a proliferative histological pattern (“crescents”) with accumulation of T cells and macrophages, and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the EGFR/ErbB1 receptor in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 days after the induction of experimental RPGN. This suggests that targeting the HB-EGF/EGFR pathway could also be beneficial for treatment of human RPGN. PMID:21946538

HBEGF promotes gliomagenesis in the context of Ink4a/Arf and Pten loss.

PubMed

Shin, C H; Robinson, J P; Sonnen, J A; Welker, A E; Yu, D X; VanBrocklin, M W; Holmen, S L

2017-08-10

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is a ligand for the EGF receptor (EGFR), one of the most commonly amplified receptor tyrosine kinases (RTKs) in glioblastoma (GBM). While HBEGF has been found to be expressed in a subset of malignant gliomas, its sufficiency for glioma initiation has not been evaluated. In this study, we demonstrate that HBEGF can initiate GBM in mice in the context of Ink4a/Arf and Pten loss, and that these tumors are similar to the classical GBM subtype observed in patients. Isogenic astrocytes from these mice showed activation not only of Egfr but also the RTK Axl in response to HBEGF stimulation. Deletion of either Egfr or Axl decreased the tumorigenic properties of HBEGF-transformed cells; however, only EGFR was able to rescue the phenotype in cells lacking both RTKs indicating that Egfr is required for activation of Axl in this context. Silencing of HBEGF in vivo resulted in tumor regression and significantly increased survival, suggesting that HBEGF may be a clinically relevant target.

HBEGF promotes gliomagenesis in the context of Ink4a/Arf and Pten loss

PubMed Central

Shin, Clifford H.; Robinson, James P.; Sonnen, Joshua A.; Welker, Adam E.; Yu, Diana X.; VanBrocklin, Matthew W.; Holmen, Sheri L.

2017-01-01

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is a ligand for the epidermal growth factor receptor (EGFR), one of the most commonly amplified receptor tyrosine kinases (RTK) in glioblastoma. While HBEGF has been found to be expressed in a subset of malignant gliomas, its sufficiency for glioma initiation has not been evaluated. In this study, we demonstrate that HBEGF can initiate glioblastoma (GBM) in mice in the context of Ink4a/Arf and Pten loss, and that these tumors are similar to the classical GBM subtype observed in patients. Isogenic astrocytes from these mice showed activation not only of Egfr but also the RTK Axl in response to HBEGF stimulation. Deletion of either Egfr or Axl decreased the tumorigenic properties of HBEGF transformed cells; however only EGFR was able to rescue the phenotype in cells lacking both RTKs indicating that Egfr is required for activation of Axl in this context. Silencing of HBEGF in vivo resulted in tumor regression and significantly increased survival suggesting that HBEGF may be a clinically relevant target. PMID:28368403

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin

PubMed Central

Singh, Bhuminder; Bogatcheva, Galina; Starchenko, Alina; Sinnaeve, Justine; Lapierre, Lynne A.; Williams, Janice A.; Goldenring, James R.; Coffey, Robert J.

2015-01-01

ABSTRACT Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically 156EEMETL161). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization. PMID:26272915

The place of targeted therapies in the management of non-small cell bronchial carcinoma. Molecular markers as predictors of tumor response and survival in lung cancer.

PubMed

Rosell, R; Moran, T; Fernanda Salazar, M; Mendez, P; De Aguirre, I; Ramirez, J-L; Isla, D; Cobo, M; Camps, C; Lopez-Vivanco, G; Alberola, V; Taron, M

2006-11-01

This review highlights the numerous molecular biology findings in the field of lung cancer with potential therapeutic impact in both the near and distant future. Abundant pre-clinical and clinical data indicate that BRCA1 mRNA expression is a differential modulator of chemotherapy sensitivity. Low levels predict cisplatin sensitivity and antimicrotubule drug resistance, and the opposite occurs with high levels. The main core of recent research has centered on epidermal growth factor receptor (EGFR) mutations and gene copy numbers. For the first time, EGFR mutations have been shown to predict dramatic responses in metastatic lung adenocarcinomas, with a threefold increase in time to progression and survival in patients receiving EGFR tyrosine-kinase inhibitors. Evidence has also been accumulated on the crosstalk between estrogen and EGFR receptor pathways, paving the way for clinical trials of EGFR tyrosine-kinase inhibitors plus aromatase inhibitors. Understanding the relevance of these findings can help to change the clinical practice in oncology towards customizing chemotherapy and targeted therapies, leading to improvement both in survival and in cost-effectiveness.

gone early, a Novel Germline Factor, Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary

PubMed Central

Matsuoka, Shinya; Gupta, Swati; Suzuki, Emiko; Hiromi, Yasushi; Asaoka, Miho

2014-01-01

In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment. PMID:25420147

Prevention of Bronchial Hyperplasia by EGFR Pathway Inhibitors in an Organotypic Culture Model

PubMed Central

Lee, Jangsoon; Ryu, Seung-Hee; Kang, Shin Myung; Chung, Wen-Cheng; Gold, Kathryn Ann; Kim, Edward S.; Hittelman, Walter N.; Hong, Waun Ki; Koo, Ja Seok

2011-01-01

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. The current study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured using the 3-dimensional organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands EGF, transforming growth factor-alpha, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MEK inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we demonstrated that bronchial hyperplasia can be modeled in vitro using primary NHTBE cells maintained in a 3-dimensional (3-D) organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role. PMID:21505178

Armored long non-coding RNA MEG3 targeting EGFR based on recombinant MS2 bacteriophage virus-like particles against hepatocellular carcinoma.

PubMed

Chang, Le; Wang, Guojing; Jia, Tingting; Zhang, Lei; Li, Yulong; Han, Yanxi; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan

2016-04-26

Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.

GH administration rescues fatty liver regeneration impairment by restoring GH/EGFR pathway deficiency.

PubMed

Collin de l'Hortet, A; Zerrad-Saadi, A; Prip-Buus, C; Fauveau, V; Helmy, N; Ziol, M; Vons, C; Billot, K; Baud, V; Gilgenkrantz, Hélène; Guidotti, Jacques-Emmanuel

2014-07-01

GH pathway has been shown to play a major role in liver regeneration through the control of epidermal growth factor receptor (EGFR) activation. This pathway is down-regulated in nonalcoholic fatty liver disease. Because regeneration is known to be impaired in fatty livers, we wondered whether a deregulation of the GH/EGFR pathway could explain this deficiency. Hepatic EGFR expression and triglyceride levels were quantified in liver biopsies of 32 obese patients with different degrees of steatosis. We showed a significant inverse correlation between liver EGFR expression and the level of hepatic steatosis. GH/EGFR down-regulation was also demonstrated in 2 steatosis mouse models, a genetic (ob/ob) and a methionine and choline-deficient diet mouse model, in correlation with liver regeneration defect. ob/ob mice exhibited a more severe liver regeneration defect after partial hepatectomy (PH) than methionine and choline-deficient diet-fed mice, a difference that could be explained by a decrease in signal transducer and activator of transcription 3 phosphorylation 32 hours after PH. Having checked that GH deficiency accounted for the GH signaling pathway down-regulation in the liver of ob/ob mice, we showed that GH administration in these mice led to a partial rescue in hepatocyte proliferation after PH associated with a concomitant restoration of liver EGFR expression and signal transducer and activator of trnascription 3 activation. In conclusion, we propose that the GH/EGFR pathway down-regulation is a general mechanism responsible for liver regeneration deficiency associated with steatosis, which could be partially rescued by GH administration.

Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garrett, T.; Burgess, A; Gan, H

2009-01-01

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR{sub 287-302} complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR.more » However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR{sub C271A/C283A} mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR{sub C271A/C283A}. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.« less

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters

PubMed Central

2017-01-01

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies. PMID:28493713

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

PubMed

Caetano-Pinto, Pedro; Jamalpoor, Amer; Ham, Janneke; Goumenou, Anastasia; Mommersteeg, Monique; Pijnenburg, Dirk; Ruijtenbeek, Rob; Sanchez-Romero, Natalia; van Zelst, Bertrand; Heil, Sandra G; Jansen, Jitske; Wilmer, Martijn J; van Herpen, Carla M L; Masereeuw, Rosalinde

2017-06-05

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

PubMed

Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi

2014-02-01

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.

Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells.

PubMed

Pi, Jiang; Jin, Hua; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Yang, Peihui; Cai, Jiye; Chen, Zheng W

2017-05-01

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pemetrexed-carboplatin with intercalated icotinib in the treatment of patient with advanced EGFR wild-type lung adenocarcinoma: A case report.

PubMed

Xu, Tongpeng; Wu, Hao; Jin, Shidai; Min, Huang; Zhang, Zhihong; Shu, Yongqian; Wen, Wei; Guo, Renhua

2017-08-01

Tyrosine kinase inhibitors (TKIs) are known to have greater efficacy in epidermal growth factor receptor (EGFR) mutation nonsmall cell lung cancer (NSCLC). However, about 10% of EGFR wild-type (wt) patients respond to TKIs. Several strategies to increase the efficacy of TKIs in wt NSCLC are the subjects of ongoing investigations. One of them is combining EGFR TKI with intercalated chemotherapy. We describe a patient with EGFR wt NSCLC, who was found with ovarian and lung metastasis, was treated with pemetrexed and intercalated icotinib. In this case, we reported the successful long-term maintenance treatment of a patient with EGFR wt NSCLC with pemetrexed and Icotinib. The patient (40-year-old female) was found with ovarian masses and lung masses. Pathological, immunohistochemical, and amplification refractory mutation system (ARMS) assay examinations of ovarian specimen suggested the expression of metastatic lung adenocarcinoma with wt EGFR. After failure treatment with paclitaxel-carboplatin, the patient received 4 cycles of pemetrexed plus platinum with intercalated icotinib and then remained on pemetrexed and icotinib. A partial response was achieved after the treatment. The patient's condition had remained stable on pemetrexed and icotinib for more than 20 months, with no evidence of progression. To our knowledge, this is the first report using the long-term maintenance treatment with pemetrexed and intercalated icotinib in EGFR wt patient. The therapeutic strategies warrant further exploration in selected populations of NSCLC.

Pemetrexed-carboplatin with intercalated icotinib in the treatment of patient with advanced EGFR wild-type lung adenocarcinoma

PubMed Central

Xu, Tongpeng; Wu, Hao; Jin, Shidai; Min, Huang; Zhang, Zhihong; Shu, Yongqian; Wen, Wei; Guo, Renhua

2017-01-01

Abstract Rationale: Tyrosine kinase inhibitors (TKIs) are known to have greater efficacy in epidermal growth factor receptor (EGFR) mutation nonsmall cell lung cancer (NSCLC). However, about 10% of EGFR wild-type (wt) patients respond to TKIs. Patient concerns: Several strategies to increase the efficacy of TKIs in wt NSCLC are the subjects of ongoing investigations. One of them is combining EGFR TKI with intercalated chemotherapy. Diagnoses: We describe a patient with EGFR wt NSCLC, who was found with ovarian and lung metastasis, was treated with pemetrexed and intercalated icotinib. Interventions: In this case, we reported the successful long-term maintenance treatment of a patient with EGFR wt NSCLC with pemetrexed and Icotinib. The patient (40-year-old female) was found with ovarian masses and lung masses. Pathological, immunohistochemical, and amplification refractory mutation system (ARMS) assay examinations of ovarian specimen suggested the expression of metastatic lung adenocarcinoma with wt EGFR. After failure treatment with paclitaxel-carboplatin, the patient received 4 cycles of pemetrexed plus platinum with intercalated icotinib and then remained on pemetrexed and icotinib. Outcomes: A partial response was achieved after the treatment. The patient's condition had remained stable on pemetrexed and icotinib for more than 20 months, with no evidence of progression. Lessons: To our knowledge, this is the first report using the long-term maintenance treatment with pemetrexed and intercalated icotinib in EGFR wt patient. The therapeutic strategies warrant further exploration in selected populations of NSCLC. PMID:28816950

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

PubMed

Asadi-Ghalehni, Majid; Rasaee, Mohamad Javad; RajabiBazl, Masoumeh; Khosravani, Masood; Motaghinejad, Majid; Javanmardi, Masoud; Khalili, Saeed; Modjtahedi, Helmout; Sadroddiny, Esmaeil

2017-12-01

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

miR-146a Suppresses Invasion of Pancreatic Cancer Cells

PubMed Central

Li, Yiwei; VandenBoom, Timothy G.; Wang, Zhiwei; Kong, Dejuan; Ali, Shadan; Philip, Philip A.; Sarkar, Fazlul H.

2010-01-01

The aggressive course of pancreatic cancer is believed to reflect its unusually invasive and metastatic nature, which is associated with epidermal growth factor receptor (EGFR) overexpression and NF-κB activation. MicroRNAs (miRNA) have been implicated in the regulation of various pathobiological processes in cancer, including metastasis in pancreatic cancer and in other human malignancies. In this study, we report lower expression of miR-146a in pancreatic cancer cells compared with normal human pancreatic duct epithelial cells. Reexpression of miR-146a inhibited the invasive capacity of pancreatic cancer cells with concomitant downregulation of EGFR and the NF-κB regulatory kinase interleukin 1 receptor–associated kinase 1 (IRAK-1). Cellular mechanism studies revealed crosstalk between EGFR, IRAK-1, IκBα, NF-κB, and MTA-2, a transcription factor that regulates metastasis. Treatment of pancreatic cancer cells with the natural products 3,3′-diinodolylmethane (DIM) or isoflavone, which increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1, and NF-κB, resulting in an inhibition of pancreatic cancer cell invasion. Our findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can block pancreatic cancer cell invasion and metastasis, offering starting points to design novel anticancer agents. PMID:20124483

Near infrared photoimmunotherapy targeting bladder cancer with a canine anti-epidermal growth factor receptor (EGFR) antibody.

PubMed

Nagaya, Tadanobu; Okuyama, Shuhei; Ogata, Fusa; Maruoka, Yasuhiro; Knapp, Deborah W; Karagiannis, Sophia N; Fazekas-Singer, Judit; Choyke, Peter L; LeBlanc, Amy K; Jensen-Jarolim, Erika; Kobayashi, Hisataka

2018-04-10

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro . In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups ( p < 0.001), and significantly prolonged survival was achieved ( p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans.

Near infrared photoimmunotherapy targeting bladder cancer with a canine anti-epidermal growth factor receptor (EGFR) antibody

PubMed Central

Nagaya, Tadanobu; Okuyama, Shuhei; Ogata, Fusa; Maruoka, Yasuhiro; Knapp, Deborah W.; Karagiannis, Sophia N.; Fazekas-Singer, Judit; Choyke, Peter L.; LeBlanc, Amy K.; Jensen-Jarolim, Erika; Kobayashi, Hisataka

2018-01-01

Anti-epidermal growth factor receptor (EGFR) antibody therapy is used in EGFR expressing cancers including lung, colon, head and neck, and bladder cancers, however results have been modest. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate which is activated by NIR light. NIR-PIT is in clinical trials in patients with recurrent head and neck cancers using cetuximab-IR700 as the conjugate. However, its use has otherwise been restricted to mouse models. This is an effort to explore larger animal models with NIR-PIT. We describe the use of a recombinant canine anti-EGFR monoclonal antibody (mAb), can225IgG, conjugated to the photo-absorber, IR700DX, in three EGFR expressing canine transitional cell carcinoma (TCC) cell lines as a prelude to possible canine clinical studies. Can225-IR700 conjugate showed specific binding and cell-specific killing after NIR-PIT on EGFR expressing cells in vitro. In the in vivo study, can225-IR700 conjugate demonstrated accumulation of the fluorescent conjugate with high tumor-to-background ratio. Tumor-bearing mice were separated into 4 groups: (1) no treatment; (2) 100 µg of can225-IR700 i.v. only; (3) NIR light exposure only; (4) 100 µg of can225-IR700 i.v., NIR light exposure. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other groups (p < 0.001), and significantly prolonged survival was achieved (p < 0.001 vs. other groups) in the treatment groups. In conclusion, NIR-PIT with can225-IR700 is a promising treatment for canine EGFR-expressing cancers, including invasive transitional cell carcinoma in pet dogs, that could provide a pathway to translation to humans. PMID:29721181

ErbB activation signatures as potential biomarkers for anti-ErbB3 treatment in HNSCC.

