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Sample records for activating egfr mutations

  1. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  2. 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

  3. Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2.

    PubMed

    Foster, Scott A; Whalen, Daniel M; Özen, Ayşegül; Wongchenko, Matthew J; Yin, JianPing; Yen, Ivana; Schaefer, Gabriele; Mayfield, John D; Chmielecki, Juliann; Stephens, Philip J; Albacker, Lee A; Yan, Yibing; Song, Kyung; Hatzivassiliou, Georgia; Eigenbrot, Charles; Yu, Christine; Shaw, Andrey S; Manning, Gerard; Skelton, Nicholas J; Hymowitz, Sarah G; Malek, Shiva

    2016-04-11

    Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy. PMID:26996308

  4. Differential Sensitivity of Glioma- versus Lung Cancer-specific EGFR mutations to EGFR Kinase Inhibitors

    PubMed Central

    Vivanco, Igor; Robins, H. Ian; Rohle, Daniel; Campos, Carl; Grommes, Christian; Nghiemphu, Phioanh Leia; Kubek, Sara; Oldrini, Barbara; Chheda, Milan G.; Yannuzzi, Nicolas; Tao, Hui; Zhu, Shaojun; Iwanami, Akio; Kuga, Daisuke; Dang, Julie; Pedraza, Alicia; Brennan, Cameron W.; Heguy, Adriana; Liau, Linda M.; Lieberman, Frank; Yung, W.K. Alfred; Gilbert, Mark R.; Reardon, David A.; Drappatz, Jan; Wen, Patrick Y.; Lamborn, Kathleen R.; Chang, Susan M.; Prados, Michael D.; Fine, Howard A.; Horvath, Steve; Wu, Nian; Lassman, Andrew B.; DeAngelis, Lisa M.; Yong, William H.; Kuhn, John G.; Mischel, Paul S.; Mehta, Minesh P.; Cloughesy, Timothy F.; Mellinghoff, Ingo K.

    2012-01-01

    Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors which bind to the inactive EGFR conformation, on the other hand, potently inhibit EGFR EC mutants and induce cell death in EGFR mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM, and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types. PMID:22588883

  5. Achievement of Cure with Gefitinib in Advanced Lung Adenocarcinoma Harboring an Activating EGFR Mutation: A Case Report

    PubMed Central

    Kuwata, Taiji; Yoneda, Kazue; Kobayashi, Kenichi; Oyama, Rintarou; Matumiya, Hiroki; Shinohara, Shuichi; Takenaka, Masaru; Oka, Soichi; Chikaishi, Yasuhiro; Imanishi, Naoko; Kuroda, Koji; Tanaka, Fumihiro

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) may achieve long-term survival in selected cases with advanced non-small cell lung cancer harboring activating mutations in the EGFR gene, but a cured case has not been reported yet. Here, we present the first case of EGFR-mutated lung adenocarcinoma cured with an EGFR-TKI, as the 75-year-old Japanese man has achieved complete response with gefitinib treatment and has survived without tumor 10 years after termination of gefitinib treatment.

  6. Activation of the BMP-BMPR pathway conferred resistance to EGFR-TKIs in lung squamous cell carcinoma patients with EGFR mutations.

    PubMed

    Wang, Zhijie; Shen, Zhirong; Li, Zhenxiang; Duan, Jianchun; Fu, Shuai; Liu, Zhentao; Bai, Hua; Zhang, Zemin; Zhao, Jun; Wang, Xiaodong; Wang, Jie

    2015-08-11

    The empirical criteria for defining a clinical subtype of lung cancer are gradually transiting from histopathology to genetic variations in driver genes. Targeting these driver mutations, such as sensitizing epidermal growth factor receptor (EGFR) mutations, has dramatically improved the prognosis of advanced non-small cell lung cancer (NSCLC). However, the clinical benefit of molecularly targeted therapy on NSCLC appears to be different between lung adenocarcinomas and squamous cell carcinomas (SqCCs). We report here that the resistance of lung SqCC harboring EGFR mutations to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was due to the activation of BMP-BMPR-Smad1/5-p70S6K. The combined treatment of these tumor cells with EGFR-TKI, together with inhibitors specific to BMPR or downstream mTOR, effectively reversed the resistance to EGFR-TKI. Moreover, blocking the whole PI3K-AKT-mTOR pathway with the PI3K/mTOR dual inhibitor BEZ235 also showed efficacy in treating this subtype of lung SqCC. This study details the empirical basis for a feasible clinical solution for squamous cell carcinomas with EGFR mutations.

  7. Activation of the BMP-BMPR pathway conferred resistance to EGFR-TKIs in lung squamous cell carcinoma patients with EGFR mutations

    PubMed Central

    Wang, Zhijie; Shen, Zhirong; Li, Zhenxiang; Duan, Jianchun; Fu, Shuai; Liu, Zhentao; Bai, Hua; Zhang, Zemin; Zhao, Jun; Wang, Xiaodong; Wang, Jie

    2015-01-01

    The empirical criteria for defining a clinical subtype of lung cancer are gradually transiting from histopathology to genetic variations in driver genes. Targeting these driver mutations, such as sensitizing epidermal growth factor receptor (EGFR) mutations, has dramatically improved the prognosis of advanced non–small cell lung cancer (NSCLC). However, the clinical benefit of molecularly targeted therapy on NSCLC appears to be different between lung adenocarcinomas and squamous cell carcinomas (SqCCs). We report here that the resistance of lung SqCC harboring EGFR mutations to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was due to the activation of BMP-BMPR-Smad1/5-p70S6K. The combined treatment of these tumor cells with EGFR-TKI, together with inhibitors specific to BMPR or downstream mTOR, effectively reversed the resistance to EGFR-TKI. Moreover, blocking the whole PI3K-AKT-mTOR pathway with the PI3K/mTOR dual inhibitor BEZ235 also showed efficacy in treating this subtype of lung SqCC. This study details the empirical basis for a feasible clinical solution for squamous cell carcinomas with EGFR mutations. PMID:26216950

  8. Activation of the BMP-BMPR pathway conferred resistance to EGFR-TKIs in lung squamous cell carcinoma patients with EGFR mutations.

    PubMed

    Wang, Zhijie; Shen, Zhirong; Li, Zhenxiang; Duan, Jianchun; Fu, Shuai; Liu, Zhentao; Bai, Hua; Zhang, Zemin; Zhao, Jun; Wang, Xiaodong; Wang, Jie

    2015-08-11

    The empirical criteria for defining a clinical subtype of lung cancer are gradually transiting from histopathology to genetic variations in driver genes. Targeting these driver mutations, such as sensitizing epidermal growth factor receptor (EGFR) mutations, has dramatically improved the prognosis of advanced non-small cell lung cancer (NSCLC). However, the clinical benefit of molecularly targeted therapy on NSCLC appears to be different between lung adenocarcinomas and squamous cell carcinomas (SqCCs). We report here that the resistance of lung SqCC harboring EGFR mutations to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was due to the activation of BMP-BMPR-Smad1/5-p70S6K. The combined treatment of these tumor cells with EGFR-TKI, together with inhibitors specific to BMPR or downstream mTOR, effectively reversed the resistance to EGFR-TKI. Moreover, blocking the whole PI3K-AKT-mTOR pathway with the PI3K/mTOR dual inhibitor BEZ235 also showed efficacy in treating this subtype of lung SqCC. This study details the empirical basis for a feasible clinical solution for squamous cell carcinomas with EGFR mutations. PMID:26216950

  9. Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry

    PubMed Central

    Greig, Michael J.; Niessen, Sherry; Weinrich, Scott L.; Feng, Jun Li; Shi, Manli; Johnson, Ted O.

    2015-01-01

    Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates. PMID:26689952

  10. A comparison of ARMS and mutation specific IHC for common activating EGFR mutations analysis in small biopsy and cytology specimens of advanced non small cell lung cancer.

    PubMed

    Wang, Xueqing; Wang, Guoqing; Hao, Yueyue; Xu, Yinhong; Zhang, Lihua

    2014-01-01

    We have compared mutation analysis by Amplification Refractory Mutation System (ARMS) and epidermal growth factor receptor (EGFR) mutant-specific antibodies for their ability to detect two common activating EGFR mutations in a cohort of 115 advanced non-small cell lung cancer (NSCLC), including cytology material, core biopsy, and bronchoscopic biopsies. Assessment of EGFR mutation status was performed by using antibodies and ARMS assay specific to the two major forms of mutant EGFR, exon 19 deletion E746-A750 (c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750 del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg). In this study the optimal buffer for antigen retrieval was sodium citrate (pH 6.0). Q score was used to evaluate the specific mutant EGFR proteins expression. Validation using clinical material showed deletions in exon 19 were detected in 19.1% and L858R mutation in 20% of all cases by ARMS assay. A cutoff value of score 1 was used as positive by IHC. No wild type cases were immuno-reactive. The antibodies performed well in cytology, core biopsies and bronchoscopic biopsies. There were only one false positive case using L858R IHC (sensitivity 100%, specificity 98.5%, positive predictive value 96%, negative predictive value 100%). All 23 E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 100%, positive predictive value 100% and negative predictive value 100%. The result of the IHC stains was finely correlated with mutations status determined by ARMS assay. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases especially where limited tumor material is available, or in situations where molecular genetic analysis is not readily available.

