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

Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line

PubMed Central

Sullivan, Ivana; Planchard, David

2017-01-01

Tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (EGFR) are now standard treatment in the clinic for patients with advanced EGFR mutant non-small-cell lung cancer (NSCLC). First-generation EGFR TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR tyrosine kinase domain, have resulted in a significant improvement in outcome for NSCLC patients with activating EGFR mutations (L858R and Del19). However, after a median duration of response of ~12 months, all patients develop tumor resistance, and in over half of these patients this is due to the emergence of the EGFR T790M resistance mutation. The second-generation EGFR/HER TKIs were developed to treat resistant disease, targeting not only T790M but EGFR-activating mutations and wild-type EGFR. Although they exhibited promising anti-T790M activity in the laboratory, their clinical activity among T790M+ NSCLC was poor mainly because of dose-limiting toxicity due to simultaneous inhibition of wild-type EGFR. The third-generation EGFR TKIs selectively and irreversibly target EGFR T790M and activating EGFR mutations, showing promising efficacy in NSCLC resistant to the first- and second-generation EGFR TKIs. They also appear to have lower incidences of toxicity due to the limited inhibitory effect on wild-type EGFR. Currently, the first-generation gefitinib and erlotinib and second-generation afatinib have been approved for first-line treatment of metastatic NSCLC with activating EGFR mutations. Among the third-generation EGFR TKIs, osimertinib is today the only drug approved by the Food and Drug Administration and the European Medicines Agency to treat metastatic EGFR T790M NSCLC patients who have progressed on or after EGFR TKI therapy. In this review, we summarize the available post-progression therapies including third-generation EGFR inhibitors and combination treatment strategies for treating patients with NSCLC harboring EGFR mutations and address the

Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates.

PubMed

Powers, Alicia D; Liu, Bi; Lee, Andrew G; Palecek, Sean P

2012-09-07

Overactive and overexpressed kinases have been implicated in the cause and progression of many cancers. Kinase inhibitors offer a targeted approach for treating cancers associated with increased or deregulated kinase activity. Often, however, cancer cells exhibit initial resistance to these inhibitors or evolve to develop resistance during treatment. Additionally, cancers of any one tissue type are typically heterogeneous in their oncogenesis mechanisms, and thus diagnosis of a particular type of cancer does not necessarily provide insight into what kinase therapies may be effective. For example, while some lung cancer cells that overexpress the epidermal growth factor receptor (EFGR) respond to treatment with EGFR kinase inhibitors, overexpression or hyperactivity of Met kinase correlates with resistance to EGFR kinase inhibitors. Here we describe a microfluidic-based assay for quantifying Met kinase activity in cancer cell lysates with the eventual goals of predicting cancer cell responsiveness to kinase inhibitors and monitoring development of resistance to these inhibitors. In this assay, we immobilized a phosphorylation substrate for Met kinase into macroporous hydrogel micropillars. We then exposed the micropillars to a cancer cell lysate and detected substrate phosphorylation using a fluorescently conjugated antibody. This assay is able to quantify Met kinase activity in whole cell lysate from as few as 150 cancer cells. It can also detect cells expressing overactive Met kinase in a background of up to 75% non-cancerous cells. Additionally, the assay can quantify kinase inhibition by the Met-specific kinase inhibitors SU11274 and PHA665752, suggesting predictive capability for cellular response to kinase inhibitors.

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

PubMed

Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

2018-03-15

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

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

Tang, Wenqing; Weng, Shuqiang; Zhang, Si

2013-05-10

Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits {sup 125}I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFRmore » in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using {sup 125}I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited {sup 125}I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation.« less

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

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.

WHSC1L1-mediated EGFR mono-methylation enhances the cytoplasmic and nuclear oncogenic activity of EGFR in head and neck cancer

PubMed Central

Saloura, Vassiliki; Vougiouklakis, Theodore; Zewde, Makda; Deng, Xiaolan; Kiyotani, Kazuma; Park, Jae-Hyun; Matsuo, Yo; Lingen, Mark; Suzuki, Takehiro; Dohmae, Naoshi; Hamamoto, Ryuji; Nakamura, Yusuke

2017-01-01

While multiple post-translational modifications have been reported to regulate the function of epidermal growth factor receptor (EGFR), the effect of protein methylation on its function has not been well characterized. In this study, we show that WHSC1L1 mono-methylates lysine 721 in the tyrosine kinase domain of EGFR, and that this methylation leads to enhanced activation of its downstream ERK cascade without EGF stimulation. We also show that EGFR K721 mono-methylation not only affects the function of cytoplasmic EGFR, but also that of nuclear EGFR. WHSC1L1-mediated methylation of EGFR in the nucleus enhanced its interaction with PCNA in squamous cell carcinoma of the head and neck (SCCHN) cells and resulted in enhanced DNA synthesis and cell cycle progression. Overall, our study demonstrates the multifaceted oncogenic function of the protein lysine methyltransferase WHSC1L1 in SCCHN, which is mediated through direct non-histone methylation of the EGFR protein with effects both in its cytoplasmic and nuclear functions. PMID:28102297

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

Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer *

PubMed Central

Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; de Castro, Gilberto

2015-01-01

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC. PMID:26398757

Identifying activating mutations in the EGFR gene: prognostic and therapeutic implications in non-small cell lung cancer.

PubMed

Lopes, Gabriel Lima; Vattimo, Edoardo Filippo de Queiroz; Castro Junior, Gilberto de

2015-01-01

Lung cancer is the leading cause of cancer-related deaths worldwide. Promising new therapies have recently emerged from the development of molecular targeted drugs; particularly promising are those blocking the signal transduction machinery of cancer cells. One of the most widely studied cell signaling pathways is that of EGFR, which leads to uncontrolled cell proliferation, increased cell angiogenesis, and greater cell invasiveness. Activating mutations in the EGFR gene (deletions in exon 19 and mutation L858R in exon 21), first described in 2004, have been detected in approximately 10% of all non-squamous non-small cell lung cancer (NSCLC) patients in Western countries and are the most important predictors of a response to EGFR tyrosine-kinase inhibitors (EGFR-TKIs). Studies of the EGFR-TKIs gefitinib, erlotinib, and afatinib, in comparison with platinum-based regimens, as first-line treatments in chemotherapy-naïve patients have shown that the EGFR-TKIs produce gains in progression-free survival and overall response rates, although only in patients whose tumors harbor activating mutations in the EGFR gene. Clinical trials have also shown EGFR-TKIs to be effective as second- and third-line therapies in advanced NSCLC. Here, we review the main aspects of EGFR pathway activation in NSCLC, underscore the importance of correctly identifying activating mutations in the EGFR gene, and discuss the main outcomes of EGFR-TKI treatment in NSCLC.

Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR mutant Lung Cancer

PubMed Central

Uddin, Sharmeen; Capelletti, Marzia; Ercan, Dalia; Ogino, Atsuko; Pratilas, Christine A.; Rosen, Neal; Gray, Nathanael S.; Wong, Kwok-Kin; Jänne, Pasi A.

2016-01-01

Irreversible pyrimidine based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR activating and EGFR inhibitor resistant T790M mutations more potently than wild type EGFR. While this class of mutant selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFR T790M, prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002 induced apoptosis and inhibits the emergence of resistance in WZ4002 sensitive models known to acquire resistance via both T790M dependent and independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial co-targeting of EGFR and MEK could significantly impede the development of acquired resistance in mutant EGFR lung cancer. PMID:26036643

Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

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

Shankaran, Harish; Zhang, Yi; Tan, Yunbing

2013-08-22

The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panelmore » of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.« less

Icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone.

PubMed

Liu, Weidong; Mao, Li; Ji, Feng; Chen, Fengli; Wang, Shouguo; Xie, Yue

2017-01-10

The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

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.

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.

Photonic modulation of EGFR: 280nm low level light arrests cancer cell activation and migration

NASA Astrophysics Data System (ADS)

Botelho, Cláudia M.; Marques, Rogério; Viruthachalam, Thiagarajan; Gonçalves, Odete; Vorum, Henrik; Gomes, Andreia C.; Neves-Petersen, Maria Teresa

2017-02-01

Overexpression of the Epidermal Growth Factor Receptor (EGFR) by cancer cells is associated with a poor prognosis for the patient. For several decades, therapies targeting EGFR have been designed, including the use of monoclonal antibodies and small molecule tyrosine kinase inhibitors. The use of these molecules had good clinical results, although its efficiency (and specificity) is still far from being optimal. In this paper, we present a new approach for a possible new cancer therapy targeting EGFR and using low intensity 280nm light. The influence of 280nm UVB illumination on cancer cells stimulated with 2nM of EGF was followed by time-lapse confocal microscopy. The 280nm illumination of the cancer cells blocks EGFR activation, inhibiting EGFR internalization and cell migration thus inhibiting the transition to the metastatic phenotype. Exposure time is a very important factor. The higher the illumination time the more significant differences were observed: 280nm light delayed or completely halted EGFR activation in the cell membrane, mainly at the cell junction level, and delayed or halted EGFR endocytic internalization, filopodia formation and cell migration.

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

Unravelling signal escape through maintained EGFR activation in advanced non-small cell lung cancer (NSCLC): new treatment options

PubMed Central

Remon, Jordi; Besse, Benjamin

2016-01-01

The discovery of activating epidermal growth factor receptor (EGFR) mutations has opened up a new era in the development of more effective treatments for patients with non-small cell lung cancer (NSCLC). However, patients with EGFR-activating mutated NSCLC treated with EGFR tyrosine kinase inhibitors (TKIs) ultimately develop acquired resistance (AR). Among known cases of patients with AR, 70% of the mechanisms involved in the development of AR to EGFR TKI have been identified and may be categorised as either secondary EGFR mutations such as the T790M mutation, activation of bypass track signalling pathways such as MET amplification, or histologic transformation. EGFR-mutant NSCLC tumours maintain oncogenic addiction to the EGFR pathway beyond progression with EGFR TKI. Clinical strategies that can be implemented in daily clinical practice to potentially overcome this resistance and prolong the outcome in this subgroup of patients are presented. PMID:27843631

Inhibition of EGFR attenuates fibrosis and stellate cell activation in diet-induced model of nonalcoholic fatty liver disease.

PubMed

Liang, Dandan; Chen, Hongjin; Zhao, Leping; Zhang, Wenxin; Hu, Jie; Liu, Zhiguo; Zhong, Peng; Wang, Wei; Wang, Jingying; Liang, Guang

2018-01-01

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. NAFLD begins with steatosis and advances to nonalcoholic steatohepatitis (NASH) and cirrhosis. The molecular mechanisms involved in NAFLD progression are not understood. Based on recent studies showing dysregulation of epidermal growth factor receptor (EGFR) in animal models of liver injury, we sought to determine if inhibition of EGFR mitigates liver fibrosis and HSC activation in NAFLD. We utilized the high fat diet (HFD)-induced murine model of liver injury to study the role of EGFR in NAFLD. The lipid accumulation, oxidative stress, hepatic stellate cell (HSC) activation and matrix deposition were examined in the liver tissues. We also evaluated the EGFR signaling pathway, ROS activation and pro-fibrogenic phenotype in oxidized low density lipoproteins (ox-LDL) challenged cultured HSCs. We demonstrate that EGFR was phosphorylated in liver tissues of HFD murine model of NAFLD. Inhibition of EGFR prevented diet-induced lipid accumulation, oxidative stress, and HSC activation and matrix deposition. In cultured HSCs, we show that ox-LDL caused rapid activation of the EGFR signaling pathway and induce the production of reactive oxygen species. EGFR also mediated HSC activation and promoted a pro-fibrogenic phenotype. In conclusion, our data demonstrate that EGFR plays an important role in NAFLD and is an attractive target for NAFLD therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

PubMed

Thai, Alesha; Chia, Puey L; Russell, Prudence A; Do, Hongdo; Dobrovic, Alex; Mitchell, Paul; John, Thomas

2017-09-01

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features. © 2017 Royal Australasian College of Physicians.

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.

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

PubMed Central

Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

2013-01-01

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

Activation of sperm EGFR by light irradiation is mediated by reactive oxygen species.

PubMed

Shahar, Shiran; Hillman, Pnina; Lubart, Rachel; Ickowicz, Debby; Breitbart, Haim

2014-01-01

To acquire fertilization competence, spermatozoa must undergo several biochemical and motility changes in the female reproductive tract, collectively called capacitation. Actin polymerization and the development of hyperactivated motility (HAM) are part of the capacitation process. In a recent study, we showed that irradiation of human sperm with visible light stimulates HAM through a mechanism involving reactive-oxygen-species (ROS), Ca(2+) influx, protein kinases A (PKA), and sarcoma protein kinase (Src). Here, we showed that this effect of light on HAM is mediated by ROS-dependent activation of the epidermal growth factor receptor (EGFR). Interestingly, ROS-mediated HAM even when the EGFR was activated by EGF, the physiological ligand of EGFR. Light irradiation stimulated ROS-dependent actin polymerization, and this effect was abrogated by PBP10, a peptide which activates the actin-severing protein, gelsolin, and causes actin-depolymerization in human sperm. Light-stimulated tyrosine phosphorylation of Src-dependent gelsolin, resulting in enhanced HAM. Thus, light irradiation stimulates HAM through a mechanism involving Src-mediated actin polymerization. Light-stimulated HAM and in vitro-fertilization (IVF) rate in mouse sperm, and these effects were mediated by ROS and EGFR. In conclusion, we show here that irradiation of sperm with visible light, enhances their fertilization capacity via a mechanism requiring ROS, EGFR and HAM. © 2014 The American Society of Photobiology.

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

Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

PubMed Central

Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

2002-01-01

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B

PubMed Central

Zhang, Hongqiao; Forman, Henry Jay

2015-01-01

Src, a non-receptor protein tyrosine kinase involved in many biological processes, can be activated through both redox-dependent and independent mechanisms. 4-Hydroxy-2-nonenal (HNE) is a lipid peroxidation product that is increased in pathophysiological conditions associated with Src activation. This study examined how HNE activates human c-Src. In the canonical pathway Src activation is initiated by dephosphorylation of pTyr530 followed by conformational change that causes Src auto-phosphorylation at Tyr419 and its activation. HNE increased Src activation in both dose- and time-dependent manner, while it also increased Src phosphorylation at Tyr530 (pTyr530 Src), suggesting that HNE activated Src via a non-canonical mechanism. Protein tyrosine phosphatase 1B inhibitor (539741), at concentrations that increased basal pTyr530 Src, also increased basal Src activity and significantly reduced HNE-mediated Src activation. The EGFR inhibitor, AG1478, and EGFR silencing, abrogated HNE-mediated EGFR activation and inhibited basal and HNE-induced Src activity. In addition, AG1478 also eliminated the increase of basal Src activation by a PTP1B inhibitor. Taken together these data suggest that HNE can activate Src partly through a non-canonical pathway involving activation of EGFR and inhibition of PTP1B. PMID:26453921

6,7-Dimorpholinoalkoxy quinazoline derivatives as potent EGFR inhibitors with enhanced antiproliferative activities against tumor cells.

PubMed

Zhang, Yaling; Chen, Li; Xu, Hongjiang; Li, Xiabing; Zhao, Lijun; Wang, Wei; Li, Baolin; Zhang, Xiquan

2018-03-10

A series of novel 6,7-dimorpholinoalkoxy quinazoline derivatives was designed, synthesized and evaluated as potent EGFR inhibitors. Most of synthesized derivatives exhibited moderate to excellent antiproliferative activities against five human tumor cell lines. Compound 8d displayed the most remarkable inhibitory activities against tumor cells expressing wild type (A431, A549 and SW480 cells) or mutant (HCC827 and NCI-H1975 cells) epidermal growth factor receptor (EGFR) (with IC 50 values in the range of 0.37-4.87 μM), as well as more potent inhibitory effects against recombinant EGFR tyrosine kinase (EGFR-TK, wt or T790M) (with the IC 50 values of 7.0 and 9.3 nM, respectively). Molecular docking showed that 8d can form four hydrogen bonds with EGFR, and two of them were located in the Asp855-Phe856-Gly857 (DFG) motif of EGFR. Meanwhile, 8d can significantly block EGF-induced EGFR activation and the phosphorylation of its downstream proteins such as Akt and Erk1/2 in human NSCLC cells. Also, 8d mediated cell apoptosis and the prolongation of cell cycle progression in G0/G1-phase in A549 cells. The work would have remarkable implications for further design and development of more potent EGFR tyrosine kinase inhibitors (TKIs). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab.

PubMed

Zhang, Kai-Liang; Zhou, Xuan; Han, Lei; Chen, Lu-Yue; Chen, Ling-Chao; Shi, Zhen-Dong; Yang, Ming; Ren, Yu; Yang, Jing-Xuan; Frank, Thomas S; Zhang, Chuan-Bao; Zhang, Jun-Xia; Pu, Pei-Yu; Zhang, Jian-Ning; Jiang, Tao; Wagner, Eric J; Li, Min; Kang, Chun-Sheng

2014-03-20

Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.

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.

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

Effects of oncogenic mutations on the conformational free-energy landscape of EGFR kinase

PubMed Central

Sutto, Ludovico; Gervasio, Francesco Luigi

2013-01-01

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are frequently found in many cancers. It has been suggested that changes in the equilibrium between its active and inactive conformations are linked to its oncogenic potential. Here, we quantify the effects of some of the most common single (L858R and T790M) and double (T790M-L858R) oncogenic mutations on the conformational free-energy landscape of the EGFR kinase domain by using massive molecular dynamics simulations together with parallel tempering, metadynamics, and one of the best force-fields available. Whereas the wild-type EGFR catalytic domain monomer is mostly found in an inactive conformation, our results show a clear shift toward the active conformation for all of the mutants. The L858R mutation stabilizes the active conformation at the expense of the inactive conformation and rigidifies the αC-helix. The T790M gatekeeper mutant favors activation by stabilizing a hydrophobic cluster. Finally, T790M with L858R shows a significant positive epistasis effect. This combination not only stabilizes the active conformation, but in nontrivial ways changes the free-energy landscape lowering the transition barriers. PMID:23754386

Effects of oncogenic mutations on the conformational free-energy landscape of EGFR kinase.

PubMed

Sutto, Ludovico; Gervasio, Francesco Luigi

2013-06-25

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase are frequently found in many cancers. It has been suggested that changes in the equilibrium between its active and inactive conformations are linked to its oncogenic potential. Here, we quantify the effects of some of the most common single (L858R and T790M) and double (T790M-L858R) oncogenic mutations on the conformational free-energy landscape of the EGFR kinase domain by using massive molecular dynamics simulations together with parallel tempering, metadynamics, and one of the best force-fields available. Whereas the wild-type EGFR catalytic domain monomer is mostly found in an inactive conformation, our results show a clear shift toward the active conformation for all of the mutants. The L858R mutation stabilizes the active conformation at the expense of the inactive conformation and rigidifies the αC-helix. The T790M gatekeeper mutant favors activation by stabilizing a hydrophobic cluster. Finally, T790M with L858R shows a significant positive epistasis effect. This combination not only stabilizes the active conformation, but in nontrivial ways changes the free-energy landscape lowering the transition barriers.

Preclinical Comparison of Osimertinib with Other EGFR-TKIs in EGFR-Mutant NSCLC Brain Metastases Models, and Early Evidence of Clinical Brain Metastases Activity.

PubMed

Ballard, Peter; Yates, James W T; Yang, Zhenfan; Kim, Dong-Wan; Yang, James Chih-Hsin; Cantarini, Mireille; Pickup, Kathryn; Jordan, Angela; Hickey, Mike; Grist, Matthew; Box, Matthew; Johnström, Peter; Varnäs, Katarina; Malmquist, Jonas; Thress, Kenneth S; Jänne, Pasi A; Cross, Darren

2016-10-15

Approximately one-third of patients with non-small cell lung cancer (NSCLC) harboring tumors with EGFR-tyrosine kinase inhibitor (TKI)-sensitizing mutations (EGFRm) experience disease progression during treatment due to brain metastases. Despite anecdotal reports of EGFR-TKIs providing benefit in some patients with EGFRm NSCLC brain metastases, there is a clinical need for novel EGFR-TKIs with improved efficacy against brain lesions. We performed preclinical assessments of brain penetration and activity of osimertinib (AZD9291), an oral, potent, irreversible EGFR-TKI selective for EGFRm and T790M resistance mutations, and other EGFR-TKIs in various animal models of EGFR-mutant NSCLC brain metastases. We also present case reports of previously treated patients with EGFRm-advanced NSCLC and brain metastases who received osimertinib in the phase I/II AURA study (NCT01802632). Osimertinib demonstrated greater penetration of the mouse blood-brain barrier than gefitinib, rociletinib (CO-1686), or afatinib, and at clinically relevant doses induced sustained tumor regression in an EGFRm PC9 mouse brain metastases model; rociletinib did not achieve tumor regression. Under positron emission tomography micro-dosing conditions, [ 11 C]osimertinib showed markedly greater exposure in the cynomolgus monkey brain than [ 11 C]rociletinib and [ 11 C]gefitinib. Early clinical evidence of osimertinib activity in previously treated patients with EGFRm-advanced NSCLC and brain metastases is also reported. Osimertinib may represent a clinically significant treatment option for patients with EGFRm NSCLC and brain metastases. Further investigation of osimertinib in this patient population is ongoing. Clin Cancer Res; 22(20); 5130-40. ©2016 AACR. ©2016 American Association for Cancer Research.

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

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.

EGFR Activation Mediates Inhibition of Axon Regeneration by Myelin and Chondroitin Sulfate Proteoglycans

NASA Astrophysics Data System (ADS)

Koprivica, Vuk; Cho, Kin-Sang; Park, Jong Bae; Yiu, Glenn; Atwal, Jasvinder; Gore, Bryan; Kim, Jieun A.; Lin, Estelle; Tessier-Lavigne, Marc; Chen, Dong Feng; He, Zhigang

2005-10-01

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.

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

ASP8273 tolerability and antitumor activity in TKI-naive Japanese patients with EGFR mutation-positive non-small cell lung cancer.

PubMed

Azuma, Koichi; Nishio, Makoto; Hayashi, Hidetoshi; Kiura, Katsuyuki; Satouchi, Miyako; Sugawara, Shunichi; Hida, Toyoaki; Iwamoto, Yasuo; Inoue, Akira; Takeda, Koji; Ikeda, Satoshi; Nakagawa, Tomoki; Takeda, Kentaro; Asahina, Seitaro; Komatsu, Kanji; Morita, Satoshi; Fukuoka, Masahiro; Nakagawa, Kazuhiko

2018-05-28

Epidermal growth factor receptor (EGFR) activating mutations occur in approximately 50% of East Asian patients with non-small cell lung cancer (NSCLC) and confer sensitivity to tyrosine kinase inhibitors (TKI). ASP8273 is an orally administered, irreversible EGFR-TKI that inhibits EGFR activating mutations and has demonstrated clinical activity in patients with EGFR mutation-positive NSCLC. EGFR-TKI-naïve Japanese adult patients (≥20 years) with NSCLC harboring EGFR mutations were enrolled in this open-label, single-arm, Phase 2 study (NCT02500927). Patients received ASP8273 300mg once daily until discontinuation criteria were met. The primary endpoint was to determine the safety of ASP8273 300mg; secondary endpoint was antitumor activity defined by RECIST v1.1. Thirty-one patients (12M/19F; median age 64 years [range: 31-82]) with EGFR mutation-positive NSCLC were enrolled; as of 23 February 2016, 25 patients (81%) were still on study. Of the 31 patients, 27 (87%) had an ex19del (n=13, 42%) or a L858R (n=14, 45%) EGFR activating mutation; 2 (7%) had L861Q mutation and 5 (16%) had other EGFR activating mutations, two had an activating mutation and the T790M resistance mutation. The most commonly reported treatment-emergent adverse event was diarrhea [n=24, 77%]. All patients had at least 1 post-baseline scan; 1 patient (3%) achieved a confirmed complete response, 13 (42%) had a confirmed partial response, and 15 (48%) had confirmed stable disease (disease control rate: 94% [n=29/31]) per investigator assessment. Once-daily ASP8273 300 mg was generally well tolerated and demonstrated antitumor activity in TKI-naïve Japanese patients with EGFR mutation-positive NSCLC. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

PubMed Central

de Jong, Petrus R.; Takahashi, Naoki; Harris, Alexandra R.; Lee, Jihyung; Bertin, Samuel; Jeffries, James; Jung, Michael; Duong, Jen; Triano, Amy I.; Lee, Jongdae; Niv, Yaron; Herdman, David S.; Taniguchi, Koji; Kim, Chang-Whan; Dong, Hui; Eckmann, Lars; Stanford, Stephanie M.; Bottini, Nunzio; Corr, Maripat; Raz, Eyal

2014-01-01

The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (ApcMin/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in ApcMin/+ mice, similar to — as well as in conjunction with — a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis. PMID:25083990

Naturally occurring phenolic acids modulate TPA-induced activation of EGFR, AP-1, and STATs in mouse epidermis.