PubMed

Alvarado, Diego; Ligon, Gwenda F; Lillquist, Jay S; Seibel, Scott B; Wallweber, Gerald; Neumeister, Veronique M; Rimm, David L; McMahon, Gerald; LaVallee, Theresa M

2017-01-01

Head and neck squamous cell carcinoma (HNSCC) accounts for 3-5% of all tumor types and remains an unmet medical need with only two targeted therapies approved to date. ErbB3 (HER3), the kinase-impaired member of the EGFR/ErbB family, has been implicated as a disease driver in a number of solid tumors, including a subset of HNSCC. Here we show that the molecular components required for ErbB3 activation, including its ligand neuregulin-1 (NRG1), are highly prevalent in HNSCC and that HER2, but not EGFR, is the major activating ErbB3 kinase partner. We demonstrate that cetuximab treatment primarily inhibits the ERK signaling pathway and KTN3379, an anti-ErbB3 monoclonal antibody, inhibits the AKT signaling pathway, and that dual ErbB receptor inhibition results in enhanced anti-tumor activity in HNSCC models. Surprisingly, we found that while NRG1 is required for ErbB3 activation, it was not sufficient to fully predict for KTN3379 activity. An evaluation of HNSCC patient samples demonstrated that NRG1 expression was significantly associated with expression of the EGFR ligands amphiregulin (AREG) and transforming growth factor α (TGFα). Furthermore, NRG1-positive HNSCC cell lines that secreted high levels of AREG and TGFα or contained high levels of EGFR homodimers (H11D) demonstrated a better response to KTN3379. Although ErbB3 and EGFR activation are uncoupled at the receptor level, their respective signaling pathways are linked through co-expression of their respective ligands. We propose that NRG1 expression and EGFR activation signatures may enrich for improved efficacy of anti-ErbB3 therapeutic mAb approaches when combined with EGFR-targeting therapies in HNSCC.

Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells.

PubMed

Tang, Zheng-Hai; Cao, Wen-Xiang; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian

2017-04-15

Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of more red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-l-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Ubiquitin Ligase Cbl-b Is Involved in Icotinib (BPI-2009H)-Induced Apoptosis and G1 Phase Arrest of EGFR Mutation-Positive Non-Small-Cell Lung Cancer

PubMed Central

Mu, Xiaodong; Zhang, Ye; Qu, Xiujuan; Hou, Kezuo; Kang, Jian; Hu, Xuejun; Liu, Yunpeng

2013-01-01

Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μM, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells. PMID:23586056

Ubiquitin ligase Cbl-b is involved in icotinib (BPI-2009H)-induced apoptosis and G1 phase arrest of EGFR mutation-positive non-small-cell lung cancer.

PubMed

Mu, Xiaodong; Zhang, Ye; Qu, Xiujuan; Hou, Kezuo; Kang, Jian; Hu, Xuejun; Liu, Yunpeng

2013-01-01

Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μ M, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

PubMed Central

Ahmad, Gulzar; Mohapatra, Bhopal; Schulte, Nancy A.; Nadeau, Scott; Luan, Haitao; Zutshi, Neha; Tom, Eric; Ortega-Cava, Cesar; Tu, Chun; Sanada, Masashi; Ogawa, Seishi; Toews, Myron L.; Band, Vimla; Band, Hamid

2014-01-01

Members of the Casitas B-Lineage Lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK. PMID:25449262

The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.

PubMed

Shao, Genbao; Wang, Ranran; Sun, Aiqin; Wei, Jing; Peng, Ke; Dai, Qian; Yang, Wannian; Lin, Qiong

2018-02-19

EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion. NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.

The gefitinib long-term responder (LTR)--a cancer stem-like cell story? Insights from molecular analyses of German long-term responders treated in the IRESSA expanded access program (EAP).

PubMed

Gottschling, Sandra; Herpel, Esther; Eberhardt, Wilfried E E; Heigener, David F; Fischer, Jürgen R; Köhne, Claus-Henning; Kortsik, Cornelius; Kuhnt, Thomas; Muley, Thomas; Meister, Michael; Bischoff, Helge G; Klein, Peter; Moldenhauer, Ines; Schnabel, Philipp A; Thomas, Michael; Penzel, Roland

2012-07-01

In selected patients with advanced non-small cell lung cancer (NSCLC) the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor (TKI) gefitinib (IRESSA) shows response rates of ≥ 70% and a significant prolongation of progression free survival (PFS). However, cogent biomarkers predicting long-term response to EGFR-TKIs are yet lacking. Cancer stem-like cells (CSC) are thought to play a pivotal role in tumor regeneration and appear to be influenced by the EGFR-pathway. This makes them a promising candidate for predicting long-term response to EGFR-TKIs. We analyzed pre-therapeutic tissue specimens of a rare and specific subset of previously treated German patients with advanced NSCLC who experienced ≥ 3 year response to gefitinib within the International IRESSA EAP. 11/20 identified long-term responders (LTRs) had appropriate tissue specimens available. Those were analyzed for EGFR and k-ras (Kirsten rat sarcoma) mutations, EGFR and c-met (met proto-oncogene) amplifications and protein expression of EGFR, E-cadherin/vimentin and the CSC antigens CD133 and BCRP1 (breast cancer resistance protein 1). The results were compared to primary resistant patients (RPs) and intermediate responders (IRs) showing a median response of 8.6 months. Each group consisted of 6 women and 5 men, with 1 squamous cell carcinoma (SCC) and 10 adenocarcinoma (AC). Along the LTRs, all but the SCC had EGFR mutations, whereas the RPs had no EGFR, but k-ras mutations in 5/11 cases. 8/11 IRs had EGFR and 3/11 k-ras mutations, of which 2 occurred concomitantly. One patient of each group had an EGFR and/or c-met amplification. EGFR and E-cadherin/vimentin expression was not different between the groups, whereas CD133 was expressed only in 4/10 LTRs and BCRP1 predominantly in responders. The LTRs showed a substantially longer mean PFS to previous therapies, a substantially lower number of metastatic sites and almost exclusively pulmonary or pleural metastasis. LTRs display established properties of EGFR-TKI responders. Antigens characterizing CSC might identify a fraction of LTRs and matter of interest for further evaluation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Early Growth Response-1 Induces and Enhances Vascular Endothelial Growth Factor-A Expression in Lung Cancer Cells

PubMed Central

Shimoyamada, Hiroaki; Yazawa, Takuya; Sato, Hanako; Okudela, Koji; Ishii, Jun; Sakaeda, Masashi; Kashiwagi, Korehito; Suzuki, Takehisa; Mitsui, Hideaki; Woo, Tetsukan; Tajiri, Michihiko; Ohmori, Takahiro; Ogura, Takashi; Masuda, Munetaka; Oshiro, Hisashi; Kitamura, Hitoshi

2010-01-01

Vascular endothelial growth factor-A (VEGF-A) is crucial for angiogenesis, vascular permeability, and metastasis during tumor development. We demonstrate here that early growth response-1 (EGR-1), which is induced by the extracellular signal–regulated kinase (ERK) pathway activation, activates VEGF-A in lung cancer cells. Increased EGR-1 expression was found in adenocarcinoma cells carrying mutant K-RAS or EGFR genes. Hypoxic culture, siRNA experiment, luciferase assays, chromatin immunoprecipitation, electrophoretic mobility shift assays, and quantitative RT-PCR using EGR-1–inducible lung cancer cells demonstrated that EGR-1 binds to the proximal region of the VEGF-A promoter, activates VEGF-A expression, and enhances hypoxia inducible factor 1α (HIF-1α)-mediated VEGF-A expression. The EGR-1 modulator, NAB-2, was rapidly induced by increased levels of EGR-1. Pathology samples of human lung adenocarcinomas revealed correlations between EGR-1/HIF-1α and VEGF-A expressions and relative elevation of EGR-1 and VEGF-A expression in mutant K-RAS- or EGFR-carrying adenocarcinomas. Both EGR-1 and VEGF-A expression increased as tumors dedifferentiated, whereas HIF-1α expression did not. Although weak correlation was found between EGR-1 and NAB-2 expressions on the whole, NAB-2 expression decreased as tumors dedifferentiated, and inhibition of DNA methyltransferase/histone deacetylase increased NAB-2 expression in lung cancer cells despite no epigenetic alteration in the NAB-2 promoter. These findings suggest that EGR-1 plays important roles on VEGF-A expression in lung cancer cells, and epigenetic silencing of transactivator(s) associated with NAB-2 expression might also contribute to upregulate VEGF-A expression. PMID:20489156

DNA methylation down-regulates EGFR expression in chicken

USDA-ARS?s Scientific Manuscript database

The epidermal growth factor receptor (EGFR), a growth-factor-receptor tyrosine kinase, was found up-regulated in numerous tumors, which provides a good target for cancer therapy. Although it was documented that oncoviruses are responsible for the activation of EGFR in tumors, the impact of Marek’s d...

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

PubMed Central

Chen, Tzu-Chi; Liu, Yu-Wen; Huang, Yei-Hsuan; Yeh, Yi-Chen; Chou, Teh-Ying; Wu, Yu-Chung; Wu, Chun-Chi; Chen, Yi-Rong; Cheng, Hui-Chuan; Lu, Pei-Jung; Lai, Jin-Mei; Huang, Chi-Ying F.

2013-01-01

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations. PMID:23520446

ADAM17 and EGFR regulate IL-6 receptor and amphiregulin mRNA expression and release in cigarette smoke-exposed primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD).

PubMed

Stolarczyk, Marta; Amatngalim, Gimano D; Yu, Xiao; Veltman, Mieke; Hiemstra, Pieter S; Scholte, Bob J

2016-08-01

Aberrant activity of a disintegrin and metalloprotease 17 (ADAM17), also known as TACE, and epidermal growth factor receptor (EGFR) has been suggested to contribute to chronic obstructive pulmonary disease (COPD) development and progression. The aim of this study was to investigate the role of these proteins in activation of primary bronchial epithelial cells differentiated at the air-liquid interface (ALI-PBEC) by whole cigarette smoke (CS), comparing cells from COPD patients with non-COPD CS exposure of ALI-PBEC enhanced ADAM17-mediated shedding of the IL-6 receptor (IL6R) and the EGFR agonist amphiregulin (AREG) toward the basolateral compartment, which was more pronounced in cells from COPD patients than in non-COPD controls. CS transiently increased IL6R and AREG mRNA in ALI-PBEC to a similar extent in cultures from both groups, suggesting that posttranslational events determine differential shedding between COPD and non-COPD cultures. We show for the first time by in situ proximity ligation (PLA) that CS strongly enhances interactions of phosphorylated ADAM17 with AREG and IL-6R in an intracellular compartment, suggesting that CS-induced intracellular trafficking events precede shedding to the extracellular compartment. Both EGFR and ADAM17 activity contribute to CS-induced IL-6R and AREG protein shedding and to mRNA expression, as demonstrated using selective inhibitors (AG1478 and TMI-2). Our data are consistent with an autocrine-positive feedback mechanism in which CS triggers shedding of EGFR agonists evoking EGFR activation, in ADAM17-dependent manner, and subsequently transduce paracrine signaling toward myeloid cells and connective tissue. Reducing ADAM17 and EGFR activity could therefore be a therapeutic approach for the tissue remodeling and inflammation observed in COPD. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Constitutively Activated STAT3 Frequently Coexpresses with Epidermal Growth Factor Receptor in High-Grade Gliomas and Targeting STAT3 Sensitizes Them to Iressa and Alkylators

PubMed Central

Lo, Hui-Wen; Cao, Xinyu; Zhu, Hu; Ali-Osman, Francis

2009-01-01

Purpose The goals of this study are to elucidate the relationship of the oncogenic transcription factor signal transducer and activator of transcription 3(STAT3) with glioma aggressiveness and to understand the role of high STAT3 activity in the resistance of malignant gliomas and medulloblastomas to chemotherapy. Experimental Design Immunohistochemical staining and biochemical methods were used to examine the extent of STAT3 activation and EGFR expression in primary specimens and cell lines, respectively. Cellular response to drug treatments was determined using cell cytotoxicity and clonogenic growth assays. Results We found STAT3 to be constitutively activated in 60% of primary high-grade/malignant gliomas and the extent of activation correlated positively with glioma grade. High levels of activated/phosphorylated STAT3 were also present in cultured human malignant glioma and medulloblastoma cells. Three STAT3-activating kinases, Janus-activated kinase 2 (JAK2), EGFR, and EGFRvIII, contributed to STAT3 activation. An inhibitor toJAK2/STAT3, JSI-124, significantly reduced expression of STAT3 target genes, suppressed cancer cell growth, and induced apoptosis. Furthermore, we found that STAT3 constitutive activation coexisted with EGFR expression in 27.2% of primary high-grade/malignant gliomas and such coexpression correlated positively with glioma grade. Combination of an anti-EGFR agent Iressa and a JAK2/STAT3 inhibitor synergistically suppressed STAT3 activation and potently killed glioblastoma cell lines that expressed EGFR or EGFRvIII. JSI-124 also sensitized malignant glioma and medulloblastoma cells to temozolomide, 1,3-bis(2-chloroethyl)-1-nitrosourea, and cisplatin in which a synergism existed between JSI-124 and cisplatin. Conclusion STAT3 constitutive activation, alone and in concurrence with EGFR expression, plays an important role in high-grade/malignant gliomas and targeting STAT3/JAK2 sensitizes these tumors to anti-EGFR and alkylating agents. PMID:18829483

Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

PubMed Central

Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

2016-01-01

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

Retrospective Molecular Epidemiology Study of PD-L1 Expression in Patients with EGFR-Mutant Non-small Cell Lung Cancer.

PubMed

Cho, Jong Ho; Zhou, Wei; Choi, Yoon-La; Sun, Jong-Mu; Choi, Hyejoo; Kim, Tae-Eun; Dolled-Filhart, Marisa; Emancipator, Kenneth; Rutkowski, Mary Anne; Kim, Jhingook

2018-01-01

Data are limited on programmed death ligand 1 (PD-L1) expression in epidermal growth factor receptor ( EGFR )-mutant non-small cell lung cancer (NSCLC). We retrospectively evaluated the relationship between PD-L1 expression and recurrence-free survival (RFS) and overall survival in 319 patients with EGFR -mutant NSCLC who were treated at Samsung Medical Center from 2006 to 2014. Membranous PD-L1 expression on tumor cells was measured using the PD-L1 IHC 22C3 pharmDx antibody and reported as tumor proportion score (TPS). Kaplan-Meier methods, log-rank test, and Cox proportional hazards models were used for survival analysis. All patients had ≥1 EGFR mutation-54% in exon 19 and 39% in exon 21. Overall, 51% of patients had PD-L1-positive tumors. The prevalence of PD-L1 positivity was higher among patients with stages II-IV versus stage I disease (64% vs. 44%) and among patients with other EGFR mutations (75%) than with L858R mutation (39%) or exon 19 deletion (52%). PD-L1 positivity was associated with shorter RFS, with an adjusted hazard ratio of 1.52 (95% confidence interval [CI], 0.81 to 2.84; median, 18 months) for the PD-L1 TPS ≥ 50% group, 1.51 (95% CI, 1.02 to 2.21; median, 31 months) for the PD-L1 TPS 1%-49% group, and 1.51 (95% CI, 1.05 to 2.18) for the combined PD-L1-positive groups (TPS ≥ 1%) compared with the PD-L1-negative group (median, 35 months). PD-L1 expression is associated with disease stage and type of EGFR mutation. PD-L1 positivity might be associated with worse RFS among patients with surgically treated EGFR -mutant NSCLC.

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

NASA Astrophysics Data System (ADS)

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-03-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR-TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure-activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

PubMed

Griffero, Fabrizio; Daga, Antonio; Marubbi, Daniela; Capra, Maria Cristina; Melotti, Alice; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adriana; Porcile, Carola; Barbieri, Federica; Favoni, Roberto E; Lo Casto, Michele; Zona, Gianluigi; Spaziante, Renato; Florio, Tullio; Corte, Giorgio

2009-03-13

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Osimertinib (AZD9291) decreases programmed death ligand-1 in EGFR-mutated non-small cell lung cancer cells.