  11. Structural investigations of T854A mutation in EGFR and identification of novel inhibitors using structure activity relationships

    PubMed Central

    2015-01-01

    Background The epidermal growth factor receptor (EGFR) is a member of the ErbB family that is involved in a number of processes responsible for cancer development and progression such as angiogenesis, apoptosis, cell proliferation and metastatic spread. Malfunction in activation of protein tyrosine kinases has been shown to result in uncontrolled cell growth. The EGFR TK domain has been identified as suitable target in cancer therapy and tyrosine kinase inhibitors such as erlotinib have been used for treatment of cancer. Mutations in the region of the EGFR gene encoding the tyrosine kinase (TK) domain causes altered responses to EGFR TK inhibitors (TKI). In this paper we perform molecular dynamics simulations and PCA analysis on wild-type and mutant (T854A) structures to gain insight into the structural changes observed in the target protein upon mutation. We also report two novel inhibitors identified by combined approach of QSAR model development. Results The wild-type and mutant structure was observed to be stable for 26 ns and 24 ns respectively. In PCA analysis, the mutant structure proved to be more flexible than wild-type. We developed a 3D-QSAR model using 38 thiazolyl-pyrazoline compounds which was later used for prediction of inhibitory activity of natural compounds of ZINC library. The 3D-QSAR model was proved to be robust by the statistical parameters such as r2 (0.9751), q2(0.9491) and pred_r2(0.9525). Conclusion Analysis of molecular dynamics simulations results indicate stability loss and increased flexibility in the mutant structure. This flexibility results in structural changes which render the mutant protein drug resistant against erlotinib. We report two novel compounds having high predicted inhibitory activity to EGFR TK domain with both wild-type and mutant structure. PMID:26041145

  12. EGFR mutation and lobar location of lung adenocarcinoma.

    PubMed

    Tseng, Chien-Hua; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Ho, Chao-Chi; Hsia, Te-Chun; Su, Kang-Yi; Wu, Ming-Fang; Chiu, Kuo-Liang; Liu, Chien-Ming; Wu, Tzu-Chin; Chen, Hung-Jen; Chen, Hsuan-Yu; Chang, Chi-Sheng; Hsu, Chung-Ping; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Lin, Chin-Hung; Chen, Jeremy J W; Chen, Kuan-Yu; Liao, Wei-Yu; Shih, Jin-Yuan; Yu, Sung-Liang; Yu, Chong-Jen; Yang, Pan-Chyr; Yang, Tsung-Ying; Chang, Gee-Chen

    2016-02-01

    The objective of this study was to investigate the associations among lung cancer location, and epidermal growth factor receptor (EGFR) mutation status. Treatment-naive, pathologically confirmed lung adenocarcinomas with tumor specimens available for genetic analysis were included from 2011 through 2014. Overall, 1771 patients with lung adenocarcinoma were included for analysis, after excluding those with carcinoma not otherwise specified, or synchronous multiple primary lung cancers. The median age was 64 years, and the female:male and never smoker:ever smoker ratios were 930:855 (52:48%) and 1167:604 (65:35%), respectively. The EGFR mutation rate was 56%. Among patients, 1093 (62%) had primary tumors in the upper lobes. Compared with the characteristics of the EGFR wild-type, tumors with EGFR activating mutations were more common in women (P < 0.001), never smokers (P < 0.001), and in the upper lobes (P = 0.004). Among EGFR activating mutations, compared with the EGFR exon 19 deletion, L858R mutation were more common in women (P = 0.002), never smokers (P = 0.038), and the upper lobes P < 0.0005). The present study is the first to address that different pulmonary lobar locations might harbor different EGFR mutation subtypes. We demonstrated that adenocarcinomas with L858R mutation, rather than exon 19 deletion or wild-type EGFR gene, prefer to locate over the upper lungs. This phenomenon was more significant in females and never-smokers, implying the result of complex interactions between genetic susceptibility and environmental factors. Therefore, EGFR L858R mutation and exon 19 deletion may not be identical disease entity from the point of carcinogenesis.

  13. Intercalated chemotherapy and erlotinib for non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations

    PubMed Central

    Zwitter, Matjaz; Rajer, Mirjana; Stanic, Karmen; Vrankar, Martina; Doma, Andrej; Cuderman, Anka; Grmek, Marko; Kern, Izidor; Kovac, Viljem

    2016-01-01

    ABSTRACT Among attempts to delay development of resistance to tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC) with activating mutations of epidermal growth factor receptor (EGFR), intercalated therapy has not been properly evaluated. In a phase II trial, 38 patients with EGFR mutated NSCLC in advanced stage were treated with 4 to 6 3-weekly cycles of intercalated schedule with gemcitabine (1250 mg/m2, days 1 and 4), cisplatin (75 mg/m2, day 2) and erlotinib (150 mg, days 5 – 15), followed by continuous erlotinib as maintenance. In addition to standard radiologic evaluation according to RECIST, PET/CT was done prior to treatment and at 6 months, using PERCIST as a method for assessment of response. The primary endpoint was progression-free survival (PFS). In general, tolerance to treatment was good, even among 8 patients with performance status 2–3 and 13 patients with brain metastases; grade 4 toxicity included 2 cases of neutropenia and 4 thrombo-embolic events. Complete response (CR) or partial response (PR) were seen in 15 (39.5%) and 17 (44.7%) cases, respectively. All cases of CR were confirmed also by PET/CT. Median PFS was 23.4 months and median overall survival (OS) was 38.3  months. After a median follow-up of 35 months, 8 patients are still in CR and on maintenance erlotinib. In conclusion, intercalated treatment for treatment-naive patients with EGFR activating mutations leads to excellent response rate and prolonged PFS and survival. Comparison of the intercalated schedule to monotherapy with TKIs in a randomized trial is warranted. PMID:27261103

  14. Low prevalence of K-RAS, EGF-R and BRAF mutations in sinonasal adenocarcinomas. Implications for anti-EGFR treatments.

    PubMed

    Franchi, Alessandro; Innocenti, Duccio Rossi Degli; Palomba, Annarita; Miligi, Lucia; Paiar, Fabiola; Franzese, Ciro; Santucci, Marco

    2014-07-01

    We have previously shown that a subset of sinonasal intestinal-type adenocarcinomas (ITAC) shows activation of the epidermal growth factor-receptor (EGFR) pathway. In this study we examine the status of the EGFR, KRAS and BRAF genes in a series of sinonasal intestinal (ITAC) and non-intestinal type adenocarcinomas (non-ITAC). Eighteen ITACs and 12 non-ITACs were studied immunohistochemically for EGFR expression. Point mutations were analyzed for EGFR exons 19 and 21, KRAS exon 2 and BRAF exon 15 by direct sequencing. Non-ITACs showed significantly higher expression of EGFR (p = 0.015). Mutation analysis revealed one ITAC with EGFR and one ITAC with KRAS mutation, while two non-ITACs presented mutation of BRAF. We conclude that a subset of sinonasal adenocarcinomas shows overexpression of EGFR, while activating mutations of the signaling cascade downstream of EGFR are rare, suggesting that these tumors could be good candidates for anti-EGFR therapies.

  15. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

    PubMed

    Young, Christian D; Zimmerman, Lisa J; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B; Gatza, Michael L; Morrison, Meghan M; Moore, Preston D; Whitwell, Corbin A; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E; Silva, Grace O; Patel, Premal; Brantley-Sieders, Dana M; Levin, Maren; Horiates, Marina; Palma, Norma A; Wang, Kai; Stephens, Philip J; Perou, Charles M; Weaver, Alissa M; O'Shaughnessy, Joyce A; Chang, Jenny C; Park, Ben Ho; Liebler, Daniel C; Cook, Rebecca S; Arteaga, Carlos L

    2015-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  16. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer*

    PubMed Central

    Young, Christian D.; Zimmerman, Lisa J.; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B.; Gatza, Michael L.; Morrison, Meghan M.; Moore, Preston D.; Whitwell, Corbin A.; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E.; Silva, Grace O.; Patel, Premal; Brantley-Sieders, Dana M.; Levin, Maren; Horiates, Marina; Palma, Norma A.; Wang, Kai; Stephens, Philip J.; Perou, Charles M.; Weaver, Alissa M.; O'Shaughnessy, Joyce A.; Chang, Jenny C.; Park, Ben Ho; Liebler, Daniel C.; Cook, Rebecca S.; Arteaga, Carlos L.