PubMed

Cichocki, Michał; Dałek, Miłosz; Szamałek, Mateusz; Baer-Dubowska, Wanda

2014-01-01

Epidermal growth factor receptor (EGFR) plays an important role in epithelial carcinogenesis and appears to be involved in STATs activation. In this study we investigated the possible interference of naturally occurring phenolic acids with EGFR, activator protein-1 (AP-1), and signal transducers and activators of transcription (STATs) pathways activated by topical application of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Balb/c mice epidermis. Pretreatment with tannic or chlorogenic acid resulted in a significant decrease in the phosphorylation of EGFR Y-1068 and Y-1173 tyrosine residues, which was accompanied by reduced activation of AP-1. Tannic acid decreased also the c-Jun AP-1 subunit level and binding to TPA response element (TRE) (3- and 2-fold in comparison with TPA-treated group respectively). Simultaneous reduction of JNK activity might be responsible for reduced activation of AP-1. In contrast to these more complex phenolics, protocatechuic acid increased the activity of JNK and was also the most efficient inhibitor of STATs activation. These results indicate that naturally occurring phenolic acids, by decreasing EGFR, AP-1, and STATs activation, may modulate other elements both upstream and downstream in these pathways and thus inhibit the tumor development. Although more complex phenolics affect mainly the EGFR/AP-1 pathway, STATs seem to be the most important targets for simple compounds, such as protocatechuic acid.

Impact of active smoking on survival of patients with metastatic lung adenocarcinoma harboring an epidermal growth factor receptor (EGFR) mutation.

PubMed

Erdogan, Bulent; Kodaz, Hilmi; Karabulut, Senem; Cinkaya, Ahmet; Tozkir, Hilmi; Tanriverdi, Ozgur; Cabuk, Devrim; Hacioglu, Muhammed Bekir; Turkmen, Esma; Hacibekiroglu, Ilhan; Uzunoglu, Sernaz; Cicin, Irfan

2016-11-10

Lung cancer in smokers and non-smokers demonstrates distinct genetic profiles, and cigarette smoking affects epidermal growth factor receptor (EGFR) function and causes secondary EGFR tyrosine kinase resistance. We evaluated the effect of active smoking in patients with metastatic lung adenocarcinoma. A total of 132 metastatic lung adenocarcinoma patients, diagnosed between 2008 and 2013, with known EGFR mutation status, were evaluated retrospectively. Among these patients, 40 had an activating EGFR mutation. Patients who continued smoking during the treatment were defined as active smokers. Former smokers and never smokers were together defined as non-smokers. The outcomes of the treatment in relation to the EGFR mutation and smoking status were evaluated. The median follow-up time was 10.5 months. The overall response rate for the first-line therapy was significantly higher among the EGFR-mutant patients (p = 0.01), however, smoking status had no impact on the response rate (p = 0.1). The EGFR-mutant active smokers progressed earlier than the non-smokers (p < 0.01). The overall survival (OS) of the non-smokers and patients treated with erlotinib was significantly longer (p = 0.02 and p = 0.01, respectively). Smoking status did not affect the OS in EGFR wild type tumors (p = 0.49) but EGFR-mutant non-smokers had a longer OS than the active smokers (p = 0.01).The active smokers treated with erlotinib had poorer survival than the non-smokers (p = 0.03). Multivariate analysis of EGFR-mutant patients showed that erlotinib treatment at any line and non-smoking were independent prognostic factors for the OS (p = 0.04 and p = 0.01, respectively). Smoking during treatment is a negative prognostic factor in metastatic lung adenocarcinoma with an EGFR mutation.

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

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

PubMed

Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation.

PubMed

Tanaka, Tomohiro; Zhou, Yue; Ozawa, Tatsuhiko; Okizono, Ryuya; Banba, Ayako; Yamamura, Tomohiro; Oga, Eiji; Muraguchi, Atsushi; Sakurai, Hiroaki

2018-02-16

The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand lead to a higher proportion of receptor that is recycled and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e. it has yet to be established what happens to unbound receptors following stimulation with ligand. Here we demonstrate that a minimal level of activation of epidermal growth factor receptor (EGFR) tyrosine kinase by low levels of ligand is sufficient to fully activate downstream mitogen-activated protein kinase (MAPK) pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated mitogen-activated protein kinase. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser-1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

NF-{kappa}B signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells

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

Sakuma, Yuji, E-mail: ysakuma@gancen.asahi.yokohama.jp; Yamazaki, Yukiko; Nakamura, Yoshiyasu

2012-07-13

Highlights: Black-Right-Pointing-Pointer EGFR-mutant cells in 3D culture resist EGFR inhibition compared with suspended cells. Black-Right-Pointing-Pointer Degradation of I{kappa}B and activation of NF-{kappa}B are observed in 3D-cultured cells. Black-Right-Pointing-Pointer Inhibiting NF-{kappa}B enhances the efficacy of the EGFR inhibitor in 3D-cultured cells. -- Abstract: Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cellsmore » cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of I{kappa}B{alpha}, the inhibitor of nuclear factor (NF)-{kappa}B, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-{kappa}B. Moreover, the inhibition of NF-{kappa}B with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-{kappa}B signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.« less

EGFR TKI as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer

PubMed Central

Nan, Xueli; Xie, Chao; Yu, Xueyan; Liu, Jie

2017-01-01

After the discovery of activating mutations in EGFR, EGFR tyrosine kinase inhibitors (TKIs) have been introduced into the first-line treatment of non-small-cell lung cancer (NSCLC). A series of studies have shown that EGFR TKI monotherapy as first-line treatment can benefit NSCLC patients harbouring EGFR mutations. Besides, combination strategies based on EGFR TKIs in the first line treatment have also been proved to delay the occurrence of resistance. In this review, we summarize the scientific literature and evidence of EGFR TKIs as first-line therapy from the first-generation EGFR TKIs to conceptually proposed fourth-generation EGFR TKI, and also recommend the application of monotherapy and combination therapies of the EGFR-based targeted therapy with other agents such as chemotherapy, anti-angiogenic drugs and immunecheckpoint inhibitors. PMID:29088904

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

Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier

PubMed Central

Wang, Xiangru; Maruvada, Ravi; Morris, Andrew J.; Liu, Jun O.; Baek, Dong Jae; Kim, Kwang Sik

2016-01-01

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood–brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics. PMID:27711202

Cancer stem cell-like population is preferentially suppressed by EGFR-TKIs in EGFR-mutated PC-9 tumor models.

PubMed

Yang, Fan; Li, Yang; Liu, Bin; You, Jiacong; Zhou, Qinghua

2018-01-01

Although the epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling systems synergistically regulate many essential developmental and regenerative processes in lung cancer, the mechanisms of their crosstalk remain poorly defined. Our study aimed to investigate an interaction between EGFR and the β-catenin signal. In this study, we described a potent activation of β-catenin by EGFR, which is dependent of the PtdIns3K/AKT pathway. We found EGF activated β-catenin signaling via phosphorylation of EGFR and AKT in EGFR-mutated PC-9 lung cancer cells. Meanwhile, EGFR tyrosine kinase inhibitors (EGFR-TKIs) regulated cancer stem-like cells (CSCs) by inhibiting autophosphorylation of EGFR and downstream signaling proteins, as well as β-catenin. Further, β-catenin depletion by RNA interference virtually eliminated cancer stem cell-like population in PC-9 cells in vitro. The nude mice transplantation model was also performed to confirm EGFR-TKIs strongly inhibited the β-catenin signal and decreased CSCs. Importantly, the reduction of CSCs that sorted out by side population (SP) cells significantly reduced the migration capability. Thus, our results improved the understanding of this process to provide insights into mechanisms of responding to EGFR-TKIs. Our discoveries raise an intriguing question of the role of β-catenin in EGFR-TKIs-treated cancer stem cell-like population(s) and its potential as a new therapeutic target for NSCLC in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2009-01-01

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced

EGFR Amplification as a Target in Gastroesophageal Adenocarcinoma: Do Anti-EGFR Therapies Deserve a Second Chance?

PubMed

Strickler, John H

2018-06-01

Anti-EGFR therapies have failed to improve survival for unselected patients with metastatic gastroesophageal cancer, but in a subset of patients, EGFR amplification may predict treatment benefit. Maron and colleagues report the clinical activity of anti-EGFR therapies in a cohort of patients with EGFR -amplified metastatic gastroesophageal cancer and utilize serial blood and tumor tissue collection to identify molecular drivers of treatment sensitivity and resistance. Their insights offer a path to overcome technical limitations associated with EGFR amplification and facilitate molecularly targeted therapeutic strategies. Cancer Discov; 8(6); 679-81. ©2018 AACR See related article by Maron et al., p. 696 . ©2018 American Association for Cancer Research.

Periodic mechanical stress activates EGFR-dependent Rac1 mitogenic signals in rat nucleus pulpous cells via ERK1/2

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

Gao, Gongming; Shen, Nan; Jiang, Xuefeng

2016-01-15

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p < 0.05 for each) and was associated with increases in the phosphorylation and activation of EGFR, Rac1, and ERK1/2 (p < 0.05 for each). Pretreatment with the ERK1/2 selective inhibitor PD98059 reduced periodic mechanical stress-induced nucleus pulpous cell proliferationmore » (p < 0.05 for each), while the activation levels of EGFR and Rac1 were not inhibited. Proliferation and phosphorylation of ERK1/2 were inhibited after pretreatment with the Rac1 inhibitor NSC23766 in nucleus pulpous cells in response to periodic mechanical stress (p < 0.05 for each), while the phosphorylation site of EGFR was not affected. Inhibition of EGFR activity with AG1478 abrogated nucleus pulpous cell proliferation (p < 0.05 for each) and attenuated Rac1 and ERK1/2 activation in nucleus pulpous cells subjected to periodic mechanical stress (p < 0.05 for each). These findings suggest that periodic mechanical stress promotes nucleus pulpous cell proliferation in part through the EGFR-Rac1-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade. - Highlights: • The mechanism involved in nucleus pulpous cells to respond to mechanical stimuli. • Periodic mechanical stress can stimulate the phosphorylation of EGFR. • EGFR activates Rac1 and leads to rat nucleus pulpous cell proliferation. • EGFR and Rac1 activate ERK1/2 mitogenic signals in nucleus pulpous cells. • EGFR-Rac1-ERK1/2 is constitutes at least one critical signal transduction pathway.« less

Integrated experimental and model-based analysis reveals the spatial aspects of EGFR activation dynamics

PubMed Central

Shankaran, Harish; Zhang, Yi; Chrisler, William B.; Ewald, Jonathan A.; Wiley, H. Steven; Resat, Haluk

2012-01-01

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models to determine the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor phosphorylation kinetics at the cell surface and early endosomes are comparable. We validated the last finding by measuring the EGFR dephosphorylation rates at various times following ligand addition both in whole cells and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. This study demonstrates that an iterative cycle of experiments and modeling can be used to gain mechanistic insight regarding complex cell signaling networks. PMID:22952062

Integrated Experimental and Model-based Analysis Reveals the Spatial Aspects of EGFR Activation Dynamics

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

Shankaran, Harish; Zhang, Yi; Chrisler, William B.

2012-10-02

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models for deconvolving the contributions of receptor dimerizationmore » and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor dephosphorylation rates at the cell surface and early endosomes are comparable. We validated the last finding by measuring EGFR dephosphorylation rates at various times following ligand addition both in whole cells, and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. Further, the mathematical model described here can be extended to determine receptor dimer abundances in cells co-expressing various levels of ErbB receptors. This study demonstrates that an iterative

Antitumor Efficacy of Dual Blockade of EGFR Signaling by Osimertinib in Combination With Selumetinib or Cetuximab in Activated EGFR Human NCLC Tumor Models.

PubMed

Della Corte, Carminia Maria; Ciaramella, Vincenza; Cardone, Claudia; La Monica, Silvia; Alfieri, Roberta; Petronini, Pier Giorgio; Malapelle, Umberto; Vigliar, Elena; Pepe, Francesco; Troncone, Giancarlo; Castellone, Maria Domenica; Troiani, Teresa; Martinelli, Erika; Ciardiello, Fortunato; Morgillo, Floriana

2018-03-08

Osimertinib showed great clinical efficacy for activated-EGFR NCLC patient treatment. The aim of this work was to test the efficacy of a complete EGFR-inhibition by osimertinib plus the monoclonal antibody cetuximab or the MEK1/2-inhibitor selumetinib in EGFR-mutated NCLC in vivo models. We evaluated combinations of osimertinib plus selumetinib/cetuximab in HCC827 (E746-A759del/T790M-), H1975 (L858R/T790M+), and PC9-T790M (E746-A759del /T790M+) xenografts in second-line therapy after the development of resistance to osimertinib, and in first-line therapy, and we explored mechanisms of resistance to these treatments. The addition of selumetinib or cetuximab to osimertinib in second-line therapy reverted the sensibility to osimertinib in the majority of mice, with a response rate (RR) of 50% to 80%, and a median progression-free survival (mPFS) of first- plus second-line of therapy of 28 weeks. The early use of combinations in first-line therapy increased the RR to 90%, with an mPFS not reached in all combination arms in the three xenografts models, with a statistically significant superiority (p < 0.005) as compared to osimertinib, achieving in first-line therapy an mPFS time of 17 to 18 weeks. Moreover, in ex vivo primary cell cultures obtained from osimertinib plus selumetinib-resistant tumors, we found Hedgehog pathway activation and we showed that therapy with an SMO inhibitor plus osimertinib and selumetinib inhibited proliferation and migratory and invasive properties of resistant cells. We showed that a dual vertical EGFR blockade with osimertinib plus selumetinib/cetuximab is a novel effective therapeutic option in EGFR-mutated NCLC and that hedgehog pathway activation and its interplay with MAPK is involved in resistance to these combination treatments. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Antitumor activity of taspine by modulating the EGFR signaling pathway of Erk1/2 and Akt in vitro and in vivo.

PubMed

Zhang, Yanmin; Zheng, Lei; Zhang, Jie; Dai, Bingling; Wang, Nan; Chen, Yinnan; He, Langchong

2011-11-01

EGFR, as a critical signaling pathway in many human tumors, has become an important target of cancer drug design. Taspine has shown meaningful angiogenesis activity in previous studies. This paper is to investigate the antitumor action of taspine by modulating the EGFR signaling pathway. The study determined the expression of key signaling molecules of EGFR (EGFR, Akt, p-Akt, Erk, and p-Erk) by Western blot and real-time PCR and analyzed their correlations with subsequent reactions. In addition, the cell proliferation, migration, and EGF production were examined by MTT, transwell system, and ELISA. The antitumor activity in vivo was carried out by xenograft in athymic mice. The results showed that taspine could inhibit A431 and Hek293/EGFR cell proliferation and A431 cell migration as well as EGF production. Compared to the negative control, EGFR, Akt, and phosphorylation of Akt were significantly inhibited by taspine treatment in A431 and HEK293/EGFR cells. Consistent with the inhibition of Akt activity, Erk1/2 and its phosphorylation were reduced. Moreover, taspine inhibited A431 xenograft tumor growth. These results suggest that EGFR activated by EGF and its downstream signaling pathways proteins could be downregulated by taspine in a dose-dependent manner. The antitumor mechanism of taspine through the EGFR pathway lies in the ability to inhibit A431 cell proliferation and migration by reducing EGF secretion. This occurs through the repression of EGFR which mediates not only MAPK (Erk1/2) but also Akt signals. © Georg Thieme Verlag KG Stuttgart · New York.

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

Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

PubMed Central

2015-01-01

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

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

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

Irreversible Inhibition of EGFR: Modeling the Combined Pharmacokinetic-Pharmacodynamic Relationship of Osimertinib and Its Active Metabolite AZ5104.

PubMed

Yates, James W T; Ashton, Susan; Cross, Darren; Mellor, Martine J; Powell, Steve J; Ballard, Peter

2016-10-01

Osimertinib (AZD9291) is a potent, selective, irreversible inhibitor of EGFR-sensitizing (exon 19 and L858R) and T790M-resistant mutation. In vivo, in the mouse, it is metabolized to an active des-methyl metabolite, AZ5104. To understand the therapeutic potential in patients, this study aimed to assess the relationship between osimertinib pharmacokinetics, the pharmacokinetics of the active metabolite, the pharmacodynamics of phosphorylated EGFR reduction, and efficacy in mouse xenograft models of EGFR-driven cancers, including two NSCLC lines. Osimertinib was dosed in xenografted models of EGFR-driven cancers. In one set of experiments, changes in phosphorylated EGFR were measured to confirm target engagement. In a second set of efficacy studies, the resulting changes in tumor volume over time after repeat dosing of osimertinib were observed. To account for the contributions of both molecules, a mathematical modeling approach was taken to integrate the resulting datasets. The model was able to describe the pharmacokinetics, pharmacodynamics, and efficacy in A431, PC9, and NCI-H1975 xenografts, with the differences in sensitivity described by the varying potency against wild-type, sensitizing, and T790M-mutant EGFR and the phosphorylated EGFR reduction required to reduce tumor volume. It was inferred that recovery of pEGFR is slower after chronic dosing due to reduced resynthesis. It was predicted and further demonstrated that although inhibition is irreversible, the resynthesis of EGFR is such that infrequent intermittent dosing is not as efficacious as once daily dosing. Mol Cancer Ther; 15(10); 2378-87. ©2016 AACR. ©2016 American Association for Cancer Research.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

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

Yu, Zhiwei; Cui, Binbin; Jin, Yinghu

2011-08-12

Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study,more » using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.« less

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

PubMed Central

Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

2017-01-01

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P < 0.05). Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P < 0.05), an indication of reduced microvessel density in tumor xenografts. Moreover, high-dose icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study. PMID:27852073

The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation.

PubMed

Garcia-Recio, Susana; Pastor-Arroyo, Eva M; Marín-Aguilera, Mercedes; Almendro, Vanessa; Gascón, Pedro

2015-01-01

Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation. Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1-10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines. Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

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

Ebi, Masahide; Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp; Shimura, Takaya

2010-11-19

Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cellmore » growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway.

PubMed

Yi, Lian; Shi, Tujin; Gritsenko, Marina A; X'avia Chan, Chi-Yuet; Fillmore, Thomas L; Hess, Becky M; Swensen, Adam C; Liu, Tao; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun

2018-04-17

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR-MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO 2 ), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO 2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 μg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 μg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25 μg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.

STAT1 Activation is Enhanced by Cisplatin and Variably Affected by EGFR Inhibition in HNSCC Cells

PubMed Central

Schmitt, Nicole C.; Trivedi, Sumita; Ferris, Robert L.

2015-01-01

Cisplatin is a cytotoxic chemotherapeutic drug frequently used to treat many solid tumors, including head and neck squamous cell carcinoma (HNSCC). EGFR inhibitors have also shown efficacy as alternatives to cisplatin in some situations. However, large clinical trials have shown no added survival benefit from the use of these two drugs in combination. Possible explanations for this include overlapping downstream signaling cascades. Using in vitro studies, we tested the hypothesis that cisplatin and EGFR inhibitors rely on the activation of the tumor suppressor STAT1, characterized by its phosphorylation at serine (S727) or tyrosine (Y701) residues. Cisplatin consistently increased the levels of p-S727-STAT1, and STAT1 siRNA knockdown attenuated cisplatin-induced cell death. EGFR stimulation also activated p-S727-STAT1 and p-Y701-STAT1 in a subset of cell lines, whereas EGFR inhibitors alone decreased levels of p-S727-STAT1 and p-Y701-STAT1 in these cells. Contrary to our hypothesis, EGFR inhibitors added to cisplatin treatment caused variable effects among cell lines, with attenuation of p-S727-STAT1 and enhancement of cisplatin-induced cell death in some cells and minimal effect in other cells. Using HNSCC tumor specimens from a clinical trial of adjuvant cisplatin plus the anti-EGFR antibody panitumumab, higher intratumoral p-S727-STAT1 appeared to correlate with worse survival. Together, these results suggest that cisplatin-induced cell death is associated with STAT1 phosphorylation, and the addition of anti-EGFR therapy to cisplatin has variable effects on STAT1 and cell death in HNSCC. PMID:26141950

CMTM7 knockdown increases tumorigenicity of human non-small cell lung cancer cells and EGFR-AKT signaling by reducing Rab5 activation.

PubMed

Liu, Baocai; Su, Yu; Li, Ting; Yuan, Wanqiong; Mo, Xiaoning; Li, Henan; He, Qihua; Ma, Dalong; Han, Wenling

2015-12-01

The dysregulation of epidermal growth factor receptor (EGFR) signaling has been well documented to contribute to the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer death in the world. EGF-stimulated EGFR activation induces receptor internalization and degradation, which plays an important role in EGFR signaling. This process is frequently deregulated in cancer cells, leading to enhanced EGFR levels and signaling. Our previous study on CMTM7 is only limited to a brief description of the relationship of overexpressed CMTM7 with EGFR-AKT signaling. The biological functions of endogenous CMTM7 and its molecular mechanism remained unclear. In this study, we show that the stable knockdown of CMTM7 augments the malignant potential of NSCLC cells and enhances EGFR-AKT signaling by decreasing EGFR internalization and degradation. Mechanistically, CMTM7 knockdown reduces the activation of Rab5, a protein known to be required for early endosome fusion. In NSCLC, the loss of CMTM7 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, our findings highlight the role of CMTM7 in the regulation of EGFR signaling in tumor cells, revealing CMTM7 as a novel molecule related to Rab5 activation.

Dynamics of EGFR Mutation Load in Plasma for Prediction of Treatment Response and Disease Progression in Patients With EGFR-Mutant Lung Adenocarcinoma.

PubMed

Taus, Álvaro; Camacho, Laura; Rocha, Pedro; Hardy-Werbin, Max; Pijuan, Lara; Piquer, Gabriel; López, Eva; Dalmases, Alba; Longarón, Raquel; Clavé, Sergi; Salido, Marta; Albanell, Joan; Bellosillo, Beatriz; Arriola, Edurne

2018-03-23

The assessment of epidermal growth factor receptor (EGFR) mutations is crucial for the management of patients with lung adenocarcinoma. Circulating tumor DNA (ctDNA)-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Consecutive patients diagnosed with EGFR-mutant lung adenocarcinoma in tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. EGFR mutations in plasma were quantified using BEAMing (beads, emulsions, amplification, and magnetics) or digital PCR and were correlated with mutations in tumor and with radiologic response and progression. Two hundred twenty-one plasma samples from 33 patients were analyzed. EGFR mutations in plasma were detected in 83% of all patients and 100% of those with extrathoracic metastases. The dynamics of the EGFR mutation load predicted response in 93% and progression in 89% of cases well in advance of radiologic evaluation. Progression-free survival for patients in whom ctDNA was not detected in plasma during treatment was significantly longer than for those in whom ctDNA remained detectable (295 vs. 55 days; hazard ratio, 17.1; P < .001). The detection of EGFR mutations in ctDNA showed good correlation with that in tumor biopsy and predicted tumor response and progression in most patients. The liquid biopsy for ctDNA-based assessment of EGFR mutations is a reliable technique for diagnosis and follow-up in patients with EGFR-mutant lung adenocarcinoma in routine clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Identification of the zinc finger 216 (ZNF216) in human carcinoma cells: a potential regulator of EGFR activity

PubMed Central

Mincione, Gabriella; Di Marcantonio, Maria Carmela; Tarantelli, Chiara; Savino, Luca; Ponti, Donatella; Marchisio, Marco; Lanuti, Paola; Sancilio, Silvia; Calogero, Antonella; Di Pietro, Roberta; Muraro, Raffaella

2016-01-01

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling. PMID:27732953

Lead acetate induces EGFR activation upstream of SFK and PKC{alpha} linkage to the Ras/Raf-1/ERK signaling

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

Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.

2009-03-01

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC {yields} ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1{sup S338} and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKC{alpha} using specific small interfering RNA blocked Pb induction ofmore » Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKC{alpha}, Ras-GTP, phospho-Raf-1{sup S338} and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKC{alpha} activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKC{alpha} activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKC{alpha} and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.« less

Intratumoral heterogeneity in EGFR mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition

PubMed Central

Soucheray, Margaret; Capelletti, Marzia; Pulido, Inés; Kuang, Yanan; Paweletz, Cloud P.; Becker, Jeffrey H.; Kikuchi, Eiki; Xu, Chunxiao; Patel, Tarun B.; Al-shahrour, Fatima; Carretero, Julián; Wong, Kwok-Kin; Jänne, Pasi A.; Shapiro, Geoffrey I.; Shimamura, Takeshi

2015-01-01

Non-small cell lung cancers (NSCLC) that have developed resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated-EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT, but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared to cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly develops EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele is lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. PMID:26282169

Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

PubMed

Michailov, Yulia; Ickowicz, Debbi; Breitbart, Haim

2014-12-15

Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

Mechanisms and Clinical Activity of an EGFR and HER2 Exon 20-selective Kinase Inhibitor in Non-small Cell Lung Cancer

PubMed Central

Robichaux, Jacqulyne P.; Elamin, Yasir Y.; Tan, Zhi; Carter, Brett W.; Zhang, Shuxing; Liu, Shengwu; Li, Shuai; Chen, Ting; Poteete, Alissa; Estrada-Bernal, Adriana; Le, Anh T.; Truini, Anna; Nilsson, Monique B.; Sun, Huiying; Roarty, Emily; Goldberg, Sarah B.; Brahmer, Julie R.; Altan, Mehmet; Lu, Charles; Papadimitrakopoulou, Vassiliki; Politi6, Katerina; Doebele, Robert C.; Wong, Kwok-Kin; Heymach, John V.

2018-01-01

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations. PMID:29686424

Sequential liquid biopsies reveal dynamic alterations of EGFR driver mutations and indicate EGFR amplification as a new mechanism of resistance to osimertinib in NSCLC.