PubMed

Jiang, Xiao-Ming; Xu, Yu-Lian; Huang, Mu-Yang; Zhang, Le-Le; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian

2017-11-01

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In NSCLC patients, an EGFR mutation is likely to be correlated with high levels of expression of programmed death ligand-1 (PD-L1). Here, we showed that osimertinib decreased PD-L1 expression in human EGFR mutant NSCLC cells in vitro. Osimertinib (125 nmol/L) markedly suppressed PD-L1 mRNA expression in both NCI-H1975 and HCC827 cells. Pretreatment with the N-linked glycosylation inhibitor tunicamycin, osimertinib clearly decreased the production of new PD-L1 protein probably due to a reduction in mRNA. After blocking transcription and translation processes with actinomycin D and cycloheximide, respectively, osimertinib continued to reduce the expression of PD-L1, demonstrating that osimertinib might degrade PD-L1 at the post-translational level, which was confirmed by a cycloheximide chase assay, revealing that osimertinib (125 nmol/L) decreased the half-life of PD-L1 from approximately 17.8 h and 13.8 h to 8.6 h and 4.6 h, respectively, in NCI-H1975 and HCC827 cells. Pretreatment with the proteasome inhibitors (MG-132 or bortezomib) blocked the osimertinib-induced degradation of PD-L1, but an inhibitor of autophagy (chloroquine) did not. In addition, inhibition of GSK3β by LiCl prevented osimertinib-induced PD-L1 degradation. The results demonstrate that osimertinib reduces PD-L1 mRNA expression and induces its protein degradation, suggesting that osimertinib may reactivate the immune activity of T cells in the tumor microenvironment in EGFR-mutated NSCLC patients.

A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

PubMed Central

Habtemichael, Negusse; Bier, Carolin; Unruhe, Britta; Weisheit, Simona; Spange, Stephanie; Nonnenmacher, Frank; Fetz, Verena; Ginter, Torsten; Reichardt, Sigrid; Liebmann, Claus; Schneider, Günter; Krämer, Oliver H.

2012-01-01

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p<0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P<0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation. PMID:22289787

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg; Zhu Congju; Wong Yinling

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival,more » {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G{sub 2}/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair.« less

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

PubMed

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem glioma cells. Gefitinib differentially enhances radiosensitivity of stem-like gliomaspheres by reducing EGFR-Akt activation and DNA-PKcs expression, accompanied by enhanced irradiation-induced DNA DSBs and inhibition of DSB repair. Copyright © 2012 Elsevier Inc. All rights reserved.

Mutual regulation of TGF-β1, TβRII and ErbB receptors expression in human thyroid carcinomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mincione, Gabriella, E-mail: g.mincione@unich.it; Center of Excellence on Aging, Ce.S.I., ‘G. d'Annunzio’ University Foundation, Chieti; Tarantelli, Chiara

2014-09-10

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC andmore » FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype. - Highlights: • We reinforce the hypothesis of a cross talk between EGF and TGF-β1 in follicular thyroid carcinoma. • Increased migration MAPK-dependent is observed after EGF+TGF-β1 treatment in follicular thyroid carcinoma cells. • EGF and TGF-β1 caused opposite effect on the migratory ability in B-CPAP and in FTC-133 cells. • TGF-β1, TβRII and EGFR are overexpressed in follicular thyroid carcinoma.« less

Significance of Epidermal Growth Factor Receptor in the Radiation Resistance of Glioblastoma Tumors

NASA Astrophysics Data System (ADS)

Petrás, Miklós; Lajtos, Tamás; Pintye, Éva; Feuerstein, Burt G.; Szöllősi, János; Vereb, György

2008-12-01

In the United States, a dramatically increased incidence and mortality of brain tumors have been observed over the past decades. Of the ˜44 thousand new cases of primary malignant and benign brain tumors diagnosed per year, high grade astrocytomas or multiform glioblastomas show particularly bad prognosis in spite of therapeutic developments. Current management of multiform glioblastoma includes the most extensive surgical resection possible, followed by adjuvant radio- and chemotherapy. However, treatment is frequently hampered by decreased radiosensitivity of the tumor. Recent studies revealed that subpopulations of glioblastoma cells show amplified checkpoint activation of the cell cycle upon ionizing radiation, which induces overactivation of DNA repair processes and leads to maintained proliferation rate as well as clinically observed radioresistance and recurrence of the tumor over time. In addition, overexpression of some transmembrane receptors has also been implicated in radioresistance. However, the role of the overexpressed proteins can only be interpreted reliably if their multi-faceted molecular interactions are properly characterized. Thus, based on recent evidence for the functional crosstalk between certain cell adhesion molecules and receptor tyrosine kinases, we have examined the molecular interactions of the receptor tyrosine kinase EGFR and the cell adhesion molecule β1-integrin using flow cytometric and microscopic fluorescence resosnance energy transfer (FRET) measurements on two cellular model systems showing similar expression patterns to low and high grade astrocytomas. On the one hand, U251 glioblastoma clones established by introducing varying amounts of extra chromosome 7 into the cells, and on the other hand stable, high and low EGFR expressing transfenctant U251 NCI sublines were investigated. The results revealed that increased EGFR and β1-integrin expression levels correlate with stronger EGFR—β1-integrin heteroassociation, while concurrently the EGFR homoassociation is decreased, suggesting that β1-integrins may dynamically modulate the homoassociation state of EGFR receptors. This functional relationship may play an important role in decreasing radiosensitivity and tumor progression, especially since the EGFR—β1-integrin molecular interaction appears to promote radioresistance via the Akt pathway.

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance.

PubMed

Sevelda, Florian; Mayr, Lisa; Kubista, Bernd; Lötsch, Daniela; van Schoonhoven, Sushilla; Windhager, Reinhard; Pirker, Christine; Micksche, Michael; Berger, Walter

2015-11-02

Enhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines. We have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed. Osteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential. Our data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.

Acquired MET expression confers resistance to EGFR inhibition in a mouse model of glioblastoma multiforme.

PubMed

Jun, H J; Acquaviva, J; Chi, D; Lessard, J; Zhu, H; Woolfenden, S; Bronson, R T; Pfannl, R; White, F; Housman, D E; Iyer, L; Whittaker, C A; Boskovitz, A; Raval, A; Charest, A

2012-06-21

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type epidermal growth factor receptor (EGFR) and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that an important component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.

Epidermal growth factor receptor inhibitors in cancer treatment: advances, challenges and opportunities.

PubMed

Modjtahedi, Helmout; Essapen, Sharadah

2009-11-01

Aberrant expression of the epidermal growth factor receptor (EGFR) system has been reported in a wide range of epithelial cancers. In some studies, this has also been associated with a poor prognosis and resistance to the conventional forms of therapies. These discoveries have led to the strategic development of several kinds of EGFR inhibitors, five of which have gained US Food and Drug Administration approval for the treatment of patients with non-small-cell lung cancer (gefitinib and erlotinib), metastatic colorectal cancer (cetuximab and panitumumab), head and neck (cetuximab), pancreatic cancer (erlotinib) and breast (lapatinib) cancer. Despite these advances and recent studies on the predictive value of activating EGFR mutation and KRAS mutations with response in non-small-cell lung cancer and colon cancer patients, there is currently no reliable predictive marker for response to therapy with the anti-EGFR monoclonal antibodies cetuximab and panitumumab or the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib. In particular, there has been no clear association between the expression of EGFR, determined by the US Food and Drug Administration-approved EGFR PharmDX kit, and response to the EGFR inhibitors. Here, we discuss some of the controversial data and explanatory factors as well as future studies for the establishment of more reliable markers for response to therapy with EGFR inhibitors. Such investigations should lead to the selection of a more specific subpopulation of cancer patients who benefit from therapy with EGFR inhibitors, but equally to spare those who will receive no benefit or a detrimental effect from such biological agents.

Activation of EGFR Bypass Signaling by TGFα Overexpression Induces Acquired Resistance to Alectinib in ALK-Translocated Lung Cancer Cells.

PubMed

Tani, Tetsuo; Yasuda, Hiroyuki; Hamamoto, Junko; Kuroda, Aoi; Arai, Daisuke; Ishioka, Kota; Ohgino, Keiko; Miyawaki, Masayoshi; Kawada, Ichiro; Naoki, Katsuhiko; Hayashi, Yuichiro; Betsuyaku, Tomoko; Soejima, Kenzo

2016-01-01

Alectinib is a highly selective ALK inhibitor and shows promising efficacy in non-small cell lung cancers (NSCLC) harboring the EML4-ALK gene rearrangement. The precise mechanism of acquired resistance to alectinib is not well defined. The purpose of this study was to clarify the mechanism of acquired resistance to alectinib in ALK-translocated lung cancer cells. We established alectinib-resistant cells (H3122-AR) from the H3122 NSCLC cell line, harboring the EML4-ALK gene rearrangement, by long-term exposure to alectinib. The mechanism of acquired resistance to alectinib in H3122-AR cells was evaluated by phospho-receptor tyrosine kinase (phospho-RTK) array screening and Western blotting. No mutation of the ALK-TK domain was found. Phospho-RTK array analysis revealed that the phosphorylation level of EGFR was increased in H3122-AR cells compared with H3122. Expression of TGFα, one of the EGFR ligands, was significantly increased and knockdown of TGFα restored the sensitivity to alectinib in H3122-AR cells. We found combination therapy targeting ALK and EGFR with alectinib and afatinib showed efficacy both in vitro and in a mouse xenograft model. We propose a preclinical rationale to use the combination therapy with alectinib and afatinib in NSCLC that acquired resistance to alectinib by the activation of EGFR bypass signaling. ©2015 American Association for Cancer Research.

Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.

PubMed

Santra, Manoranjan; Chopp, Michael; Santra, Sutapa; Nallani, Ankita; Vyas, Shivam; Zhang, Zheng Gang; Morris, Daniel C

2016-01-01

Thymosin beta 4 (Tβ4), a secreted 43 amino acid peptide, promotes oligodendrogenesis, and improves neurological outcome in rat models of neurologic injury. We demonstrated that exogenous Tβ4 treatment up-regulated the expression of the miR-200a in vitro in rat brain progenitor cells and in vivo in the peri-infarct area of rats subjected to middle cerebral artery occlusion (MCAO). The up-regulation of miR-200a down-regulated the expression of the following targets in vitro and in vivo models: (i) growth factor receptor-bound protein 2 (Grb2), an adaptor protein involved in epidermal growth factor receptor (EGFR)/Grb2/Ras/MEK/ERK1/c-Jun signaling pathway, which negatively regulates the expression of myelin basic protein (MBP), a marker of mature oligodendrocyte; (ii) ERRFI-1/Mig-6, an endogenous potent kinase inhibitor of EGFR, which resulted in activation/phosphorylation of EGFR; (iii) friend of GATA 2, and phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which are potent inhibitors of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, and resulted in marked activation of AKT; and (iv) transcription factor, p53, which induces pro-apoptotic genes, and possibly reduced apoptosis of the progenitor cells subjected to oxygen glucose deprivation (OGD). Anti-miR-200a transfection reversed all the effects of Tβ4 treatment in vitro. Thus, Tβ4 up-regulated MBP synthesis, and inhibited OGD-induced apoptosis in a novel miR-200a dependent EGFR signaling pathway. Our findings of miR-200a-mediated protection of progenitor cells may provide a new therapeutic importance for the treatment of neurologic injury. Tβ4-induced micro-RNA-200a (miR-200a) regulates EGFR signaling pathways for MBP synthesis and apoptosis: up-regulation of miR-200a after Tβ4 treatment, increases MBP synthesis after targeting Grb2 and thereby inactivating c-Jun from inhibition of MBP synthesis; and also inhibits OGD-mediated apoptosis after targeting EGFR inhibitor (Mig-6), PI3K inhibitors (FOG2 and Pten) and an inducer (p53) of pro-apoptotic genes, for AKT activation and down-regulation of p53. These findings may contribute the therapeutic benefits for stroke and other neuronal diseases associated with demyelination disorders. © 2015 International Society for Neurochemistry.

Apatinib enhances antitumour activity of EGFR-TKIs in non-small cell lung cancer with EGFR-TKI resistance.

PubMed

Li, Fang; Zhu, Tengjiao; Cao, Baoshan; Wang, Jiadong; Liang, Li

2017-10-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)-rechallenged therapy for EGFR-mutant non-small cell lung cancer (NSCLC) patients who acquired resistance showed moderate efficacy. Considering the high interrelation between EGFR and vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) pathways, we firstly evaluated EGFR-TKI combined with apatinib (a highly selective VEGFR2 inhibitor) in EGFR-TKI-resistant model and patients. Effects of apatinib, gefitinib and gefitinib plus apatinib were assessed on four NSCLC cell lines (A549 with wild-type EGFR, H1975 harbouring L858R and T790M, H1650 and HCC827 harbouring E746_A750 deletion) and xenograft model of acquired resistance that was established by injecting H1975 cells. Furthermore, we retrospectively evaluated EGFR-TKI rechallenge with apatinib in 16 patients. Gefitinib plus apatinib strengthened the effect of gefitinib and apatinib alone on the four NSCLC cell lines, and H1975 was the most susceptible one. Co-administration delayed the tumour growth than mono-therapy in the xenograft model and had better effect on inhibiting the activation of EGFR and VEGFR2 and expression of CD31 (an angiogenesis marker) and vascular endothelial growth factor A (an important pro-angiogenesis factor in the tumour microenvironment). Changes in protein expression of protein kinase B/mammalian target of rapamycin and extracellular signal-regulated kinase pathways demonstrated the potent inhibitory effect on the pro-survival signalling pathways by combined therapy. EGFR-TKI rechallenge with apatinib achieved a median progression-free survival of 4.60 months (95% confidence interval, 2.23-12.52 months) in the patients. Apatinib significantly potentiated the antitumour effect of gefitinib in NSCLC with T790M-related EGFR-TKI resistance both in vivo and vitro. EGFR-TKI rechallenge with apatinib might represent a new option for NSCLC with T790M or unknown resistance mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genistein enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nuclear factor kappa B in nonsmall cell lung cancer cell lines.

PubMed

Gadgeel, Shirish M; Ali, Shadan; Philip, Philip A; Wozniak, Antoinette; Sarkar, Fazlul H

2009-05-15

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown modest clinical benefit in patients with relapsed nonsmall cell lung cancer (NSCLC). Down-regulation of Akt appears to correlate with the antitumor activity of EGFR-TKIs. Akt activates nuclear factor kappa B (NF-kappaB), which transcribes genes important for cell survival, invasion, and metastasis. The authors hypothesized that genistein, through the inhibition of NF-kappaB, could enhance the activity of EGFR-TKIs in NSCLCs. Three NSCLC cell lines with various EGFR mutation status and sensitivities to EGFR-TKIs were selected: H3255 (L858R), H1650 (del E746-A750), and H1781 (wild-type EGFR). Cells were treated with erlotinib, gefitinib, genistein, or the combination of each of the EGFR-TKIs with genistein. Cell survival and apoptosis were assessed, and expression levels of EGFR, pAkt, cyclooxygenase-2 (COX-2), E-cadherin, prostaglandin E(2) (PGE(2)), and NF-kappaB were measured. Both EGFR-TKIs demonstrated growth inhibition and apoptosis in each of the cell lines, but H1650 and H1781 were much less sensitive. Genistein demonstrated some antitumor activity in all cell lines, but enhanced growth inhibition and apoptosis when combined with the EGFR-TKIs in each of the cell lines. Both combinations down-regulated NF-kappaB significantly more than either agent alone in H3255. In addition, the combinations reduced the expression of EGFR, pAkt, COX-2, and PGE(2,) consistent with inactivation of NF-kappaB. The authors concluded that genistein enhances the antitumor effects of EGFR-TKIs in 3 separate NSCLC cell lines. This enhanced activity is in part because of greater reduction in the DNA-binding activity of NF-kappaB when EGFR-TKIs were combined with genistein.

Association of BIM Deletion Polymorphism and BIM-γ RNA Expression in NSCLC with EGFR Mutation.