    2015-01-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  17. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

    PubMed

    Young, Christian D; Zimmerman, Lisa J; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B; Gatza, Michael L; Morrison, Meghan M; Moore, Preston D; Whitwell, Corbin A; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E; Silva, Grace O; Patel, Premal; Brantley-Sieders, Dana M; Levin, Maren; Horiates, Marina; Palma, Norma A; Wang, Kai; Stephens, Philip J; Perou, Charles M; Weaver, Alissa M; O'Shaughnessy, Joyce A; Chang, Jenny C; Park, Ben Ho; Liebler, Daniel C; Cook, Rebecca S; Arteaga, Carlos L

    2015-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

  18. EGFR Mutation Analysis of Circulating Tumor DNA Using an Improved PNA-LNA PCR Clamp Method

    PubMed Central

    Watanabe, Kana; Fukuhara, Tatsuro; Tsukita, Yoko; Morita, Mami; Suzuki, Aya; Tanaka, Nobuyuki; Terasaki, Hiroshi; Nukiwa, Toshihiro

    2016-01-01

    Introduction. Rebiopsies have become more crucial in non-small cell lung cancer (NSCLC). Instead of invasive biopsies, development of collecting biological data of the tumor from blood samples is expected. We conducted a prospective study to assess the feasibility of detection of epidermal growth factor receptor (EGFR) mutation in plasma samples. Method. NSCLC patients harboring EGFR activating mutations, who were going to receive EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatment, were enrolled in this study. Plasma EGFR activating mutations and the T790M resistance mutation were analyzed by an improved PNA-LNA PCR clamp method, characterized by a 10-fold or more sensitivity compared with the original methods. Result. Six patients with wild-type EGFR and 24 patients with EGFR mutations were enrolled in this study. Pretreatment plasma samples achieved sensitivity of 79%. The 6 patients with wild-type EGFR were all negative for plasma EGFR mutations. At the time of disease progression, plasma T790M mutation was detected in 8 of 16 cases. Absence of T790M before and during TKI treatment and disappearance of activating mutations during TKI treatment were considered as predictors of EGFR-TKIs efficacy. Conclusion. We were able to detect EGFR mutations in plasma samples by using an improved PNA-LNA PCR clamp method. PMID:27478396

  19. Mutation status of somatic EGFR and KRAS genes in Chinese patients with prostate cancer (PCa).

    PubMed

    Fu, Meng; Zhang, Wei; Shan, Ling; Song, Jian; Shang, Donghao; Ying, Jianming; Zhao, Jimao

    2014-05-01

    Activating mutations of the epidermal growth factor receptor (EGFR) confers sensitivity to tyrosine kinase inhibitors (TKIs). In colorectal cancer and in lung adenocarcinomas, clinical trials have shown a lack of response to anti-EGFR therapy when KRAS gene mutations are present. In this study, the mutation status of specified exons of the EGFR and KRAS genes was profiled in patients with prostate cancer (PCa). Direct Sanger sequencing was used to screen for mutations in exons 19-21 of EGFR and in exon 2 of KRAS in 88 Chinese patients diagnosed with prostate adenocarcinomas. Mutations were detected in 11 patients. In nine cases (10 %), activating mutations in the region of EGFR encoding the tyrosine kinase (TK) domain were present. Deletions in exon 19 and the L858R substitution in exon 21 were "hotspot" mutations, together accounting for five (55 %) of nine cases. Many synonymous substitutions were also detected. KRAS mutations were found in two cases (2.3 % of 88). There were no cases with mutations in both EGFR and KRAS, suggesting that mutations in the two genes might be mutually exclusive. Although prognostic relevance of EGFR expression by immunohistochemistry (IHC) was observed in PCa patients in previous studies, we found no statistically significant association between EGFR or KRAS mutations and clinicopathological features (including age, smoking status, preoperative prostate-specific antigen, Gleason scores, and tumor stage). We contend that accurate profiling of the mutation status of EGFR and KRAS could improve prognostic stratification, and we suggest a potential anti-EGFR therapy for patients with PCa with EGFR mutations. PMID:24595526

  20. EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma.

    PubMed

    Zhang, Wei; Jiang, Yinghao; Yu, Qingmiao; Qiang, Shaoying; Liang, Ping; Gao, Yane; Zhao, Xingye; Liu, Wenchao; Zhang, Ju

    2015-10-01

    Therapy strategy toward epidermal growth factor receptor (EGFR) inhibition in cervical cancer has been ongoing. EGFR promoter methylation status and EGFR tyrosine kinase inhibitor-sensitive mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. In this study, EGFR tyrosine kinase inhibitor-sensitive mutations, EGFR exons 18, 19, and 21 mutations, were detected by sequencing in a total of 293 Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation status was detected by an EGFR asymmetric PCR and hybridization-fluorescence polarization assay and sequencing in 293 Chinese cervical squamous cell carcinoma tissue samples. High-risk human papillomavirus (HPV) genotypes in 293 Chinese cervical squamous cell carcinoma tissue samples were detected by an asymmetric GP5+/6+ PCR and hybridization-fluorescence polarization assay. No EGFR exons 18, 19, and 21 mutations were detected, EGFR promoter methylation status was identified in 98 samples, and HPV 16 infection was the first frequent HPV genotype. The methylated EGFR promoter was identified most frequently in cervical squamous cell carcinoma samples with HPV 16 infection (53.4%). Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 and other HPV genotypes (P<0.01). This study suggested that there was no EGFR tyrosine kinase inhibitor-sensitive mutation in EGFR exons 18, 19, and 21 in Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation was common and it might be associated with HPV 16 infection in Chinese cervical squamous cell carcinoma. The results provided a novel understanding and an applicable pharmacogenomic tool for individualized management of cervical cancer patients.

  1. 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

  2. Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

    PubMed Central

    KIM, HEE JOUNG; OH, SEO YOUNG; KIM, WAN SEOP; KIM, SUN JONG; YOO, GWANG HA; KIM, WON DONG; LEE, KYE YOUNG

    2013-01-01

    Direct sequencing is the standard method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer, however, its relatively low sensitivity limits its clinical use. Pyrosequencing is a bioluminometric, real-time non-electrophoretic DNA sequencing technique with a number of advantages compared with direct sequencing, including higher sensitivity, speed, automation and cost-effectiveness. Clinical specimens from 202 lung cancer patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21 using the pyrosequencing method following genomic DNA extraction from paraffin-embedded tissue specimens. All clinical data and tumor specimens were obtained from the Konkuk University Hospital (Korea) between July 2006 and December 2008. The results and clinical responses to EGFR-tyrosine kinase inhibitors (TKIs) were compared. Overall, EGFR mutation-positive rate was 26.7% (54/202). Activating EGFR mutations were observed more frequently in female (52.1 vs. 13.0%), non-smoking (47.8 vs. 15.8%) and adenocarcinoma (35.2 vs. 5.2%) patients. However, significant numbers of EGFR mutation-positive patients were identified as male, former or current smokers and non-adenocarcinoma patients. The combinations of favorable clinicopathological factors, including female, non-smoking and adenocarcinoma, were not identified to significantly increase the positive EGFR mutation rate (female, 52.1%; female and non-smoker, 52.6%; female, non-smoker and adenocarcinoma, 51.9%). The present findings indicate that EGFR mutation analysis is a highly useful method for the prediction of response to EGFR-TKI and the use of favorable clinicopathological factors to perform this analysis is not suitable. Exon 19 deletion was the most common mutation (63.6%) and exon 21 L858R substitution was measured at 32.7%. The exon 20 T790M mutation was identified in 1 patient prior to EGFR-TKI treatment. EGFR mutation status is associated with response to EGFR-TKI and the overall

  3. 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.

  4. A Novel Technique to Detect EGFR Mutations in Lung Cancer.

    PubMed

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-05-23

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples.