PubMed

Knebel, Franciele H; Bettoni, Fabiana; Shimada, Andrea K; Cruz, Manoel; Alessi, João Victor; Negrão, Marcelo V; Reis, Luiz Fernando L; Katz, Artur; Camargo, Anamaria A

2017-06-01

Osimertinib is an EGFR-T790M-specific TKI, which has demonstrated impressive response rates in NSCLC, after failure to first-line anti-EGFR TKIs. However, acquired resistance to osimertinib is also observed and the molecular mechanisms of resistance are not yet fully understood. Monitoring and managing NSCLC patients who progressed on osimertinib is, therefore, emerging as an important clinical challenge. Sequential liquid biopsies were used to monitor a patient with EGFR-exon19del positive NSCLC, who received erlotinib and progressed through the acquisition of the EGFR-T790M mutation. Erlotinib was discontinued and osimertinib was initiated. Blood samples were collected at erlotinib progression and during osimertinib treatment for the detection of the activating (EGFR-exon19del) and resistance mutations (EGFR-T790M, EGFR-C797S, BRAF-V600E, METamp and ERBB2amp) in the plasma DNA using digital droplet PCR. Plasma levels of the activating EGFR-exon19del accurately paralleled the clinical and radiological progression of disease and allowed early detection of AR to osimertinib. Resistance to osimertinib coincided with the emergence of a small tumor cell subpopulation carrying the known EGFR-C797S resistance mutation and an additional subpopulation carrying amplified copies of EGFR-exon19del. Given the existence of multiple AR mechanisms, quantification of the original EGFR activation mutation, instead of the resistance mutations, can be efficiently used to monitor response to osimertinib, allowing early detection of AR. Absolute quantification of both activation and resistance mutations can provide important information on tumor clonal evolution upon progression to osimertinib. Selective amplification of the EGFR-exon19del allele may represent a novel resistance mechanism to osimertinib. Copyright © 2017 Elsevier B.V. All rights reserved.

Epiregulin and EGFR interactions are involved in pain processing

PubMed Central

Martin, Loren J.; Smith, Shad B.; Khoutorsky, Arkady; Magnussen, Claire A.; Samoshkin, Alexander; Sorge, Robert E.; Cho, Chulmin; Yosefpour, Noosha; Sivaselvachandran, Sivaani; Tohyama, Sarasa; Cole, Tiffany; Khuong, Thang M.; Mir, Ellen; Gibson, Dustin G.; Wieskopf, Jeffrey S.; Sotocinal, Susana G.; Austin, Jean Sebastien; Meloto, Carolina B.; Gitt, Joseph H.; Sonenberg, Nahum; Greenspan, Joel D.; Fillingim, Roger B.; Slade, Gary D.; Knott, Charles; Dubner, Ronald; Nackley, Andrea G.; Ribeiro-da-Silva, Alfredo; Neely, G. Gregory; Maixner, William; Zaykin, Dmitri V.; Mogil, Jeffrey S.; Diatchenko, Luda

2017-01-01

The EGFR belongs to the well-studied ErbB family of receptor tyrosine kinases. EGFR is activated by numerous endogenous ligands that promote cellular growth, proliferation, and tissue regeneration. In the present study, we have demonstrated a role for EGFR and its natural ligand, epiregulin (EREG), in pain processing. We show that inhibition of EGFR with clinically available compounds strongly reduced nocifensive behavior in mouse models of inflammatory and chronic pain. EREG-mediated activation of EGFR enhanced nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. Moreover, EREG application potentiated capsaicin-induced calcium influx in a subset of sensory neurons. Both the EGFR and EREG genes displayed a genetic association with the development of chronic pain in several clinical cohorts of temporomandibular disorder. Thus, EGFR and EREG may be suitable therapeutic targets for persistent pain conditions. PMID:28783046

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.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

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

Zhou, Wenjun; Ercan, Dalia; Chen, Liang

2010-01-12

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potentmore » against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.« less

[Regulation on EGFR function via its interacting proteins and its potential application].

PubMed

Zheng, Jun-Fang; Chen, Hui-Min; He, Jun-Qi

2013-12-01

Epidermal growth factor receptor (EGFR) is imptortant for cell activities, oncogenesis and cell migration, and EGFR inhibitor can treat cancer efficiently, but its side effects, for example, in skin, limited its usage. On the other hand, EGFR interacting proteins may also lead to oncogenesis and its interacting protein as drug targets can avoid cutaneous side effect, which implies possibly a better outcome and life quality of cancer patients. For the multiple EGFR interaction proteins, B1R enhances Erk/MAPK signaling, while PTPN12, Kek1, CEACAM1 and NHERF repress Erk/MAPK signaling. CaM may alter charge of EGFR juxamembrane domain and regulate activation of PI3K/Akt and PLC-gamma/PKC. STAT1, STAT5b are widely thought to be activated by EGFR, while there is unexpectedly inhibiting sequence within EGFR to repress the activity of STATs. LRIG1 and ACK1 enhance the internalization and degration of EGFR, while NHERF and HIP1 repress it. In this article, proteins interacting with EGFR, their interacting sites and their regulation on EGFR signal transduction will be reviewed.

Triple Inhibition of EGFR, Met, and VEGF Suppresses Regrowth of HGF-Triggered, Erlotinib-Resistant Lung Cancer Harboring an EGFR Mutation

PubMed Central

Nakade, Junya; Takeuchi, Shinji; Nakagawa, Takayuki; Ishikawa, Daisuke; Sano, Takako; Nanjo, Shigeki; Yamada, Tadaaki; Ebi, Hiromichi; Zhao, Lu; Yasumoto, Kazuo; Matsumoto, Kunio; Yonekura, Kazuhiko

2014-01-01

Introduction: Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer. Methods: Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene–transfected PC-9 (PC-9/HGF) cells were examined. Results: Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment. Conclusion: These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis. PMID:24828661

A comparison of ARMS-Plus and droplet digital PCR for detecting EGFR activating mutations in plasma

PubMed Central

Zhang, Xinxin; Chang, Ning; Yang, Guohua; Zhang, Yong; Ye, Mingxiang; Cao, Jing; Xiong, Jie; Han, Zhiping; Wu, Shuo; Shang, Lei; Zhang, Jian

2017-01-01

In this study, we introduce a novel amplification refractory mutation system (ARMS)-based assay, namely ARMS-Plus, for the detection of epidermal growth factor receptor (EGFR) mutations in plasma samples. We evaluated the performance of ARMS-Plus in comparison with droplet digital PCR (ddPCR) and assessed the significance of plasma EGFR mutations in predicting efficacy of EGFR-tyrosine kinase inhibitor (TKI) regimen. A total of 122 advanced non-small cell lung cancer (NSCLC) patients were enrolled in this study. The tumor tissue samples from these patients were evaluated by conventional ARMS PCR method to confirm their EGFR mutation status. For the 116 plasma samples analyzed by ARMS-Plus, the sensitivity, specificity, and concordance rate were 77.27% (34/44), 97.22% (70/72), and 89.66% (104/116; κ=0.77, P<0.0001), respectively. Among the 71 plasma samples analyzed by both ARMS-Plus and ddPCR, ARMS-Plus showed a higher sensitivity than ddPCR (83.33% versus 70.83%). The presence of EGFR activating mutations in plasma was not associated with the response to EGFR-TKI, although further validation with a larger cohort is required to confirm the correlation. Collectively, the performance of ARMS-Plus and ddPCR are comparable. ARMS-Plus could be a potential alternative to tissue genotyping for the detection of plasma EGFR mutations in NSCLC patients. PMID:29340107

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.

Celecoxib induces proliferation and Amphiregulin production in colon subepithelial myofibroblasts, activating erk1-2 signaling in synergy with EGFR.

PubMed

Benelli, Roberto; Venè, Roberta; Minghelli, Simona; Carlone, Sebastiano; Gatteschi, Beatrice; Ferrari, Nicoletta

2013-01-01

The COX-2 inhibitor Celecoxib, tested in phase III trials for the prevention of sporadic colon adenomas, reduced the appearance of new adenomas, but was unable to affect the incidence of colon cancer. Moreover the 5years follow-up showed that patients discontinuing Celecoxib treatment had an increased incidence of adenomas as compared to the placebo arm. In the APC(min/+) mouse model short term treatment with Celecoxib reduced gut adenomas, but a prolonged administration of the drug induced fibroblast activation and intestinal fibrosis with a final tumor burden. The way Celecoxib could directly activate human colon myofibroblasts (MF) has not yet been investigated. We found that MF are activated by non toxic doses of Celecoxib. Celecoxib induces erk1-2 and Akt phosphorylation within 5'. This short term activation is apparently insufficient to cause phenotypic changes, but the contemporary triggering of EGFR causes an impressive synergic effect inducing MF proliferation and the neo-expression and release of Amphiregulin (AREG), a well known EGFR agonist involved in colon cancer progression. As a confirm to these observations, the erk inhibitor U0126 and the EGFR inhibitors Tyrphostin and Cetuximab were able to contrast AREG induction. Our data provide evidence that Celecoxib directly activates MF empowering EGFR signaling. According to these results the association with EGFR (or erk1-2) inhibitors could abolish the off-target activity of Celecoxib, possibly extending the potential of this drug for colon cancer prevention. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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.

HDAC9 promotes glioblastoma growth via TAZ-mediated EGFR pathway activation.

PubMed

Yang, Rui; Wu, Yanan; Wang, Mei; Sun, Zhongfeng; Zou, Jiahua; Zhang, Yundong; Cui, Hongjuan

2015-04-10

Histone deacetylase 9 (HDAC9), a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions. We found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.

Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

PubMed

Takeuchi, Shinji; Wang, Wei; Li, Qi; Yamada, Tadaaki; Kita, Kenji; Donev, Ivan S; Nakamura, Takahiro; Matsumoto, Kunio; Shimizu, Eiji; Nishioka, Yasuhiko; Sone, Saburo; Nakagawa, Takayuki; Uenaka, Toshimitsu; Yano, Seiji

2012-09-01

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode

PubMed Central

Chen, Cheng; Yu, Kailin; Zou, Fengming; Wang, Wenchao; Wang, Wei; Wu, Jiaxin; Liu, Juan; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shanchun; Yun, Cai-Hong; Liu, Jing; Liu, Qingsong

2017-01-01

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose. PMID:28407693

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.

Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells

PubMed Central

Law, Mary E.; Davis, Bradley J.; Bartley, Ashton N.; Higgins, Paul J.; Kilberg, Michael S.; Santostefano, Katherine E.; Terada, Naohiro; Heldermon, Coy D.; Castellano, Ronald K.; Law, Brian K.

2017-01-01

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and “Triple-Negative” Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance. PMID:28423644

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

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

PubMed Central

Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

2016-01-01

Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

Stress-induced EGFR trafficking: mechanisms, functions, and therapeutic implications

PubMed Central

Tan, Xiaojun; Lambert, Paul F.; Rapraeger, Alan C.; Anderson, Richard A.

2016-01-01

Epidermal growth factor receptor (EGFR) has fundamental roles in normal physiology and in cancer, making it a rational target for cancer therapy. Surprisingly, however, inhibitors that target canonical, ligand-stimulated EGFR signaling have proven to be largely ineffective in treating many EGFR-dependent cancers. Recent evidence indicates that both intrinsic and therapy-induced cellular stress triggers robust, non-canonical pathways of ligand-independent EGFR trafficking and signaling, which provides cancer cells with a survival advantage and resistance to therapeutics. Here we review the mechanistic regulation of non-canonical EGFR trafficking and signaling, the pathological and therapeutic stresses that activate it, and discuss the implications of this pathway in clinical treatment of EGFR-overexpressing cancers. PMID:26827089

Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations.

PubMed

Lou, Na-Na; Zhang, Xu-Chao; Chen, Hua-Jun; Zhou, Qing; Yan, Li-Xu; Xie, Zhi; Su, Jian; Chen, Zhi-Hong; Tu, Hai-Yan; Yan, Hong-Hong; Wang, Zhen; Xu, Chong-Rui; Jiang, Ben-Yuan; Wang, Bin-Chao; Bai, Xiao-Yan; Zhong, Wen-Zhao; Wu, Yi-Long; Yang, Jin-Ji

2016-10-04

The co-occurrence of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements constitutes a rare molecular subtype of non-small-cell lung cancer (NSCLC). Herein, we assessed the clinical outcomes and incidence of acquired resistance to tyrosine kinase inhibitors (TKIs) in this subtype. So we enrolled 118 advanced NSCLC treated with TKIs. EGFR mutations and ALK rearrangements were detected by DNA sequencing or Scorpion amplification refractory mutation system and fluorescence in situ hybridization respectively. Immunohistochemistry was used to evaluate the activation of associated proteins. We found that nine in ten patients with EGFR/ALK co-alterations had good response with first-line EGFR TKI, and the objective response rate (ORR) of EGFR TKIs was 80% (8/10) for EGFR/ALK co-altered and 65.5% (55/84) for EGFR-mutant (P = 0.57), with a median progression-free survival (PFS) of 11.2 and 13.2 months, (hazard ratio [HR]=0.95, 95% [CI], 0.49-1.84, P= 0.87). ORR of crizotinib was 40% (2/5) for EGFR/ALK co-altered and 73.9% (17/23) for ALK-rearranged (P= 0.29), with a median PFS of 1.9 and 6.9 months (hazard ratio [HR], 0.40; 95% [CI] 0.15-1.10, P = 0.08). The median overall survival (OS) was 21.3, 23.7, and 18.5 months in EGFR-mutant, ALK-rearranged, and EGFR/ALK co-altered (P= 0.06), and there existed a statistically significant difference in OS between ALK-rearranged and EGFR/ALK co-altered (P=0.03). Taken together, the first-line EGFR-TKI might be the reasonable care for advanced NSCLC harbouring EGFR/ALK co-alterations, whether or nor to use sequential crizotinib should be guided by the status of ALK rearrangement and the relative level of phospho-EGFR and phospho-ALK.

Sapanisertib and Osimertinib in Treating Patients With Stage IV EGFR Mutation Positive Non-small Cell Lung Cancer After Progression on a Previous EGFR Tyrosine Kinase Inhibitor

ClinicalTrials.gov

2018-04-25

EGFR Activating Mutation; EGFR Exon 19 Deletion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage III Non-Small Cell Lung Cancer AJCC v7; Stage IIIA Non-Small Cell Lung Cancer AJCC v7; Stage IIIB Non-Small Cell Lung Cancer AJCC v7; Stage IV Non-Small Cell Lung Cancer AJCC v7

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

PubMed Central

2011-01-01

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series. PMID:21266046

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

PubMed

Peraldo-Neia, Caterina; Migliardi, Giorgia; Mello-Grand, Maurizia; Montemurro, Filippo; Segir, Raffaella; Pignochino, Ymera; Cavalloni, Giuliana; Torchio, Bruno; Mosso, Luciano; Chiorino, Giovanna; Aglietta, Massimo

2011-01-25

Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

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

EGFR mediates astragaloside IV-induced Nrf2 activation to protect cortical neurons against in vitro ischemia/reperfusion damages

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

Gu, Da-min; Lu, Pei-Hua, E-mail: lphty1_1@163.com; Zhang, Ke

In this study, we tested the potential role of astragaloside IV (AS-IV) against oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced damages in murine cortical neurons, and studied the associated signaling mechanisms. AS-IV exerted significant neuroprotective effects against OGD/R by reducing reactive oxygen species (ROS) accumulation, thereby attenuating oxidative stress and neuronal cell death. We found that AS-IV treatment in cortical neurons resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by Nrf2 Ser-40 phosphorylation, and its nuclear localization, as well as transcription of antioxidant-responsive element (ARE)-regulated genes: heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO-1) and sulphiredoxin 1 (SRXN-1). Knockdown of Nrf2 throughmore » lentiviral shRNAs prevented AS-IV-induced ARE genes transcription, and abolished its anti-oxidant and neuroprotective activities. Further, we discovered that AS-IV stimulated heparin-binding-epidermal growth factor (HB-EGF) release to trans-activate epidermal growth factor receptor (EGFR) in cortical neurons. Blockage or silencing EGFR prevented Nrf2 activation by AS-IV, thus inhibiting AS-IV-mediated anti-oxidant and neuroprotective activities against OGD/R. In summary, AS-IV protects cortical neurons against OGD/R damages through activating of EGFR-Nrf2 signaling. - Highlights: • Pre-treatment of astragaloside IV (AS-IV) protects murine cortical neurons from OGD/R. • AS-IV activates Nrf2-ARE signaling in murine cortical neurons. • Nrf2 is required for AS-IV-mediated anti-oxidant and neuroprotective activities. • AS-IV stimulates HB-EGF release to trans-activate EGFR in murine cortical neurons. • EGFR mediates AS-IV-induced Nrf2 activation and neuroprotection against OGD/R.« less

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.

Biodistribution and Pharmacokinetic Analysis of Combination Lonidamine and Paclitaxel Delivery in an Orthotopic Animal Model of Multi-drug Resistant Breast Cancer Using EGFR-Targeted Polymeric Nanoparticles

PubMed Central

Milane, Lara; Duan, Zhen-feng; Amiji, Mansoor

2011-01-01

The aim of this study was to assess the biodistribution and pharmacokinetics of epidermal growth factor receptor (EGFR)-targeted polymer blend nanoparticles loaded with the anticancer drugs lonidamine and paclitaxel. Plasma, tumor, and tissue distribution profiles were quantified in an orthotopic animal model of multi-drug resistant (MDR) breast cancer and were compared to treatment with non-targeted nanoparticles and to treatment with drug solution. Poly(D,L-lactide-co-glycolide)/poly(ethylene glycol)/EGFR targeting peptide (PLGA/PEG/EFGR peptide) construct was synthesized for incorporation in poly(ε-caprolactone) (PCL) particles to achieve active EGFR targeting. An isocratic HPLC method was developed to quantify lonidamine and paclitaxel in mice plasma, tumors, and vital organs. The targeted nanoparticles demonstrated superior pharmacokinetic profile relative to drug solution and non-targeted nanoparticles, particularly for lonidamine delivery. The first target site of accumulation is the liver, followed by the kidneys, and then the tumor mass; maximal tumor accumulation occurs at 3 hours post-administration. Lonidamine/paclitaxel combination therapy administered via EGFR-targeted polymer blend nanocarriers may become a viable platform for the future treatment of MDR cancer. PMID:21220050

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.

Emerging functions of the EGFR in cancer.

PubMed

Sigismund, Sara; Avanzato, Daniele; Lanzetti, Letizia

2018-01-01

The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis. In pathological settings, mostly in lung and breast cancer and in glioblastoma, the EGFR is a driver of tumorigenesis. Inappropriate activation of the EGFR in cancer mainly results from amplification and point mutations at the genomic locus, but transcriptional upregulation or ligand overproduction due to autocrine/paracrine mechanisms has also been described. Moreover, the EGFR is increasingly recognized as a biomarker of resistance in tumors, as its amplification or secondary mutations have been found to arise under drug pressure. This evidence, in addition to the prominent function that this receptor plays in normal epithelia, has prompted intense investigations into the role of the EGFR both at physiological and at pathological level. Despite the large body of knowledge obtained over the last two decades, previously unrecognized (herein defined as 'noncanonical') functions of the EGFR are currently emerging. Here, we will initially review the canonical ligand-induced EGFR signaling pathway, with particular emphasis to its regulation by endocytosis and subversion in human tumors. We will then focus on the most recent advances in uncovering noncanonical EGFR functions in stress-induced trafficking, autophagy, and energy metabolism, with a perspective on future therapeutic applications. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

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

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

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

PubMed

Zhou, Xiangjun; Zhang, Wei; Yao, Qisheng; Zhang, Hao; Dong, Guie; Zhang, Ming; Liu, Yutao; Chen, Jian-Kang; Dong, Zheng

2017-06-01

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing. Copyright © 2017 the American Physiological Society.

Gemcitabine enhances cell invasion via activating HAb18G/CD147-EGFR-pSTAT3 signaling.

PubMed

Xu, Bao-Qing; Fu, Zhi-Guang; Meng, Yao; Wu, Xiao-Qing; Wu, Bo; Xu, Liang; Jiang, Jian-Li; Li, Ling; Chen, Zhi-Nan

2016-09-20

Pancreatic cancer, one of the most lethal cancers, has very poor 5-year survival partly due to gemcitabine resistance. Recently, it was reported that chemotherapeutic agents may act as stressors to induce adaptive responses and to promote chemoresistance in cancer cells. During long-term drug treatment, the minority of cancer cells survive and acquire an epithelial-mesenchymal transition phenotype with increased chemo-resistance and metastasis. However, the short-term response of most cancer cells remains unclear. This study aimed to investigate the short-term response of pancreatic cancer cells to gemcitabine stress and to explore the corresponding mechanism. Our results showed that gemcitabine treatment for 24 hours enhanced pancreatic cancer cell invasion. In gemcitabine-treated cells, HAb18G/CD147 was up-regulated; and HAb18G/CD147 down-regulation or inhibition attenuated gemcitabine-enhanced invasion. Mechanistically, HAb18G/CD147 promoted gemcitabine-enhanced invasion by activating the EGFR (epidermal growth factor receptor)-STAT3 (signal transducer and activator of transcription 3) signaling pathway. Inhibition of EGFR-STAT3 signaling counteracted gemcitabine-enhanced invasion, and which relied on HAb18G/CD147 levels. In pancreatic cancer tissues, EGFR was highly expressed and positively correlated with HAb18G/CD147. These data indicate that pancreatic cancer cells enhance cell invasion via activating HAb18G/CD147-EGFR-pSTAT3 signaling. Our findings suggest that inhibiting HAb18G/CD147 is a potential strategy for overcoming drug stress-associated resistance in pancreatic cancer.

EGFR transactivation: mechanisms, pathophysiology and potential therapies in cardiovascular system

PubMed Central

Forrester, Steven J.; Kawai, Tatsuo; Elliott, Katherine J.; O’Brien, Shannon; Thomas, Walter; Harris, Raymond C.; Eguchi, Satoru

2017-01-01

Accumulating studies suggest that the epidermal growth factor receptor (EGFR) activation is associated with the physiology and pathophysiology of the cardiovascular system, and inhibition of EGFR activity is emerging as a potential therapeutic strategy to treat diseases, including hypertension, cardiac hypertrophy, renal fibrosis and abdominal aortic aneurysm. The capacity of G protein-coupled receptor (GPCR) agonists, such as angiotensin II (AngII), to promote EGFR signaling is well described – a process termed EGFR “transactivation” – yet delineating the molecular processes and functional relevance of this crosstalk has been challenging. Moreover, these critical findings are dispersed among many different fields. The aim of our review is to highlight the recent advancement of the signaling cascades and downstream consequences of EGFR transactivation within the cardiovascular renal system in vitro and in vivo. We will also focus on linking EGFR transactivation to animal models of the disease as well as the potential therapeutic applications. PMID:26566153

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.

Redox Regulation of EGFR Signaling Through Cysteine Oxidation1

PubMed Central

Truong, Thu H.; Carroll, Kate S.

2012-01-01

Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes including growth, proliferation and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H2O2 by NADPH-dependent oxidases. In turn, H2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for development of new therapeutic strategies to target this and other H2O2-modulated pathways. PMID:23186290

Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1

PubMed Central

Pirazzoli, Valentina; Nebhan, Caroline; Song, Xiaoling; Wurtz, Anna; Walther, Zenta; Cai, Guoping; Zhao, Zhongming; Jia, Peilin; de Stanchina, Elisa; Shapiro, Erik M.; Gale, Molly; Yin, Ruonan; Horn, Leora; Carbone, David P.; Stephens, Philip J; Miller, Vincent; Gettinger, Scott; Pao, William; Politi, Katerina

2014-01-01

SUMMARY Patients with EGFR-mutant lung adenocarcinomas (LUADs) who initially respond to first-generation TKIs develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. Addition of rapamycin reversed resistance in vivo. Analysis of afatinib+cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib+cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs. PMID:24813888

Role of Epidermal Growth Factor Receptor (EGFR) Inhibitors and Radiation in the Management of Brain Metastases from EGFR Mutant Lung Cancers.

PubMed

Khandekar, Melin J; Piotrowska, Zofia; Willers, Henning; Sequist, Lecia V

2018-04-27

The growth of genotype-directed targeted therapies, such as inhibitors of the epidermal growth factor receptor (EGFR), has revolutionized treatment for some patients with oncogene-addicted lung cancer. However, as systemic control for these patients has improved, brain metastases remain an important source of morbidity and mortality. Traditional treatment for brain metastases has been radiotherapy, either whole-brain radiation or stereotactic radiosurgery. The growing availability of drugs that can cross the blood-brain barrier and have activity in the central nervous system (CNS) has led to many studies investigating whether targeted therapy can be used in combination with or in lieu of radiation. In this review, we summarize the key literature about the incidence and nature of EGFR-mutant brain metastases (EGFR BMs), the data about the activity of EGFR inhibitors in the CNS, and whether they can be used as front-line therapy for brain metastases. Although initial use of tyrosine kinase inhibitors for EGFR BMs can often be an effective treatment strategy, multidisciplinary evaluation is critical, and prospective studies are needed to clarify which patients may benefit from early radiotherapy. Management of brain metastases in epidermal growth factor receptor (EGFR) mutant lung cancer is a common clinical problem. The question of whether to start initial therapy with an EGFR inhibitor or radiotherapy (either whole-brain radiotherapy or stereotactic radiosurgery) is controversial. The development of novel EGFR inhibitors with enhanced central nervous system (CNS) penetration is an important advance in the treatment of CNS disease. Multidisciplinary evaluation and evaluation of extracranial disease status are critical to choosing the best treatment option for each patient. © AlphaMed Press 2018.