PubMed

Isobe, Kazutoshi; Kakimoto, Atsushi; Mikami, Tetsuo; Kaburaki, Kyohei; Kobayashi, Hiroshi; Yoshizawa, Takahiro; Makino, Takashi; Otsuka, Hajime; Sano, G O; Sugino, Keishi; Sakamoto, Susumu; Takai, Yujiro; Tochigi, Naobumi; Iyoda, Akira; Homma, Sakae

This pilot study assessed the association of BIM deletion polymorphism and BIM RNA isoform in patients with EGFR-positive non-small cell lung cancer (NSCLC). The study included 33 patients with EGFR-positive NSCLC treated with gefitinib. BIM deletion polymorphism and BIM RNA isoform (EL/L/S/γ) were determined by polymerase chain reaction (PCR). BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism inside tumors (p=0.038) and around tumors (p=0.0024). Relative BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism (p=0.0017). Patients with BIM-γ had significantly shorter progression-free survival than those without BIM-γ (median: 304 vs. 732 days; p=0.023). Expression of BIM-γ mRNA and BIM deletion polymorphism were strongly associated. BIM-γ overexpression may have a role in apoptosis related to EGFR-tyrosine kinase inhibitor. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castillo, Gaelle del; Murillo, Miguel M.; IDIBELL-Institut de Recerca Oncologica, Gran Via s/n, Km 2.7, 08907 L'Hospitalet, Barcelona

Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by themore » proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.« less

[Current Possibilities for Predicting Responses to EGFR Blockade in Metastatic Colorectal Cancer].

PubMed

Němeček, R; Svoboda, M; Slabý, O

2016-01-01

The combination of modern systemic chemotherapy and anti-EGFR monoclonal antibodies improves overall survival and quality of life for patients with metastatic colorecal cancer. By contrast, the addition of anti-EGFR therapy to the treatment regime of resistant patients may lead to worse progression-free survival and overall survival. Therefore, identifying sensitive and resistant patients prior to targeted therapy of metastatic colorecal cancer is a key point during the initial decision making process. Previous research shows that primary resistance to EGFR blockade is in most cases caused by constitutive activation of signaling pathways downstream of EGFR. Of all relevant factors (mutation of KRAS, NRAS, BRAF, and PIK3CA oncogenes, inactivation of tumor suppressors PTEN and TP53, amplification of EGFR and HER2, and expression of epiregulin and amphiregulin, mikroRNA miR-31-3p, and miR-31-5p), only evaluation of KRAS and NRAS mutations has entered routine clinical practice. The role of the other markers still needs to be validated. The ongoing benefit of anti-EGFR therapy could be indicated by specific clinical parameters measured after the initiation of targeted therapy, including early tumor shrinkage, the deepness of the response, or hypomagnesemia. The accuracy of predictive dia-gnostic tools could be also increased by examining a combination of predictive markers using next generation sequencing methods. However, unjustified investigation of many molecular markers should be resisted as this may complicate interpretation of the results, particularly in terms of their specific clinical relevance. The aim of this review is to describe current possibilities with respect to predicting responses to EGFR blockade in the context of the EGFR pathway, and the utilization of such results in routine clinical practice.

Targeting EGF-receptor(s) - STAT1 axis attenuates tumor growth and metastasis through downregulation of MUC4 mucin in human pancreatic cancer.

PubMed

Seshacharyulu, Parthasarathy; Ponnusamy, Moorthy P; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Haridas, Dhanya; Yan, Ying; Ganti, Apar K; Batra, Surinder K

2015-03-10

Transmembrane proteins MUC4, EGFR and HER2 are shown to be critical in invasion and metastasis of pancreatic cancer. Besides, we and others have demonstrated de novo expression of MUC4 in ~70-90% of pancreatic cancer patients and its stabilizing effects on HER2 downstream signaling in pancreatic cancer. Here, we found that use of canertinib or afatinib resulted in reduction of MUC4 and abrogation of in vitro and in vivo oncogenic functions of MUC4 in pancreatic cancer cells. Notably, silencing of EGFR family member in pancreatic cancer cells decreased MUC4 expression through reduced phospho-STAT1. Furthermore, canertinib and afatinib treatment also inhibited proliferation, migration and survival of pancreatic cancer cells by attenuation of signaling events including pERK1/2 (T202/Y204), cyclin D1, cyclin A, pFAK (Y925) and pAKT (Ser473). Using in vivo bioluminescent imaging, we demonstrated that canertinib treatment significantly reduced tumor burden (P=0.0164) and metastasis to various organs. Further, reduced expression of MUC4 and EGFR family members were confirmed in xenografts. Our results for the first time demonstrated the targeting of EGFR family members along with MUC4 by using pan-EGFR inhibitors. In conclusion, our studies will enhance the translational acquaintance of pan-EGFR inhibitors for combinational therapies to combat against lethal pancreatic cancer.

Biological characterization of cetuximab-conjugated gold nanoparticles in a tumor animal model

NASA Astrophysics Data System (ADS)

Kao, Hao-Wen; Lin, Yi-Yu; Chen, Chao-Cheng; Chi, Kwan-Hwa; Tien, Der-Chi; Hsia, Chien-Chung; Lin, Wuu-Jyh; Chen, Fu-Du; Lin, Ming-Hsien; Wang, Hsin-Ell

2014-07-01

Gold nanoparticles (AuNPs) are widely applied to the diagnosis and treatment of cancer and can be modified to contain target-specific ligands via gold-thiolate bonding. This study investigated the pharmacokinetics and microdistribution of antibody-mediated active targeting gold nanoparticles in mice with subcutaneous lung carcinoma. We conjugated AuNPs with cetuximab (C225), an antibody-targeting epidermal growth factor receptor (EGFR), and then labeled with In-111, which created EGFR-targeted AuNPs. In vitro studies showed that after a 2 h incubation, the uptake of C225-conjugated AuNPs in high EGFR-expression A549 cells was 14.9-fold higher than that of PEGylated AuNPs; furthermore, uptake was also higher at 3.8-fold when MCF7 cells with lower EGFR-expression were used. MicroSPECT/CT imaging and a biodistribution study conducted by using a A549 tumor xenograft mouse model provided evidence of elevated uptake of the C225-conjugated AuNPs into the tumor cells as a result of active targeting. Moreover, the microdistribution of PEGylated AuNPs revealed that a large portion of AuNPs remained in the tumor interstitium, whereas the C225-conjugated AuNPs displayed enhanced internalization via antibody-mediated endocytosis. Our findings suggest that the anti-EGFR antibody-conjugated AuNPs are likely to be a plausible nano-sized vehicle for drug delivery to EGFR-expressing tumors.

Genetic risk variants in the CDKN2A/B, RTEL1 and EGFR genes are associated with somatic biomarkers in glioma.

PubMed

Ghasimi, Soma; Wibom, Carl; Dahlin, Anna M; Brännström, Thomas; Golovleva, Irina; Andersson, Ulrika; Melin, Beatrice

2016-05-01

During the last years, genome wide association studies have discovered common germline genetic variants associated with specific glioma subtypes. We aimed to study the association between these germline risk variants and tumor phenotypes, including copy number aberrations and protein expression. A total of 91 glioma patients were included. Thirteen well known genetic risk variants in TERT, EGFR, CCDC26, CDKN2A, CDKN2B, PHLDB1, TP53, and RTEL1 were selected for investigation of possible correlations with the glioma somatic markers: EGFR amplification, 1p/19q codeletion and protein expression of p53, Ki-67, and mutated IDH1. The CDKN2A/B risk variant, rs4977756, and the CDKN2B risk variant, rs1412829 were inversely associated (p = 0.049 and p = 0.002, respectively) with absence of a mutated IDH1, i.e., the majority of patients homozygous for the risk allele showed no or low expression of mutated IDH1. The RTEL1 risk variant, rs6010620 was associated (p = 0.013) with not having 1p/19q codeletion, i.e., the majority of patients homozygous for the risk allele did not show 1p/19q codeletion. In addition, the EGFR risk variant rs17172430 and the CDKN2B risk variant rs1412829, both showed a trend for association (p = 0.055 and p = 0.051, respectively) with increased EGFR copy number, i.e., the majority of patients homozygote for the risk alleles showed chromosomal gain or amplification of EGFR. Our findings indicate that CDKN2A/B risk genotypes are associated with primary glioblastoma without IDH mutation, and that there is an inverse association between RTEL1 risk genotypes and 1p/19q codeletion, suggesting that these genetic variants have a molecular impact on the genesis of high graded brain tumors. Further experimental studies are needed to delineate the functional mechanism of the association between genotype and somatic genetic aberrations.

Predictive value of EGFR-PI3K-pAKT-mTOR-pS6 pathway in sinonasal squamous cell carcinomas.

PubMed

Muñoz-Cordero, María Gabriela; López, Fernando; García-Inclán, Cristina; López-Hernández, Alejandro; Potes-Ares, Sira; Fernández-Vañes, Laura; Llorente, José Luis; Hermsen, Mario

2018-03-21

We have previously indicated that EGFR has a role in carcinogenesis in a subgroup of sinonasal squamous cell carcinomas (SNSCC). In addition, EGFR activates 2 of the most important intracellular signalling pathways: PI3K/pAKT/mTOR/pS6 and MAP pathway kinases. The objective of this study was to evaluate the involvement of the EGFR/PI3K/pAKT/mTOR/pS6 pathway and its relationship with clinical-pathological parameters and follow-up of sinonasal squamous cell carcinoma. The immunohistochemical expression of different components of the PI3K/AKT/mTOR/pS6 pathway and its relationship with various clinical-pathological parameters was studied in a series of 54 patients with SNSCC. Loss of PTEN expression was observed in 33/54 cases (61%) and pAKT, mTOR and pS6 pre-expression was observed in 19/54 cases (35%), 8/54 cases (15%), and 47/54 cases (87%), respectively. Loss of PTEN expression was related to intracranial invasion and development of regional metastases (p=0.005). Overexpression of pS6 was associated with a decrease in survival (p=0.008), presence of local recurrences (p=0.055), and worsening of overall prognosis (p=0.007). No significant relationships were observed between pAKT and mTOR expression and the clinicopathological parameters studied. Alterations in the expression of EGFR/PI3K/pAKT/mTOR/pS6 pathway components are common in a subgroup of SNSCC. This study reveals that the absence of pS6 overexpression is associated with better clinical outcomes. Therefore, pS6 expression could be considered as an unfavourable prognostic marker. Copyright © 2018. Publicado por Elsevier España, S.L.U.

JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor

PubMed Central

Duan, Shan-zhou; Xia, Ying-chen; Zhu, Rong-ying; Chen, Yong-bing

2015-01-01

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment. PMID:25869210

Cigarette smoke enhances oncogene addiction to c-MET and desensitizes EGFR-expressing non-small cell lung cancer to EGFR TKIs.

PubMed

Tu, Chih-Yen; Cheng, Fang-Ju; Chen, Chuan-Mu; Wang, Shu-Ling; Hsiao, Yu-Chun; Chen, Chia-Hung; Hsia, Te-Chun; He, Yu-Hao; Wang, Bo-Wei; Hsieh, I-Shan; Yeh, Yi-Lun; Tang, Chih-Hsin; Chen, Yun-Ju; Huang, Wei-Chien

2018-05-01

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke-derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine-derived nitrosamine ketone (NNK), reduced the sensitivity of wild-type EGFR-expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P-treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c-MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c-MET reduces B[α]P-induced Akt activation. The CSE-treated NSCLC cells are sensitive to the c-MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c-MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

PubMed

Banisadr, Arsham; Safdari, Yaghoub; Kianmehr, Anvarsadat; Pourafshar, Mahdieh

2018-04-03

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly 4 Ser) 3 ] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Usefulness of predictive tests for cancer treatment.

PubMed

Rosell, R; Cuello, M; Cecere, F; Santarpia, M; Reguart, N; Felip, E; Taron, M

2006-08-01

This review highlights the numerous molecular biology findings in the field of lung cancer with potential therapeutic impact in both the near and distant future. At least six lines of research have recently emerged as potential contributors to changes in clinical practice. Abundant pre-clinical and clinical data indicate that BRCA1 mRNA expression is a differential modulator of chemotherapy sensitivity. Low levels predict cisplatin sensitivity and antimicrotubule drug resistance, and the opposite occurs with high levels. Secondly, single nucleotide polymorphisms in the ERCC1 gene influence survival and toxicity with cisplatin-based chemotherapy. The main core of recent research has centered on EGFR mutations and gene copy numbers. For the first time, EGFR mutations have been shown to predict dramatic responses in metastatic lung adenocarcinomas, with a threefold increase in time to progression and survival in patients receiving EGFR tyrosine kinase inhibitors. In contrast, K-ras mutations confer a negative effect in these patients. Evidence has also been accumulated on the crosstalk between estrogen and EGFR receptor pathways, paving the way for clinical trials of EGFR tyrosine kinase inhibitors plus aromatase inhibitors. microRNAs control the expression of cognate target genes, and downregulation of Dicer has been shown to be a strong predictor of relapse in surgically resected non-small-cell lung cancer patients. Finally, overexpression of the Wingless-type (Wnt) genes and methylation of Wnt antagonists like WIF and secreted frizzled related proteins have been documented in non-small-cell lung cancer and are believed to be an important mechanism of cancer stem cell maintenance.

Type I γ Phosphatidylinositol Phosphate 5-Kinase i5 Controls the Ubiquitination and Degradation of the Tumor Suppressor Mitogen-inducible Gene 6.

PubMed

Sun, Ming; Cai, Jinyang; Anderson, Richard A; Sun, Yue

2016-10-07

Mitogen-inducible gene 6 (Mig6) is a tumor suppressor, and the disruption of Mig6 expression is associated with cancer development. Mig6 directly interacts with epidermal growth factor receptor (EGFR) to suppress the activation and downstream signaling of EGFR. Therefore, loss of Mig6 enhances EGFR-mediated signaling and promotes EGFR-dependent carcinogenesis. The molecular mechanism modulating Mig6 expression in cancer remains unclear. Here we demonstrate that type I γ phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), stabilizes Mig6 expression. Knockdown of PIPKIγi5 leads to the loss of Mig6 expression, which dramatically enhances and prolongs EGFR-mediated cell signaling. Loss of PIPKIγi5 significantly promotes Mig6 protein degradation via proteasomes, but it does not affect the Mig6 mRNA level. PIPKIγi5 directly interacts with the E3 ubiquitin ligase neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1). The C-terminal domain of PIPKIγi5 and the WW1 and WW2 domains of NEDD4-1 are required for their interaction. The C2 domain of NEDD4-1 is required for its interaction with PtdIns(4,5)P 2 By binding with NEDD4-1 and producing PtdIns(4,5)P 2 , PIPKIγi5 perturbs NEDD4-1-mediated Mig6 ubiquitination and the subsequent proteasomal degradation. Thus, loss of NEDD4-1 can rescue Mig6 expression in PIPKIγi5 knockdown cells. In this way, PIPKIγi5, NEDD4-1, and Mig6 form a novel molecular nexus that controls EGFR activation and downstream signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Combinatorial Effects of Lapatinib and Rapamycin in Triple-Negative Breast Cancer Cells

PubMed Central

Liu, Tongrui; Yacoub, Rami; Taliaferro-Smith, LaTonia D.; Sun, Shi-Yong; Graham, Tisheeka R.; Dolan, Ryan; Lobo, Christine; Tighiouart, Mourad; Yang, Lily; Adams, Amy; O'Regan, Ruth M.