  5. A Novel Technique to Detect EGFR Mutations in Lung Cancer.

    PubMed

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-01-01

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples. PMID:27223277

  6. A Novel Technique to Detect EGFR Mutations in Lung Cancer

    PubMed Central

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-01-01

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples. PMID:27223277

  7. Pooled analysis of clinical outcome for EGFR TKI-treated patients with EGFR mutation-positive NSCLC.

    PubMed

    Paz-Ares, Luis; Soulières, Denis; Moecks, Joachim; Bara, Ilze; Mok, Tony; Klughammer, Barbara

    2014-08-01

    Patients with non-small-cell lung cancer (NSCLC) appear to gain particular benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKI) if their disease tests positive for EGFR activating mutations. Recently, several large, controlled, phase III studies have been published in NSCLC patients with EGFR mutation-positive tumours. Given the increased patient dataset now available, a comprehensive literature search for EGFR TKIs or chemotherapy in EGFR mutation-positive NSCLC was undertaken to update the results of a previously published pooled analysis. Pooling eligible progression-free survival (PFS) data from 27 erlotinib studies (n = 731), 54 gefitinib studies (n = 1802) and 20 chemotherapy studies (n = 984) provided median PFS values for each treatment. The pooled median PFS was: 12.4 months (95% accuracy intervals [AI] 11.6-13.4) for erlotinib-treated patients; 9.4 months (95% AI 9.0-9.8) for gefitinib-treated patients; and 5.6 months (95% AI 5.3-6.0) for chemotherapy. Both erlotinib and gefitinib resulted in significantly longer PFS than chemotherapy (permutation testing; P = 0.000 and P = 0.000, respectively). Data on more recent TKIs (afatinib, dacomitinib and icotinib) were insufficient at this time-point to carry out a pooled PFS analysis on these compounds. The results of this updated pooled analysis suggest a substantial clear PFS benefit of treating patients with EGFR mutation-positive NSCLC with erlotinib or gefitinib compared with chemotherapy.

  8. [Afatinib as first-line therapy in mutation-positive EGFR. Results by type of mutation].

    PubMed

    Vidal, Óscar Juan

    2016-04-01

    The discovery of endothelial growth factor receptor (EGFR) mutations has laid the foundations for personalized medicine in non-small cell lung carcinoma (NSCLC). In phase III trials, the first-generation tyrosine kinase inhibitors (TKI), gefitinib and erlotinib, demonstrated greater efficacy compared with chemotherapy in patients with EGFR mutations, achieving progression-free survival of 8-13.5 months. Afatinib, a second-generation irreversible pan-ErbB inhibitor, is the first TKI that has shown a benefit in overall survival (OS) compared with chemotherapy in EGFR mutation-positive NSCLC when used as first-line treatment. Exon 19 deletion (Del19) and the single-point substitution mutation (L858R) in exon 21, called activating mutations due to their ability to confer sensitivity to TKI, represent approximately 90% of the EGFR mutations in NSCLC. Distinct sensitivity to TKI has been observed depending on the type of mutation, with greater progression-free survival in patients with the Del19 mutation. The analysis of OS in the LUX-Lung 3 and LUX-Lung 6 trials showed a statistically significant increase in survival in afatinib-treated patients with the Del 19 mutation, but no significant increase in that of patients with the L858R mutation. Direct comparison of afatinib and gefitinib as first-line therapy (LUX-Lung 7 trial) showed a statistically-significant increase in progression-free survival (hazard ratio: 0.73; 95% confidence interval, 0.57-0.95; p=0.0165) with afatinib. In the analysis by type of mutation, this benefit was observed for both the Del19 and the L858R mutations. PMID:27426243

  9. [Afatinib as first-line therapy in mutation-positive EGFR. Results by type of mutation].

    PubMed

    Vidal, Óscar Juan

    2016-04-01

    The discovery of endothelial growth factor receptor (EGFR) mutations has laid the foundations for personalized medicine in non-small cell lung carcinoma (NSCLC). In phase III trials, the first-generation tyrosine kinase inhibitors (TKI), gefitinib and erlotinib, demonstrated greater efficacy compared with chemotherapy in patients with EGFR mutations, achieving progression-free survival of 8-13.5 months. Afatinib, a second-generation irreversible pan-ErbB inhibitor, is the first TKI that has shown a benefit in overall survival (OS) compared with chemotherapy in EGFR mutation-positive NSCLC when used as first-line treatment. Exon 19 deletion (Del19) and the single-point substitution mutation (L858R) in exon 21, called activating mutations due to their ability to confer sensitivity to TKI, represent approximately 90% of the EGFR mutations in NSCLC. Distinct sensitivity to TKI has been observed depending on the type of mutation, with greater progression-free survival in patients with the Del19 mutation. The analysis of OS in the LUX-Lung 3 and LUX-Lung 6 trials showed a statistically significant increase in survival in afatinib-treated patients with the Del 19 mutation, but no significant increase in that of patients with the L858R mutation. Direct comparison of afatinib and gefitinib as first-line therapy (LUX-Lung 7 trial) showed a statistically-significant increase in progression-free survival (hazard ratio: 0.73; 95% confidence interval, 0.57-0.95; p=0.0165) with afatinib. In the analysis by type of mutation, this benefit was observed for both the Del19 and the L858R mutations.

  10. Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

    PubMed

    Furuyama, Kazuto; Harada, Taishi; Iwama, Eiji; Shiraishi, Yoshimasa; Okamura, Kyoko; Ijichi, Kayo; Fujii, Akiko; Ota, Keiichi; Wang, Shuo; Li, Heyan; Takayama, Koichi; Giaccone, Giuseppe; Nakanishi, Yoichi

    2013-05-01

    The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

  11. Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cell lung cancer harboring EGFR mutations: a review of the evidence.

    PubMed

    Wang, Xiaochun; Goldstein, David; Crowe, Philip J; Yang, Jia-Lin

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) against human epidermal growth factor receptor (EGFR/HER) family have been introduced into the clinic to treat cancers, particularly non-small-cell lung cancer (NSCLC). There have been three generations of the EGFR/HER-TKIs. First-generation EGFR/HER-TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR TK domain, show a significant breakthrough treatment in selected NSCLC patients with activating EGFR mutations (actEGFRm) EGFR (L858R) and EGFR (Del19), in terms of safety, efficacy, and quality of life. However, all those responders inevitably develop acquired resistance within 12 months, because of the EGFR (T790M) mutation, which prevents TKI binding to ATP-pocket of EGFR by steric hindrance. The second-generation EGFR/HER-TKIs were developed to prolong and maintain more potent response as well as overcome the resistance to the first-generation EGFR/HER-TKIs. They are different from the first-generation EGFR/HER-TKIs by covalently binding to the ATP-binding site, irreversibly blocking enzymatic activation, and targeting EGFR/HER family members, including EGFR, HER2, and HER4. Preclinically, these compounds inhibit the enzymatic activation for actEGFRm, EGFR (T790M), and wtEGFR. The second-generation EGFR/HER-TKIs improve overall survival in cancer patients with actEGFRm in a modest way. However, they are not clinically active in overcoming EGFR (T790M) resistance, mainly because of dose-limiting toxicity due to simultaneous inhibition against wtEGFR. The third-generation EGFR/HER-TKIs selectively and irreversibly target EGFR (T790M) and actEGFRm while sparing wtEGFR. They yield promising efficacy in NSCLC patients with actEGFRm as well as EGFR (T790M) resistant to the first- and second-generation EGFR-TKIs. They also appear to have a lower incidence of toxicity due to the reduced inhibitory effect on wtEGFR. Currently, the first-generation EGFR/HER-TKIs gefitinib and erlotinib and second-generation EGFR

  12. Kinase inhibitor-responsive genotypes in EGFR mutated lung adenocarcinomas: moving past common point mutations or indels into uncommon kinase domain duplications and rearrangements

    PubMed Central

    2016-01-01

    The most frequent epidermal growth factor receptor (EGFR) mutations found by traditional or comprehensive molecular profiling of lung adenocarcinomas include indels of exon 19 (the exon 19 deletion delE746_A750 being the most common) and the exon 21 L858R point mutation. The current approval labels for first line palliative gefitinib 250 mg/day, erlotinib 150 mg/day and afatinib 40 mg/day for advanced lung cancers require the presence of the aforementioned classical/sensitizing EGFR mutations. Other gefitinib, erlotinib and afatinib sensitizing mutations include exon 18 indels, G719X, exon 19 insertions, A763_Y764insFQEA, S768I and L861Q; for which off-label EGFR kinase inhibitor use is generally agreed upon by thoracic oncologists. The main biological mechanism of resistance to approved first line EGFR inhibitors is the selection/acquisition of EGFR-T790M that in itself can be inhibited by osimertinib 80 mg/day, a 3rd generation EGFR inhibitor that is bypassed by EGFR-C797X mutations. Another class of de novo inhibitor insensitive mutation includes EGFR exon 20 insertions. More recently, the dichotomy of only point mutations or indels explaining aberrant kinase activation of EGFR plus inhibitor response has been shattered by the discovery of uncommon (<0.5% of all EGFR mutations) genomic events involving exon 18–25 kinase domain duplications (KDD) and rearrangements (EGFR-RAD51 or EGFR-PURB). The latter lead to oncogene addiction, enhanced sensitivity to kinase inhibitors in vitro and clinical responses to approved EGFR inhibitors. The enhanced landscape of EGFR inhibitor-responsive genotypes highlights that comprehensive molecular profiling may be necessary to maximize the identification of all cases that can benefit from precision oncology. PMID:27413714