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

2D-QSAR and 3D-QSAR Analyses for EGFR Inhibitors

PubMed Central

Zhao, Manman; Zheng, Linfeng; Qiu, Chun

2017-01-01

Epidermal growth factor receptor (EGFR) is an important target for cancer therapy. In this study, EGFR inhibitors were investigated to build a two-dimensional quantitative structure-activity relationship (2D-QSAR) model and a three-dimensional quantitative structure-activity relationship (3D-QSAR) model. In the 2D-QSAR model, the support vector machine (SVM) classifier combined with the feature selection method was applied to predict whether a compound was an EGFR inhibitor. As a result, the prediction accuracy of the 2D-QSAR model was 98.99% by using tenfold cross-validation test and 97.67% by using independent set test. Then, in the 3D-QSAR model, the model with q2 = 0.565 (cross-validated correlation coefficient) and r2 = 0.888 (non-cross-validated correlation coefficient) was built to predict the activity of EGFR inhibitors. The mean absolute error (MAE) of the training set and test set was 0.308 log units and 0.526 log units, respectively. In addition, molecular docking was also employed to investigate the interaction between EGFR inhibitors and EGFR. PMID:28630865

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

Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs.

PubMed

Zhang, Chengjuan; Wei, Bing; Li, Peng; Yang, Ke; Wang, Zhizhong; Ma, Jie; Guo, Yongjun

2017-01-01

Epidermal growth factor receptor (EGFR) specific mutations have been known to improve survival of patients with non-small-cell lung carcinoma (NSCLC). However, whether there are any changes of EGFR mutations after targeted therapy and its clinical significance is unclear. This study was to identify the status of EGFR mutations after targeted therapy and predict the prognostic significance for NSCLC patients. A total of forty-five (45) NSCLC patients who received EGFR-TKI therapy were enrolled. We identified the changes of EGFR mutations in plasma ctDNA by Amplification Refractory Mutation System (ARMS) PCR technology. In the 45 cases of NSCLC with EGFR mutations, the EGFR mutation status changed in 26 cases, in which, 12 cases (26.7%) from positive to negative, and 14 cases (31.1%) from T790M mutation negative to positive after TKI targeted therapy. The T790M occurance group had a shorter Progression -Free-Survival (PFS) than the groups of EGFR mutation undetected and EGFR mutation turned out to have no change after EGFR-TKI therapy (p < 0.05). According to this study, it's necessary to closely monitor EGFR mutations during follow-up to predict the prognosis of NSCLC patients who are to receive the TKI targeted therapy.

Drosophila Hox and Sex-Determination Genes Control Segment Elimination through EGFR and extramacrochetae Activity

PubMed Central

Foronda, David; Martín, Paloma; Sánchez-Herrero, Ernesto

2012-01-01

The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. We show here that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. Our results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change. PMID:22912593

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR

PubMed Central

Edwards, Vonetta L.; Wang, Liang-Chun; Dawson, Valerie; Stein, Daniel C.; Song, Wenxia

2017-01-01

Summary Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell–cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the ‘fence’ function of the apical junction but not its ‘gate’ function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced β-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium. PMID:23279089

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...

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2016-11-01

available to the research community. Similarly, any cell lines generated in our studies will also be shared. The EGFR transgenic mouse models used in...Lines and Transgenic Mice Active Completed – May 31, 2015 NIH/NCI R01CA121210 Overcoming Acquired Resistance to EGFR Inhibitors in Lung Cancer...Active Active Labrecque Foundation Not Applicable A Translational Pilot Study on Serum Biomarkers of Lung Cancer Using Transgenic Mouse Models of

EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling

PubMed Central

Baumdick, Martin; Brüggemann, Yannick; Schmick, Malte; Xouri, Georgia; Sabet, Ola; Davis, Lloyd; Chin, Jason W; Bastiaens, Philippe IH

2015-01-01

Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR’s capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF. DOI: http://dx.doi.org/10.7554/eLife.12223.001 PMID:26609808

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

NF-κB activating complex engaged in response to EGFR oncogene inhibition drives tumor cell survival and residual disease in lung cancer

PubMed Central

Blakely, Collin M.; Pazarentzos, Evangelos; Olivas, Victor; Asthana, Saurabh; Yan, Jenny Jiacheng; Tan, Irena; Hrustanovic, Gorjan; Chan, Elton; Lin, Luping; Neel, Dana S.; Newton, William; Bobb, Kathryn; Fouts, Timothy; Meshulam, Jeffrey; Gubens, Matthew A.; Jablons, David M.; Johnson, Jeffrey R.; Bandyopadhyay, Sourav; Krogan, Nevan J.; Bivona, Trever G.

2015-01-01

Summary Although oncogene-targeted therapy often elicits profound initial tumor responses in patients, responses are generally incomplete because some tumor cells survive initial therapy as residual disease that enables eventual acquired resistance. The mechanisms underlying tumor cell adaptation and survival during initial therapy are incompletely understood. Here, through the study of EGFR-mutant lung adenocarcinoma we show that NF-κB signaling is rapidly engaged upon initial EGFR inhibitor treatment to promote tumor cell survival and residual disease. EGFR oncogene inhibition induced an EGFR-TRAF2-RIP1-IKK complex that stimulated an NF-κB-mediated transcriptional survival program. The direct NF-κB inhibitor PBS-1086 suppressed this adaptive survival program and increased the magnitude and duration of initial EGFR inhibitor response in multiple NSCLC models, including a patient-derived xenograft. These findings unveil NF-κB activation as a critical adaptive survival mechanism engaged by EGFR oncogene inhibition and provide rationale for EGFR and NF-κB co-inhibition to eliminate residual disease and enhance patient responses. PMID:25843712

EGFR-TKI-induced HSP70 degradation and BER suppression facilitate the occurrence of the EGFR T790 M resistant mutation in lung cancer cells.

PubMed

Cao, Xiang; Zhou, Yi; Sun, Hongfang; Xu, Miao; Bi, Xiaowen; Zhao, Zhihui; Shen, Binghui; Wan, Fengyi; Hong, Zhuan; Lan, Lei; Luo, Lan; Guo, Zhigang; Yin, Zhimin

2018-06-28

Non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations initially respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and have shown favorable outcomes. However, acquired drug resistance to EGFR-TKIs develops in almost all patients mainly due to the EGFR T790 M mutation. Here, we show that treatment with low-dose EGFR-TKI results in the emergence of the EGFR T790 M mutation and in the reduction of HSP70 protein levels in HCC827 cells. Erlotinib treatment inhibits HSP70 phosphorylation at tyrosine 41 and increases HSP70 ubiquitination, resulting in HSP70 degradation. We show that EGFR-TKI treatment causes increased DNA damage and enhanced gene mutation rates, which are secondary to the EGFR-TKI-induced reduction of HSP70 protein. Importantly, HSP70 overexpression delays the occurrence of Erlotinib-induced EGFR T790 M mutation. We further demonstrate that HSP70 interacts with multiple enzymes in the base excision repair (BER) pathway and promotes not only the efficiency but also the fidelity of BER. Collectively, our findings show that EGFR-TKI treatment facilitates gene mutation and the emergence of EGFR T790 M secondary mutation by the attenuation of BER via induction of HSP70 protein degradation. Copyright © 2018. Published by Elsevier B.V.

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

PubMed

Mehrabi, Masomeh; Mahdiuni, Hamid; Rasouli, Hassan; Mansouri, Kamran; Shahlaei, Mohsen; Khodarahmi, Reza

2018-04-14

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln 43 , Tyr 44 , Arg 45 and Asp 46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings

EGFR-TKIs plus chemotherapy demonstrated superior efficacy than EGFR-TKIs alone as first-line setting in advanced NSCLC patients with EGFR mutation and BIM deletion polymorphism.

PubMed

Liu, Sangtian; He, Yayi; Jiang, Tao; Ren, Shengxiang; Zhou, Fei; Zhao, Chao; Li, Xuefei; Zhang, Jie; Su, Chunxia; Chen, Xiaoxia; Cai, Weijing; Gao, Guanghui; Li, Wei; Wu, Fengying; Li, Jiayu; Zhao, Jing; Hu, Qiong; Zhao, Mingchuan; Zhou, Caicun; Hirsch, Fred R

2018-06-01

Non-small-cell lung cancer (NSCLC) patients with both epidermal growth factor receptor (EGFR) positive mutation and B-cell chronic lymphocytic leukemia/lymphoma-like 11 (BIM) deletion polymorphism had a poor clinical response to EGFR-tyrosine kinase inhibitors (TKIs). The current study aimed to investigate the clinical efficacy and tolerability of EGFR-TKIs plus chemotherapy versus EGFR-TKIs alone as first-line treatment in advanced NSCLC patients with EGFR mutations and BIM deletion polymorphism. A retrospective, non-randomized analysis was conducted. BIM deletion polymorphism was detected using polymerase chain reaction (PCR) analysis and direct sequencing of DNA from peripheral blood cells. Clinical characteristics, overall survival (OS), progress-free-survival (PFS), objective response rate (ORR) and treatment-related adverse events were compared between EGFR-TKIs alone versus EGFR-TKIs plus chemotherapy group. 65 patients were enrolled. 36 of them received EGFR-TKIs and 29 received EGFR-TKIs plus chemotherapy. EGFR-TKIs plus chemotherapy had significantly higher ORR than TKIs alone (65.5% vs. 38.9%, P = 0.046). Median PFS was significantly longer in EGFR-TKIs plus chemotherapy group than in TKIs group (7.2 vs 4.7 m; P = 0.008). Median OS was numerically longer in EGFR-TKIs plus chemotherapy group than in TKIs alone (18.5 vs 14.2 m; P = 0.107). EGFR-TKIs plus chemotherapy was associated with more grade 3 or 4 hematological toxic effects than EGFR-TKIs alone. EGFR-TKIs plus chemotherapy conferred a significantly higher ORR, prolonged PFS and numerically longer OS in advanced NSCLC patients with EGFR mutation and BIM deletion polymorphism. Further prospective studies are needed to validate these findings. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non-small cell lung cancer.

PubMed

Ali, Azhar; Levantini, Elena; Teo, Jun Ting; Goggi, Julian; Clohessy, John G; Wu, Chan Shuo; Chen, Leilei; Yang, Henry; Krishnan, Indira; Kocher, Olivier; Zhang, Junyan; Soo, Ross A; Bhakoo, Kishore; Chin, Tan Min; Tenen, Daniel G

2018-02-15

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16-C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI-resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA-approved anti-obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI-resistant EGFR mutant NSCLC patients. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Surmounting the resistance against EGFR inhibitors through the development of thieno[2,3-d]pyrimidine-based dual EGFR/HER2 inhibitors.

PubMed

Milik, Sandra N; Abdel-Aziz, Amal Kamal; Lasheen, Deena S; Serya, Rabah A T; Minucci, Saverio; Abouzid, Khaled A M

2018-06-06

In light of the emergence of resistance against the currently available EGFR inhibitors, our study focuses on tackling this problem through the development of dual EGFR/HER2 inhibitors with improved enzymatic affinities. Guided by the binding mode of the marketed dual EGFR/HER2 inhibitor, Lapatinib, we proposed the design of dual EGFR/HER2 inhibitors based on the 6-phenylthieno[2,3-d]pyrimidine as a core scaffold and hinge binder. After two cycles of screening aiming to identify the optimum aniline headgroup and solubilizing group, we eventually identified 27b as a dual EGFR/HER2 inhibitor with IC 50 values of 91.7 nM and 1.2 μM, respectively. Notably, 27b dramatically reduced the viability of various patient-derived cancer cells preferentially overexpressing EGFR/HER2 (A431, MDA-MBA-361 and SKBr3 with IC 50 values of 1.45, 3.5 and 4.83 μM, respectively). Additionally, 27b efficiently thwarted the proliferation of lapatinib-resistant human non-small lung carcinoma (NCI-H1975) cells, harboring T790 M mutation, with IC 50 of 4.2 μM. Consistently, 27b significantly blocked EGF-induced EGFR activation and inactivated its downstream AKT/mTOR/S6 signalling pathway triggering apoptotic cell death in NCI-H1975 cells. The present study presents a promising candidate for further design and development of novel EGFR/HER2 inhibitors capable of overcoming EGFR TKIs resistance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Antitumor efficacy of triple monoclonal antibody inhibition of epidermal growth factor receptor (EGFR) with MM151 in EGFR-dependent and in cetuximab-resistant human colorectal cancer cells

PubMed Central

Napolitano, Stefania; Martini, Giulia; Martinelli, Erika; Della Corte, Carminia Maria; Morgillo, Floriana; Belli, Valentina; Cardone, Claudia; Matrone, Nunzia; Ciardiello, Fortunato; Troiani, Teresa

2017-01-01

Purpose We investigated the effect of triple monoclonal antibody inhibition of EGFR to overcome acquired resistance to first generation of anti-EGFR inhibitors. Experimental design MM151 is a mixture of three different monoclonal IgG1 antibodies directed toward three different, non-overlapping, epitopes of the EGFR. We performed an in vivo study by using human CRC cell lines (SW48, LIM 1215 and CACO2) which are sensitive to EGFR inhibitors, in order to evaluate the activity of MM151 as compared to standard anti-EGFR mAbs, such as cetuximab, as single agent or in a sequential strategy of combination MM151 with irinotecan (induction therapy) followed by MM151 with a selective MEK1/2 inhibitor (MEKi) (maintenance therapy). Furthermore, the ability of MM151 to overcome acquired resistance to cetuximab has been also evaluated in cetuximab-refractory CRC models. Results MM151 shown stronger antitumor activity as compared to cetuximab. The maintenance treatment with MM151 plus MEKi resulted the most effective therapeutic modality. In fact, this combination caused an almost complete suppression of tumor growth in SW48, LIM 1215 and CACO2 xenografts model at 30 week. Moreover, in this treatment group, mice with no evidence of tumor were more than double as compared to single agent treated mice. Its superior activity has also been demonstrated, in cetuximab-refractory CRC models. Conclusions These results provide experimental evidence that more efficient and complete EGFR blockade may determine better antitumor activity and could contribute to prevent and/or overcome acquired resistance to EGFR inhibitors. PMID:29137301

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

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.

β-catenin contributes to lung tumor development induced by EGFR mutations

PubMed Central

Nakayama, Sohei; Sng, Natasha; Carretero, Julian; Welner, Robert; Hayashi, Yuichiro; Yamamoto, Mihoko; Tan, Alistair J.; Yamaguchi, Norihiro; Yasuda, Hiroyuki; Li, Danan; Soejima, Kenzo; Soo, Ross A.; Costa, Daniel B.; Wong, Kwok-Kin; Kobayashi, Susumu S.

2014-01-01

The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and development of EGFR tyrosine kinase inhibitors (TKIs) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. β-catenin was upregulated and activated in EGFR mutated cells. Mutant EGFR preferentially bound to and tyrosine-phosphorylated β-catenin, leading to increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacological inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs. PMID:25164010

Interactions between EGFR and PD-1/PD-L1 pathway: Implications for treatment of NSCLC.

PubMed

Li, Xue; Lian, Zhen; Wang, Shuai; Xing, Ligang; Yu, Jinming

2018-04-01

Immune checkpoint inhibitors targeting the programmed cell death receptor/ligand 1 (PD-1/PD-L1) pathway displayed striking and durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, it is still undefined about the efficacy of PD-1/PD-L1 inhibitors in NSCLC patients with EGFR activating mutations. Preclinical studies indicate the immune modulatory effect of EGFR signaling by regulating expression of MHC I/II and PD-L1 on tumor cells and activity of lymphocytes. Thus, it might be practicable for the use of PD-1/PD-L1 inhibitors as monotherapy or combined with EGFR-TKIs in patients with EGFR activating mutations. In this review, we discussed the regulation effect of EGFR signaling on PD-1/PD-L1 pathway and the potential mechanisms behind combing EGFR-TKIs with PD-1/PD-L1 inhibitors. We also reviewed current available data on PD-1/PD-L1 inhibitors as monotherapy or combined with EGFR-TKIs in NSCLC with EGFR activating mutations, and explored possible factors influence its efficacy, which would be important considerations for future clinical trial designs. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway

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

Yi, Lian; Shi, Tujin; Gritsenko, Marina A.

2018-03-27

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichmentmore » methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

Gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancers by accelerating EGFR turnover.

PubMed

Nam, Boas; Rho, Jin Kyung; Shin, Dong-Myung; Son, Jaekyoung

2016-10-01

Gallic acid is a common botanic phenolic compound, which is present in plants and foods worldwide. Gallic acid is implicated in various biological processes such as cell growth and apoptosis. Indeed, gallic acid has been shown to induce apoptosis in many cancer types. However, the molecular mechanisms of gallic acid-induced apoptosis in cancer, particularly lung cancer, are still unclear. Here, we report that gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancer (NSCLC) cells, but not in EGFR-WT NSCLC cells. Treatment with gallic acid resulted in a significant reduction in proliferation and induction of apoptosis, only in EGFR-mutant NSCLC cells. Interestingly, treatment with gallic acid led to a robust decrease in EGFR levels, which is critical for NSCLC survival. Treatment with gallic acid had no significant effect on transcription, but induced EGFR turnover. Indeed, treatment with a proteasome inhibitor dramatically reversed gallic acid-induced EGFR downregulation. Moreover, treatment with gallic acid induced EGFR turnover leading to apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Thus, these studies suggest that gallic acid can induce apoptosis in EGFR-dependent lung cancers that are dependent on EGFR for growth and survival via acceleration of EGFR turnover. Copyright © 2016 Elsevier Ltd. All rights reserved.

EGFR gene copy number alterations are not a useful screening tool for predicting EGFR mutation status in lung adenocarcinoma.

PubMed

Russell, Prudence A; Yu, Yong; Do, Hongdo; Clay, Timothy D; Moore, Melissa M; Wright, Gavin M; Conron, Matthew; Wainer, Zoe; Dobrovic, Alexander; McLachlan, Sue-Anne

2014-01-01

We investigated if gene copy number (GCN) alterations of the epidermal growth factor receptor (EGFR), as detected by silver enhanced in situ hybridisation (SISH), could be used to select patients for EGFR mutation testing. Resected lung adenocarcinoma specimens with adequate tumour were identified. EGFR SISH was performed using the Ventana Benchmark Ultra platform. EGFR GCN was classified according to the Colorado Classification System. EGFR mutations were scanned by high resolution melting and confirmed by Sanger sequencing. Thirty-four of 96 tumours were EGFR SISH positive (35%), and 31 of 96 tumours harboured one or more EGFR mutations (32%). Of 31 EGFR-mutant tumours, 18 were EGFR SISH positive (58%). There was a statistically significant relationship between the presence of an EGFR mutation and EGFR GCN (p = 0.003). Thirteen of 31 EGFR-mutant tumours were EGFR SISH negative (42%), and 16 of 65 EGFR-wild type tumours were EGFR SISH positive (24%). The sensitivity, specificity, positive predictive value and negative predictive value were 58%, 75%, 52.9% and 79%, respectively. Despite a significant relationship between EGFR GCN alterations and EGFR mutations, our results indicate that EGFR GCN as detected by SISH is not a suitable way to select patients for EGFR mutation testing.

Development of the Third Generation EGFR Tyrosine Kinase Inhibitors for Anticancer Therapy.

PubMed

Cheng, Weiyan; Zhou, Jianhua; Tian, Xin; Zhang, Xiaojian

2016-01-01

Epidermal growth factor receptor (EGFR) is one of the most important targets in anticancer therapy. Till date, a large number of first and second generation EGFR tyrosine kinase inhibitors (TKIs) have been marketed or advanced into clinical studies. However, the occurrence of TKI-resistant mutations has led to the loss of efficacy of these inhibitors. In the purpose of overcoming resistant mutations and reducing side effects, lots of third generation EGFR inhibitors are explored with promising potencies against EGFR mutations while sparing wild-type EGFR. This review outlines the current landscape of the development of third generation EGFR inhibitors, mainly focusing on the biological properties, clinical status and structure-activity relationships.

Characterization of In Vivo Resistance to Osimertinib and JNJ-61186372, an EGFR/Met Bispecific Antibody, Reveals Unique and Consensus Mechanisms of Resistance.

PubMed

Emdal, Kristina B; Dittmann, Antje; Reddy, Raven J; Lescarbeau, Rebecca S; Moores, Sheri L; Laquerre, Sylvie; White, Forest M

2017-11-01

Approximately 10% of non-small cell lung cancer (NSCLC) patients in the United States and 40% of NSCLC patients in Asia have activating epidermal growth factor receptor (EGFR) mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be cotargeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry-based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy-resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third-generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase (SFK) substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as SFK signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages. Mol Cancer Ther; 16(11); 2572-85. ©2017 AACR . ©2017 American Association for Cancer Research.

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.

Activation of the EGFR/p38/JNK Pathway by Mitochondrial-Derived Hydrogen Peroxide Contributes To Oxygen-induced Contraction Of Ductus Arteriosus

PubMed Central

Hong, Zhigang; Cabrera, Jésus A; Mahapatra, Saswati; Kutty, Shelby; Weir, E. Kenneth; Archer, Stephen L.

2014-01-01

Oxygen-induced contraction of the ductus arteriosus (DA) involves a mitochondrial oxygen-sensor, which signals pO2 in the DA smooth muscle cell (DASMC) by increasing production of diffusible hydrogen peroxide (H2O2). H2O2 stimulates vasoconstriction by regulating ion channels and rho kinase, leading to calcium influx and calcium sensitization. Because epidermal growth factor receptor (EGFR) signaling is also redox regulated and participates in oxygen sensing and vasoconstriction in other systems, we explored the role of the EGFR and its signaling cascade (p38 and JNK) in DA contraction. Experiments were performed in DA rings isolated from full-term New Zealand White rabbits and human DASMC. In human DASMCs increasing pO2 from hypoxia to normoxia (40 to 100 mmHg) significantly increased cytosolic calcium, p<0.01. This normoxic rise in intracellular calcium was mimicked by EGF and inhibited by EGFR siRNA. In DA rings, EGF caused contraction whilst the specific EGFR inhibitor (AG1478) and the tyrosine kinase inhibitors (genistein or tyrphostin A23) selectively attenuated oxygen-induced contraction (p <0.01). Conversely, orthovanadate, a tyrosine phosphatase inhibitor known to activate EGFR signaling, caused dose-dependent contraction of hypoxic DA and superimposed increases in oxygen caused minimal additional contraction. Ansomycin, an activator of EGFR’s downstream kinases, p38 and JNK, caused DA contraction; conversely, oxygen-induced DA contraction was blocked by inhibitors of p38 MAPK (SB203580) or JNK (JNK inhibitor II). O2-induced phosphorylation of EGFR occurred within 5-minutes of increasing pO2 and was inhibited by mitochondrial-targeted overexpression of catalase. AG1478 prevented the oxygen-induced p38 and JNK phosphorylation. In conclusion, O2-induced EGFR transactivation initiates p38/JNK-mediated increases in cytosolic calcium and contributes to DA contraction. The EGFR/p38/JNK pathway is regulated by mitochondrial redox signaling and is a promising

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.

Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells.

PubMed

Chen, Z; Chen, J; Gu, Y; Hu, C; Li, J-L; Lin, S; Shen, H; Cao, C; Gao, R; Li, J; Ha, P K; Kaye, F J; Griffin, J D; Wu, L

2014-07-17

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1-MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1-MAML2-positive MEC.

Resistance to EGFR inhibitors in non-small cell lung cancer: Clinical management and future perspectives.

PubMed

Tomasello, Chiara; Baldessari, Cinzia; Napolitano, Martina; Orsi, Giulia; Grizzi, Giulia; Bertolini, Federica; Barbieri, Fausto; Cascinu, Stefano

2018-03-01

In the last few years, the development of targeted therapies for non-small cell lung cancer (NSCLC) expressing oncogenic driver mutations (e.g. EGFR) has changed the clinical management and the survival outcomes of this specific minority of patients. Several phase III trials demonstrated the superiority of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) over chemotherapy in EGFR-mutant NSCLC patients. However, in the vast majority of cases EGFR TKIs lose their clinical activity within 8-12 months. Many genetic aberrations have been described as possible mechanisms of EGFR TKIs acquired resistance and can be clustered in four main sub-groups: 1. Development of secondary EGFR mutations; 2. Activation of parallel signaling pathways; 3. Histological transformation; 4. Activation of downstream signaling pathways. In this review we will describe the molecular alterations underlying each of these EGFR TKIs resistance mechanisms, focusing on the currently available and future therapeutic strategies to overcome these phenomena. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR.

PubMed

Hwang, Ki-Eun; Kwon, Su-Jin; Kim, Young-Suk; Park, Do-Sim; Kim, Byoung-Ryun; Yoon, Kwon-Ha; Jeong, Eun-Taik; Kim, Hak-Ryul

2014-05-01

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib-simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction. Copyright © 2014 Elsevier Inc. All rights reserved.

Benzo[g]quinazolin-based scaffold derivatives as dual EGFR/HER2 inhibitors.

PubMed

Ghorab, Mostafa M; Alsaid, Mansour S; Soliman, Aiten M; Al-Mishari, Abdullah A

2018-12-01

Targeting EGFR has proven to be beneficial in the treatment of several types of solid tumours. So, a series of novel 2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio)-N-substituted acetamide 5-19 were synthesised from the starting material 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4, to be evaluated as dual EGFR/HER2 inhibitors. The target compounds 5-19, were screened for their cytotoxic activity against A549 lung cancer cell line. The percentage inhibition of EGFR enzyme was measured and compared with erlotinib as the reference drug. Compounds 6, 8, 10, and 16 showed excellent EGFR inhibitory activity and were further selected for screening as dual EGFR/HER2 inhibitors. The four selected compounds showed IC 50 ranging from 0.009 to 0.026 µM for EGFR and 0.021 to 0.069 µM for the HER2 enzyme. Compound 8 was found to be the most potent in this study with IC 50 0.009 and 0.021 µM for EGFR and HER2, respectively.

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.