2016-01-01

Triple-negative breast cancers, which lack estrogen receptor, progesterone receptor, and HER2/neu overexpression, account for approximately 15% of breast cancers, but occur more commonly in African Americans. The poor survival outcomes seen with triple-negative breast cancers patients are, in part, due to a lack of therapeutic targets. Epidermal growth factor receptor (EGFR) is overexpressed in 50% of triple-negative breast cancers, but EGFR inhibitors have not been effective in patients with metastatic breast cancers. However, mTOR inhibition has been shown to reverse resistance to EGFR inhibitors. We examined the combination effects of mTOR inhibition with EGFR inhibition in triple-negative breast cancer in vitro and in vivo. The combination of EGFR inhibition by using lapatinib and mTOR inhibition with rapamycin resulted in significantly greater cytotoxicity than the single agents alone and these effects were synergistic in vitro. The combination of rapamycin and lapatinib significantly decreased growth of triple-negative breast cancers in vivo compared with either agent alone. EGFR inhibition abrogated the expression of rapamycin-induced activated Akt in triple-negative breast cancer cells in vitro. The combination of EGFR and mTOR inhibition resulted in increased apoptosis in some, but not all, triple-negative cell lines, and these apoptotic effects correlated with a decrease in activated eukaryotic translation initiation factor (eIF4E). These results suggest that mTOR inhibitors could sensitize a subset of triple-negative breast cancers to EGFR inhibitors. Given the paucity of effective targeted agents in triple-negative breast cancers, these results warrant further evaluation. PMID:21690228

Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation

PubMed Central

Wang, Wenchao; Li, Zhiyuan; Liu, Juanjuan; Yang, Xingxing; Ji, Xinmiao; Luo, Yan; Hu, Chen; Hou, Yubin; He, Qianqian; Fang, Jun; Wang, Junfeng; Liu, Qingsong; Li, Guohui; Lu, Qingyou; Zhang, Xin

2016-01-01

Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs. PMID:27223425

Inhibition of EGFR Induces a c-MET Driven Stem Cell Population in Glioblastoma

PubMed Central

Jun, Hyun Jung; Bronson, Roderick T.; Charest, Al

2015-01-01

Glioblastoma multiforme (GBM) is the most lethal form of primary brain tumors, characterized by highly invasive and aggressive tumors that are resistant to all current therapeutic options. GBMs are highly heterogeneous in nature and contain a small but highly tumorigenic and self-renewing population of stem or initiating cells (Glioblastoma stem cells or GSCs). GSCs have been shown to contribute to tumor propagation and resistance to current therapeutic modalities. Recent studies of human GBMs have elucidated the genetic alterations common in these tumors, but much remains unknown about specific signaling pathways that regulate GSCs. Here we identify a distinct fraction of cells in a genetically engineered mouse model of EGFR-driven GBM that respond to anti-EGFR therapy by inducing high levels of c-MET expression. The MET positive cells displayed clonogenic potential and long-term self-renewal ability in vitro and are capable of differentiating into multiple lineages. The MET positive GBM cells are resistant to radiation and highly tumorigenic in vivo. Activation of MET signaling led to an increase in expression of the stemness transcriptional regulators Oct4, Nanog and Klf4. Pharmacological inhibition of MET activity in GSCs prevented the activation of Oct4, Nanog and Klf4 and potently abrogated stemness. Finally, the MET expressing cells were preferentially localized in perivascular regions of mouse tumors consistent with their function as GSCs. Together, our findings indicate that EGFR inhibition in GBM induces MET activation in GSCs, which is a functional requisite for GSCs activity and thus represents a promising therapeutic target. PMID:24115218

ROS mediated EGFR/MEK/ERK/HIF-1α Loop Regulates Glucose metabolism in pancreatic cancer.

PubMed

Wang, Gang; Li, Yifeng; Yang, Zeyu; Xu, Weina; Yang, Yifan; Tan, Xiaodong

2018-06-12

To investigate the glycometabolism associated mechanism in invasion and metastasis of pancreatic cancer, We screened out genes involved in anaerobic glycolysis headed by HIF-1α,using pre-established a pair of pancreatic cancer cell lines. In this study, we further detected the glucose metabolism state not only in the cells but all also in two groups of patients with different SUVmax on 18 F-FDG PET/CT. The data suggests that ROS mediated EGFR/MEK/ERK/HIF-1α loop is activated in high glucose metabolic samples both in vitro and in vivo: The increasing of HIF-1α expression is controlled by activation of EGFR/MEK/ERK pathway in hypoxia condition, HIF-1α inhibits excessive release of ROS, the reduction of ROS further activates EGFR to form a positive feedback loop. This difference is closely related to invasion and metastasis capacity of pancreatic cancer, and can be rescued by separate or combined inhibition of EGFR or HIF-1α in various degree. These results indicate a new clue to develop therapy of pancreatic cancer by regulating the glucose metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

PubMed Central

Tomas, Alejandra; Vaughan, Simon O.; Burgoyne, Thomas; Sorkin, Alexander; Hartley, John A.; Hochhauser, Daniel; Futter, Clare E.

2015-01-01

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance. PMID:26066081

Histogram analysis of ADC in rectal cancer: associations with different histopathological findings including expression of EGFR, Hif1-alpha, VEGF, p53, PD1, and KI 67. A preliminary study.

PubMed

Meyer, Hans Jonas; Höhn, Annekathrin; Surov, Alexey

2018-04-06

Functional imaging modalities like Diffusion-weighted imaging are increasingly used to predict tumor behavior like cellularity and vascularity in different tumors. Histogram analysis is an emergent imaging analysis, in which every voxel is used to obtain a histogram and therefore statistically information about tumors can be provided. The purpose of this study was to elucidate possible associations between ADC histogram parameters and several immunhistochemical features in rectal cancer. Overall, 11 patients with histologically proven rectal cancer were included into the study. There were 2 (18.18%) females and 9 males with a mean age of 67.1 years. KI 67-index, expression of p53, EGFR, VEGF, and Hif1-alpha were semiautomatically estimated. The tumors were divided into PD1-positive and PD1-negative lesions. ADC histogram analysis was performed as a whole lesion measurement using an in-house matlab application. Spearman's correlation analysis revealed a strong correlation between EGFR expression and ADCmax (p=0.72, P=0.02). None of the vascular parameters (VEGF, Hif1-alpha) correlated with ADC parameters. Kurtosis and skewness correlated inversely with p53 expression (p=-0.64, P=0.03 and p=-0.81, P=0.002, respectively). ADCmedian and ADCmode correlated with Ki67 (p=-0.62, P=0.04 and p=-0.65, P=0.03, respectively). PD1-positive tumors showed statistically significant lower ADCmax values in comparison to PD1-negative tumors, 1.93 ± 0.36 vs 2.32 ± 0.47×10 -3 mm 2 /s, p=0.04. Several associations were identified between histogram parameter derived from ADC maps and EGFR, KI 67 and p53 expression in rectal cancer. Furthermore, ADCmax was different between PD1 positive and PD1 negative tumors indicating an important role of ADC parameters for possible future treatment prediction.

Histogram analysis of ADC in rectal cancer: associations with different histopathological findings including expression of EGFR, Hif1-alpha, VEGF, p53, PD1, and KI 67. A preliminary study

PubMed Central

Meyer, Hans Jonas; Höhn, Annekathrin; Surov, Alexey

2018-01-01

Functional imaging modalities like Diffusion-weighted imaging are increasingly used to predict tumor behavior like cellularity and vascularity in different tumors. Histogram analysis is an emergent imaging analysis, in which every voxel is used to obtain a histogram and therefore statistically information about tumors can be provided. The purpose of this study was to elucidate possible associations between ADC histogram parameters and several immunhistochemical features in rectal cancer. Overall, 11 patients with histologically proven rectal cancer were included into the study. There were 2 (18.18%) females and 9 males with a mean age of 67.1 years. KI 67-index, expression of p53, EGFR, VEGF, and Hif1-alpha were semiautomatically estimated. The tumors were divided into PD1-positive and PD1-negative lesions. ADC histogram analysis was performed as a whole lesion measurement using an in-house matlab application. Spearman's correlation analysis revealed a strong correlation between EGFR expression and ADCmax (p=0.72, P=0.02). None of the vascular parameters (VEGF, Hif1-alpha) correlated with ADC parameters. Kurtosis and skewness correlated inversely with p53 expression (p=-0.64, P=0.03 and p=-0.81, P=0.002, respectively). ADCmedian and ADCmode correlated with Ki67 (p=-0.62, P=0.04 and p=-0.65, P=0.03, respectively). PD1-positive tumors showed statistically significant lower ADCmax values in comparison to PD1-negative tumors, 1.93 ± 0.36 vs 2.32 ± 0.47×10−3mm2/s, p=0.04. Several associations were identified between histogram parameter derived from ADC maps and EGFR, KI 67 and p53 expression in rectal cancer. Furthermore, ADCmax was different between PD1 positive and PD1 negative tumors indicating an important role of ADC parameters for possible future treatment prediction. PMID:29719621

Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors.

PubMed

Ghosh, Gargi; Lian, Xiaojun; Kron, Stephen J; Palecek, Sean P

2012-03-20

Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown. An erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity in vitro were assessed in erlotinib resistant H1650-ER1 cells. The erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib. Our findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.

CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

PubMed Central

Wang, Tianxiao; Yang, Jingxuan; Xu, Jianwei; Li, Jian; Cao, Zhe; Zhou, Li; You, Lei; Shu, Hong; Lu, Zhaohui; Li, Huihua; Li, Min; Zhang, Taiping; Zhao, Yupei

2014-01-01

Carboxyl terminus of heat shock protein 70-interacting protein (CHIP) is an E3 ubiquitin ligase that is involved in protein quality control and mediates several tumor-related proteins in many cancers, but the function of CHIP in pancreatic cancer is not known. Here we show that CHIP interacts and ubiquitinates epidermal growth factor receptor (EGFR) for proteasome-mediated degradation in pancreatic cancer cells, thereby inhibiting the activation of EGFR downstream pathways. CHIP suppressed cell proliferation, anchor-independent growth, invasion and migration, as well as enhanced apoptosis induced by erlotinib in vitro and in vivo. The expression of CHIP was decreased in pancreatic cancer tissues or sera. Low CHIP expression in tumor tissues was correlated with tumor differentiation and shorter overall survival. These observations indicate that CHIP serves as a novel tumor suppressor by down-regulating EGFR pathway in pancreatic cancer cells, decreased expression of CHIP was associated with poor prognosis in pancreatic cancer. PMID:24722501

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

PubMed Central

Auth, Marcus K.H.; Boost, Kim A.; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H.; Bechstein, Wolf-Otto; Blaheta, Roman A.

2005-01-01

AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors. PMID:15810072

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.

PubMed

Auth, Marcus-K H; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H; Bechstein, Wolf-Otto; Blaheta, Roman A

2005-04-14

Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.

Angiodrastic Chemokines in Colorectal Cancer: Clinicopathological Correlations.

PubMed

Emmanouil, George; Ayiomamitis, George; Zizi-Sermpetzoglou, Adamantia; Tzardi, Maria; Moursellas, Andrew; Voumvouraki, Argyro; Kouroumalis, Elias

2018-01-01

To study the expression of angiodrastic chemokines in colorectal tumors and correlate findings with clinicopathological parameters and survival. The proangiogenic factor VEGF, the angiogenic chemokines CXCL8 and CXCL6, and the angiostatic chemokine CXCL4 were measured by ELISA in tumor and normal tissue of 35 stage II and III patients and correlated with the histopathology markers Ki67, p53, p21, bcl2, EGFR, and MLH1 and 5-year survival. The Wilcoxon and chi-square tests were used for statistical comparisons. There was a significant increase of CXCL6 ( p = 0.005) and VEGF ( p = 0.003) in cancerous tissue compared to normal. Patients with lower levels of CXCL8 and CXCL4 lived significantly longer. Patients with loss of EGFR expression had higher levels of CXCL8 while p21 loss was associated with higher levels of CXCL6. Chemokine levels were not correlated with TNM or Dukes classification. Strong expression of p53 was accompanied by decreased survival. (1) The angiogenic factors CXCL6 and VEGF are increased in colorectal cancer tissue with no association with the clinical stage of the disease or survival. (2) However, increased levels of tissue CXCL8 and CXCL4 are associated with poor survival. (3) Strong expression of p53 is found in patients with poor survival.

MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands

PubMed Central

CHUNG, EUN JOO; URICK, MARY ELLEN; KURSHAN, NAAMIT; SHIELD, WILLIAM; ASANO, HIROAKI; SMITH, PAUL D.; SCROGGINS, BRADLEY S.; BURKEEN, JEFFREY; CITRIN, DEBORAH E.

2013-01-01

The inhibition of the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway through the suppression of mutated Ras or MAPK/extracellular signal-regulated kinase 1/2 (MEK1/2) has been shown to sensitize tumor cells to ionizing radiation (IR). The molecular mechanisms of this sensitization however, are not yet fully understood. In this study, we investigated the role of transforming growth factor-α (TGF-α) in the radiosensitizing effects of selumetinib, a selective inhibitor of MEK1/2. The expression of epidermal growth factor receptor (EGFR) ligands was assessed by ELISA in both Ras wild-type and Ras mutant cells that were exposed to radiation with or without selumetinib. The effects of selumetinib on the TGF-α/EGFR signaling cascade in response to radiation were examined by western blot analysis, clonogenic assay and by determing the yield of mitotic catastrophe. The treatment of cells with selumetinib reduced the basal and IR-induced secretion of TGF-α in both Ras wild-type and Ras mutant cell lines in vitro and in vivo. The reduction of TGF-α secretion was accompanied with a reduction in phosphorylated tumor necrosis factor-α converting enzyme (TACE) in the cells treated with selumetinib with or without IR. The treatment of cells with selumetinib with or without IR inhibited the phosphorylation of EGFR and check-point kinase 2 (Chk2), and reduced the expression of survivin. Supplementation with exogenous TGF-α partially rescued the selumetinib-treated cells from IR-induced cell death, restored EGFR and Chk2 phosphorylation and increased survivin expression. These data suggest that the inhibition of MEK1/2 with selumetinib may provide a mechanism to sensitize tumor cells to IR in a fashion that prevents the activation of the TGF-α autocrine loop following IR. PMID:23588995

Immunohistochemical analysis of EGFR and hyaluronan in tongue cancer and the development of regional recurrence in patients initially diagnosed N0.

PubMed

Lindell Jonsson, Eva; Nylander, Karin; Hallén, Lars; Laurell, Göran

2017-08-01

To investigate whether the extent of expression of hyaluronan (HA) and epidermal growth factor receptor (EGFR) in squamous cell carcinoma of the mobile tongue can predict the risk of cervical metastasis and survival. Retrospective histopathologic study. Surgical specimens from 64 patients who had undergone surgery for squamous cell carcinoma of the mobile tongue were assessed using immunohistochemistry to investigate the expression of HA and EGFR in the primary tumours, and the data were then correlated to cervical metastasis and survival. There was a significant correlation between the intensity of HA staining and patient survival (p .024), and a weak correlation between the staining proportion of EGFR and the risk for regional recurrence (AUC 66). This study indicates that immunoscoring using HA could be used to provide prognostic tools for tongue cancer, and that it might be of interest to study the prognostic properties of EGFR further concerning the risk for regional recurrence after the primary treatment.

Combination therapy of potential gene to enhance oral cancer therapeutic effect

NASA Astrophysics Data System (ADS)

Yeh, Chia-Hsien; Hsu, Yih-Chih

2015-03-01

The epidermal growth factor receptor (EGFR) over-regulation related to uncontrolled cell division and promotes progression in tumor. Over-expression of human epidermal growth factor receptor (EGFR) has been detected in oral cancer cells. EGFR-targeting agents are potential therapeutic modalities for treating oral cancer based on our in vitro study. Liposome nanotechnology is used to encapsulate siRNA and were modified with target ligand to receptors on the surface of tumor cells. We used EGFR siRNA to treat oral cancer in vitro.

[Clinical significance and mechanism of upregulation of PI3Kp110α in non-small cell lung carcinoma].

PubMed

Xiong, Y; Qu, L L; Li, D; Wang, Y; Li, T

2017-10-23

Objective: To investigate the clinical significance and mechanism of upregulation of phosphoinositide 3-kinase p110α(PI3Kp110α)in non-small cell lung carcinoma (NSCLC). Methods: Expressions of PI3Kp110α and other components in PI3K signaling pathway (including phospho-Akt (p-Akt, Ser 473), MET, ROS1, HER-2, ALK, total EGFR and mutant EGFR) and p53 (the transcription factor of PIK3CA) mutation in NSCLC were detected by immunohistochemistry. The relationships between PI3Kp110α expression and clinicopathological characteristics, expressions of other proteins in PI3K pathway and p53 mutation were analyzed. Results: In 170 NSCLC patients, 72 cases (42.4%) showed lower expression and 98 cases (57.6%) showed higher expression of PI3Kp110α. Upregulation of PI3Kp110α was not significantly associated with gender, age, T stage and pathologic grade ( P >0.05). While upregulation of PI3Kp110α was significantly associated with smoking status of patients, pathologic classification, N stage, TNM stage and Ki-67 index ( P <0.05). Expression of PI3Kp110α was positively correlated with expressions of MET ( P <0.05) and mutant EGFR ( P =0.018), while not significantly related with expressions of p-Akt(Ser473), HER-2, ALK, ROS1, total EGFR or p53 mutation ( P >0.05). Conclusions: Upregulation of PI3Kp110α is closely related with tumorigenesis of non-smoking lung adenocarcinoma. MET overexpression and EGFR mutation may be crucial to upregulate expression of PI3Kp110α in NSCLC. Overexpression of PI3Kp110α may inhibit tumor cell proliferation in NSCLC through a different pathway other than classical PI3K pathway. Upregulation of PI3Kp110α may predict favorable prognosis of NSCLC patients.