  13. Identification of a novel HER3 activating mutation homologous to EGFR-L858R in lung cancer

    PubMed Central

    Umelo, Ijeoma; Noeparast, Amir; Chen, Gang; Renard, Marleen; Geers, Caroline; Vansteenkiste, Johan; Giron, Philippe; De Wever, Olivier; Teugels, Erik; De Grève, Jacques

    2016-01-01

    Somatic mutations found within the tyrosine kinase domain (TKD) of the human epidermal growth factor (HER) family of receptors have been implicated in the development and progression of non-small cell lung cancer (NSCLC). However, no conclusive reports have described pathogenic mutations in kinase-impaired HER3. Here, we report a case of an advanced chemotherapy-resistant NSCLC, harboring a novel HER3V855A somatic mutation homologous to the EGFRL858Ractivating mutation. Co-expression of HER3V855A and wild-type HER2 enhances ligand-induced transformation of murine and human cell lines, while HER-targeted inhibitors potently suppress mutant HER3 activity. Consistent with these observations, in silico computational modeling predicts that mutant V855A alters the kinase domain and c-terminal end of the HER3 protein. Taken together, these findings provide a basis for the clinical exploration of targeted therapies in HER3 mutant NSCLC and by extrapolation, in other cancers that more frequently carry somatic HER3 mutations. PMID:26689995

  14. Nickel may contribute to EGFR mutation and synergistically promotes tumor invasion in EGFR-mutated lung cancer via nickel-induced microRNA-21 expression.

    PubMed

    Chiou, Yu-Hu; Liou, Saou-Hsing; Wong, Ruey-Hong; Chen, Chih-Yi; Lee, Huei

    2015-08-19

    We recently reported that nickel accumulation in lung tissues may be associated with an increased in p53 mutation risk via reduced DNA repair activity. Here, we hypothesized that nickel accumulation in lung tissues could contribute to EGFR mutations in never-smokers with lung cancer. We enrolled 76 never-smoking patients to evaluate nickel level in adjacent normal lung tissues by ICP-MS. The prevalence of EGFR mutations was significantly higher in the high-nickel subgroup than in the low-nickel subgroup. Intriguingly, the OR for the occurrence of EGFR mutations in female, adenocarcinoma, and female adenocarcinoma patients was higher than that of all patients. Mechanistically, SPRY2 and RECK expressions were decreased by nickel-induced miR-21 via activation of the EGFR/NF-κB signaling pathway, which promoted invasiveness in lung cancer cells, and particularly in the cells with EGFR L858R expression vector transfection. The patients' nickel levels were associated with miR-21 expression levels. Kaplan-Meier analysis revealed poorer overall survival (OS) and shorter relapse free survival (RFS) in the high-nickel subgroup than in low-nickel subgroup. The high-nickel/high-miR-21 subgroup had shorter OS and RFS periods when compared to the low-nickel/low-miR-21 subgroup. Our findings support previous epidemiological studies indicating that nickel exposure may not only contribute to cancer incidence but also promote tumor invasion in lung cancer.

  15. Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients

    PubMed Central

    PAPADOPOULOU, EIRINI; TSOULOS, NIKOLAOS; TSIRIGOTI, ANGELIKI; APESSOS, ANGELA; AGIANNITOPOULOS, KONSTANTINOS; METAXA-MARIATOU, VASILIKI; ZAROGOULIDIS, KONSTANTINOS; ZAROGOULIDIS, PAVLOS; KASARAKIS, DIMITRIOS; KAKOLYRIS, STYLIANOS; DAHABREH, JUBRAIL; VLASTOS, FOTIS; ZOUBLIOS, CHARALAMPOS; RAPTI, AGGELIKI; PAPAGEORGIOU, NIKI GEORGATOU; VELDEKIS, DIMITRIOS; GAGA, MINA; ARAVANTINOS, GERASIMOS; KARAVASILIS, VASILEIOS; KARAGIANNIDIS, NAPOLEON; NASIOULAS, GEORGE

    2015-01-01

    It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of

  16. EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

    PubMed

    Liu, Feng; Hon, Gary C; Villa, Genaro R; Turner, Kristen M; Ikegami, Shiro; Yang, Huijun; Ye, Zhen; Li, Bin; Kuan, Samantha; Lee, Ah Young; Zanca, Ciro; Wei, Bowen; Lucey, Greg; Jenkins, David; Zhang, Wei; Barr, Cathy L; Furnari, Frank B; Cloughesy, Timothy F; Yong, William H; Gahman, Timothy C; Shiau, Andrew K; Cavenee, Webster K; Ren, Bing; Mischel, Paul S

    2015-10-15

    Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.

  17. EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

    PubMed

    Liu, Feng; Hon, Gary C; Villa, Genaro R; Turner, Kristen M; Ikegami, Shiro; Yang, Huijun; Ye, Zhen; Li, Bin; Kuan, Samantha; Lee, Ah Young; Zanca, Ciro; Wei, Bowen; Lucey, Greg; Jenkins, David; Zhang, Wei; Barr, Cathy L; Furnari, Frank B; Cloughesy, Timothy F; Yong, William H; Gahman, Timothy C; Shiau, Andrew K; Cavenee, Webster K; Ren, Bing; Mischel, Paul S

    2015-10-15

    Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy. PMID:26455392

  18. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  19. Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

    PubMed Central

    Ulivi, Paola; Delmonte, Angelo; Chiadini, Elisa; Calistri, Daniele; Papi, Maximilian; Mariotti, Marita; Verlicchi, Alberto; Ragazzini, Angela; Capelli, Laura; Gamboni, Alessandro; Puccetti, Maurizio; Dubini, Alessandra; Burgio, Marco Angelo; Casanova, Claudia; Crinò, Lucio; Amadori, Dino; Dazzi, Claudio

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity. PMID:25561229

  20. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M.

    PubMed

    Thress, Kenneth S; Paweletz, Cloud P; Felip, Enriqueta; Cho, Byoung Chul; Stetson, Daniel; Dougherty, Brian; Lai, Zhongwu; Markovets, Aleksandra; Vivancos, Ana; Kuang, Yanan; Ercan, Dalia; Matthews, Sarah E; Cantarini, Mireille; Barrett, J Carl; Jänne, Pasi A; Oxnard, Geoffrey R

    2015-06-01

    Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

  1. LDA-SVM-based EGFR mutation model for NSCLC brain metastases: an observational study.

    PubMed

    Hu, Nan; Wang, Ge; Wu, Yu-Hao; Chen, Shi-Feng; Liu, Guo-Dong; Chen, Chuan; Wang, Dong; He, Zhong-Shi; Yang, Xue-Qin; He, Yong; Xiao, Hua-Liang; Huang, Ding-De; Xiong, Kun-Lin; Wu, Yan; Huang, Ming; Yang, Zhen-Zhou

    2015-02-01

    Epidermal growth factor receptor (EGFR) activating mutations are a predictor of tyrosine kinase inhibitor effectiveness in the treatment of non-small-cell lung cancer (NSCLC). The objective of this study is to build a model for predicting the EGFR mutation status of brain metastasis in patients with NSCLC. Observation and model set-up. This study was conducted between January 2003 and December 2011 in 6 medical centers in Southwest China. The study included 31 NSCLC patients with brain metastases. Eligibility requirements were histological proof of NSCLC, as well as sufficient quantity of paraffin-embedded lung and brain metastases specimens for EGFR mutation detection. The linear discriminant analysis (LDA) method was used for analyzing the dimensional reduction of clinical features, and a support vector machine (SVM) algorithm was employed to generate an EGFR mutation model for NSCLC brain metastases. Training-testing-validation (3 : 1 : 1) processes were applied to find the best fit in 12 patients (validation test set) with NSCLC and brain metastases treated with a tyrosine kinase inhibitor and whole-brain radiotherapy. Primary and secondary outcome measures: EGFR mutation analysis in patients with NSCLC and brain metastases and the development of a LDA-SVM-based EGFR mutation model for NSCLC brain metastases patients. EGFR mutation discordance between the primary lung tumor and brain metastases was found in 5 patients. Using LDA, 13 clinical features were transformed into 9 characteristics, and 3 were selected as primary vectors. The EGFR mutation model constructed with SVM algorithms had an accuracy, sensitivity, and specificity for determining the mutation status of brain metastases of 0.879, 0.886, and 0.875, respectively. Furthermore, the replicability of our model was confirmed by testing 100 random combinations of input values. The LDA-SVM-based model developed in this study could predict the EGFR status of brain metastases in this small cohort of