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.

Discovery of a series of novel phenylpiperazine derivatives as EGFR TK inhibitors

NASA Astrophysics Data System (ADS)

Sun, Juan; Wang, Xin-Yi; Lv, Peng-Cheng; Zhu, Hai-Liang

2015-09-01

Human epidermal growth factor receptor (EGFR) is an important drug target that plays a fundamental role in signal transduction pathways in oncology. We report herein the discovery of a novel class of phenylpiperazine derivatives with improved potency toward EGFR. The biological activity of compound 3p as inhibitor of EGFR was further investigated both in vitro and in vivo. Notably, compound 3p exhibited an IC50 in the nanomolar range in A549 cell cultures and induced a cessation of tumor growth with no toxicity, as determined by loss of body weight and death of treated mice. Compoutational docking studies also showed that compound 3p has interaction with EGFR key residues in the active site.

Loss of EGFR confers acquired resistance to AZD9291 in an EGFR-mutant non-small cell lung cancer cell line with an epithelial-mesenchymal transition phenotype.

PubMed

Xu, Jing; Zhao, Xiaoting; He, Dengfeng; Wang, Jinghui; Li, Weiying; Liu, Yinghui; Ma, Li; Jiang, Mei; Teng, Yu; Wang, Ziyu; Gu, Meng; Wu, Jianbin; Wang, Yue; Yue, Wentao; Zhang, Shucai

2018-05-24

AZD9291 is an irreversible, small-molecule inhibitor which has potency against mutant EGFR- and T790M-resistant mutation. Despite the encouraging efficacy in clinical, the acquired resistance will finally occur. Further study will need to be done to identify the acquired resistance mechanisms and determine the next treatment. We established an AZD9291-resistant cell line (HCC827/AZDR) from parental HCC827 cell line through stepwise pulsed selection of AZD9291. The expression of EGFR and its downstream pathways were determined by western blot analysis or immunofluorescence assay. The sensitivity to indicated agents were evaluated by MTS. Compared with parental HCC827 cells, the HCC827/AZDR cells showed high resistance to AZD9291 and other EGFR-TKIs, and exhibited a mesenchymal-like phenotype. Almost complete loss of EGFR expression was observed in HCC827/AZDR cells. But the activation of downstream pathway, MAPK signaling, was found in HCC827/AZDR cells even in the presence of AZD9291. Inhibition of MAPK signaling had no effect on cell viability of HCC827/AZDR and could not reverse AZD9291 resistance because of the subsequent activation of AKT signaling. When treated with the combination of AKT and MAPK inhibitor, HCC827/AZDR showed remarkable growth inhibition. Loss of EGFR could be proposed as a potential acquired resistance mechanism of AZD9291 in EGFR-mutant NSCLC cells with an EMT phenotype. Despite the loss of EGFR, the activation of MAPK pathway which had crosstalk with AKT pathway could maintain the proliferation and survival of resistant cells. Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

PubMed

Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

2005-05-01

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

PubMed

Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

2004-08-01

To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

PubMed Central

Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

2009-01-01

PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma.

PubMed

Zhou, Jin; Wu, Zhong; Wong, Gabrielle; Pectasides, Eirini; Nagaraja, Ankur; Stachler, Matthew; Zhang, Haikuo; Chen, Ting; Zhang, Haisheng; Liu, Jie Bin; Xu, Xinsen; Sicinska, Ewa; Sanchez-Vega, Francisco; Rustgi, Anil K; Diehl, J Alan; Wong, Kwok-Kin; Bass, Adam J

2017-01-06

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial-mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.

Transforming growth factor-{alpha} enhances corneal epithelial cell migration by promoting EGFR recycling.

PubMed

McClintock, Jennifer L; Ceresa, Brian P

2010-07-01

PURPOSE. The goal of this study was to determine the molecular mechanism by which transforming growth factor-alpha (TGF-alpha) is a more potent activator of epidermal growth factor receptor (EGFR)-mediated corneal wound healing than epidermal growth factor (EGF). METHODS. Telomerase immortalized human corneal epithelial (hTCEpi) cells and primary human corneal epithelial cells were tested for their ability to migrate in response to EGF and TGF-alpha. In parallel, the endocytic trafficking of the EGFR in response to these same ligands was examined using indirect immunofluorescence, immunoblots, and radioligand binding. RESULTS. TGF-alpha, compared with EGF, is a more potent activator of corneal epithelial cell migration. Although both TGF-alpha and EGF were able to induce EGFR internalization and phosphorylation, only those receptors that were stimulated with EGF progressed to lysosomal degradation. EGFRs stimulated with TGF-alpha recycled back to the plasma membrane, where they could be reactivated with ligand. CONCLUSIONS. This study reveals that EGFR-mediated cell migration is limited by ligand-stimulated downregulation of the EGFR. This limitation can be overcome by treating cells with TGF-alpha because TGF-alpha stimulates EGFR endocytosis, but not degradation. After internalization of the TGF-alpha/EGFR complex, EGFR recycles back to the plasma membrane, where it can be restimulated. This sequence of events provides the receptor multiple opportunities for stimulation. Thus, stimulation with TGF-alpha prolongs EGFR signaling compared with EGF.

Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma

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

Xu, Jie; Zeng, Li-Fan; Shen, Weihua

Highlights: •SHP2 is required for EGFR inhibitor resistant NSCLC H1975 cell proliferation. •SHP2 inhibitor blocks EGF-stimulated ERK1/2 activation and proliferation. •SHP2 inhibitor exhibits marked anti-tumor activity in H1975 xenograft mice. •SHP2 inhibitor synergizes with PI3K inhibitor in suppressing cell growth. •Targeting SHP2 represents a novel strategy for EGFR inhibitor resistant NSCLCs. -- Abstract: Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFRmore » (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.« less

Functional cooperation between HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR.

PubMed

Meng, Shuyan; Wang, Guorui; Lu, Yang; Fan, Zhen

2018-07-01

Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are important transcription factors regulating expression of genes involved in cell survival. HIF-1α and c-Jun are key components of HIF-1 and AP-1, respectively, and are regulated by epidermal growth factor receptor (EGFR)-mediated cell signaling and tumor microenvironmental cues. The roles of HIF-1α and c-Jun in development of resistance to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) with activating mutation of EGFR have not been explored. In this study, we investigated the roles of HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR. Changes in HIF-1α protein and in total and phosphorylated c-Jun levels in relation to changes in total and phosphorylated EGFR levels before and after gefitinib treatment were measured using Western blot analysis in NSCLC cells sensitive or resistant to gefitinib. The impact of overexpression of a constitutively expressed HIF-1α (HIF-1α/ΔODD) or a constitutively active c-Jun upstream regulator (SEK1 S220E/T224D mutant) on cell response to gefitinib was also examined. The effect of pharmacological inhibition of SEK1-JNK-c-Jun pathway on cell response to gefitinib was evaluated. Downregulation of HIF-1α and total and phosphorylated c-Jun levels correlated with cell inhibitory response to gefitinib better than decrease in phosphorylated EGFR did in NSCLC cells with intrinsic or acquired resistance to gefitinib. Overexpression of HIF-1α/ΔODD or SEK1 S220E/T224D mutant conferred resistance to gefitinib. There exists a positive feed-forward regulation loop between HIF-1 and c-Jun. The JNK inhibitor SP600125 sensitized gefitinib-resistant NSCLC cells to gefitinib. HIF-1α and c-Jun functionally cooperate in development of resistance to gefitinib in NSCLC cells. The translational value of inhibiting HIF-1α/c-Jun cooperation in overcoming resistance to EGFR TKI

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

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

Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast,more » amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.« less

Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors.

PubMed

Gao, Hongying; Yang, Zimo; Yang, Xinglin; Rao, Yu

2017-09-01

Osimertinib has been identified as a promising therapeutic drug targeting for EGFR T790M mutant non-small cell lung cancer (NSCLC). A new series of N-oxidized and fluorinated osimertinib derivatives were designed and synthesized. The cellular anti-proliferative activity, kinase inhibitory activity and the activation of EGFR signaling pathways of 1-6 in vitro were determined against L858R/T790M and wild-type EGFR, the antitumor efficacy in NCI-H1975 xenografts in vivo were further studied. Compound 2, the newly synthesized N-oxide metabolite in N,N,N'-trimethylethylenediamine side chain of osimertinib, showed a comparable kinase selectivity in vitro and a slightly better antitumor efficacy in vivo to osimertinib, making it valuable and suitable for the potential lung cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bioinformatics-driven discovery of rational combination for overcoming EGFR-mutant lung cancer resistance to EGFR therapy.

PubMed

Kim, Jihye; Vasu, Vihas T; Mishra, Rangnath; Singleton, Katherine R; Yoo, Minjae; Leach, Sonia M; Farias-Hesson, Eveline; Mason, Robert J; Kang, Jaewoo; Ramamoorthy, Preveen; Kern, Jeffrey A; Heasley, Lynn E; Finigan, James H; Tan, Aik Choon

2014-09-01

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Targeted tyrosine kinase inhibitors (TKIs) directed against the epidermal growth factor receptor (EGFR) have been widely and successfully used in treating NSCLC patients with activating EGFR mutations. Unfortunately, the duration of response is short-lived, and all patients eventually relapse by acquiring resistance mechanisms. We performed an integrative systems biology approach to determine essential kinases that drive EGFR-TKI resistance in cancer cell lines. We used a series of bioinformatics methods to analyze and integrate the functional genetics screen and RNA-seq data to identify a set of kinases that are critical in survival and proliferation in these TKI-resistant lines. By connecting the essential kinases to compounds using a novel kinase connectivity map (K-Map), we identified and validated bosutinib as an effective compound that could inhibit proliferation and induce apoptosis in TKI-resistant lines. A rational combination of bosutinib and gefitinib showed additive and synergistic effects in cancer cell lines resistant to EGFR TKI alone. We have demonstrated a bioinformatics-driven discovery roadmap for drug repurposing and development in overcoming resistance in EGFR-mutant NSCLC, which could be generalized to other cancer types in the era of personalized medicine. K-Map can be accessible at: http://tanlab.ucdenver.edu/kMap. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

PubMed

Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

2014-10-01

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

High ratio of T790M to EGFR activating mutations correlate with the osimertinib response in non-small-cell lung cancer.

PubMed

Ariyasu, Ryo; Nishikawa, Shingo; Uchibori, Ken; Oh-Hara, Tomoko; Yoshizawa, Takahiro; Dotsu, Yosuke; Koyama, Junji; Saiki, Masafumi; Sonoda, Tomoaki; Kitazono, Satoru; Yanagitani, Noriko; Horiike, Atsushi; Inase, Naohiko; Kasahara, Kazuo; Nishio, Makoto; Katayama, Ryohei

2018-03-01

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that can overcome resistance due to the Thr790Met (T790M) mutation. However, osimertinib occasionally shows limited efficacy in a small population of patients. We investigated the correlation between the ratio of T790M to EGFR activating mutation and the response to osimertinib. Between April 2016 and April 2017, 44 patients started osimertinib therapy at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research. We performed EGFR mutation analysis of cytological samples from 33 patients using droplet digital PCR. We calculated the ratio of T790M to EGFR activating mutations and correlated it with the systemic response to osimertinib. In tumors from the 33 patients, the average ratio of T790M to EGFR activating mutations was 0.420. Twenty-one of the 33 patients had tumors with a T790M ratio of ≥0.4. The osimertinib response rate was significantly higher (92.3%) in patients with a T790M ratio of ≥0.4 than in those with a T790M ratio of <0.4 (52.6%; p = 0.0237). We examined the correlation between the T790M ratio and the tumor reduction rate and obtained a coefficient of r = 0.417 (p = 0.0175). In patients with a T790M ratio of ≥0.4, the median progression-free survival was 355 days, which was longer, but not significant, than that in patients with a T790M ratio of <0.4 (median: 264 days). In patients with a T790M ratio of ≥0.4, the median treatment duration from first-line therapy onward was 931 days, which was significantly longer than that in patients with a T790M ratio of <0.4 (median, 567.5 days) (p = 0.044). The T790M ratio to EGFR activating mutation in tumor may correlate with the response to osimertinib, and patients with a higher T790M ratio have a longer treatment history. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Dual silencing of EGFR and HER2 enhances the sensitivity of gastric cancer cells to gefitinib.

PubMed

Wang, Liying; Zhang, Hongfeng; Zheng, Jiaxin; Wei, Xiaoli; Du, Jingwen; Lu, Haibo; Sun, Qiuying; Zhou, Weiyu; Zhang, Rui; Han, Yu

2018-04-10

Gefitinib exhibits very limited efficacy in gastric cancer (GC). Indeed, the limited clinical results obtained with gefitinib alone justify investigation of additional therapeutic strategies. Here, we demonstrate the importance of EGFR and HER2 in GC malignancy using RNA interference (RNAi). Additionally, we explored the ability of RNAi targeting EGFR and HER2 to enhance the sensitivity of GC cells to gefitinib. Specific small interfering RNAs (siRNAs) significantly inhibited mRNA and protein expression of target genes. EGFR-specific siRNA, EGFR/HER2 siRNAs, and gefitinib inhibited growth and induced apoptosis in GC cell lines in a dose-dependent manner. In contrast, resistance to HER2-siRNA-induced growth inhibition and apoptosis was linked to compensatory activation of EGFR. Moreover, gefitinib dramatically reduced p-EGFR and p-HER2 levels in the cell lines tested, and sensitivity to gefitinib was enhanced through dual silencing of EGFR and HER2 via suppression of AKT and ERK activation. These findings are in agreement with the profound inhibitory effect of gefitinib on activation of both EGFR and HER2. Overall, EGFR/HER2 knockdown by siRNAs further decreased the growth of GC cells treated with gefitinib alone, confirming that single-agent drug targeting does not achieve a maximal biological effect. The combination of gefitinib with EGFR/HER2 siRNAs should be further investigated as a new strategy for the treatment of GC and other EGFR/HER2-dependent cancers. © 2018 Wiley Periodicals, Inc.

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.

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

PubMed

Ward, Richard A; Anderton, Mark J; Ashton, Susan; Bethel, Paul A; Box, Matthew; Butterworth, Sam; Colclough, Nicola; Chorley, Christopher G; Chuaqui, Claudio; Cross, Darren A E; Dakin, Les A; Debreczeni, Judit É; Eberlein, Cath; Finlay, M Raymond V; Hill, George B; Grist, Matthew; Klinowska, Teresa C M; Lane, Clare; Martin, Scott; Orme, Jonathon P; Smith, Peter; Wang, Fengjiang; Waring, Michael J

2013-09-12

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Consolidative local ablative therapy improves the survival of patients with synchronous oligometastatic NSCLC harboring EGFR activating mutation treated with first-line EGFR-TKIs.

PubMed

Xu, Qinghua; Zhou, Fei; Liu, Hui; Jiang, Tao; Li, Xuefei; Xu, Yaping; Zhou, Caicun

2018-05-28

The aim of the current study was to investigate whether consolidative local ablative therapy (LAT) can improve the survival of patients with stage IV EGFR-mutant NSCLC who have oligometastatic disease treated with first-line EGFR-TKI therapy. Patients with stage IV EGFR-mutant NSCLC and no more than five metastases within 2 months of diagnosis were identified. All patients were treated with first-line EGFR-TKIs. Consolidative LAT included radiotherapy, surgery or both. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier curves. From October 2010 to May 2016, 145 patients were enrolled, including 51 (35.2%) who received consolidative LAT to all oligometastatic sites (All-LAT group), 55 (37.9%) who received consolidative LAT to either primary tumor or oligometastatic sites (Part-LAT group), and 39 (26.9%) who did not receive any consolidative LAT (Non-LAT group). The median PFS in All-LAT, Part-LAT, and Non-LAT group were 20.6, 15.6, and 13.9 months, respectively (P<0.001). The median OS in All-LAT, Part-LAT, and Non-LAT group were 40.9, 34.1, and 30.8 months, respectively (P<0.001). The difference was statistically significant between All-LAT group and Part-LAT or Non-LAT group but was not between Part-LAT and Non-LAT group. The median OS was significantly improved with consolidative LAT for primary tumor (40.5 versus 31.5 months, P<0.001), brain metastases (38.2 versus 29.2 months, P=0.002), adrenal metastases (37.1 versus 29.2 months, P =0.032). Adverse events (Grade ≥ 3) due to radiotherapy included pneumonitis (7.7%) and esophagitis (16.9%). The current study demonstrated that consolidative LAT to all metastatic sites was a feasible option for patients with EGFR-mutant oligometastatic NSCLC during first-line EGFR-TKI treatment, with significantly improved PFS and OS compared with consolidative LAT to partial sites or observation alone. Copyright © 2018. Published by Elsevier Inc.

Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation.

PubMed

Osoegawa, Atsushi; Hashimoto, Takafumi; Takumi, Yohei; Abe, Miyuki; Yamada, Tomonori; Kobayashi, Ryoji; Miyawaki, Michiyo; Takeuchi, Hideya; Okamoto, Tatsuro; Sugio, Kenji

2018-03-28

Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma

PubMed Central

Schneeberger, Valentina E.; Ren, Yuan; Luetteke, Noreen; Huang, Qingling; Chen, Liwei; Lawrence, Harshani R.; Lawrence, Nicholas J.; Haura, Eric B.; Koomen, John M.; Coppola, Domenico; Wu, Jie

2015-01-01

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma. PMID:25730908

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

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.

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.

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

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR.

PubMed

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-08-21

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration.

PubMed

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A

2017-05-09

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration

PubMed Central

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A.

2017-01-01

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair. PMID:28485389

Prevalence of EGFR Mutations in Lung Cancer in Uruguayan Population

PubMed Central

Touya, Diego; Bertoni, Bernardo; Osinaga, Eduardo; Varangot, Mario

2017-01-01

Background Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR) gene into routine clinical practice represents a milestone for personalized therapy of the non-small-cell lung cancer (NSCLC). However, the genetic testing of EGFR mutations has not yet become a routine clinical practice in developing countries. In view of different prevalence of such mutations among different ethnicities and geographic regions, as well as the limited existing data from Latin America, our aim was to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients from Uruguay. Methods We examined EGFR mutations in exons 18 through 21 in 289 NSCLC Uruguayan patients by PCR-direct sequencing. Results EGFR mutations were detected in 53 of the 289 (18.3%) patients, more frequently in women (23.4%) than in men (14.5%). The distribution by exon was similar to that generally reported in the literature. Conclusions This first epidemiological study of EGFR mutations in Uruguay reveals a wide spectrum of mutations and an overall prevalence of 18.3%. The background ethnic structure of the Uruguayan population could play an important role in explaining our findings. PMID:28744312

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment.

PubMed

Lauriola, Mattia; Enuka, Yehoshua; Zeisel, Amit; D'Uva, Gabriele; Roth, Lee; Sharon-Sevilla, Michal; Lindzen, Moshit; Sharma, Kirti; Nevo, Nava; Feldman, Morris; Carvalho, Silvia; Cohen-Dvashi, Hadas; Kedmi, Merav; Ben-Chetrit, Nir; Chen, Alon; Solmi, Rossella; Wiemann, Stefan; Schmitt, Fernando; Domany, Eytan; Yarden, Yosef

2014-10-03

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Known and putative mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations—a review

PubMed Central

Stewart, Erin L.; Tan, Samuel Zhixing; Liu, Geoffrey

2015-01-01

Lung cancer is the leading cause of cancer related deaths in Canada with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Tumor characterization can identify cancer-driving mutations as treatment targets. One of the most successful examples of cancer targeted therapy is inhibition of mutated epidermal growth factor receptor (EGFR), which occurs in ~10-30% of NSCLC patients. While this treatment has benefited many patients with activating EGFR mutations, almost all who initially benefited will eventually acquire resistance. Approximately 50% of cases of acquired resistance (AR) are due to a secondary T790M mutation in exon 20 of the EGFR gene; however, many of the remaining mechanisms of resistance are still unknown. Much work has been done to elucidate the remaining mechanisms of resistance. This review aims to highlight both the mechanisms of resistance that have already been identified in patients and potential novel mechanisms identified in preclinical models which have yet to be validated in the patient settings. PMID:25806347

The phosphatase inhibitor menadione (vitamin K3) protects cells from EGFR inhibition by erlotinib and cetuximab.

PubMed

Perez-Soler, Roman; Zou, Yiyu; Li, Tianhong; Ling, Yi He

2011-11-01

Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect. Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays. Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two-hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells. Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. ©2011 AACR

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.

EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

PubMed Central

Arasada, Rajeswara Rao; Amann, Joseph M.; Rahman, Mohammad A; Huppert, Stacey S.; Carbone, David P.

2014-01-01

Mutations in the epidermal growth factor receptor (EGFR) are the most common actionable genetic abnormalities yet discovered in lung cancer. However, targeting these mutations with kinase inhibitors is not curative in advanced disease and has yet to demonstrate an impact on potentially curable, early-stage disease, with some data suggesting adverse outcomes. Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, enriches the ALDH+ stem-like cells through EGFR-dependent activation of Notch3. Additionally, we demonstrate that erlotinib treatment increases the clonogenicity of lung cancer cells in a sphere-forming assay, suggesting increased stem-like cell potential. We demonstrate that inhibition of EGFR kinase activity leads to activation of Notch transcriptional targets in a gamma secretase inhibitor sensitive manner and causes Notch activation. leading to an increase in ALDH high+ cells. We also find a kinase-dependent physical association between the Notch3 and EGFR receptors and tyrosine phosphorylation of Notch3. This could explain the worsened survival observed in some studies of erlotinib treatment at early-stage disease, and suggests that specific dual targeting might overcome this adverse effect. PMID:25125655

Anti-EGFR monoclonal antibody in cancer treatment: in vitro and in vivo evidence

PubMed

Quatrale, Anna Elisa; Petriella, Daniela; Porcelli, Letizia; Tommasi, Stefania; Silvestris, Nicola; Colucci, Giuseppe; Angelo, Angelo; Azzariti, Amalia

2011-01-01

The complexity of EGFR signaling network suggests that the receptor could be promising targets for new personalised therapy. In clinical practice two strategies targeting the receptor are available; they utilise monoclonal antibodies, directed towards the extracellular domain of EGFR, and small molecule tyrosine kinase inhibitors, which bind the catalytic kinase domain of the receptor. In this review, we summarise currently known pre-clinical data on the antitumor effects of monoclonal antibodies, which bind to EGFR in its inactive configuration, competing for ligand binding and thereby blocking ligand-induced EGFR tyrosine kinase activation. As a consequence of treatment, key EGFR-dependent intracellular signals in cancer cells are affected. Data explaining the mechanisms of action of anti-EGFR monoclonal antibodies, currently used in clinical setting and under development for the treatment of solid tumors, are revised with the aim to provide an overview of the most important preclinical studies showing the impact of this class of EGFR targeted agents on tumor biology.

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR

PubMed Central

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-01-01

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance. PMID:26015401

Cationic Polyamidoamine Dendrimers as Modulators of EGFR Signaling In Vitro and In Vivo

PubMed Central

Akhtar, Saghir; Al-Zaid, Bashayer; El-Hashim, Ahmed Z.; Chandrasekhar, Bindu; Attur, Sreeja; Yousif, Mariam H. M.; Benter, Ibrahim F.

2015-01-01

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling—an important signaling cascade that regulates cell growth, survival and apoptosis- in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM

EGFR Targeted Theranostic Nanoemulsion For Image-Guided Ovarian Cancer Therapy

PubMed Central

Ganta, Srinivas; Singh, Amit; Kulkarni, Praveen; Keeler, Amanda W.; Piroyan, Aleksandr; Sawant, Rupa R.; Patel, Niravkumar R.; Davis, Barbara; Ferris, Craig; O’Neal, Sara; Zamboni, William; Amiji, Mansoor M.; Coleman, Timothy P.

2015-01-01

Purpose Platinum-based therapies are the first line treatments for most types of cancer including ovarian cancer. However, their use is associated with dose-limiting toxicities and resistance. We report initial translational studies of a theranostic nanoemulsion loaded with a cisplatin derivative, myrisplatin and pro-apoptotic agent, C6-ceramide. Methods The surface of the nanoemulsion is annotated with an endothelial growth factor receptor (EGFR) binding peptide to improve targeting ability and gadolinium to provide diagnostic capability for image-guided therapy of EGFR overexpressing ovarian cancers. A high shear microfludization process was employed to produce the formulation with particle size below 150 nm. Results Pharmacokinetic study showed a prolonged blood platinum and gadolinium levels with nanoemulsions in nu/nu mice. The theranostic nanoemulsions also exhibited less toxicity and enhanced the survival time of mice as compared to an equivalent cisplatin treatment. Conclusions Magnetic resonance imaging (MRI) studies indicate the theranostic nanoemulsions were effective contrast agents and could be used to track accumulation in a tumor. The MRI study additionally indicate that significantly more EGFR-targeted theranostic nanoemulsion accumulated in a tumor than non-targeted nanoemulsuion providing the feasibility of using a targeted theranostic agent in conjunction with MRI to image disease loci and quantify the disease progression. PMID:25732960

Monitoring of Circulating Tumor Cells and Their Expression of EGFR/Phospho-EGFR During Combined Radiotherapy Regimens in Locally Advanced Squamous Cell Carcinoma of the Head and Neck

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

Tinhofer, Ingeborg, E-mail: ingeborg.tinhofer@charite.de; Hristozova, Tsvetana; Stromberger, Carmen

2012-08-01

Purpose: The numbers of circulating tumor cells (CTCs) and their expression/activation of epidermal growth factor receptor (EGFR) during the course of combined chemo- or bioradiotherapy regimens as potential biomarkers of treatment efficacy in squamous cell carcinoma of the head and neck (SCCHN) were determined. Methods and Materials: Peripheral blood samples from SCCHN patients with locally advanced stage IVA/B disease who were treated with concurrent radiochemotherapy or induction chemotherapy followed by bioradiation with cetuximab were included in this study. Using flow cytometry, the absolute number of CTCs per defined blood volume as well as their expression of EGFR and its phosphorylatedmore » form (pEGFR) during the course of treatment were assessed. Results: Before treatment, we detected {>=}1 CTC per 3.75 mL blood in 9 of 31 patients (29%). Basal expression of EGFR was detected in 100% and pEGFR in 55% of the CTC+ cases. The frequency of CTC detection was not influenced by induction chemotherapy. However, the number of CTC+ samples significantly increased after radiotherapy. This radiation-induced increase in CTC numbers was less pronounced when radiotherapy was combined with cetuximab compared to its combination with cisplatin/5-fluorouracil. The former treatment regimen was also more effective in reducing pEGFR expression in CTCs. Conclusions: Definitive radiotherapy regimens of locally advanced SCCHN can increase the number of CTCs and might thus contribute to a systemic spread of tumor cells. Further studies are needed to evaluate the predictive value of the radiation-induced increase in CTC numbers and the persistent activation of the EGFR signalling pathway in individual CTC+ cases.« less

Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells.