Genomic and Expression Profiling of Benign and Malignant Nerve Sheath Tumors in Neurofibromatosis Patients

DTIC Science & Technology

2008-05-01

DAMD17-03-1-0297 Title: Genomic and Expression Pr ofiling of Benign and Malignant Nerve Sheath Tumors in Neurofibromatosis Patients...have determined the gene expression signature for benign and malignant peripheral nerve sheath tumors and found that the major trend in transformation...However, EGFR data in soft tissue neoplasms is limited. Using a variety of benign and malignant spindle cell neoplasms, we assessed EGFR status by

Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury.

PubMed

Qu, Wen-Sheng; Tian, Dai-Shi; Guo, Zhi-Bao; Fang, Jun; Zhang, Qiang; Yu, Zhi-Yuan; Xie, Min-Jie; Zhang, Hua-Qiu; Lü, Jia-Gao; Wang, Wei

2012-07-23

Emerging evidence indicates that reactive microglia-initiated inflammatory responses are responsible for secondary damage after primary traumatic spinal cord injury (SCI); epidermal growth factor receptor (EGFR) signaling may be involved in cell activation. In this report, we investigate the influence of EGFR signaling inhibition on microglia activation, proinflammatory cytokine production, and the neuronal microenvironment after SCI. Lipopolysaccharide-treated primary microglia/BV2 line cells and SCI rats were used as model systems. Both C225 and AG1478 were used to inhibit EGFR signaling activation. Cell activation and EGFR phosphorylation were observed after fluorescent staining and western blot. Production of interleukin-1 beta (IL-1 β) and tumor necrosis factor alpha (TNF α) was tested by reverse transcription PCR and ELISA. Western blot was performed to semi-quantify the expression of EGFR/phospho-EGFR, and phosphorylation of Erk, JNK and p38 mitogen-activated protein kinases (MAPK). Wet-dry weight was compared to show tissue edema. Finally, axonal tracing and functional scoring were performed to show recovery of rats. EGFR phosphorylation was found to parallel microglia activation, while EGFR blockade inhibited activation-associated cell morphological changes and production of IL-1 β and TNF α. EGFR blockade significantly downregulated the elevated MAPK activation after cell activation; selective MAPK inhibitors depressed production of cytokines to a certain degree, suggesting that MAPK mediates the depression of microglia activation brought about by EGFR inhibitors. Subsequently, seven-day continual infusion of C225 or AG1478 in rats: reduced the expression of phospho-EGFR, phosphorylation of Erk and p38 MAPK, and production of IL-1 β and TNF α; lessened neuroinflammation-associated secondary damage, like microglia/astrocyte activation, tissue edema and glial scar/cavity formation; and enhanced axonal outgrowth and functional recovery. These findings indicate that inhibition of EGFR/MAPK suppresses microglia activation and associated cytokine production; reduces neuroinflammation-associated secondary damage, thus provides neuroprotection to SCI rats, suggesting that EGFR may be a therapeutic target, and C225 and AG1478 have potential for use in SCI treatment.

Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hisatsune, Akinori, E-mail: hisatsun@kumamoto-u.ac.jp; Nakayama, Hideki; Kawasaki, Mitsuru

2011-02-18

Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalizedmore » by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.« less

Src mediates cigarette smoke-induced resistance to tyrosine kinase inhibitors in NSCLC cells.

PubMed

Filosto, Simone; Baston, David S; Chung, Samuel; Becker, Cathleen R; Goldkorn, Tzipora

2013-08-01

The EGF receptor (EGFR) is a proto-oncogene commonly dysregulated in several cancers including non-small cell lung carcinoma (NSCLC) and, thus, is targeted for treatment using tyrosine kinase inhibitors (TKI) such as erlotinib. However, despite the efficacy observed in patients with NSCLC harboring oncogenic variants of the EGFR, general ineffectiveness of TKIs in patients with NSCLC who are current and former smokers necessitates identification of novel mechanisms to overcome this phenomenon. Previously, we showed that NSCLC cells harboring either wild-type (WT) EGFR or oncogenic mutant (MT) L858R EGFR become resistant to the effects of TKIs when exposed to cigarette smoke, evidenced by their autophosphorylation and prolonged downstream signaling. Here, we present Src as a target mediating cigarette smoke-induced resistance to TKIs in both WT EGFR- and L858R MT EGFR-expressing NSCLC cells. First, we show that cigarette smoke exposure of A549 cells leads to time-dependent activation of Src, which then abnormally binds to the WT EGFR causing TKI resistance, contrasting previous observations of constitutive binding between inactive Src and TKI-sensitive L858R MT EGFR. Next, we show that Src inhibition restores TKI sensitivity in cigarette smoke-exposed NSCLC cells, preventing EGFR autophosphorylation in the presence of erlotinib. Furthermore, we show that overexpression of a dominant-negative Src (Y527F/K295R) restores TKI sensitivity to A549 exposed to cigarette smoke. Importantly, the TKI resistance that emerges even in cigarette smoke-exposed L858R EGFR-expressing NSCLC cells could be eliminated with Src inhibition. Together, these findings offer new rationale for using Src inhibitors for treating TKI-resistant NSCLC commonly observed in smokers.

Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

2017-12-01

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Bombesin receptor-activated protein regulates neutrophil elastase-induced mucin5AC hypersecretion in human bronchial epithelial cells.

PubMed

Xu, Qing; Chen, Ling-Xiu; Ran, Dan-Hua; Xie, Wen-Yue; Li, Qi; Zhou, Xiang-Dong

2017-08-15

Bombesin receptor-activated protein (BRAP) is highly expressed in human bronchial epithelial cells. Recent studies have shown that BRAP reduces oxidative stress, inhibits airway inflammation and suppresses nuclear factor kappaB (NF-κB) activity. Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. Neutrophil elastase (NE) is a potent inducer of mucin5AC (MUC5AC), which is considered the predominant mucin secreted by human airway epithelial cells. Here, we hypothesize that BRAP may regulate NE-induced MUC5AC hypersecretion in a bronchial epithelial cell line (HBE16). We also investigated the underlying mechanism involved in the process. In this study, we found that BRAP was present in HBE16 human bronchial epithelial cells and was significantly increased by NE. Next, we found that the up-regulation of BRAP by pEGFP-N1-BRAP caused a significant decrease in the increased levels of MUC5AC expression, NF-κB activity, and the phosphorylation of extracellular signal-regulated kinases (ERK) and epidermal growth factor receptor (EGFR) induced by NE. Meanwhile, there was a significant decrease in ROS, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels when BRAP was up-regulated by pEGFP-N1-BRAP. Moreover, when cells were transfected with pEGFP-N1-BRAP and pretreated with NF-κB, ERK or EGFR inhibitors before the NE stimulation, there were further decreased in MUC5AC expression, NF-κB activity, and the phosphorylation of ERK and EGFR. These results suggest that BRAP plays an important role in airway inflammation and its overexpression may regulate NE-induced MUC5AC hypersecretion in HBE16 cells via the EGFR/ERK/NF-κB signaling pathway. Copyright © 2017. Published by Elsevier Inc.

Correlation of EGFR or KRAS mutation status with 18F-FDG uptake on PET-CT scan in lung adenocarcinoma.

PubMed

Takamochi, Kazuya; Mogushi, Kaoru; Kawaji, Hideya; Imashimizu, Kota; Fukui, Mariko; Oh, Shiaki; Itoh, Masayoshi; Hayashizaki, Yoshihide; Ko, Weijey; Akeboshi, Masao; Suzuki, Kenji

2017-01-01

18F-fluoro-2-deoxy-glucose (18F-FDG) positron emission tomography (PET) is a functional imaging modality based on glucose metabolism. The correlation between EGFR or KRAS mutation status and the standardized uptake value (SUV) of 18F-FDG PET scanning has not been fully elucidated. Correlations between EGFR or KRAS mutation status and clinicopathological factors including SUVmax were statistically analyzed in 734 surgically resected lung adenocarcinoma patients. Molecular causal relationships between EGFR or KRAS mutation status and glucose metabolism were then elucidated in 62 lung adenocarcinomas using cap analysis of gene expression (CAGE), a method to determine and quantify the transcription initiation activities of mRNA across the genome. EGFR and KRAS mutations were detected in 334 (46%) and 83 (11%) of the 734 lung adenocarcinomas, respectively. The remaining 317 (43%) patients had wild-type tumors for both genes. EGFR mutations were more frequent in tumors with lower SUVmax. In contrast, no relationship was noted between KRAS mutation status and SUVmax. CAGE revealed that 4 genes associated with glucose metabolism (GPI, G6PD, PKM2, and GAPDH) and 5 associated with the cell cycle (ANLN, PTTG1, CIT, KPNA2, and CDC25A) were positively correlated with SUVmax, although expression levels were lower in EGFR-mutated than in wild-type tumors. No similar relationships were noted with KRAS mutations. EGFR-mutated adenocarcinomas are biologically indolent with potentially lower levels of glucose metabolism than wild-type tumors. Several genes associated with glucose metabolism and the cell cycle were specifically down-regulated in EGFR-mutated adenocarcinomas.

Mig6 Puts the Brakes on Mutant EGFR-Driven Lung Cancer | Center for Cancer Research

Cancer.gov

Lung cancer is the most common cause of cancer-related death worldwide. These cancers are often induced by mutations in the epidermal growth factor receptor (EGFR), resulting in constitutive activation of the protein’s tyrosine kinase domain. Lung cancers expressing these EGFR mutants are initially sensitive to tyrosine kinase inhibitors (TKIs), such as erlotinib, but often become resistant by developing compensatory mutations in EGFR or other growth-promoting pathways. To better understand how mutant EGFR initiates and maintains tumor growth in the hopes of identifying novel targets for drug development, Udayan Guha, M.D., Ph.D., of CCR’s Thoracic and Gastrointestinal Oncology Branch, and his colleagues examined the landscape of proteins phosphorylated in EGFR wild type and mutant cells. One protein hyper-phosphorylated in mutant EGFR cells was Mig6, a putative tumor suppressor.

Loss of Sh3gl2/Endophilin A1 Is a Common Event in Urothelial Carcinoma that Promotes Malignant Behavior12

PubMed Central

Majumdar, Shyama; Gong, Edward M; Di Vizio, Dolores; Dreyfuss, Jonathan; DeGraff, David J; Hager, Martin H; Park, Peter J; Bellmunt, Joaquim; Matusik, Robert J; Rosenberg, Jonathan E; Adam, Rosalyn M

2013-01-01

Urothelial carcinoma (UC) causes substantial morbidity and mortality worldwide. However, the molecular mechanisms underlying urothelial cancer development and tumor progression are still largely unknown. Using informatics analysis, we identified Sh3gl2 (endophilin A1) as a bladder urothelium-enriched transcript. The gene encoding Sh3gl2 is located on chromosome 9p, a region frequently altered in UC. Sh3gl2 is known to regulate endocytosis of receptor tyrosine kinases implicated in oncogenesis, such as the epidermal growth factor receptor (EGFR) and c-Met. However, its role in UC pathogenesis is unknown. Informatics analysis of expression profiles as well as immunohistochemical staining of tissue microarrays revealed Sh3gl2 expression to be decreased in UC specimens compared to nontumor tissues. Loss of Sh3gl2 was associated with increasing tumor grade and with muscle invasion, which is a reliable predictor of metastatic disease and cancer-derived mortality. Sh3gl2 expression was undetectable in 19 of 20 human UC cell lines but preserved in the low-grade cell line RT4. Stable silencing of Sh3gl2 in RT4 cells by RNA interference 1) enhanced proliferation and colony formation in vitro, 2) inhibited EGF-induced EGFR internalization and increased EGFR activation, 3) stimulated phosphorylation of Src family kinases and STAT3, and 4) promoted growth of RT4 xenografts in subrenal capsule tissue recombination experiments. Conversely, forced re-expression of Sh3gl2 in T24 cells and silenced RT4 clones attenuated oncogenic behaviors, including growth and migration. Together, these findings identify loss of Sh3gl2 as a frequent event in UC development that promotes disease progression. PMID:23814487

EGFR Ligands Drive Multipotential Stromal Cells to Produce Multiple Growth Factors and Cytokines via Early Growth Response-1

PubMed Central

Kerpedjieva, Svetoslava S.; Kim, Duk Soo; Barbeau, Dominique J.

2012-01-01

Cell therapy with adult bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) presents a promising approach to promote wound healing and tissue regeneration. The strong paracrine capability of various growth factors and cytokines is a key mechanism of MSC-mediated wound healing and tissue regeneration, and the goal of this study is to understand the underlying mechanism that supports the strong paracrine machineries in MSCs. Microarray database analyses revealed that early growth response-1 (EGR1) is highly expressed in MSCs. Our previous studies showed that epidermal growth factor (EGF) treatment induces growth factor production in MSCs in vitro. Since EGF strongly upregulates EGR1, we hypothesized that EGF receptor (EGFR)–EGR1 signaling plays a pivotal role in MSC paracrine activity. EGF treatment upregulated the gene expression of growth factors and cytokines, including EGFR ligands in a protein kinase C (PKC)- and/or mitogen-activated protein kinase–extracellular-signal-regulated kinase-dependent manner, and it was reversed by shRNA against EGR1. PKC activator phorbol 12-myristate 13-acetate enhanced EGFR tyrosyl phosphorylation and upregulated the gene expression of growth factors and cytokines in a heparin-binding EGF-like growth factor (HBEGF) inhibitor CRM197 sensitive manner, indicating an involvement of autocrined HBEGF in the downstream of PKC signaling. Moreover, stimulation with growth factors and cytokines induced the expression of EGFR ligands, presumably via EGR1 upregulation. These data indicate EGR1 as a convergence point of multiple signaling pathways, which in turn augments the production of multiple growth factors and cytokines by enhancing the autocrine signaling with EGFR ligands. PMID:22316125

EGFR ligands drive multipotential stromal cells to produce multiple growth factors and cytokines via early growth response-1.

PubMed

Kerpedjieva, Svetoslava S; Kim, Duk Soo; Barbeau, Dominique J; Tamama, Kenichi

2012-09-01

Cell therapy with adult bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) presents a promising approach to promote wound healing and tissue regeneration. The strong paracrine capability of various growth factors and cytokines is a key mechanism of MSC-mediated wound healing and tissue regeneration, and the goal of this study is to understand the underlying mechanism that supports the strong paracrine machineries in MSCs. Microarray database analyses revealed that early growth response-1 (EGR1) is highly expressed in MSCs. Our previous studies showed that epidermal growth factor (EGF) treatment induces growth factor production in MSCs in vitro. Since EGF strongly upregulates EGR1, we hypothesized that EGF receptor (EGFR)-EGR1 signaling plays a pivotal role in MSC paracrine activity. EGF treatment upregulated the gene expression of growth factors and cytokines, including EGFR ligands in a protein kinase C (PKC)- and/or mitogen-activated protein kinase-extracellular-signal-regulated kinase-dependent manner, and it was reversed by shRNA against EGR1. PKC activator phorbol 12-myristate 13-acetate enhanced EGFR tyrosyl phosphorylation and upregulated the gene expression of growth factors and cytokines in a heparin-binding EGF-like growth factor (HBEGF) inhibitor CRM197 sensitive manner, indicating an involvement of autocrined HBEGF in the downstream of PKC signaling. Moreover, stimulation with growth factors and cytokines induced the expression of EGFR ligands, presumably via EGR1 upregulation. These data indicate EGR1 as a convergence point of multiple signaling pathways, which in turn augments the production of multiple growth factors and cytokines by enhancing the autocrine signaling with EGFR ligands.