  2. LDA-SVM-Based EGFR Mutation Model for NSCLC Brain Metastases

    PubMed Central

    Hu, Nan; Wang, Ge; Wu, Yu-Hao; Chen, Shi-Feng; Liu, Guo-Dong; Chen, Chuan; Wang, Dong; He, Zhong-Shi; Yang, Xue-Qin; He, Yong; Xiao, Hua-Liang; Huang, Ding-De; Xiong, Kun-Lin; Wu, Yan; Huang, Ming; Yang, Zhen-Zhou

    2015-01-01

    Abstract Epidermal growth factor receptor (EGFR) activating mutations are a predictor of tyrosine kinase inhibitor effectiveness in the treatment of non–small-cell lung cancer (NSCLC). The objective of this study is to build a model for predicting the EGFR mutation status of brain metastasis in patients with NSCLC. Observation and model set-up. This study was conducted between January 2003 and December 2011 in 6 medical centers in Southwest China. The study included 31 NSCLC patients with brain metastases. Eligibility requirements were histological proof of NSCLC, as well as sufficient quantity of paraffin-embedded lung and brain metastases specimens for EGFR mutation detection. The linear discriminant analysis (LDA) method was used for analyzing the dimensional reduction of clinical features, and a support vector machine (SVM) algorithm was employed to generate an EGFR mutation model for NSCLC brain metastases. Training-testing-validation (3 : 1 : 1) processes were applied to find the best fit in 12 patients (validation test set) with NSCLC and brain metastases treated with a tyrosine kinase inhibitor and whole-brain radiotherapy. Primary and secondary outcome measures: EGFR mutation analysis in patients with NSCLC and brain metastases and the development of a LDA-SVM-based EGFR mutation model for NSCLC brain metastases patients. EGFR mutation discordance between the primary lung tumor and brain metastases was found in 5 patients. Using LDA, 13 clinical features were transformed into 9 characteristics, and 3 were selected as primary vectors. The EGFR mutation model constructed with SVM algorithms had an accuracy, sensitivity, and specificity for determining the mutation status of brain metastases of 0.879, 0.886, and 0.875, respectively. Furthermore, the replicability of our model was confirmed by testing 100 random combinations of input values. The LDA-SVM-based model developed in this study could predict the EGFR status of brain metastases in this

  3. Selectivity profile of afatinib for EGFR-mutated non-small-cell lung cancer.

    PubMed

    Wang, Debby D; Lee, Victor H F; Zhu, Guangyu; Zou, Bin; Ma, Lichun; Yan, Hong

    2016-04-26

    EGFR-mutated non-small-cell lung cancer (NSCLC) has long been a research focus in lung cancer studies. Besides reversible tyrosine kinase inhibitors (TKIs), new-generation irreversible inhibitors, such as afatinib, embark on playing an important role in NSCLC treatment. To achieve an optimal application of these inhibitors, the correlation between the EGFR mutation status and the potency of such an inhibitor should be decoded. In this study, the correlation was profiled for afatinib, based on a cohort of patients with the EGFR-mutated NSCLC. Relying on extracted DNAs from the paraffin-embedded tumor samples, EGFR mutations were detected by direct sequencing. Progression-free survival (PFS) and the response level were recorded as study endpoints. These PFS and response values were analyzed and correlated to different mutation types, implying a higher potency of afatinib to classic activation mutations (L858R and deletion 19) and a lower one to T790M-related mutations. To further bridge the mutation status with afatinib-related response or PFS, we conducted a computational study to estimate the binding affinity in a mutant-afatinib system, based on molecular structural modeling and dynamics simulations. The derived binding affinities were well in accordance with the clinical response or PFS values. At last, these computational binding affinities were successfully mapped to the patient response or PFS according to linear models. Consequently, a detailed mutation-response or mutation-PFS profile was drafted for afatinib, implying the selective nature of afatinib to various EGFR mutants and further encouraging the design of specialized therapies or innovative drugs. PMID:26961138

  4. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations

    PubMed Central

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-01-01

    Abstract This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer. This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated. Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23–52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2–26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7–14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856). Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  5. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations.

    PubMed

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-06-01

    This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer.This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated.Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23-52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2-26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7-14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856).Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  6. EGFR-Mutated Breast Metastasis of Lung Adenocarcinoma: A Case Report.

    PubMed

    Dansin, Eric; Carnot, Aurélien; Servent, Véronique; Daussay, Dorothée; Robin, Yves-Marie; Surmei-Pintilie, Ecaterina; Lauridant, Géraldine; Descarpentries, Clothilde; Révillion, Françoise; Delattre, Claire

    2015-01-01

    Breast metastasis from other primary carcinoma is very rare and could be difficult to identify despite immunohistochemistry analysis. Breast metastasis from lung adenocarcinoma can mimic triple-negative breast cancer. Given the prognosis and therapeutic challenges, a correct diagnosis appears essential, and molecular biomarkers could be useful. We report the case of a 52-year-old woman with a breast mass initially diagnosed as primary breast cancer and secondarily attached to breast metastasis from an EGFR-mutated lung adenocarcinoma. The same activating EGFR mutations were identified in both the primary lung carcinoma and the breast metastasis.

  7. EGFR-Mutated Breast Metastasis of Lung Adenocarcinoma: A Case Report

    PubMed Central

    Dansin, Eric; Carnot, Aurélien; Servent, Véronique; Daussay, Dorothée; Robin, Yves-Marie; Surmei-Pintilie, Ecaterina; Lauridant, Géraldine; Descarpentries, Clothilde; Révillion, Françoise; Delattre, Claire

    2015-01-01

    Breast metastasis from other primary carcinoma is very rare and could be difficult to identify despite immunohistochemistry analysis. Breast metastasis from lung adenocarcinoma can mimic triple-negative breast cancer. Given the prognosis and therapeutic challenges, a correct diagnosis appears essential, and molecular biomarkers could be useful. We report the case of a 52-year-old woman with a breast mass initially diagnosed as primary breast cancer and secondarily attached to breast metastasis from an EGFR-mutated lung adenocarcinoma. The same activating EGFR mutations were identified in both the primary lung carcinoma and the breast metastasis. PMID:25873885

  8. Concordance of KRAS/BRAF Mutation Status in Metastatic Colorectal Cancer before and after Anti-EGFR Therapy.

    PubMed

    Gattenlöhner, S; Etschmann, B; Kunzmann, V; Thalheimer, A; Hack, M; Kleber, G; Einsele, H; Germer, C; Müller-Hermelink, H-K

    2009-01-01

    Anti-EGFR targeted therapy is a potent strategy in the treatment of metastatic colorectal cancer (mCRC) but activating mutations in the KRAS gene are associated with poor response to this treatment. Therefore, KRAS mutation analysis is employed in the selection of patients for EGFR-targeted therapy and various studies have shown a high concordance between the mutation status in primary CRC and corresponding metastases. However, although development of therapy related resistance occurs also in the context of novel drugs such as tyrosine kinase-inhibitors the effect of the anti-EGFR treatment on the KRAS/BRAF mutation status itself in recurrent mCRC has not yet been clarified. Therefore, we analyzed 21 mCRCs before/after anti-EGFR therapy and found a pre-/posttherapeutic concordance of the KRAS/BRAF mutation status in 20 of the 21 cases examined. In the one discordant case, further analyses revealed that a tumor mosaicism or multiple primary tumors were present, indicating that anti-EGFR therapy has no influence on KRAS/BRAF mutation status in mCRC. Moreover, as the preselection of patients with a KRAS(wt) genotype for anti-EGFR therapy has become a standard procedure, sample sets such ours might be the basis for future studies addressing the identification of potential anti-EGFR therapy induced genetic alterations apart from KRAS/BRAF mutations.

  9. Uncommon epidermal growth factor receptor mutations in non-small cell lung cancer and their mechanisms of EGFR tyrosine kinase inhibitors sensitivity and resistance.