PubMed

Song, Xiulong; Wei, Zhengxi; Shaikh, Zahir A

2015-08-15

Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1-3μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

The APPLE Trial: Feasibility and Activity of AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients. EORTC 1613.

PubMed

Remon, Jordi; Menis, Jessica; Hasan, Baktiar; Peric, Aleksandra; De Maio, Eleonora; Novello, Silvia; Reck, Martin; Berghmans, Thierry; Wasag, Bartosz; Besse, Benjamin; Dziadziuszko, Rafal

2017-09-01

The AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients (APPLE) trial is a randomized, open-label, multicenter, 3-arm, phase II study in advanced, epidermal growth factor receptor (EGFR)-mutant and EGFR tyrosine kinase inhibitor (TKI)-naive non-small-cell lung cancer (NSCLC) patients, to evaluate the best strategy for sequencing gefitinib and osimertinib treatment. Advanced EGFR-mutant NSCLC patients, with World Health Organization performance status 0-2 who are EGFR TKI treatment-naive and eligible to receive first-line treatment with EGFR TKI will be randomized to: In all arms, a plasmatic ctDNA T790M test will be performed by a central laboratory at the Medical University of Gdansk (Poland) but will be applied as a predictive marker for making treatment decisions only in arm B. The primary objective is to evaluate the best strategy for sequencing of treatment with gefitinib and osimertinib in advanced NSCLC patients with common EGFR mutations, and to understand the value of liquid biopsy for the decision-making process. The progression-free survival rate at 18 months is the primary end point of the trial. The activity of osimertinib versus gefitinib to prevent brain metastases will be evaluated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Clinical outcomes of EGFR-TKI treatment and genetic heterogeneity in lung adenocarcinoma patients with EGFR mutations on exons 19 and 21.

PubMed

Yu, Jiang-Yong; Yu, Si-Fan; Wang, Shu-Hang; Bai, Hua; Zhao, Jun; An, Tong-Tong; Duan, Jian-Chun; Wang, Jie

2016-03-21

Epidermal growth factor receptor (EGFR) mutations, including a known exon 19 deletion (19 del) and exon 21 L858R point mutation (L858R mutation), are strong predictors of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment in lung adenocarcinoma. However, whether patients carrying EGFR 19 del and L858R mutations exhibit different responsiveness to EGFR-TKIs and what are the potential mechanism for this difference remain controversial. This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes. Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858R mutations, 532 received EGFR-TKI treatment and were included in this study. EGFR 19 del and L858R mutations were detected by using denaturing high-performance liquid chromatography (DHPLC). T790M mutation, which is a common resistant mutation on exon 20 of EGFR, was detected by amplification refractory mutation system (ARMS). Next-generation sequencing (NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858R mutations. Of the 532 patients, 319 (60.0%) had EGFR 19 del, and 213 (40.0%) had L858R mutations. The patients with EGFR 19 del presented a significantly higher overall response rate (ORR) for EGFR-TKI treatment (55.2% vs. 43.7%, P = 0.017) and had a longer progression-free survival (PFS) after first-line EGFR-TKI treatment (14.4 vs. 11.4 months, P = 0.034) compared with those with L858R mutations. However, no statistically significant difference in overall survival (OS) was observed between the two groups of patients. T790M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment, and there was no significant difference in the co-existence of T790M mutation with EGFR 19 del and L858R mutations before EGFR-TKI treatment (5.6% vs. 8.8%, P = 0.554) or after

TCDD promoted EMT of hFPECs via AhR, which involved the activation of EGFR/ERK signaling

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

Gao, Zhan; The Fifth Affiliated Hospital, Zhengzhou University, 450052; Bu, Yongjun

2016-05-01

One critical step of second palatal fusion is the newly formed medial epithelia seam (MES) disintegration, which involves apoptosis, epithelial to mesenchymal transition (EMT), and cell migration. Although the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate at high rates, little is known about the effects of TCDD exposure on the fate of palatal epithelial cells. By using primary epithelial cells isolated from human fetal palatal shelves (hFPECs), we show that TCDD increased cell proliferation and EMT, as demonstrated by increased the epithelial markers (E-cadherin and cytokeratin14) and enhanced the mesenchymal markers (vimentin and fibronectin), but had no effect on cellmore » migration and apoptosis. TCDD exposure led to a dose-dependent increase in Slug protein expression. Coimmunoprecipitation revealed that TCDD promoted AhR to form a protein complex with Slug. ChIP assay confirmed that TCDD exposure recruited AhR to the xenobiotic responsive element of Slug promoter. Knockdown of AhR by siRNA remarkably weakened TCDD-induced binding of AhR to the XRE promoter of slug, thereby suppressed TCDD-induced vimentin. Further experiment showed that TCDD stimulated EGFR phosphorylation did not influence the TGFβ3/Smad signaling; whereas TCDD increased phosphorylation of ERK1/2 and p38 with no effect on activation of JNK. By using varieties of inhibitors, we confirmed that TCDD promoted proliferation and EMT of hFPECs via activation of EGFR/ERK pathway. These data make a novel contribution to the molecular mechanism of cleft palate by TCDD. - Highlights: • TCDD exposure promoted cell proliferation and EMT of hFPECs; • AhR signaling was activated and required for TCDD-induced EMT; • TCDD-mediated EMT of hFPECs involved the activation of EGFR/ERK signaling; • TCDD exposure had no effect on TGFβ3/Smad pathway.« less

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

Co-targeting of EGFR and autophagy signaling is an emerging treatment strategy in metastatic colorectal cancer.

PubMed

Koustas, Evangelos; Karamouzis, Michalis V; Mihailidou, Chrysovalantou; Schizas, Dimitrios; Papavassiliou, Athanasios G

2017-06-28

The epidermal growth factor receptor (EGFR) and its associated pathway is a critical key regulator of CRC development and progression. The monoclonal antibodies (MoAbs) cetuximab and panitumumab, directed against EGFR, represent a major step forward in the treatment of metastatic colorectal cancer (mCRC), in terms of progression-free survival and overall survival in several clinical trials. However, the activity of anti-EGFR MoAbs appears to be limited to a subset of patients with mCRC. Studies have highlighted that acquired-resistance to anti-EGFR MoAbs biochemically converge into Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways. Recent data also suggest that acquired-resistance to anti-EGFR MoAbs is accompanied by inhibition of EGFR internalization, ubiqutinization, degradation and prolonged downregulation. It is well established that autophagy, a self-cannibalization process, is considered to be associated with resistance to the anti-EGFR MoAbs therapy. Additionally, autophagy induced by anti-EGFR MoAbs acts as a protective response in cancer cells. Thus, inhibition of autophagy after treatment with EGFR MoAbs can result in autophagic cell death. A combination therapy comprising of anti-EGFR MoAbs and autophagy inhibitors would represent a multi-pronged approach that could be evolved into an active therapeutic strategy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop.

PubMed

Pirker, Robert; Herth, Felix J F; Kerr, Keith M; Filipits, Martin; Taron, Miquel; Gandara, David; Hirsch, Fred R; Grunenwald, Dominique; Popper, Helmut; Smit, Egbert; Dietel, Manfred; Marchetti, Antonio; Manegold, Christian; Schirmacher, Peter; Thomas, Michael; Rosell, Rafael; Cappuzzo, Federico; Stahel, Rolf

2010-10-01

Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations. The "EGFR testing in NSCLC: from biology to clinical practice" International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group. Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests. The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.

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.

Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

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

Backer, Joseph M.

2009-07-12

In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need formore » information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for 1) rational patient stratification, 2) rapid monitoring of responses to therapy, and 3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg’s group at

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.

Are EGFR tyrosine kinase inhibitors effective in elderly patients with EGFR-mutated non-small cell lung cancer?

PubMed

Roviello, Giandomenico; Zanotti, Laura; Cappelletti, Maria Rosa; Gobbi, Angela; Dester, Martina; Paganini, Giovanni; Pacifico, Chiara; Generali, Daniele; Roudi, Raheleh

2018-02-01

EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib changed dramatically the history of metastatic non-small cell lung cancer (NSCLC) harbouring EGFR mutations. However, not enough data are available on the efficacy of these targeted drugs in elderly patients. The aim of this study is to analyse the available clinical data evaluating the efficacy of anti-EGFR therapies in elderly patients with advanced NSCLC carrying EGFR mutations. A literature-based meta-analysis of the results of randomized clinical trials was undertaken. Relevant publications from PubMed, the Cochrane Library, and abstracts from American Society of Clinical Oncology meetings were searched. Progression-free survival (PFS), as a measure of the efficacy of treatment, was the primary outcome investigated. The pooled analysis revealed an overall significant improvement in PFS (HR = 0.44, 95% CI 0.28-0.69; p = 0.0004) with the use of EGFR TKIs in EGFR-mutated NSCLC. The data stratification per age subgroups showed that EGFR TKIs were more effective in prolonging PFS in elderly patients, with HR 0.39 (p = 0.008), in comparison with young patients (HR = 0.48; p = 0.04). The results of this study suggest that EGFR TKIs have a significant effect in slowing down diseases progression in elderly patients with advanced NSCLC, therefore representing a valid therapeutic option in this age group.

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma.

PubMed

Komuro, Akiyoshi; Raja, Erna; Iwata, Caname; Soda, Manabu; Isogaya, Kazunobu; Yuki, Keiko; Ino, Yasushi; Morikawa, Masato; Todo, Tomoki; Aburatani, Hiroyuki; Suzuki, Hiromichi; Ranjit, Melissa; Natsume, Atsushi; Mukasa, Akitake; Saito, Nobuhito; Okada, Hitoshi; Mano, Hiroyuki; Miyazono, Kohei; Koinuma, Daizo

2018-04-15

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma. © 2017 UICC.

Icotinib in Patients with Pretreated Advanced Esophageal Squamous Cell Carcinoma with EGFR Overexpression or EGFR Gene Amplification: A Single-Arm, Multicenter Phase 2 Study.

PubMed

Huang, Jing; Fan, Qingxia; Lu, Ping; Ying, Jianming; Ma, Changwu; Liu, Wei; Liu, Ying; Tan, Fenlai; Sun, Yan

2016-06-01

Epidermal growth factor receptor (EGFR) has been reported to be overexpressed and amplified in a high percentage of patients with esophageal squamous cell carcinoma (ESCC). The activity of icotinib, an EGFR tyrosine kinase inhibitor, was assessed in previously treated ESCC with EGFR overexpression or amplification. For this phase 2, single-arm, multicenter trial undertaken at six hospitals in China, we included Chinese patients with previously treated, histologically confirmed advanced ESCC and EGFR overexpression (immunohistochemical staining sore of 3+) or amplification (positive fluorescence in situ hybridization result). These patients received oral icotinib (250 mg, three times daily).The primary end point was the proportion of patients with objective responses as assessed by an independent radiology review committee. Between December 5, 2013, and May 28, 2015, a total of 281 patients were screened. Fifty-four eligible patients were enrolled. Nine responses were observed, including one complete response and eight partial responses, and 16 patients had stable disease, resulting in a 16.7% objective response rate (95% confidence interval [CI]: 6.7-26.6) and 46.3% disease control rate (95% CI: 33.0-59.6). The median progression-free survival and overall survival times were 52 (95% CI: 40-95) days and 153 (95% CI: 139-218) days, respectively. A total of 43 patients experienced at least one adverse event, but most were only grade 1 to 2 in severity. The most frequent was rash (48.1%), followed by diarrhea (22.2%). Icotinib showed favorable activity in patients with advanced, previously treated ESCC with EGFR overexpression or amplification. These findings suggest further research into EGFR overexpression or amplification for selecting responsive patients. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

MLH1 V384D polymorphism associates with poor response to EGFR tyrosine kinase inhibitors in patients with EGFR L858R-positive lung adenocarcinoma.

PubMed

Chiu, Chao-Hua; Ho, Hsiang-Ling; Doong, Howard; Yeh, Yi-Chen; Chen, Mei-Yu; Chou, Teh-Ying; Tsai, Chun-Ming

2015-04-10

A significant fraction of patients with lung adenocarcinomas harboring activating epidermal growth factor receptor (EGFR) mutations do not experience clinical benefits from EGFR tyrosine kinase inhibitor (TKI) therapy. Using next-generation sequencing, we screened 739 mutation hotspots in 46 cancer-related genes in EGFR L858R-mutant lung adenocarcinomas from 29 patients who received EGFR-TKI therapy; 13 had short (< 3 months) and 16 had long (> 1 year) progression-free survival (PFS). We discovered MLH1 V384D as a genetic variant enriched in the group of patients with short PFS. Next, we investigated this genetic variation in 158 lung adenocarcinomas with the EGFR L858R mutation and found 14 (8.9%) patients had MLH1 V384D; available blood or non-tumor tissues from patients were also tested positive for MLH1 V384D. Patients with MLH1 V384D had a significantly shorter median PFS than those without (5.1 vs. 10.6 months; P= 0.001). Multivariate analysis showed that MLH1 V384D polymorphism was an independent predictor for a reduced PFS time (hazard ratio, 3.5; 95% confidence interval, 1.7 to 7.2; P= 0.001). In conclusion, MLH1 V384D polymorphism is associated with primary resistance to EGFR-TKIs in patients with EGFR L858R-positive lung adenocarcinoma and may potentially be a novel biomarker to guide treatment decisions.

EGFR, HER2 and VEGF pathways: validated targets for cancer treatment.

PubMed

Press, Michael F; Lenz, Heinz-Josef

2007-01-01

Targeted therapies are rationally designed to interfere with specific molecular events that are important in tumour growth, progression or survival. Several targeted therapies with anti-tumour activity in human cancer cell lines and xenograft models have now been shown to produce objective responses, delay disease progression and, in some cases, improve survival of patients with advanced malignancies. These targeted therapies include cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody; gefitinib and erlotinib, EGFR-specific tyrosine kinase inhibitors; trastuzumab, an anti-human EGFR type 2 (HER2)-related monoclonal antibody; lapatinib, a dual inhibitor of both EGFR- and HER2-associated tyrosine kinases; and bevacizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody. On the basis of preclinical and clinical evidence, EGFR, HER2 and VEGF represent validated targets for cancer therapy and remain the subject of intensive investigation. Both EGFR and HER2 are targets found on cancer cells, whereas VEGF is a target that acts in the tumour microenvironment. Clinical studies are focusing on how to best incorporate targeted therapy into current treatment regimens and other studies are exploring whether different strategies for inhibiting these targets will offer greater benefit. It is clear that optimal use of targeted therapy will depend on understanding how these drugs work mechanistically, and recognising that their activities may differ across patient populations, tumour types and disease stages, as well as when and how they are used in cancer treatment. The results achieved with targeted therapies to date are promising, although they illustrate the need for additional preclinical and clinical study.

Structural insights into drug development strategy targeting EGFR T790M/C797S.

PubMed

Zhu, Su-Jie; Zhao, Peng; Yang, Jiao; Ma, Rui; Yan, Xiao-E; Yang, Sheng-Yong; Yang, Jing-Wen; Yun, Cai-Hong

2018-03-02

Treatment of non-small-cell lung cancers (NSCLCs) harboring primary EGFR oncogenic mutations such as L858R and exon 19 deletion delE746_A750 (Del-19) using gefitinib/erlotinib ultimately fails due to the emergence of T790M mutation. Though WZ4002/CO-1686/AZD9291 are effective in overcoming EGFR T790M by targeting Cys797 via covalent bonding, their efficacy is again limited due to the emergence of C797S mutation. New agents effectively inhibiting EGFR T790M without covalent linkage through Cys 797 may solve this problem. We presented here crystal structures of EGFR activating/drug-resistant mutants in complex with a panel of reversible inhibitors along with mutagenesis and enzyme kinetic data. These data revealed a previously un-described hydrophobic clamp structure in the EGFR kinase which may be exploited to facilitate development of next generation drugs targeting EGFR T790M with or without concomitant C797S. Interestingly, mutations in the hydrophobic clamp that hinder drug binding often also weaken ATP binding and/or abolish kinase activity, thus do not readily result in resistance to the drugs.

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.

Continued EGFR-TKI with concurrent radiotherapy to improve time to progression (TTP) in patients with locally progressive non-small cell lung cancer (NSCLC) after front-line EGFR-TKI treatment.

PubMed

Wang, Y; Li, Y; Xia, L; Niu, K; Chen, X; Lu, D; Kong, R; Chen, Z; Sun, J

2018-03-01

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is the optimal treatment for EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, most patients developed systemic or local progression due to acquired EGFR-TKI resistance. This retrospective study aimed to evaluate the feasibility of continued EGFR-TKI with concurrent radiotherapy (CTCRT) in patients with local progression after front-line EGFR-TKI treatment. Advanced NSCLC patients with active EGFR mutation who received EGFR-TKI were treated with CTCRT after local progression. Medical data were analyzed for time to progression (TTP), progression-free survival (PFS), tumor response rate, overall survival (OS) and adverse events. A total of 50 irradiated lesions from 44 patients were included. Median TTP and PFS of measurable lesions (n = 31) were both significantly prolonged after local radiotherapy (TTP1 + TTP2 vs. TTP1: 21.7 vs. 16.0 months, P = 0.010; PFS1 + PFS2 vs. PFS1: 21.3 vs. 16.0 months, P = 0.027). For all lesions (n = 50), objective response rate (ORR) and local tumor control rate (LCR) were 54.0 and 84.0%, respectively. Median OS was 26.6 months. There were no serious adverse events before or after radiotherapy. The treatment modality of CTCRT is considerable and effective for EGFR-mutant NSCLC patients even with local failure from front-line EGFR-TKI treatment.

Therapeutic value of EGFR inhibition in CRC and NSCLC: 15 years of clinical evidence.

PubMed

Troiani, Teresa; Napolitano, Stefania; Della Corte, Carminia Maria; Martini, Giulia; Martinelli, Erika; Morgillo, Floriana; Ciardiello, Fortunato

2016-01-01

Epidermal growth factor receptor (EGFR) plays a key role in tumour evolution, proliferation and immune evasion, and is one of the most important targets for biological therapy, especially for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). In the past 15 years, several EGFR antagonists have been approved for the treatment of NSCLC and metastatic CRC (mCRC). To optimise the use of anti-EGFR agents in clinical practice, various clinical and molecular biomarkers have been investigated, thus moving their indication from unselected to selected populations. Nowadays, anti-EGFR drugs represent a gold-standard therapy for metastatic NSCLC harbouring EGFR activating mutation and for RAS wild-type mCRC. Their clinical efficacy is limited by the presence of intrinsic resistance or the onset of acquired resistance. In this review, we provide an overview of the antitumour activity of EGFR inhibitors in NSCLC and CRC and of mechanisms of resistance, focusing on the development of a personalised approach through 15 years of preclinical and clinical research.

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

NASA Astrophysics Data System (ADS)

Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

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.

Molecular modeling and description of a newly characterized activating mutation of the EGFR gene in non-small cell lung cancer.

PubMed

Otto, Claudia; Csanadi, Agnes; Fisch, Paul; Werner, Martin; Kayser, Gian

2012-10-22

Lung cancer is the leading cause of death among malignant diseases in humans worldwide. In the last decade development of new targeted drugs for the treatment of non-small cell lung cancer proved to be a promising approach to prolong the otherwise very poor prognosis of patients with advanced UICC stages. Epidermal growth factor receptor (EGFR) has been in the focus of this lung cancer science and specific activating mutations are eligible for the treatment with specific tyrosine kinase inhibitors like gefitinib or erlotinib. Beside typical deletions in exon 19 and point mutations in exons 18 and 21 several insertions in exon 19 have been described and attributed activating properties as well. This is the first European and overall the 5th description in English literature of one of these specific insertions. To elucidate its structural changes leading to the activating properties we performed molecular modeling studies. These revealed conformational and electrostatic force field changes in the kinase domain of EGFR. To not miss uncommon mutations thorough and precise characterization of EGFR hotspots, i. e. at least exons 18, 19 and 21, should therefore be conducted to provide best medical care and to offer lung cancer patients appropriate cancer treatment. The vistual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2209889658102062.

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.

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.

Osimertinib for EGFR T790M mutation-positive non-small cell lung cancer.

PubMed

Soejima, Kenzo; Yasuda, Hiroyuki; Hirano, Toshiyuki

2017-01-01

Significant advances have been made since the development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) targeting EGFR mutations in non-small-cell lung cancer (NSCLC), however, lung cancer cells eventually acquire resistance to those agents. Osimertinib (AZD9291) has been developed as 3 rd generation EGFR-TKI with activities against sensitizing mutations and T790 M resistance mutation, which account for about 50% of the mechanisms of acquired resistance to 1 st or 2 nd generation EGFR-TKIs. A recent phase I/II clinical trial with osimertinib for advanced NSCLC patients with known sensitizing EGFR mutations and documented disease progression on prior EGFR-TKIs revealed promising effect with acceptable toxicities. Areas covered: This article summarizes current understanding and available preclinical and clinical data on osimertinib and also discusses future directions. The literature search included PubMed and the latest articles from international conferences. Expert commentary: The development of osimertinib has provided new therapeutic options for NSCLC patients harboring T790 M. Compared with other EGFR-TKIs including rociletinib, osimertinib seems to possess an advantage with respect to the effect and safety profile among existing EGFR-TKIs. However, tumor progression still occurs even when treating with osimertinib. A further understanding of the mechanisms of resistance is eagerly anticipated in order to develop next generation EGFR-TKIs.

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

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

PubMed Central

Yasuda, Hiroyuki; Hamamoto, Junko; Oashi, Ayano; Ishioka, Kota; Arai, Daisuke; Nukaga, Shigenari; Miyawaki, Masayoshi; Kawada, Ichiro; Naoki, Katsuhiko; Costa, Daniel B.; Kobayashi, Susumu S.; Betsuyaku, Tomoko; Soejima, Kenzo

2015-01-01

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations. PMID:26515464

INHIBITION OF PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

EPA Science Inventory

A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden particulate matter inhibits protein tyrosine phosphatase activity in HAEC and leads to Src-dependent activation of EGFR sign...

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

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.

w09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway.

PubMed

Zhang, Pinghu; Zheng, Zuguo; Ling, Li; Yang, Xiaohui; Zhang, Ni; Wang, Xue; Hu, Maozhi; Xia, Yu; Ma, Yiwen; Yang, Haoran; Wang, Yunyi; Liu, Hongqi

2017-07-03

The EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

Renal toxicity of anticancer agents targeting HER2 and EGFR.

PubMed

Cosmai, Laura; Gallieni, Maurizio; Porta, Camillo

2015-12-01

EGFR and HER2 are found overexpressed and/or activated in many different human malignancies (e.g. breast and colon cancer), and a number of drugs specifically targeting these two tyrosine kinases have been developed over the years as anticancer agents. In the present review, the renal safety profile of presently available agents targeting either HER2 or EGFR will be discussed, together with the peculiarities related to their clinical use in patients with impaired renal function, or even in dialysis. Indeed, even though renal toxicity is not so common with these agents, it may nevertheless happen, especially when these agents are combined with traditional chemotherapeutic agents. As a whole, kidney impairment or dialysis should not be regarded per se as reasons not to administer or to stop an active anti-HER or anti-EGFR anticancer treatment, especially given the possibility of significantly improving the life expectancy of many cancer patients with the use of these agents.

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.

SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

PubMed

Thomas, J Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R; Moos, Malcolm

2016-01-01

In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

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.