Mig6 Puts the Brakes on Mutant EGFR-Driven Lung Cancer | Center for Cancer Research

Cancer.gov

Lung cancer is the most common cause of cancer-related death worldwide. These cancers are often induced by mutations in the epidermal growth factor receptor (EGFR), resulting in constitutive activation of the protein’s tyrosine kinase domain. Lung cancers expressing these EGFR mutants are initially sensitive to tyrosine kinase inhibitors (TKIs), such as erlotinib, but often

Driving Neurofibroma Formation in Mice

DTIC Science & Technology

2006-08-01

pirfenidone . Ratner, Nancy DAMD17-02-1-0679 5 BODY Task 1: Analysis of EGFR-expressing mouse. a. Define abnormalities in...EGFR-overexpressing mouse nerves (Year 1). b. Evaluate effects of: a specific EGFR receptor antagonist and c. an anti-fibrotic drug, pirfenidone ...considered significant. Task 1c, In Progress: Evaluate effects of an anti-fibrotic drug, pirfenidone . Our move to the Children’s Hospital Research

EGFR-dependent signalling reduced and p38 dependent apoptosis required by Gallic acid in Malignant Mesothelioma cells.

PubMed

Demiroglu-Zergeroglu, Asuman; Candemir, Gulsife; Turhanlar, Ebru; Sagir, Fatma; Ayvali, Nurettin

2016-12-01

The unrestrained EGFR signalling contributes to malignant phenotype in a number of cancers including Malignant Mesotheliomas. Present study was designed to evaluate EGFR-dependent anti-proliferative and apoptotic effects of Gallic acid in transformed Mesothelial (MeT-5A) and Malignant Mesothelioma (SPC212) cells. Gallic acid reduced the viability of Malignant Mesothelioma cells in a concentration and time-dependent manner. However, viability of mesothelial cells reduced only at high concentration and longer time periods. Gallic acid restrained the activation of EGFR, ERK1/2 and AKT proteins and down regulated expression of Cyclin D and Bcl-2 genes, but upregulated the expression of p21 gene in EGF-induced SPC212 cells. GA-induced transitory G1 arrest and triggered mitochondrial and death receptor mediated apoptosis, which requires p38MAPK activation. The data provided here indicate that GA is able to inhibit EGFR dependent proliferation and survival signals and induces p38 pathway dependent apoptosis in Malignant Mesothelioma cells. On the basis of these experimental findings it is worthwhile to investigate further the biological activity of Gallic acid on other Mesothelioma cell lines harbouring aberrant EGFR signals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

PubMed Central

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-01-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR. PMID:28287083

Differentiated thyroid cancer cell invasion is regulated through epidermal growth factor receptor-dependent activation of matrix metalloproteinase (MMP)-2/gelatinase A

PubMed Central

Yeh, Michael W; Rougier, Jean-Philippe; Park, Jin-Woo; Duh, Quan-Yang; Wong, Mariwil; Werb, Zena; Clark, Orlo H

2008-01-01

Mechanisms of invasion in thyroid cancer remain poorly understood. We hypothesized that signaling via the epidermal growth factor receptor (EGFR) stimulates thyroid cancer cell invasion by altering the expression and cleavage of matrix metalloproteinases (MMPs). Papillary and follicular carcinoma cell lines were treated with EGF, the EGFR tyrosine kinase inhibitor AG1478, and the MMP inhibitors GM-6001 and Col-3. Flow cytometry was used to detect EGFR. In vitro invasion assays, gelatin zymography, and quantitative reverse transcription-PCR were used to assess the changes in invasive behavior and MMP expression and activation. All cell lines were found to overexpress functional EGFR. EGF stimulated invasion by thyroid cancer cells up to sevenfold (P<0.0001), a process that was antagonized completely by AG1478 and Col-3, partially by GM-6001, but not by the serine protease inhibitor aprotinin. EGF upregulated expression of MMP-9 (2.64– to 8.89-fold, P<0.0001) and membrane type-1 MMP (MT1-MMP, 1.97- to 2.67-fold, P<0.0001). This effect was blocked completely by AG1478 and partially by Col-3. The activation of MMP-2 paralleled MT1-MMP expression. We demonstrate that MMPs are critical effectors of invasion in the papillary and follicular thyroid cancer cell lines studied. Invasion is regulated by signaling through EGFR, an effect mediated by augmentation of gelatinase expression and activation. MMP inhibitors and growth factor antagonists may be effective tumoristatic agents for the treatment of aggressive thyroid carcinomas. PMID:17158762

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.

PubMed

Taron, Miguel; Ichinose, Yukito; Rosell, Rafael; Mok, Tony; Massuti, Bartomeu; Zamora, Lurdes; Mate, Jose Luis; Manegold, Christian; Ono, Mayumi; Queralt, Cristina; Jahan, Thierry; Sanchez, Jose Javier; Sanchez-Ronco, Maria; Hsue, Victor; Jablons, David; Sanchez, Jose Miguel; Moran, Teresa

2005-08-15

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non-small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non-small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention

PubMed Central

Davis, Nicole M.; Sokolosky, Melissa; Stadelman, Kristin; Abrams, Stephen L.; Libra, Massimo; Candido, Saverio; Nicoletti, Ferdinando; Polesel, Jerry; Maestro, Roberta; D’Assoro, Antonino; Drobot, Lyudmyla; Rakus, Dariusz; Gizak, Agnieszka; Laidler, Piotr; Dulińska-Litewka, Joanna; Basecke, Joerg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Montalto, Giuseppe; Cervello, Melchiorre; Fitzgerald, Timothy L.; Demidenko, Zoya N.; Martelli, Alberto M.; Cocco, Lucio; Steelman, Linda S.; McCubrey, James A.

2014-01-01

The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway. PMID:25051360

Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention.

PubMed

Davis, Nicole M; Sokolosky, Melissa; Stadelman, Kristin; Abrams, Steve L; Libra, Massimo; Candido, Saverio; Nicoletti, Ferdinando; Polesel, Jerry; Maestro, Roberta; D'Assoro, Antonino; Drobot, Lyudmyla; Rakus, Dariusz; Gizak, Agnieszka; Laidler, Piotr; Dulińska-Litewka, Joanna; Basecke, Joerg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Montalto, Giuseppe; Cervello, Melchiorre; Fitzgerald, Timothy L; Demidenko, Zoya; Martelli, Alberto M; Cocco, Lucio; Steelman, Linda S; McCubrey, James A

2014-07-15

The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway.

Targeting EGF-receptor(s) - STAT1 axis attenuates tumor growth and metastasis through downregulation of MUC4 mucin in human pancreatic cancer

PubMed Central

Seshacharyulu, Parthasarathy; Ponnusamy, Moorthy P.; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Haridas, Dhanya; Yan, Ying; Ganti, Apar K.; Batra, Surinder K.

2015-01-01

Transmembrane proteins MUC4, EGFR and HER2 are shown to be critical in invasion and metastasis of pancreatic cancer. Besides, we and others have demonstrated de novo expression of MUC4 in ~70-90% of pancreatic cancer patients and its stabilizing effects on HER2 downstream signaling in pancreatic cancer. Here, we found that use of canertinib or afatinib resulted in reduction of MUC4 and abrogation of in vitro and in vivo oncogenic functions of MUC4 in pancreatic cancer cells. Notably, silencing of EGFR family member in pancreatic cancer cells decreased MUC4 expression through reduced phospho-STAT1. Furthermore, canertinib and afatinib treatment also inhibited proliferation, migration and survival of pancreatic cancer cells by attenuation of signaling events including pERK1/2 (T202/Y204), cyclin D1, cyclin A, pFAK (Y925) and pAKT (Ser473). Using in vivo bioluminescent imaging, we demonstrated that canertinib treatment significantly reduced tumor burden (P=0.0164) and metastasis to various organs. Further, reduced expression of MUC4 and EGFR family members were confirmed in xenografts. Our results for the first time demonstrated the targeting of EGFR family members along with MUC4 by using pan-EGFR inhibitors. In conclusion, our studies will enhance the translational acquaintance of pan-EGFR inhibitors for combinational therapies to combat against lethal pancreatic cancer. PMID:25686822

Imaging and targeted therapy of pancreatic ductal adenocarcinoma using the theranostic sodium iodide symporter (NIS) gene

PubMed Central

Trajkovic-Arsic, Marija; Klutz, Kathrin; Braren, Rickmer; Schwaiger, Markus; Nelson, Peter J.; Ogris, Manfred; Wagner, Ernst; Siveke, Jens T.; Spitzweg, Christine

2017-01-01

The theranostic sodium iodide symporter (NIS) gene allows detailed molecular imaging of transgene expression and application of therapeutic radionuclides. As a crucial step towards clinical application, we investigated tumor specificity and transfection efficiency of epidermal growth factor receptor (EGFR)-targeted polyplexes as systemic NIS gene delivery vehicles in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) that closely reflects human disease. PDAC was induced in mice by pancreas-specific activation of constitutively active KrasG12D and deletion of Trp53. We used tumor-targeted polyplexes (LPEI-PEG-GE11/NIS) based on linear polyethylenimine, shielded by polyethylene glycol and coupled with the EGFR-specific peptide ligand GE11, to target a NIS-expressing plasmid to high EGFR-expressing PDAC. In vitro iodide uptake studies in cell explants from murine EGFR-positive and EGFR-ablated PDAC lesions demonstrated high transfection efficiency and EGFR-specificity of LPEI-PEG-GE11/NIS. In vivo 123I gamma camera imaging and three-dimensional high-resolution 124I PET showed significant tumor-specific accumulation of radioiodide after systemic LPEI-PEG-GE11/NIS injection. Administration of 131I in LPEI-PEG-GE11/NIS-treated mice resulted in significantly reduced tumor growth compared to controls as determined by magnetic resonance imaging, though survival was not significantly prolonged. This study opens the exciting prospect of NIS-mediated radionuclide imaging and therapy of PDAC after systemic non-viral NIS gene delivery. PMID:28380420

Evaluation of [(18)F]gefitinib as a molecular imaging probe for the assessment of the epidermal growth factor receptor status in malignant tumors.

PubMed

Su, Helen; Seimbille, Yann; Ferl, Gregory Z; Bodenstein, Claudia; Fueger, Barbara; Kim, Kevin J; Hsu, Yu-Tien; Dubinett, Steven M; Phelps, Michael E; Czernin, Johannes; Weber, Wolfgang A

2008-06-01

Gefitinib, an inhibitor of the epidermal growth factor receptor-tyrosine kinase (EGFR-TK), has shown potent effects in a subset of patients carrying specific EGFR-TK mutations in advanced non-small-cell lung cancer. In this study, we asked whether PET with [(18)F]gefitinib may be used to study noninvasively the pharmacokinetics of gefitinib in vivo and to image the EGFR status of cancer cells. Synthesis of [(18)F]gefitinib has been previously described. The biodistribution and metabolic stability of [(18)F]gefitinib was assessed in mice and vervet monkeys for up to 2 h post injection by both micropositron emission tomography (PET)/computed tomography (CT) scans and postmortem ex vivo tissue harvesting. Uptake levels of radiolabeled gefitinib in EGFR-expressing human cancer cell lines with various levels of EGFR expression or mutation status were evaluated both in vivo and in vitro. MicroPET/CT scans in two species demonstrated a rapid and predominantly hepatobiliary clearance of [(18)F]gefitinib in vivo. However, uptake levels of radiolabeled gefitinib, both in vivo and in vitro, did not correlate with EGFR expression levels or functional status. This unexpected observation was due to high nonspecific, nonsaturable cellular uptake of gefitinib. The biodistribution of the drug analogue [(18)F]gefitinib suggests that it may be used to assess noninvasively the pharmacokinetics of gefitinib in patients by PET imaging. This is of clinical relevance, as insufficient intratumoral drug concentrations are considered to be a factor for resistance to gefitinib therapy. However, the highly nonspecific cellular binding of [(18)F]gefitinib may preclude the use of this imaging probe for noninvasive assessment of EGFR receptor status in patients.

Immuno- and gene expression analysis of EGFR and Nestin during mice skin development.

PubMed

Falodah, Fawaz Adnan; Al-Karim, Saleh

2016-06-01

Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. The main objectives of the present study were to identify the precise anatomical localization of stem cells in mice during skin developing; and to determine the expression levels by using immuno- and gene expression analysis. In this comparative cross sectional study, six ages been chosen and divided into: embryonic days (E12.5, E14.5 and E19.5) and litter days (L7, L14 and L19). Skin were removed from the back side and processed to assess both immuno- and gene-expression of EGFR and Nestin surface antigen markers. Data of the different studied age groups was compared using the SPSS software. EGFR was mainly expressed in the outer root sheath (ORS), in basal and, to a lesser extent, in suprabasal keratinocytes and tend to lie where the dermis comes closest to the skin surface, while Nestin expressed throughout the dermis in the early embryo, but it is subsequently restricted to the follicular connective tissue sheaths later in development and to hair follicles after birth. Immunoexpression analysis showed a strong EGFR expression in all group ages except E12.5 which recorded as moderate, while Nestin showed strong expression level for all embryonic stages, while in the litters it was moderate. The qRT-PCR results were consistent with those of the immunohistochemical study. The Pearson correlation analyze present a correlation between the cases of study with age (p≤0.01), which indicated to the effect of age to mice development. EGFR and Nestin showed to have vital role during mice development, and considered to be suitable markers for the study of skin stem cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

64Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor-Expressing Tumors.

PubMed

Pyo, Ayoung; Yun, Misun; Kim, Hyeon Sik; Kim, Tae-Yoon; Lee, Joong-Jae; Kim, Jung Young; Lee, Sunwoo; Kwon, Seong Young; Bom, Hee-Seung; Kim, Hak-Sung; Kim, Dong-Yeon; Min, Jung-Joon

2018-02-01

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64 Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-( p -isothiocyanatobenzyl)-1,4,7-triazacyclononane- N,N',N,″- triacetic acid trihydrochloride ([ p -SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA- N -hydroxysuccinimide ester), or 1-( p -isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([ p -SCN-Bn]-DTPA) in 1.0 M NaHCO 3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64 Cu in 0.1 M NH 4 OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Results: Radiochemical yields of the 64 Cu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). Conclusion: The 3 64 Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity.

PubMed

Basu, D; Nguyen, T-T K; Montone, K T; Zhang, G; Wang, L-P; Diehl, J A; Rustgi, A K; Lee, J T; Weinstein, G S; Herlyn, M

2010-07-22

Variable drug responses among malignant cells within individual tumors may represent a barrier to their eradication using chemotherapy. Carcinoma cells expressing mesenchymal markers resist conventional and epidermal growth factor receptor (EGFR)-targeted chemotherapy. In this study, we evaluated whether mesenchymal-like sub-populations within human squamous cell carcinomas (SCCs) with predominantly epithelial features contribute to overall therapy resistance. We identified a mesenchymal-like subset expressing low E-cadherin (Ecad-lo) and high vimentin within the upper aerodigestive tract SCCs. This subset was both isolated from the cell lines and was identified in xenografts and primary clinical specimens. The Ecad-lo subset contained more low-turnover cells, correlating with resistance to the conventional chemotherapeutic paclitaxel in vitro. Epidermal growth factor induced less stimulation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways in Ecad-lo cells, which was likely due to lower EGFR expression in this subset and correlated with in vivo resistance to the EGFR-targeted antibody, cetuximab. The Ecad-lo and high E-cadherin subsets were dynamic in phenotype, showing the capacity to repopulate each other from single-cell clones. Taken together, these results provide evidence for a low-turnover, mesenchymal-like sub-population in SCCs with diminished EGFR pathway function and intrinsic resistance to conventional and EGFR-targeted chemotherapies.

Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

PubMed Central

TU, DOM-GENE; YU, YUN; LEE, CHE-HSIN; KUO, YU-LIANG; LU, YIN-CHE; TU, CHI-WEN; CHANG, WEN-WEI

2016-01-01

Hinokitiol, alternatively known as β-thujaplicin, is a tropolone-associated natural compound with antimicrobial, anti-inflammatory and antitumor activity. Breast cancer stem/progenitor cells (BCSCs) are a subpopulation of breast cancer cells associated with tumor initiation, chemoresistance and metastatic behavior, and may be enriched by mammosphere cultivation. Previous studies have demonstrated that BCSCs exhibit vasculogenic mimicry (VM) activity via the epidermal growth factor receptor (EGFR) signaling pathway. The present study investigated the anti-VM activity of hinokitiol in BCSCs. At a concentration below the half maximal inhibitory concentration, hinokitiol inhibited VM formation of mammosphere cells derived from two human breast cancer cell lines. Hinokitiol was additionally indicated to downregulate EGFR protein expression in mammosphere-forming BCSCs without affecting the expression of messenger RNA. The protein stability of EGFR in BCSCs was also decreased by hinokitiol. The EGFR protein expression and VM formation capability of hinokitiol-treated BCSCs were restored by co-treatment with MG132, a proteasome inhibitor. In conclusion, the present study indicated that hinokitiol may inhibit the VM activity of BCSCs through stimulating proteasome-mediated EGFR degradation. Hinokitiol may act as an anti-VM agent, and may be useful for the development of novel breast cancer therapeutic agents. PMID:27073579

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

PubMed

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting. Copyright © 2015 Elsevier GmbH. All rights reserved.

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

PubMed

Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

2017-02-04

Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel therapeutic strategy to treat advanced bladder cancer.

TP53, STK11 and EGFR Mutations Predict Tumor Immune Profile and the Response to anti-PD-1 in Lung Adenocarcinoma.

PubMed

Biton, Jerome; Mansuet-Lupo, Audrey; Pécuchet, Nicolas; Alifano, Marco; Ouakrim, Hanane; Arrondeau, Jennifer; Boudou-Rouquette, Pascaline; Goldwasser, Francois; Leroy, Karen; Goc, Jeremy; Wislez, Marie; Germain, Claire; Laurent-Puig, Pierre; Dieu-Nosjean, Marie-Caroline; Cremer, Isabelle; Herbst, Ronald; Blons, Hélène F; Damotte, Diane

2018-05-15

By unlocking anti-tumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non-small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed. We performed in depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and to PD-L1 expression, and a public clinical database was used to validate the results obtained. We showed that distinct combinations of STK11 , EGFR and TP53 mutations, were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations ( TP53 -mut/ STK11 - EGFR -WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T cell density and PD-L1 expression. In this tumor subtype, pathways related to T cell chemotaxis, immune cell cytotoxicity, and antigen processing were up-regulated. Finally, a prolonged progression-free survival (PFS: HR=0.32; 95% CI, 0.16-0.63, p <0.001) was observed in anti-PD-1 treated patients harboring TP53 -mut/ STK11 - EGFR -WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression. Our study reveals that different combinations of TP53 , EGFR and STK11 mutations , together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Copyright ©2018, American Association for Cancer Research.

MUC4 overexpression augments cell migration and metastasis through EGFR family proteins in triple negative breast cancer cells.

PubMed

Mukhopadhyay, Partha; Lakshmanan, Imayavaramban; Ponnusamy, Moorthy P; Chakraborty, Subhankar; Jain, Maneesh; Pai, Priya; Smith, Lynette M; Lele, Subodh M; Batra, Surinder K

2013-01-01

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs. In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining. MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue. MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.

MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

PubMed Central

Mukhopadhyay, Partha; Lakshmanan, Imayavaramban; Ponnusamy, Moorthy P.; Chakraborty, Subhankar; Jain, Maneesh; Pai, Priya; Smith, Lynette M.; Lele, Subodh M.; Batra, Surinder K.

2013-01-01

Introduction Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs. Method In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining. Results MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue. Conclusions MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling. PMID:23408941

Evaluation of four affibody-based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors.

PubMed

Qi, Shibo; Miao, Zheng; Liu, Hongguang; Xu, Yingding; Feng, Yaqing; Cheng, Zhen

2012-06-20

The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes. This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.

EGF-Receptor Phosphorylation and Downstream Signaling are Activated by Benzo[a]pyrene 3,6-quinone and Benzo[a]pyrene 1,6-quinone in Human Mammary Epithelial Cells

PubMed Central

Rodríguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie; Lauer, Fredine T.; Burchiel, Scott W.

2013-01-01

Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo(a)pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-γ1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 μM), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-γ1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-γ1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the pattern of phosphorylation at EGFR, PLC-γ1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways. PMID:19166869

EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells.

PubMed

Rodríguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G; Lauer, Fredine T; Burchiel, Scott W

2009-03-15

Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-gamma1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 muM), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-gamma1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-gamma1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-gamma1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

EGFR-SGLT1 interaction does not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors.

PubMed

Ren, Jiangong; Bollu, Lakshmi R; Su, Fei; Gao, Guang; Xu, Lei; Huang, Wei-Chien; Hung, Mien-Chie; Weihua, Zhang

2013-09-01

Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in malignant tumors. Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that is overexpressed in many cancers including prostate cancer. Previously, we found that EGFR interacts with and stabilizes SGLT1 in cancer cells. In this study, we determined the micro-domain of EGFR that is required for its interaction with SGLT1 and the effects of activation/inactivation of EGFR on EGFR-SGLT1 interaction, measured the expression of EGFR and SGLT1 in prostate cancer tissues, and tested the effect of inhibition of SGLT1 on the sensitivity of prostate cancer cells to EGFR tyrosine inhibitors. We found that the autophosphorylation region (978-1210 amino acids) of EGFR was required for its sufficient interaction with SGLT1 and that this interaction was independent of EGFR's tyrosine kinase activity. Most importantly, the EGFR-SGLT1 interaction does not respond to EGFR tyrosine kinase modulators (EGF and tyrosine kinase inhibitors). EGFR and SGLT1 co-localized in prostate cancer tissues, and inhibition of SGLT1 by a SGLT1 inhibitor (Phlorizin) sensitized prostate cancer cells to EGFR inhibitors (Gefitinib and Erlotinib). These data suggest that EGFR in cancer cells can exist as either a tyrosine kinase modulator responsive status or an irresponsive status. SGLT1 is a protein involved in EGFR's functions that are irresponsive to EGFR tyrosine kinase inhibitors and, therefore, the EGFR-SGLT1 interaction might be a novel target for prostate cancer therapy. © 2013 Wiley Periodicals, Inc. This article is a U.S. Government work and is in the public domain in the USA.

Dynamic Bayesian Network Modeling of the Interplay between EGFR and Hedgehog Signaling.

PubMed

Fröhlich, Holger; Bahamondez, Gloria; Götschel, Frank; Korf, Ulrike

2015-01-01

Aberrant activation of sonic Hegdehog (SHH) signaling has been found to disrupt cellular differentiation in many human cancers and to increase proliferation. The SHH pathway is known to cross-talk with EGFR dependent signaling. Recent studies experimentally addressed this interplay in Daoy cells, which are presumable a model system for medulloblastoma, a highly malignant brain tumor that predominately occurs in children. Currently ongoing are several clinical trials for different solid cancers, which are designed to validate the clinical benefits of targeting the SHH in combination with other pathways. This has motivated us to investigate interactions between EGFR and SHH dependent signaling in greater depth. To our knowledge, there is no mathematical model describing the interplay between EGFR and SHH dependent signaling in medulloblastoma so far. Here we come up with a fully probabilistic approach using Dynamic Bayesian Networks (DBNs). To build our model, we made use of literature based knowledge describing SHH and EGFR signaling and integrated gene expression (Illumina) and cellular location dependent time series protein expression data (Reverse Phase Protein Arrays). We validated our model by sub-sampling training data and making Bayesian predictions on the left out test data. Our predictions focusing on key transcription factors and p70S6K, showed a high level of concordance with experimental data. Furthermore, the stability of our model was tested by a parametric bootstrap approach. Stable network features were in agreement with published data. Altogether we believe that our model improved our understanding of the interplay between two highly oncogenic signaling pathways in Daoy cells. This may open new perspectives for the future therapy of Hedghog/EGF-dependent solid tumors.

Effect of neoadjuvant chemotherapy and its correlation with HPV status, EGFR, Her-2-neu, and GADD45 expression in oral squamous cell carcinoma.

PubMed

Pandey, Manoj; Kannepali, Krishna Kiran; Dixit, Ruhi; Kumar, Mohan

2018-01-31

Head and neck cancers are the commonest cancer in Southeast Asia. Despite being a surface cancer, it is associated with significant morbidity as despite early detection by the patients they often report for treatment late and hence are associated with poor prognosis. The role of neoadjuvant chemotherapy in head and neck cancer is still under evaluation; there is a large subgroup of population that does not respond to chemotherapy, and hence, most studies have failed to show any survival benefit. This study evaluated the role of neoadjuvant therapy with docetaxel and carboplatin in patients with oral cancer and correlated the response to human papilloma virus, EGFR1, EGFR2, and GADD45 expression. A total of 24 locally advanced, non-metastatic oral cancer patients were included in the study. Tumor biopsies were taken prior to the start of neoadjuvant therapy for expression of EGFR, Her-2-Neu, and GADD45 by immunohistochemistry and for HPV by PCR. The response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) criteria after three cycles of chemotherapy. Statistical analysis was performed using correlation and Kaplan-Meier analysis; the difference in survival was calculated with log rank test. A total of 21 male and 3 female with a mean age of 53.12 years were enrolled. Sixty-five percent of these received three cycles of chemotherapy. Five patients were positive for HPV 16 and none for HPV 18. Twenty-two of 24 patients showed GADD45 expression, 3 showed expression of Her-2-Neu while all 24 showed expression for EGFR1 protein. Two-year overall survival was 81%; GADD45 expressions were found to significantly affect the overall and disease-free survival, while any of the other protein expression studied and HPV status was not significant. The result of the present study shows significant downgrading of the oral cancers with neoadjuvant chemotherapy suggesting its utility in borderline operable cases. However, the response of chemotherapy does not appear to be related to the expression of EGFR, Her-2-Neu, and GADD45 protein or presence of HPV. Bone involvement, perineural invasion, and GADD45 expression significantly predict OS and DFS. All patients who did not express Gadd45 died before 2 years. Study with more subjects and longer follow-up should be carried out to elucidate this relation further.

Curcumin targets FOLFOX-surviving colon cancer cells via inhibition of EGFRs and IGF-1R.

PubMed

Patel, Bhaumik B; Gupta, Deepshika; Elliott, Althea A; Sengupta, Vivek; Yu, Yingjie; Majumdar, Adhip P N

2010-02-01

Curcumin (diferuloylmethane), which has no discernible toxicity, inhibits initiation, promotion and progression of carcinogenesis. 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but produces an incomplete response resulting in survival of cells (chemo-surviving cells) that may lead to cancer recurrence. The present investigation was, therefore, undertaken to examine whether addition of curcumin to FOLFOX is a superior therapeutic strategy for chemo-surviving cells. Forty-eight-hour treatment of colon cancer HCT-116 and HT-29 cells with FOLFOX resulted in 60-70% survival, accompanied by a marked activation of insulin like growth factor-1 receptor (IGF-1R) and minor to moderate increase in epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2) as well as v-akt murine thymoma viral oncogene homolog 1 (AKT), cyclooxygenase-2 (COX-2) and cyclin-D1. However, inclusion of curcumin to continued FOLFOX treatment for another 48 h greatly reduced the survival of these cells, accompanied by a concomitant reduction in activation of EGFR, HER-2, IGF-1R and AKT, as well as expression of COX-2 and cyclin-D1. More importantly, EGFR tyrosine kinase inhibitor gefitinib or attenuation of IGF-1R expression by the corresponding si-RNA caused a 30-60% growth inhibition of chemo-surviving HCT-116 cells. However, curcumin alone was found to be more effective than both gefitinib and IGF-1R si-RNA mediated growth inhibition of chemo-surviving HCT-116 cells and addition of FOLFOX to curcumin did not increase the growth inhibitory effect of curcumin. Our data suggest that inclusion of curcumin in conventional chemotherapeutic regimens could be an effective strategy to prevent the emergence of chemoresistant colon cancer cells.

Paired-agent imaging for resection during surgery (PAIRS) of head and neck squamous cell carcinomas (Conference Presentation)

NASA Astrophysics Data System (ADS)

Samkoe, Kimberley S.; Tichauer, Kenneth M.; Chen, Eunice; Gunn, Jason R.; Hoopes, P. Jack; Wells, Wendy A.; Hasan, Tayyaba; Pogue, Brian W.

2016-03-01

Ninety percent of patients with head and neck squamous cell carcinomas (HNSCC) have overexpression of epidermal growth factor receptor (EGFR), which is correlated with poor prognosis. Complete surgical resection of HNSCC tumors has a large impact on patient survival, where detection of tumor at or close to surgical margins increases the risk of death at 5-years by 90%. In addition, large surgical margins can greatly increase the morbidity experienced by the patient due to functional and cosmetic damage of oral and facial structures. Single fluorescence targeting agents are often used for tumor detection in in vivo pre-clinical imaging; however, the arising signal is qualitative at best because it is a complex mixture of vascular perfusion, vascular leakage, inhibited lymphatic clearance, and receptor binding. In vivo ratiometric receptor concentration imaging (RCI) allows quantification of receptor expression (hence identification of cancerous tissue) by utilizing co-administered paired-agents consisting of a targeted agent and non-targeted perfusion agent to reference the plasma delivery and leakage. A panel of HNSCC tumors with varying levels of EGFR expression (SCC-15 >SCC-25 > SCC-09) have been imaged using ABY-029, a clinically relevant anti-EGFR affibody labeled with IRDye 800CW, and affibody control imaging agent labeled with IRDye 680RD. RCI maps of in vivo tissue have been created and are spatially correlated with EGFR and CD31 immunohistochemistry and basic H and E staining. The RCI threshold parameters for distinguishing tumor from normal tissues (skin and muscle) and the accuracy of margin detection in these tumors will be presented. RCI surgical resection will be further developed using a novel multi-channel, gated fluorescence-guided surgery (FGS) imaging system that is capable of performing RCI in normal room light.

Preclinical In Vitro, In Vivo, and Pharmacokinetic Evaluations of FLLL12 for the Prevention and Treatment of Head and Neck Cancers.

PubMed

Anisuzzaman, Abu Syed Md; Haque, Abedul; Rahman, Mohammad Aminur; Wang, Dongsheng; Fuchs, James R; Hurwitz, Selwyn; Liu, Yuan; Sica, Gabriel; Khuri, Fadlo R; Chen, Zhuo Georgia; Shin, Dong M; Amin, A R M Ruhul

2016-01-01

Despite its high promise for cancer prevention and therapy, the potential utility of curcumin in cancer is compromised by its low bioavailability and weak potency. The purpose of the current study was to assess the in vitro and in vivo efficacy and pharmacokinetic parameters of the potent curcumin analogue FLLL12 in SCCHN and identify the mechanisms of its antitumor effect. IC50 values against a panel of one premalignant and eight malignant head and neck cancer cell lines as well as apoptosis assay results suggested that FLLL12 is 10- to 24-fold more potent than natural curcumin depending on the cell line and induces mitochondria-mediated apoptosis. In vivo efficacy (xenograft) and pharmacokinetic studies also suggested that FLLL12 is significantly more potent and has more favorable pharmacokinetic properties than curcumin. FLLL12 strongly inhibited the expression of p-EGFR, EGFR, p-AKT, AKT, Bcl-2, and Bid and increased the expression of Bim. Overexpression of constitutively active AKT or Bcl-2 or ablation of Bim or Bid significantly inhibited FLLL12-induced apoptosis. Further mechanistic studies revealed that FLLL12 regulated EGFR and AKT at transcriptional levels, whereas Bcl-2 was regulated at the translational level. Finally, FLLL12 strongly inhibited the AKT downstream targets mTOR and FOXO1a and 3a. Taken together, our results strongly suggest that FLLL12 is a potent curcumin analogue with more favorable pharmacokinetic properties that induces apoptosis of head and neck cancer cell lines by inhibition of survival proteins including EGFR, AKT, and Bcl-2 and increasing of the proapoptotic protein Bim. ©2015 American Association for Cancer Research.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

PubMed

Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

2015-11-01

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. © 2015 Hughes, Oudin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).