    PubMed

    Massarelli, Erminia; Johnson, Faye M; Erickson, Heidi S; Wistuba, Ignacio I; Papadimitrakopoulou, Vassiliki

    2013-06-01

    Therapy targeted against the epidermal growth factor receptor (EGFR) has demonstrated dramatic tumor responses and favorable clinical outcomes in a select group of non-small cell lung cancer (NSCLC) patients whose tumors harbor EGFR activating mutations. The best characterized of the mutations conferring sensitivity to EGFR tyrosine kinase inhibitors (TKIs) are deletions in exon 19 and a point mutation in exon 21 (L858R). Likewise, the most common mutation that confers resistance is the T790M point mutation. However several other mutations have been reported and several have been characterized as regards their role in sensitivity or resistance to EGFR TKIs. Resistance to the EGFR TKIs erlotinib and gefitinib, and the newer irreversible EGFR TKIs is a problem of fundamental importance. Recognition of the presence and significance of specific EGFR mutations is important for appropriate therapeutic implementation of EGFR TKIs and research and development of mutation-specific inhibitors. We summarize the literature and present an overview of the subject of less common EGFR mutations and their clinical significance, with an emphasis on EGFR TKI sensitivity or resistance.

  10. Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cell lung cancer harboring EGFR mutations: a review of the evidence

    PubMed Central

    Wang, Xiaochun; Goldstein, David; Crowe, Philip J; Yang, Jia-Lin

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) against human epidermal growth factor receptor (EGFR/HER) family have been introduced into the clinic to treat cancers, particularly non-small-cell lung cancer (NSCLC). There have been three generations of the EGFR/HER-TKIs. First-generation EGFR/HER-TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR TK domain, show a significant breakthrough treatment in selected NSCLC patients with activating EGFR mutations (actEGFRm) EGFRL858R and EGFRDel19, in terms of safety, efficacy, and quality of life. However, all those responders inevitably develop acquired resistance within 12 months, because of the EGFRT790M mutation, which prevents TKI binding to ATP-pocket of EGFR by steric hindrance. The second-generation EGFR/HER-TKIs were developed to prolong and maintain more potent response as well as overcome the resistance to the first-generation EGFR/HER-TKIs. They are different from the first-generation EGFR/HER-TKIs by covalently binding to the ATP-binding site, irreversibly blocking enzymatic activation, and targeting EGFR/HER family members, including EGFR, HER2, and HER4. Preclinically, these compounds inhibit the enzymatic activation for actEGFRm, EGFRT790M, and wtEGFR. The second-generation EGFR/HER-TKIs improve overall survival in cancer patients with actEGFRm in a modest way. However, they are not clinically active in overcoming EGFRT790M resistance, mainly because of dose-limiting toxicity due to simultaneous inhibition against wtEGFR. The third-generation EGFR/HER-TKIs selectively and irreversibly target EGFRT790M and actEGFRm while sparing wtEGFR. They yield promising efficacy in NSCLC patients with actEGFRm as well as EGFRT790M resistant to the first- and second-generation EGFR-TKIs. They also appear to have a lower incidence of toxicity due to the reduced inhibitory effect on wtEGFR. Currently, the first-generation EGFR/HER-TKIs gefitinib and erlotinib and second-generation EGFR/HER-TKI afatinib have

  11. Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cell lung cancer harboring EGFR mutations: a review of the evidence

    PubMed Central

    Wang, Xiaochun; Goldstein, David; Crowe, Philip J; Yang, Jia-Lin

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) against human epidermal growth factor receptor (EGFR/HER) family have been introduced into the clinic to treat cancers, particularly non-small-cell lung cancer (NSCLC). There have been three generations of the EGFR/HER-TKIs. First-generation EGFR/HER-TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR TK domain, show a significant breakthrough treatment in selected NSCLC patients with activating EGFR mutations (actEGFRm) EGFRL858R and EGFRDel19, in terms of safety, efficacy, and quality of life. However, all those responders inevitably develop acquired resistance within 12 months, because of the EGFRT790M mutation, which prevents TKI binding to ATP-pocket of EGFR by steric hindrance. The second-generation EGFR/HER-TKIs were developed to prolong and maintain more potent response as well as overcome the resistance to the first-generation EGFR/HER-TKIs. They are different from the first-generation EGFR/HER-TKIs by covalently binding to the ATP-binding site, irreversibly blocking enzymatic activation, and targeting EGFR/HER family members, including EGFR, HER2, and HER4. Preclinically, these compounds inhibit the enzymatic activation for actEGFRm, EGFRT790M, and wtEGFR. The second-generation EGFR/HER-TKIs improve overall survival in cancer patients with actEGFRm in a modest way. However, they are not clinically active in overcoming EGFRT790M resistance, mainly because of dose-limiting toxicity due to simultaneous inhibition against wtEGFR. The third-generation EGFR/HER-TKIs selectively and irreversibly target EGFRT790M and actEGFRm while sparing wtEGFR. They yield promising efficacy in NSCLC patients with actEGFRm as well as EGFRT790M resistant to the first- and second-generation EGFR-TKIs. They also appear to have a lower incidence of toxicity due to the reduced inhibitory effect on wtEGFR. Currently, the first-generation EGFR/HER-TKIs gefitinib and erlotinib and second-generation EGFR/HER-TKI afatinib have

  12. Fighting cancer drug resistance: Opportunities and challenges for mutation-specific EGFR inhibitors.

    PubMed

    Juchum, Michael; Günther, Marcel; Laufer, Stefan A

    2015-05-01

    Multiple mutations in the EGFR gene are a major cause for the failure of Erlotinib and Gefitinib in the treatment of patients harboring non-small-cell lung cancer (NSCLC) who initially responded to this therapy. The development of these tyrosine kinase inhibitors (TKIs) is going back to the early 90s, where cancer was widely considered and fully treated as a disease of an organ. Fundamental gain of knowledge in cell biology in general and cancer genetics in particular led us to where we currently stand: cancer is a disease that originates in the genome. Fast and affordable gene sequencing paved the way and opened our eyes for the genetic instability of many cancers, particularly EGFR driven NSCLC. This might allow highly rational and personal therapies by aiming at a very particular wild type and mutant kinase pattern. However, the paradigm "one disease - one target - one drug" is currently challenged. Both activating and deactivating EGFR mutations are known to render the development of novel targeted drugs difficult. Among all lung adenocarcinomas, only 20% are driven by EGFR and only a subpopulation has an activating mutation (e.g. L858R), making them sensitive to first generation EGFR inhibitors. Unfortunately, most of them acquire second deactivating mutations (e.g. T790M) during treatment, leading to a complete loss of response. Are specific inhibitors of the double EGFR mutant L858R/T790M the magic bullet? Much scientific evidence but also high expectations justify this approach. Structural biology of EGFR mutants constitutes the basis for highly rational approaches. Second generation pan EGFR inhibitors inhibiting wild type (WT) and mutant EGFR like Afatinib suffer from dose-limiting adverse effects. Inhibition of WT EGFR is considered to be the culprit. Third generation EGFR inhibitors follow two strategies. Mutant selectivity and improved target residential time. These inhibitors display high mutant selectivity and irreversible binding patterns while

  13. The epidermal growth factor receptor (EGFR / HER-1) gatekeeper mutation T790M is present in European patients with early breast cancer.

    PubMed

    Bemanian, Vahid; Sauer, Torill; Touma, Joel; Lindstedt, Bjørn Arne; Chen, Ying; Ødegård, Hilde Presterud; Vetvik, Katja Marjaana; Bukholm, Ida Rashida; Geisler, Jürgen

    2015-01-01

    The epidermal growth factor receptor (EGFR) is one of the major oncogenes identified in a variety of human malignancies including breast cancer (BC). EGFR-mutations have been studied in lung cancer for some years and are established as important markers in guiding therapy with tyrosine kinase inhibitors (TKIs). In contrast, EGFR-mutations have been reported to be rare if not absent in human BC, although recent evidence has suggested a significant worldwide variation in somatic EGFR-mutations. Therefore, we investigated the presence of EGFR-mutations in 131 norwegian patients diagnosed with early breast cancer using real-time PCR methods. In the present study we identified three patients with an EGFR-T790M-mutation. The PCR-findings were confirmed by direct Sanger sequencing. Two patients had triple-negative BC (TNBC) while the third was classified as luminal-A subtype. The difference in incidence of T790M mutations comparing the TNBC subgroup with the other BC subgroups was statistical significant (P = 0.023). No other EGFR mutations were identified in the entire cohort. Interestingly, none of the patients had received any previous cancer treatment. To our best knowledge, the EGFR-T790M-TKI-resistance mutation has not been previously detected in breast cancer patients. Our findings contrast with the observations made in lung cancer patients where the EGFR-T790M-mutation is classified as a typical "second mutation"causing resistance to TKI-therapy during ongoing anticancer therapy. In conclusion, we have demonstrated for the first time that the EGFR-T790M-mutation occurs in primary human breast cancer patients. In the present study the EGFR-T790M mutation was not accompanied by any simultaneous EGFR-activating mutation.