Blocking of the EGFR-STAT3 signaling pathway through afatinib treatment inhibited the intrahepatic cholangiocarcinoma

PubMed Central

Zhang, Changhe; Xu, Hong; Zhou, Zhenping; Tian, Ye; Cao, Xiaofei; Cheng, Guochang; Liu, Qinghong

2018-01-01

Epidermal growth factor receptor (EGFR) and downstream signal transducer and activator of transcription 3 (STAT3) signaling have been extensively implicated in various human neoplasms. Recently, a novel EGFR inhibitor, known as afatinib, has exhibited broad antitumor activities in a variety of tumors. Therefore, the present study attempted to investigate the impact of this agent on intrahepatic cholangiocarcinoma (ICC). Initially, immunohistochemical assays were performed on 15 human ICC specimens and their adjacent tissues in order to assess the protein levels of phosphorylated EGFR (pEGFR) and pSTAT3. Subsequently, the human ICC cell lines JCK and OZ were exposed to different doses of afatinib, and then cell viability and apoptosis were determined by MTT assay and flow cytometry, respectively. Furthermore, immunoblotting was applied to detect any variations in the phosphorylated protein levels of EGFR and STAT3 in afatinib-treated ICC cells. The results of the current study demonstrated that ICC specimens had evidently increased pEGFR and pSTAT3 protein levels as compared with the adjacent noncancerous tissues. Further in vitro experiments indicated that afatinib evidently blocked ICC cell growth and induced cell apoptosis. At the protein level, pEGFR and pSTAT3 were evidently attenuated by afatinib-administration. In conclusion, the present study clearly determined that afatinib exerts an antitumor effect on ICC cells by silencing the EGFR-STAT3 signaling pathway. This novel agent deserves further investigation as a potential therapeutic strategy for ICC. PMID:29805522

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

PubMed

Peng, Wei; de Bruijn, Henriette S; Farrell, Eric; Sioud, Mouldy; Mashayekhi, Vida; Oliveira, Sabrina; van Dam, Go M; Roodenburg, Jan L N; Witjes, Max J H; Robinson, Dominic J

2018-05-19

The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm -2 (at 5 mW.cm -2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density

Mechanisms of skin aging induced by EGFR inhibitors.

PubMed

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner U; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E; Homey, Bernhard

2016-10-01

The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls. EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.

Mechanisms of skin aging induced by EGFR inhibitors

PubMed Central

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E.; Homey, Bernhard

2017-01-01

BACKGROUND The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. OBJECTIVE To evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. PATIENTS AND METHODS Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (< 25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. RESULTS Progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR-density in aged skin, when compared to young controls. CONCLUSIONS EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents. PMID:27165055

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

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

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon

2011-11-11

Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation.more » Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.« less

Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics.

PubMed

Jeuken, Judith; Sijben, Angelique; Alenda, Cristina; Rijntjes, Jos; Dekkers, Marieke; Boots-Sprenger, Sandra; McLendon, Roger; Wesseling, Pieter

2009-10-01

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

Anti-EGFR Targeted Monoclonal Antibody Isotype Influences Antitumor Cellular Immunity in Head and Neck Cancer Patients.

PubMed

Trivedi, Sumita; Srivastava, Raghvendra M; Concha-Benavente, Fernando; Ferrone, Soldano; Garcia-Bates, Tatiana M; Li, Jing; Ferris, Robert L

2016-11-01

EGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity. We measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab. Both monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype-dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8 + T cells in patients treated with cetuximab compared with those treated with panitumumab. Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229-37. ©2016 AACR. ©2016 American Association for Cancer Research.

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

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.

Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

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

Shi, T.; Niepel, M.; McDermott, J. E.

It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundancemore » of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.« less

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.

HER2 and EGFR overexpression support metastatic progression of prostate cancer to bone

PubMed Central

Day, Kathleen C.; Hiles, Guadalupe Lorenzatti; Kozminsky, Molly; Dawsey, Scott J.; Paul, Alyssa; Broses, Luke J.; Shah, Rajal; Kunja, Lakshmi P.; Hall, Christopher; Palanisamy, Nallasivam; Daignault-Newton, Stephanie; El-Sawy, Layla; Wilson, Steven James; Chou, Andrew; Ignatoski, Kathleen Woods; Keller, Evan; Thomas, Dafydd; Nagrath, Sunitha; Morgan, Todd; Day, Mark L.

2016-01-01

Activation of the epidermal growth factor receptors EGFR (ErbB1) and HER2 (ErbB2) drive the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. PMID:27793843

Association between BIM deletion polymorphism and clinical outcome of EGFR-mutated NSCLC patient with EGFR-TKI therapy: A meta-analysis.

PubMed

Ma, Ji-Yong; Yan, Hai-Jun; Gu, Wei

2015-01-01

BIM deletion polymorphism was deemed to be associated with downregulation of BIM, resulting in a decreased apoptosis induced by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutation-positive non-small cell lung cancer (NSCLC). However, accumulating evidences concerning the association between BIM deletion polymorphism and efficacy of EGFR-TKI and survival in EGFR-mutation-driven NSCLC patient reported contradictory results. A meta-analysis was conducted by combing six original eligible studies including 871 NSCLC patients. Our study showed that BIM deletion polymorphism was significantly associated with poor response to EGFR-TKI therapy in mutant EGFRNSCLC patients (P(h) = 0.309, P(z) = 0.001, OR = 0.39, 95% confidence interval (CI) = 0.23-0.67). Disease control rate (DCR) in mutant EGFRNSCLC patient with treatment of EGFR-TKI was significantly decreased in patients with BIM deletion polymorphism comparing to patients harbored BIM wild variant (P(h) = 0.583, P(Z) = 0.007, OR = 0.46, 95%CI = 0.25-0.85). EGFR mutation-derived NSCLC patient carrying BIM deletion polymorphism had a shorter progression-free survival (PFS; P(h) < 0.001, P(z) < 0.001, hazard ratio (HR) = 1.37, 95%CI = 1.09-1.71) and overall survival (OS; P(h) = 0.90, P(z) = 0.003, HR = 1.25, 95%CI = 1.08-1.45), than those harbored BIM wild variant. These results suggested that BIM deletion polymorphism might be a cause that contributes to primary EGFR-TKI resistance, and it could be used as a genetic predictor for EGFR-TKI outcome and an independent prognostic factor of EGFR mutation-driven NSCLC patient.

Graf regulates hematopoiesis through GEEC endocytosis of EGFR.

PubMed

Kim, Sungdae; Nahm, Minyeop; Kim, Najin; Kwon, Yumi; Kim, Joohyung; Choi, Sukwoo; Choi, Eun Young; Shim, Jiwon; Lee, Cheolju; Lee, Seungbok

2017-11-15

GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. We show that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. We also find that loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on our findings, we propose a model in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation. © 2017. Published by The Company of Biologists Ltd.

EGFR-targeted therapies in the post-genomic era.

PubMed

Xu, Mary Jue; Johnson, Daniel E; Grandis, Jennifer R

2017-09-01

Over 90% of head and neck cancers overexpress the epidermal growth factor receptor (EGFR). In diverse tumor types, EGFR overexpression has been associated with poorer prognosis and outcomes. Therapies targeting EGFR include monoclonal antibodies, tyrosine kinase inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, and antisense gene therapy. Few EGFR-targeted therapeutics are approved for clinical use. The monoclonal antibody cetuximab is a Food and Drug Administration (FDA)-approved EGFR-targeted therapy, yet has exhibited modest benefit in clinical trials. The humanized monoclonal antibody nimotuzumab is also approved for head and neck cancers in Cuba, Argentina, Colombia, Peru, India, Ukraine, Ivory Coast, and Gabon in addition to nasopharyngeal cancers in China. Few other EGFR-targeted therapeutics for head and neck cancers have led to as significant responses as seen in lung carcinomas, for instance. Recent genome sequencing of head and neck tumors has helped identify patient subgroups with improved response to EGFR inhibitors, for example, cetuximab in patients with the KRAS-variant and the tyrosine kinase inhibitor erlotinib for tumors harboring MAPK1 E322K mutations. Genome sequencing has furthermore broadened our understanding of dysregulated pathways, holding the potential to enhance the benefit derived from therapies targeting EGFR.

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

Synergistic antitumor effect between gefitinib and fractionated irradiation in anaplastic oligodendrogliomas cannot be predicted by the Egfr signaling activity.

PubMed

Pinel, Sophie; Mriouah, Jihane; Vandamme, Marc; Chateau, Alicia; Plénat, François; Guérin, Eric; Taillandier, Luc; Bernier-Chastagner, Valérie; Merlin, Jean-Louis; Chastagner, Pascal

2013-01-01

In high-grade gliomas, the identification of patients that could benefit from EGFR inhibitors remains a challenge, hindering the use of these agents. Using xenografts models, we evaluated the antitumor effect of the combined treatment "gefitinib + radiotherapy" and aimed to identify the profile of responsive tumors. Expression of phosphorylated proteins involved in the EGFR-dependent signaling pathways was analyzed in 10 glioma models. We focused on three models of anaplastic oligodendrogliomas (TCG2, TCG3 and TCG4) harboring high levels of phospho-EGFR, phospho-AKT and phospho-MEK1. They were treated with gefitinib (GEF 75 mg/kg/day x 5 days/week, for 2 weeks) and/or fractionated radiotherapy (RT: 5x2Gy/week for 2 weeks). Our results showed that GEF and/or RT induced significant tumor growth delays. However, only the TCG3 xenografts were highly responsive to the combination GEF+RT, with ∼50% of tumor cure. Phosphoproteins analysis five days after treatment onset demonstrated in TCG3 xenografts, but not in TCG2 model, that the EGFR-dependent pathways were inhibited after GEF treatment. Moreover, TCG3-bearing mice receiving GEF monotherapy exhibited a transient beneficial therapeutic response, rapidly followed by tumor regrowth, along with a major vascular remodeling. Taken together, our data evoked an "EGFR-addictive" behavior for TCG3 tumors. This study confirms that combination of gefitinib with fractionated irradiation could be a potent therapeutic strategy for anaplastic oligodendrogliomas harboring EGFR abnormalities but this treatment seems mainly beneficial for "EGFR-addictive" tumors. Unfortunately, neither the usual molecular markers (EGFR amplification, PTEN loss) nor the basal overexpression of phosphoproteins were useful to distinguish this responsive tumor. Evaluating the impact of TKIs on the EGFR-dependent pathways during the treatment might be more relevant, and requires further validation.

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.

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

PubMed Central

Sun, Jian; Li, Jing-Ran; Fang, Fei; Du, Qian-Ru; Qian, Yong; Gong, Hai-Bin; Zhu, Hai-Liang

2013-01-01

4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC50 values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor. PMID:23936329

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells

PubMed Central

2014-01-01

Background In hepatocellular carcinoma (HCC), different signaling pathways are de-regulated, and among them, the expression of the epidermal growth factor receptor (EGFR). Tyrphostin AG-1478 is a lipophilic low molecular weight inhibitor of EGFR, preferentially acting on liver tumor cells. In order to overcome its poor drug solubility and thus improving its anticancer activity, it was entrapped into nanostructured lipid carriers (NLC) by using safe ingredients for parenteral delivery. Results Nanostructured lipid carriers (NLC) carrying tyrphostin AG-1478 were prepared by using the nanoprecipitation method and different matrix compositions. The best system in terms of mean size, PDI, zeta potential, drug loading and release profile was chosen to evaluate the anti-proliferative effect of drug-loaded NLC versus free drug on human hepatocellular carcinoma HA22T/VGH cells. Conclusions Thanks to the entrapment into NLC systems, tyrphostin AG-1478 shows an enhanced in vitro anti-tumor activity compared to free drug. These finding raises hope of future drug delivery strategy of tyrphostin AG-1478 -loaded NLC targeted to the liver for the HCC treatment. PMID:24886097

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

PubMed

Hong, Kwang-Won; Kim, Chang-Goo; Lee, Seung-Hyun; Chang, Ki-Hwan; Shin, Yong Won; Ryoo, Kyung-Hwan; Kim, Se-Ho; Kim, Yong-Sung

2010-01-01

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

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.

EGFR targeted PLGA nanoparticles using gemcitabine for treatment of pancreatic cancer.

PubMed

Aggarwal, Sahil; Yadav, Sachin; Gupta, Swati

2011-02-01

The present study aimed to prepare and characterize anti EGFR monoclonal antibody (mab) conjugated Gemcitabine loaded PLGA nanoparticles for their selective delivery to pancreatic cells and evaluation of the systems in vitro. It was observed that direct covalent coupling of antibodies to glutaraldehyde activated nanoparticles is an appropriate method to achieve cell-type specific drug carrier systems based on polymeric nanoparticles that have potential to be applied for targeted chemotherapy in EGFR positive cancer.

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.

An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

PubMed Central

Li, Li; Chakraborty, Sharmistha; Yang, Chin-Rang; Hatanpaa, Kimmo J.; Cipher, Daisha J.; Puliyappadamba, Vineshkumar Thidil; Rehman, Alizeh; Jiwani, Ameena J.; Mickey, Bruce; Madden, Christopher; Raisanen, Jack; Burma, Sandeep; Saha, Debabrata; Wang, Zhixiang; Pingle, Sandeep C.; Kesari, Santosh; Boothman, David A.; Habib, Amyn A.

2014-01-01

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention. PMID:24077285

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.

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.

CONTRIBUTION OF INSPIRATORY FLOW TO ACTIVATION OF EGFR, RAS, MAPK, ATF-2 AND C-JUN DURING LUNG STRETCH

EPA Science Inventory

Contribution of Inspiratory Flow to Activation of EGFR, Ras, MAPK, ATF-2 and c-Jun during Lung Stretch

R. Silbajoris 1, Z. Li 2, J. M. Samet 1 and Y. C. Huang 1. 1 NHEERL, ORD, US EPA, RTP, NC and 2 CEMALB, UNC-CH, Chapel Hill, NC .

Mechanical ventilation with larg...

Molecular alterations of EGFR and PIK3CA in uterine serous carcinoma.

PubMed

Hayes, Monica Prasad; Douglas, Wayne; Ellenson, Lora Hedrick

2009-06-01

Uterine serous carcinoma (USC) is an aggressive endometrial cancer associated with poor prognosis despite comprehensive surgical staging and adjuvant chemotherapy and radiation therapy. Biologic targets have yet to be fully explored in this disease and research on such targets could lead to clinical trials utilizing a new class of therapeutics. This study sought to evaluate primary USC tumors for molecular alterations in epidermal growth factor receptor (EGFR) and the recently characterized oncogene PIK3CA, which encodes the catalytic p110-alpha subunit of phosphatidylinositol 3-kinase (PI3K) and thus activates the AKT-mTOR oncogenic pathway. Paraffin-embedded archival tissue of 45 primary USC tumors was utilized in this study. Immunohistochemical analysis of EGFR was performed and cases given a score of 0 to 12 calculated as the product of staining intensity (0 to 3+) and the percentage of positively stained cells (0-4), with 1=1-25%, 2=26-50%, 3=51-75%, and 4=76-100%. For mutational analysis, neoplastic tissue was microdissected and DNA was extracted with phenol-chloroform. Exons 18 through 21 of EGFR and exons 9 and 20 of PIK3CA, the most commonly mutated exons of these genes, were amplified and directly sequenced. When EGFR was evaluated, moderate or strong EGFR membranous staining was observed in 25/45 (56%) USC cases. Thus, a mutational analysis was performed on 35 cases, including all cases with moderate and strong EGFR staining. No mutations were identified in EGFR. In contrast, PIK3CA mutations were confirmed in 5/34 (15%) of USC cases. Four cases were mutated in exon 20 and one case was mutated in exon 9. Since optimal treatment of uterine serous carcinoma remains unknown, novel therapeutic approaches need to be actively pursued. In the current study of primary USC tumors, oncogenic mutations of the PIK3CA gene were seen in 15% of USC cases. This represents the first report of this gene mutation in USC. In addition, EGFR stained positively in the majority

Can EGFR-Tyrosine Kinase Inhibitors (TKI) Alone Without Talc Pleurodesis Prevent Recurrence of Malignant Pleural Effusion (MPE) in Lung Adenocarcinoma.

PubMed

Verma, Akash; Chopra, Akhil; Lee, Yeo W; Bharwani, Lavina D; Asmat, Atasha B; Aneez, Dokeu B A; Akbar, Fazuludeen A; Lim, Albert Y H; Chotirmall, Sanjay H; Abisheganaden, John

2016-01-01

Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors (EGFR-TKIs) are effective against lung adenocarcinoma. However, limited data is available assessing the effectiveness of EGFR-TKI use in preventing re-accumulation of MPE. To our knowledge, there is no literature on comparison of talc pleurodesis with EGFR-TKIs alone on re-accumulation of MPE in Asian population. We investigated if EGFR-TKI therapy for advanced lung adenocarcinoma with malignant pleural effusion (MPE) is also successful in preventing pleural fluid re-accumulation following initial drainage. An observational cohort study of patients with lung adenocarcinoma and MPE in the year 2012 was conducted. 70 patients presented with MPE from lung adenocarcinoma. Fifty six underwent EGFR mutation testing of which 39 (69.6%) had activating EGFR mutation and 34 (87.1%) received TKI. 20 were managed by pleural fluid drainage only whereas 14 underwent talc pleurodesis following pleural fluid drainage. Time taken for the pleural effusion to re-accumulate in those with and without pleurodesis was 9.9 vs. 11.7 months, p=0.59 respectively. More patients (n=10, 25.6%) with activating EGFR mutation presented with complete opacification (white-out) of the hemithorax compared to none without activating EGFR mutation (p=0.02). In TKI eligible patients, early talc pleurodesis may not confer additional benefit in preventing re-accumulation of pleural effusion and may be reserved for non-adenocarcinoma histology, or EGFR negative adenocarcinoma. Complete opacification of the hemithorax on presentation may serve as an early radiographic signal of positive EGFR mutation status.

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.

Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance.

PubMed

Bagchi, Atrish; Haidar, Jaafar N; Eastman, Scott W; Vieth, Michal; Topper, Michael; Iacolina, Michelle D; Walker, Jason M; Forest, Amelie; Shen, Yang; Novosiadly, Ruslan D; Ferguson, Kathryn M

2018-02-01

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR . ©2017 American Association for Cancer Research.

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

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

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito

2010-09-03

Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored.more » Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.« less

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

EGFR TKIs plus WBRT Demonstrated No Survival Benefit Other Than That of TKIs Alone in Patients with NSCLC and EGFR Mutation and Brain Metastases.

PubMed

Jiang, Tao; Su, Chunxia; Li, Xuefei; Zhao, Chao; Zhou, Fei; Ren, Shengxiang; Zhou, Caicun; Zhang, Jun

2016-10-01

Whether EGFR tyrosine kinase inhibitors (TKIs) plus whole brain radiation therapy (WBRT) provide a better survival benefit than EGFR TKIs alone remains undetermined in patients with NSCLC with EGFR mutation and brain metastases (BMs). A total of 230 patients with NSCLC with EGFR mutation and BM were identified. Within this group, 116 patients received EGFR TKIs alone (as first-line therapy in 91 cases) and 51 patients received EGFR TKIs plus WBRT therapy (as first-line treatment in 30 cases). Compared with TKIs alone, EGFR TKIs plus WBRT had no superior intracranial progression-free survival (PFS) (6.9 versus 7.4 months [p = 0.232]) and systemic PFS (7.5 versus 7.9 months [p = 0.546]) but were associated with worse overall survival (OS) (21.6 versus 26.4 months [p = 0.049]) in NSCLC with EGFR mutation and BM. Chemotherapy plus WBRT was shown to have an intracranial PFS (5.2 versus 5.9 months [p = 0.339]) and OS (10.5 versus 11.0 months [p = 0.977]) similar to those with chemotherapy alone in patients with EGFR of unknown or wild-type status. In multivariate analysis, EGFR mutation was found to be an independent risk factor for BM (hazard ratio = 1.476, p = 0.039) and also a significant independent prognostic factor for OS in patients with NSCLC with BM (hazard ratio = 0.601, p = 0.028). The addition of WBRT to EGFR TKIs did not appear to have survival benefit superior to that of EGFR TKIs alone in with EGFR-mutant NSCLC with BM. WBRT also did not bring additional benefit to chemotherapy in patients with BM and EGFR of wild-type or unknown status. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells

PubMed Central

Capuani, Fabrizio; Conte, Alexia; Argenzio, Elisabetta; Marchetti, Luca; Priami, Corrado; Polo, Simona; Di Fiore, Pier Paolo; Sigismund, Sara; Ciliberto, Andrea

2015-01-01

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination. PMID:26264748

Third generation EGFR TKIs: current data and future directions.

PubMed

Tan, Chee-Seng; Kumarakulasinghe, Nesaretnam Barr; Huang, Yi-Qing; Ang, Yvonne Li En; Choo, Joan Rou-En; Goh, Boon-Cher; Soo, Ross A

2018-02-19

Acquired T790 M mutation is the commonest cause of resistance for advanced non-small cell lung cancer (NSCLC) epidermal growth factor receptor (EGFR) mutant patients who had progressed after first line EGFR TKI (tyrosine kinase inhibitor). Several third generation EGFR TKIs which are EGFR mutant selective and wild-type (WT) sparing were developed to treat these patients with T790 M acquired resistant mutation. Osimertinib is one of the third generation EGFR TKIs and is currently the most advanced in clinical development. Unfortunately, despite good initial response, patients who was treated with third generation EGFR TKI would develop acquired resistance and several mechanisms had been identified and the commonest being C797S mutation at exon 20. Several novel treatment options were being developed for patients who had progressed on third generation EGFR TKI but they are still in the early phase of development. Osimertinib under FLAURA study had been shown to have better progression-free survival over first generation EGFR TKI in the first line setting and likely will become the new standard of care.

Mutation analysis of the EGFR pathway genes, EGFR, RAS, PIK3CA, BRAF, and AKT1, in salivary gland adenoid cystic carcinoma.

PubMed

Saida, Kosuke; Murase, Takayuki; Ito, Mayuko; Fujii, Kana; Takino, Hisashi; Masaki, Ayako; Kawakita, Daisuke; Ijichi, Kei; Tada, Yuichiro; Kusafuka, Kimihide; Iida, Yoshiyuki; Onitsuka, Tetsuro; Yatabe, Yasushi; Hanai, Nobuhiro; Hasegawa, Yasuhisa; Shinomiya, Hitomi; Nibu, Ken-Ichi; Shimozato, Kazuo; Inagaki, Hiroshi

2018-03-30

Adenoid cystic carcinoma (AdCC), one of the most common salivary gland carcinomas, usually has a fatal outcome. Epidermal growth factor receptor (EGFR) pathway gene mutations are important in predicting a patient's prognosis and estimating the efficacy of molecular therapy targeting the EGFR pathway. In this study of salivary gland AdCC (SAdCC), we looked for gene mutations in EGFR, RAS family ( KRAS, HRAS, and NRAS ), PIK3CA, BRAF, and AKT1 , using a highly sensitive single-base extension multiplex assay, SNaPshot. Out of 70 cases, EGFR pathway missense mutations were found in 13 (18.6%): RAS mutations in 10 (14.3%), EGFR in one (1.4%), and PIK3CA in 5 (7.1%). None of the cases showed an EGFR deletion by direct sequencing. Concurrent gene mutations were found in three cases (4.3%). EGFR pathway mutations were significantly associated with a shorter disease-free ( p = 0.011) and overall survival ( p = 0.049) and RAS mutations were as well; ( p = 0.010) and ( p = 0.024), respectively. The gene fusion status as determined by a FISH assay had no significant association with mutations of the genes involved in the EGFR pathway. In conclusion, EGFR pathway mutations, especially RAS mutations, may be frequent in SAdCC, and associated with a poor prognosis for the patient.

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

PubMed Central

Kerner, Gerald S. M. A.; Schuuring, Ed; Sietsma, Johanna; Hiltermann, Thijo J. N.; Pieterman, Remge M.; de Leede, Gerard P. J.; van Putten, John W. G.; Liesker, Jeroen; Renkema, Tineke E. J.; van Hengel, Peter; Platteel, Inge; Timens, Wim; Groen, Harry J. M.

2013-01-01

Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival. PMID:23922984

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells.

PubMed

Sathya, Shanmugaraj; Sudhagar, Selvaraj; Sarathkumar, Baskaran; Lakshmi, Baddireddi Subhadra

2014-01-24

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro. Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes. We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation. Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

In Vivo Evidence for Epidermal Growth Factor Receptor (EGFR)-mediated Release of Prolactin from the Pituitary Gland

PubMed Central

Dahlhoff, Maik; Blutke, Andreas; Wanke, Rüdiger; Wolf, Eckhard; Schneider, Marlon R.

2011-01-01

Members of the epidermal growth factor receptor (EGFR/ERBB) system are essential local regulators of mammary gland development and function. Emerging evidence suggests that EGFR signaling may also influence mammary gland activity indirectly by promoting the release of prolactin from the pituitary gland in a MAPK and estrogen receptor-α (ERα)-dependent manner. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) causes a lactating-like phenotype in the mammary gland of virgin female mice including the major hallmarks of lactogenesis. BTC transgenic (BTC-tg) females showed reduced levels of prolactin in the pituitary gland and increased levels of the hormone in the circulation. Furthermore, treatment of BTC-tg females with bromocriptine, an inhibitor of prolactin secretion, blocked the development of the lactation-like phenotype, suggesting that it is caused by central release of prolactin rather than by local actions of BTC in the mammary gland. Introduction of the antimorphic Egfr allele Wa5 also blocked the appearance of the mammary gland alterations, revealing that the phenotype is EGFR-dependent. We detected an increase in MAPK activity, but unchanged phosphorylation of ERα in the pituitary gland of BTC-tg females as compared with control mice. These results provide the first functional evidence in vivo for a role of the EGFR system in regulating mammary gland activity by modulating prolactin release from the pituitary gland. PMID:21914800

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.

Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening.

PubMed

Li, Siyuan; Sun, Xianqiang; Zhao, Hongli; Tang, Yun; Lan, Minbo

2012-06-15

By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand–dependent terminal keratinocyte differentiation

PubMed Central

Cobzaru, Cristina; Triantafyllopoulou, Antigoni; Löffek, Stefanie; Horiuchi, Keisuke; Threadgill, David W.; Kurz, Thomas; van Rooijen, Nico; Bruckner-Tuderman, Leena

2012-01-01

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17ΔKC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17ΔKC skin, and topical treatment of A17ΔKC mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (EgfrΔKC) closely resembled A17ΔKC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17–EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects. PMID:22565824

Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.

PubMed

Monslow, James; Sato, Nobuyuki; Mack, Judith A; Maytin, Edward V

2009-08-01

Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by > or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.

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.

[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.

[Efficacy of icotinib for advanced non-small cell lung cancer patients with EGFR status identified].

PubMed

Song, Zhengbo; Yu, Xinmin; Cai, Jufen; Shao, Lan; Lin, Baochai; He, Chunxiao; Zhang, Beibei; Zhang, Yiping

2013-03-01

As the first epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in China, icotinib shows promising anticancer activity in vitro and vivo. The phase III clinical study (ICOGEN) showed that icotinib has a good efficacy and tolerability in Chinese patients with advanced non-small cell lung cancer (NSCLC) compared with gefitinib. This retrospective study aims to evaluate the efficacy and tolerability of icotinib monotherapy for advanced NSCLC patients with EGFR mutation and wild-type patients in our hospital. Patients with advanced NSCLC who were treated with icotinib in Zhejiang Cancer Hospital were retrospectively analyzed from August, 2011 to August, 2012. Survival was estimated using Kaplan-Meier analysis and Log-rank tests. The clinical data of 49 patients (13 with wild-type and 36 with EGFR mutation) with NSCLC were enrolled in the current study. The patients' overall objective response rate (ORR) was 58.3% and the disease control rate (DCR) in 36 EGFR mutation patients was 88.9%. The ORR was 7.7% and DCR was 53.8% in the wild-type patients. Median progression-free survival (PFS) with icotinib treatment in EGFR mutation patients was 9.5 months and 2.2 months in wild-type patients (P<0.001). Nineteen patients with EGFR mutation received icotinib as first-line and 17 in further-line treatment. The PFS was 9.5 months in the first-line and 8.5 months for second-line or further-line patients (P=0.41). Median overall survival (OS) in EGFR mutation patients was not reached, but was 12.6 months in wild-type patients. Most of the drug-related adverse events were mild (grade I or II) and reversible with no grade IV toxicity. Icotinib monotherapy showed significant antitumor activity in advanced NSCLC EGFR mutation patients. The toxicity was well tolerated and acceptable.

The impact of common and rare EGFR mutations in response to EGFR tyrosine kinase inhibitors and platinum-based chemotherapy in patients with non-small cell lung cancer.

PubMed

Arrieta, Oscar; Cardona, Andrés Felipe; Corrales, Luis; Campos-Parra, Alma Delia; Sánchez-Reyes, Roberto; Amieva-Rivera, Eduardo; Rodríguez, July; Vargas, Carlos; Carranza, Hernán; Otero, Jorge; Karachaliou, Nikki; Astudillo, Horacio; Rosell, Rafael

2015-02-01

In non-small cell lung cancer (NSCLC), the association between common EGFR mutations (Del EX19/L858R) with EGFR tyrosine kinase inhibitors (EGFR-TKIs) has been well established. However, this has not been investigated for rare EGFR mutations or their impact on treatment response and outcome to EGFR TKIs (primary objective) and chemotherapy (secondary objective). In an observational prospective cohort, we analyzed 188 NSCLC patients from Mexico, Colombia and Costa Rica with EGFR mutations. As a first line of treatment, 66.5% received platinum-based chemotherapy. All patients received TKIs in first-line treatment or after progression to chemotherapy. The clinical-pathological characteristics as well as the f of common and rare EGFR mutations associated with treatment response were analyzed. Of all patients, 79.5% had common and 20.5% had rare EGFR mutations. Lepidic and acinar adenocarcinomas were associated with common EGFR mutations (p=0.010). Patients with common EGFR mutations had higher response rates to EGFR-TKIs than those who had rare EGFR mutations (63.8 vs 32.4%, p<0.001). Women had increased progression-free survival (PFS) to EGFR-TKIs than men (16.4 vs 9.5 months, p=0.02). The median PFS and overall survival (OS) were better in patients with common EGFR mutations (15.5 vs 3.9 months, p<0.001; and 37.3 vs 17.4 months, p<0.001) respectively. Our findings suggested that only patients with rare EGFR mutations could receive platinum-based chemotherapy as a first-line treatment, due to their low response rates and short PFS in response to EGFR-TKIs. Consequently, EGFR-TKIs could be reserved as a second- or third-line treatment. In patients with EGFR mutations, women have better PFS to EGFR-TKIs than men, and rare EGFR mutations are more frequent in high grade adenocarcinomas than in low grade tumors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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.

Gefitinib and EGFR Gene Copy Number Aberrations in Esophageal Cancer.

PubMed

Petty, Russell D; Dahle-Smith, Asa; Stevenson, David A J; Osborne, Aileen; Massie, Doreen; Clark, Caroline; Murray, Graeme I; Dutton, Susan J; Roberts, Corran; Chong, Irene Y; Mansoor, Wasat; Thompson, Joyce; Harrison, Mark; Chatterjee, Anirban; Falk, Stephen J; Elyan, Sean; Garcia-Alonso, Angel; Fyfe, David Walter; Wadsley, Jonathan; Chau, Ian; Ferry, David R; Miedzybrodzka, Zosia

2017-07-10

Purpose The Cancer Esophagus Gefitinib trial demonstrated improved progression-free survival with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib relative to placebo in patients with advanced esophageal cancer who had disease progression after chemotherapy. Rapid and durable responses were observed in a minority of patients. We hypothesized that genetic aberration of the EGFR pathway would identify patients benefitting from gefitinib. Methods A prespecified, blinded molecular analysis of Cancer Esophagus Gefitinib trial tumors was conducted to compare efficacy of gefitinib with that of placebo according to EGFR copy number gain (CNG) and EGFR, KRAS, BRAF, and PIK3CA mutation status. EGFR CNG was determined by fluorescent in situ hybridization (FISH) using prespecified criteria and EGFR FISH-positive status was defined as high polysomy or amplification. Results Biomarker data were available for 340 patients. In EGFR FISH-positive tumors (20.2%), overall survival was improved with gefitinib compared with placebo (hazard ratio [HR] for death, 0.59; 95% CI, 0.35 to 1.00; P = .05). In EGFR FISH-negative tumors, there was no difference in overall survival with gefitinib compared with placebo (HR for death, 0.90; 95% CI, 0.69 to 1.18; P = .46). Patients with EGFR amplification (7.2%) gained greatest benefit from gefitinib (HR for death, 0.21; 95% CI, 0.07 to 0.64; P = .006). There was no difference in overall survival for gefitinib versus placebo for patients with EGFR, KRAS, BRAF, and PIK3CA mutations, or for any mutation versus none. Conclusion EGFR CNG assessed by FISH appears to identify a subgroup of patients with esophageal cancer who may benefit from gefitinib as a second-line treatment. Results of this study suggest that anti-EGFR therapies should be investigated in prospective clinical trials in different settings in EGFR FISH-positive and, in particular, EGFR-amplified esophageal cancer.

[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.

Physical Activity and Change in Estimated GFR among Persons with CKD

PubMed Central

Littman, Alyson J.; Duncan, Glen E.; Weiss, Noel S.; Sachs, Michael C.; Ruzinski, John; Kundzins, John; Rock, Denise; de Boer, Ian H.; Ikizler, T. Alp; Himmelfarb, Jonathan; Kestenbaum, Bryan R.

2014-01-01

Physical activity may counteract metabolic disturbances that promote the progression of CKD. To address this concept, we performed a longitudinal cohort study of 256 participants in the Seattle Kidney Study, a clinic-based study of CKD. Participants with an estimated GFR (eGFR) of 15–59 ml/min per 1.73 m2 at baseline were eligible for the study. Physical activity was quantified using the Four-Week Physical Activity History Questionnaire. We used generalized estimating equations to test associations of physical activity with change in eGFR determined by longitudinal measurements of serum cystatin C. Mean baseline eGFR was 42 ml/min per 1.73 m2. During a median 3.7 years of follow-up, the mean change in eGFRcystatin C was −7.6% per year (interquartile range, −16.8%, 4.9% per year). Participants who reported >150 minutes of physical activity per week had the lowest rate of eGFRcystatin C loss (mean −6.2% per year compared with −9.6% per year among inactive participants). In adjusted analyses, each 60-minute increment in weekly physical activity duration associated with a 0.5% slower decline per year in eGFR (95% confidence interval, 0.02 to 0.98; P=0.04). Results were similar in sensitivity analyses restricted to participants without cardiovascular disease or diabetes, or to participants with moderate/high physical function. After adjustment for eGFR at the time of questionnaire completion, physical activity did not associate with the incidence of ESRD (n=34 events). In summary, higher physical activity levels associated with slower rates of eGFR loss in persons with established CKD. PMID:24335971

Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02).

PubMed

Lee, Ji Yun; Qing, Xu; Xiumin, Wei; Yali, Bai; Chi, Sangah; Bak, So Hyeon; Lee, Ho Yun; Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Cho, Eun Kyung; Kim, Dong-Wan; Kim, Hye Ryun; Min, Young Joo; Jung, Sin-Ho; Park, Keunchil; Mao, Mao; Ahn, Myung-Ju

2016-02-09

We hypothesized that plasma-based EGFR mutation analysis for NSCLC may be feasible for monitoring treatment response to EGFR TKIs and also predict drug resistance.Clinically relevant mutations including exon 19 deletion (ex19del), L858R and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples (n = 367) from 81 NSCLC patients treated with EGFR TKI. Of a total 58 baseline cell-free DNA (cfDNA) samples available for ddPCR analysis, 43 (74.1%) had the same mutation in the matched tumors (clinical sensitivity: 70.8% [17/24] for L858R and 76.5% [26/34] for ex19del). The concordance rates of plasma with tissue-based results of EGFR mutations were 87.9% for L858R and 86.2% for ex19del. All 40 patients who were detected EGFR mutations at baseline showed a dramatic decrease of mutant copies (>50%) in plasma during the first two months after treatment. Median progression-free survival (PFS) was 10.1 months for patients with undetectable EGFR v 6.3 months for detectable EGFR mutations in blood after two-month treatment (HR 3.88, 95% CI 1.48-10.19, P = 0.006). We observed emerging resistance with early detection of T790M as a secondary mutation in 14 (28.6%) of 49 patients. Plasma-based EGFR mutation analysis using ddPCR can monitor treatment response to EGFR TKIs and can lead to early detection of EGFR TKIs resistance. Further studies confirming clinical implications of EGFR mutation in plasma are warranted to guide optimal therapeutic strategies upon knowledge of treatment response and resistance.

GPCRs and EGFR - Cross-talk of membrane receptors in cancer.

PubMed

Köse, Meryem

2017-08-15

G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) are two important classes of cell surface receptors proven to be highly tractable as drug targets. Both receptor classes are involved in various complex (patho-) physiological processes in the human body including cellular growth and differentiation. More recently, accumulating data suggest that GPCR-induced activation of EGFR, the prototyp of RTKs represents a major mechanism in various cancers. The present review will focus on this cross-talk with particular emphasis on intracellular scaffold proteins regulating EGFR transactivation. It will give an overview about the current status of the research and future directions, highlight recent trends in the field, and discuss the potential of therapeutic strategies combining GPCR and EGFR targeting on the one hand and specific targeting of the cross-talk on the other hand in cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

PubMed

Ramirez, Ursula D; Nikonova, Anna S; Liu, Hanqing; Pecherskaya, Anna; Lawrence, Sarah H; Serebriiskii, Ilya G; Zhou, Yan; Robinson, Matthew K; Einarson, Margret B; Golemis, Erica A; Jaffe, Eileen K

2015-05-28

Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

Osimertinib and Necitumumab in Treating Patients With EGFR-Mutant Stage IV or Recurrent Non-small Cell Lung Cancer Who Have Progressed on a Previous EGFR Tyrosine Kinase Inhibitor

ClinicalTrials.gov

2018-03-07

EGFR Exon 19 Deletion Mutation; EGFR Exon 20 Insertion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR NP_005219.2:p.T790M; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage IV Non-Small Cell Lung Cancer AJCC v7

Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells.

PubMed

Codony-Servat, Jordi; García-Roman, Silvia; Molina-Vila, Miguel Ángel; Bertran-Alamillo, Jordi; Giménez-Capitán, Ana; Viteri, Santiago; Cardona, Andrés F; d'Hondt, Erik; Karachaliou, Niki; Rosell, Rafael

2018-05-08

Mutations in EGFR correlate with impaired response to immune checkpoint inhibitors and the development of novel immunotherapeutic approaches for EGFR mutant non-small cell lung cancer (NSCLC) is of particular interest. Immunization against EGF has demonstrated efficacy in a phase III trial including unselected NSCLC patients, but little was known about the mechanisms involved in the effects of the anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) or their activity in tumor cells with EGFR mutations. The EGFR-mutant, NSCLC cell lines H1975 and PC9, together with several gefitinib and osimertinib-resistant cells derived from PC9, were treated with anti-EGF VacAbs and/or EGFR tyrosine kinase inhibitors (TKIs). Cell viability was analyzed by proliferation assays, cell cycle by fluorescence-activated cell sorting analysis and levels of RNA and proteins by quantitative retro-transcription PCR and Western blotting. Anti-EGF VacAbs generated in rabbits suppressed EGF-induced cell proliferation and cycle progression and inhibited downstream EGFR signaling in EGFR-mutant cells. Sera from patients immunized with an EGF vaccine were also able to block activation of EGFR effectors. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of all TKIs tested, suppressed Erk1/2 phosphorylation, blocked the activation of signal transducer and activator of transcription 3 (STAT3) and downregulated the expression of AXL. Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones. EGFR-mutant patients can derive benefit from immunization against EGF, particularly if combined with EGFR TKIs. A Phase I trial of an EGF vaccine in combination with afatinib has been initiated. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

The Significance of MMP-1 in EGFR-TKI-Resistant Lung Adenocarcinoma: Potential for Therapeutic Targeting.

PubMed

Saito, Ryoko; Miki, Yasuhiro; Ishida, Naoya; Inoue, Chihiro; Kobayashi, Masayuki; Hata, Shuko; Yamada-Okabe, Hisafumi; Okada, Yoshinori; Sasano, Hironobu

2018-02-18

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance is one of the most important problems in lung cancer therapy. Lung adenocarcinoma with EGFR-TKI resistance was reported to have higher abilities of invasion and migration than cancers sensitive to EGFR-TKI, but the function of matrix metalloproteinases (MMPs) has not been explored in EGFR-TKI-resistant lung adenocarcinoma. This study aims to clarify the significance of MMP-1 in EGFR-TKI-resistant lung adenocarcinoma. From the results of in vitro studies of migration and invasion assays using EGFR-TKI-sensitive and -resistant cell lines and phosphorylation antibody arrays using EGF and rapamycin, we first demonstrate that overexpression of MMP-1, which might follow activation of a mammalian target of rapamycin (mTOR) pathway, plays an important role in the migration and invasion abilities of EGFR-TKI-resistant lung adenocarcinoma. Additionally, immunohistochemical studies using 89 cases of lung adenocarcinoma demonstrate that high expression of MMP-1 is significantly correlated with poor prognosis and factors such as smoking history and the subtype of invasive mucinous adenocarcinoma. These are consistent with the results of this in vitro study. To conclude, this study provides insights into the development of a possible alternative therapy manipulating MMP-1 and the mTOR signaling pathway in EGFR-TKI-resistant lung adenocarcinoma.

The CRISPR/Cas9 system targeting EGFR exon 17 abrogates NF-κB activation via epigenetic modulation of UBXN1 in EGFRwt/vIII glioma cells.

PubMed

Huang, Kai; Yang, Chao; Wang, Qi-Xue; Li, Yan-Sheng; Fang, Chuan; Tan, Yan-Li; Wei, Jian-Wei; Wang, Yun-Fei; Li, Xin; Zhou, Jun-Hu; Zhou, Bing-Cong; Yi, Kai-Kai; Zhang, Kai-Liang; Li, Jie; Kang, Chun-Sheng

2017-03-01

Worldwide, glioblastoma (GBM) is the most lethal and frequent intracranial tumor. Despite decades of study, the overall survival of GBM patients remains unchanged. epidermal growth factor receptor (EGFR) amplification and gene mutation are thought to be negatively correlated with prognosis. In this study, we used proteomics to determine that UBXN1 is a negative downstream regulator of the EGFR mutation vIII (EGFRvIII). Via bioinformatics analysis, we found that UBXN1 is a factor that can improve glioma patients' overall survival time. We also determined that the down-regulation of UBXN1 is mediated by the upregulation of H3K27me3 in the presence of EGFRvIII. Because NF-κB can be negatively regulated by UBXN1, we believe that EGFRwt/vIII activates NF-κB by suppressing UBXN1 expression. Importantly, we used the latest genomic editing tool, CRISPR/Cas9, to knockout EGFRwt/vIII on exon 17 and further proved that UBXN1 is negatively regulated by EGFRwt/vIII. Furthermore, knockout of EGFR/EGFRvIII could benefit GBM in vitro and in vivo, indicating that CRISPR/Cas9 is a promising therapeutic strategy for both EGFR amplification and EGFR mutation-bearing patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR

PubMed Central

Fang, Yuanzhang; Vaughn, Amanda; Cai, Xiaopan; Xu, Leqin; Wan, Wei; Li, Zhenxi; Chen, Shijie; Yang, Xinghai; Wu, Song; Xiao, Jianru

2015-01-01

The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC. PMID:25730907

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

PubMed Central

Tao, Jessica J.; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S.; Carey, Lisa A.; Perou, Charles M.; Baselga, José; Scaltriti, Maurizio

2014-01-01

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting. PMID:24667376

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

PubMed

Tao, Jessica J; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S; Carey, Lisa A; Perou, Charles M; Baselga, José; Scaltriti, Maurizio

2014-03-25

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent.

PubMed

Liu, Wei; Ning, Jin-Feng; Meng, Qing-Wei; Hu, Jing; Zhao, Yan-Bin; Liu, Chao; Cai, Li

2015-01-01

The epidermal growth factor receptor (EGFR) family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC), particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB). Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10) against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase-ligand interaction space in the PDB.

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

EGFR Mutation Testing Practices within the Asia Pacific Region

PubMed Central

Kerr, Keith M.; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma. Luisa D.; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

2015-01-01

Introduction: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non–small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. Methods: A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Results: Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%–32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. Conclusions: In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia. PMID:25376513

Interrogation of EGFR Targeted Uptake of TiO2 Nanoconjugates by X-ray Fluorescence Microscopy.

PubMed

Yuan, Ye; Paunesku, Tatjana; Arora, Hans; Ward, Jesse; Vogt, Stefan; Woloschak, Gayle

2011-09-01

We are developing TiO 2 nanoconjugates that can be used as therapeutic and diagnostic agents. Nanoscale TiO 2 can be surface conjugated with various molecules and has the unique ability to induce the production of reactive oxygen species after radiation activation. One way to improve the potential clinical usefulness of TiO 2 nanoparticles is to control their delivery to malignant cells by targeting them to cancer cell specific antigens. Epidermal Growth Factor Receptor is one potential target that is enriched in epithelial cancers and is rapidly internalized after ligand binding. Hence, we have synthesized TiO 2 nanoparticles and functionalized them with a short EGFR binding peptide to create EGFR-targeted NCs. X-ray Fluorescence Microscopy was used to image nanoconjugates within EGFR positive HeLa cells. Further labeling of fixed cells with antibodies against EGFR and Protein A nanogold showed that TiO 2 nanoconjugates can colocalize with receptors at the cell's plasma membrane. Interestingly, with increased incubation times, EGFR targeted nanoconjugates could also be found colocalized with EGFR within the cell nucleus. This suggests that EGFR-targeted nanoconjugates can bind the receptor at the cell membrane, which leads to the internalization of NC-receptor complexes and the subsequent transport of nanoconjugates into the nucleus.

Targeted treatment of mutated EGFR-expressing non-small-cell lung cancer: focus on erlotinib with companion diagnostics

PubMed Central

Karachaliou, Niki; Rosell, Rafael

2014-01-01

Deeper understanding of the pathobiology of non-small-cell lung cancer (NSCLC) has led to the development of small molecules that target genetic mutations known to play critical roles in the progression to metastatic disease. The discovery of epidermal growth factor receptor (EGFR) mutations in 15%–20% of lung adenocarcinomas and the associated response to EGFR tyrosine kinase inhibitors have provided a successful avenue of attack in late-stage adenocarcinomas. Use of the EGFR tyrosine kinase inhibitors gefitinib, erlotinib, and afatinib is limited to patients who have adenocarcinomas with known activating EGFR mutations. However, the EGFR mutation testing landscape is varied and includes many screening and targeted methods, each with its own benefits and limitations. These tests can simplify the drug discovery process, make clinical trials more efficient and informative, and individualize cancer therapy. In practice, the choice of method should be determined by the nature of the sample to be tested, the testing laboratory’s expertise and access to equipment, and whether the detection of only known activating EGFR mutations, or of all possible mutations, is required. Development of companion diagnostic tests for this identification is advancing; nevertheless, the use of such tests merits greater attention. PMID:28210145

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress*

PubMed Central

Tong, Jiefei; Taylor, Paul; Moran, Michael F.

2014-01-01

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S1104. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation. PMID:24797263

Temporal Resolution of Autophosphorylation for Normal and Oncogenic Forms of EGFR and Differential Effects of Gefitinib†

PubMed Central

Kim, Youngjoo; Li, Zhimin; Apetri, Mihaela; Luo, BeiBei; Settleman, Jeffrey E.; Anderson, Karen S.

2012-01-01

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of non-small cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time resolved electrospray mass spectrometry (ESI-MS) and western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa ™) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib; distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR, the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors. PMID:22657099

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

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

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.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR

PubMed Central

Henze, Anne-Theres; Garvalov, Boyan K.; Seidel, Sascha; Cuesta, Angel M.; Ritter, Mathias; Filatova, Alina; Foss, Franziska; Dopeso, Higinio; Essmann, Clara L.; Maxwell, Patrick H.; Reifenberger, Guido; Carmeliet, Peter; Acker-Palmer, Amparo; Acker, Till

2014-01-01

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability. PMID:25420773

The safety and efficacy of osimertinib for the treatment of EGFR T790M mutation positive non-small-cell lung cancer

PubMed Central

Gao, Xin; Le, Xiuning; Costa, Daniel B.

2016-01-01

First- and second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the evidence-based first-line treatment for metastatic non-small-cell lung cancers (NSCLCs) that harbor sensitizing EGFR mutations (i.e., exon 19 deletions or L858R). However, acquired resistance to EGFR TKI monotherapy occurs invariably within a median time frame of one year. The most common form of biological resistance is through the selection of tumor clones harboring the EGFR T790M mutation, present in >50% of repeat biopsies. The presence of the EGFR T790M mutation negates the inhibitory activity of gefitinib, erlotinib, and afatinib. A novel class of third-generation EGFR TKIs has been identified by probing a series of covalent pyrimidine EGFR inhibitors that bind to amino-acid residue C797 of EGFR and preferentially inhibit mutant forms of EGFR versus the wild-type receptor. We review the rapid clinical development and approval of the third-generation EGFR TKI osimertinib for treatment of NSCLCs with EGFR-T790M. PMID:26943236

α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation.

PubMed

Yang, Lan; Wen, Ya; Lv, Guoqing; Lin, Yuntao; Tang, Junlong; Lu, Jingxiao; Zhang, Manqiao; Liu, Wen; Sun, Xiaojuan

2017-12-09

Alpha lipoic acid (α -LA) is a naturally occurring antioxidant and metabolic enzyme co-factor. Recently, α -LA has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of α -LA on non-small cell lung cancer (NSCLC) development remain unclear. The CCK-8 assay was used to assess cell proliferation in NSCLC cell lines after α -LA treatment. The expression of growth factor receptor-bound protein 2 (Grb2), cyclin-dependent kinase (CDK)-2, CDK4, CDK6, Cyclin D3, Cyclin E1, Ras, c-Raf, epidermal growth factor receptor (EGFR), ERK1/2 and activated EGFR and ERK1/2 was evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in NSCLC cells via specific siRNA-mediated knockdown. α -LA dramatically decreased NSCLC cell proliferation by downregulating Grb2; in contrast, Grb2 overexpression significantly prevented α-LA-induced decrease in cell growth in vitro. Western blot analysis indicated that α-LA decreased the levels of phospho-EGFR, CDK2/4/6, Cyclins D3 and E1, which are associated with the inhibition of G1/S-phase transition. Additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. In addition, α-LA exerted greater inhibitory effects than gefitinib on NSCLC cells by preventing EGF-induced EGFR activation. For the first time, these findings provide the first evidence that α-LA inhibits cell proliferation through Grb2 by suppressing EGFR phosphorylation and that MAPK/ERK is involved in this pathway. Copyright © 2017. Published by Elsevier Inc.

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.

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

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the

Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NF{kappa}B and AhR and EGFR-ERK pathway

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

Potapovich, Alla I.; Biology Department, Belarus State University, Skorina Prosp. 10, Minsk 220050; Lulli, Daniela

Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA + UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure,more » the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50 {mu}M resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR. - Graphical abstract: Display Omitted

Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes

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

Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang

2013-02-15

Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg{sup 2+}. Intracellular reactive oxygen species (ROS)more » significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg{sup 2+} inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg{sup 2+}. These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS

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

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

Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent

PubMed Central

Liu, Wei; Ning, Jin-Feng; Meng, Qing-Wei; Hu, Jing; Zhao, Yan-Bin; Liu, Chao; Cai, Li

2015-01-01

The epidermal growth factor receptor (EGFR) family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC), particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB). Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10) against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase–ligand interaction space in the PDB. PMID:26229444