  14. Presence of the minor EGFR T790M mutation is associated with drug-sensitive EGFR mutations in lung adenocarcinoma patients.

    PubMed

    Hashida, Shinsuke; Soh, Junichi; Toyooka, Shinichi; Tanaka, Tomoaki; Furukawa, Masashi; Shien, Kazuhiko; Yamamoto, Hiromasa; Asano, Hiroaki; Tsukuda, Kazunori; Hagiwara, Koichi; Miyoshi, Shinichiro

    2014-07-01

    The T790M mutation in the epidermal growth factor receptor (EGFR) gene is known to be associated with the acquired resistance of lung adenocarcinoma patients to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). The minor T790M mutant allele is occasionally detected in EGFR-TKI-naive tumor samples, yet findings concerning the clinical impact of the minor T790M mutation vary among previous studies. In the present study, we assessed the clinical impact of the minor T790M mutation using a novel, highly sensitive assay combining high-resolution melting (HRM), mutant-enriched PCR and co-amplification at a lower denaturation temperature (COLD)-PCR. We determined the T790M mutational status in 146 surgically resected lung adenocarcinomas without a history of EGFR-TKI treatment using mutant-enriched COLD-HRM (MEC-HRM) and standard HRM assays. The sensitivities of the MEC-HRM and standard HRM assays for the detection of T790M-mutant alleles among wild-type alleles were 0.01 and 10%, respectively. Although the T790M mutation was not detected using a standard HRM assay, we identified 19 (13%) T790M mutations using the MEC-HRM assay and defined these 19 mutations as minor T790M mutations. The proportion of T790M alleles was <0.1% in 17 (84%) of the 19 samples. Multivariate analyses revealed that a minor T790M mutation was significantly associated with the presence of EGFR exon 19 deletions or the L858R mutation (both of which are drug-sensitive EGFR mutations) (P=0.04). In conclusion, the minor EGFR T790M mutations were present in 13% of EGFR-TKI-naive surgically resected lung adenocarcinomas and were associated with drug-sensitive EGFR mutations.

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

    PubMed

    Hirano, Toshiyuki; Yasuda, Hiroyuki; Tani, Tetsuo; 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

    2015-11-17

    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.

  16. Impact of bevacizumab in combination with erlotinib on EGFR-mutated non-small cell lung cancer xenograft models with T790M mutation or MET amplification.

    PubMed

    Furugaki, Koh; Fukumura, Junko; Iwai, Toshiki; Yorozu, Keigo; Kurasawa, Mitsue; Yanagisawa, Mieko; Moriya, Yoichiro; Yamamoto, Kaname; Suda, Kenichi; Mizuuchi, Hiroshi; Mitsudomi, Tetsuya; Harada, Naoki

    2016-02-15

    Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL.

  17. EGFR mutations cause a lethal syndrome of epithelial dysfunction with progeroid features

    PubMed Central

    Ganetzky, Rebecca; Finn, Erin; Bagchi, Atrish; Zollo, Ornella; Conlin, Laura; Deardorff, Matthew; Harr, Margaret; Simpson, Michael A; McGrath, John A; Zackai, Elaine; Lemmon, Mark A; Sondheimer, Neal

    2015-01-01

    The epidermal growth factor receptor (EGFR) is part of a large family of receptors required for communicating extracellular signals through internal tyrosine kinases. Epidermal growth factor (EGF) signaling is required for tissue development, whereas constitutive activation of this signaling pathway is associated with oncogenic transformation. We identified homozygous c.1283G>A (p.Gly428Asp) mutations in the extracellular domain of EGFR in two siblings. The children were born prematurely, had abnormalities in skin and hair, suffered multisystem organ failure, and died in the neonatal period from intestinal perforation. EGF failed to induce mutated receptor phosphorylation in patient-derived fibroblasts and activation of downstream targets was suppressed. The heterologously expressed extracellular domain was impaired in stability and the binding of EGF. Cells from the affected patient undergo early senescence with accelerated expression of β-galactosidase and shortened telomeres at all passages when compared to controls. A comparison of homozygous inherited regions from a separate report of a patient from the same ethnic background and EGFR genotype confirms the pathogenicity of EGFR mutations in congenital disease. PMID:26436111

  18. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

    PubMed

    Chia, Puey Ling; Do, Hongdo; Morey, Adrienne; Mitchell, Paul; Dobrovic, Alexander; John, Thomas

    2016-08-01

    AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression. PMID:27393503

  19. EGFR mutation testing on cytological and histological samples in non-small cell lung cancer: a Polish, single institution study and systematic review of European incidence.

    PubMed

    Szumera-Ciećkiewicz, Anna; Olszewski, Włodzimierz T; Tysarowski, Andrzej; Kowalski, Dariusz M; Głogowski, Maciej; Krzakowski, Maciej; Siedlecki, Janusz A; Wągrodzki, Michał; Prochorec-Sobieszek, Monika

    2013-01-01

    The targeted treatment of advanced non-small-cell lung cancer (NSCLC) depends on confirmation of activating somatic EGFR mutation. The aim of the study was to evaluate the incidence of EGFR mutations in NSCLC detected in cytological and histological material and present literature review on European EGFR mutation incidence. 273 patients with confirmed NSCLC were entered into the study: 189 histological, paraffin-embedded materials, 12 fresh and 72 fixed cytological specimens. DNA was extracted from both types of material and the EGFR mutation in exons 18-21 was analyzed by direct sequencing. In addition the EGFR gene copy number in cases with sufficient histological material (110 patients) was evaluated by fluorescent in situ hybridization (FISH) technique. The percentage of EGFR somatic mutations was 10.62%. FISH positive results (amplification or high polysomy of EGFR gene) were identified in 33 patients (30.0%). The strongest clinicopathological correlation with the EGFR mutation was found for histological type (adenocarcinoma; p < 0.01), gender (females; p < 0.01) and FISH positive result (p < 0.05). This is the first, single institution study that estimates the EGFR mutation incidence in the Polish population. Cytological material recovered from fixed preparations and stained with hematoxylin and eosin showed DNA quality comparable to fresh tumor cells and histological samples.

  20. ERβ localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations.

    PubMed

    Wang, Zhijie; Li, Zhenxiang; Ding, Xiaosheng; Shen, Zhirong; Liu, Zhentao; An, Tongtong; Duan, Jianchun; Zhong, Jia; Wu, Meina; Zhao, Jun; Zhuo, Minglei; Wang, Yuyan; Wang, Shuhang; Sun, Yu; Bai, Hua; Wang, Jie

    2015-06-22

    Effects of estrogen receptorβ (ERβ) localization on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC) are unknown. First, we analyzed the relationship between ERβ localization determined by immunohistochemistry and EGFR-TKI outcomes in 184 patients with advanced NSCLC and found that ERβ expression localized in the cytoplasm and/or nucleus. The frequency of cytoplasmic ERβ (c-ERβ) and nuclear ERβ (n-ERβ) co-expression was 12% (22/184). C-ERβ and n-ERβ co-expression was correlated with poor median progression-free survival compared to patients without co-expression. In subsequent in vitro experiments, PC9 cells transfected with ERβ isoform1 (ERβ1, strong expression of both c-ERβ and n-ERβ) were more resistant to gefitinib than PC9 cells transfected with ERβ isoform2 or 5 (ERβ2 or ERβ5, strong expression of ERβ in cytoplasm but not nucleus). Resistance was identified due to interactions between ERβ1 and other isoforms, and mediated by activation of non-genomic pathways. Moreover, gefitinib resistance was reversed by a combination treatment with gefitinib and fulvestrant, both in cell lines and in one NSCLC patient. These results suggested that c-ERβ and n-ERβ co-expression was a potential molecular indicator of EGFR-TKI resistance, which might be overcome by combining EGFR-TKI and ER antagonist.

  1. Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma

    PubMed Central

    Li, Ming; Deng, Fang; Qian, Li-Ting; Meng, Shui-Ping; Zhang, Yang; Shan, Wu-Lin; Zhang, Xiao-Lei; Wang, Bao-Long

    2016-01-01

    Previous studies have demonstrated an association between human papillomavirus (HPV) and mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer patients; however, few studies have investigated this association in advanced lung adenocarcinoma patients undergoing gefitinib treatment. The present study investigated the association between HPV and EGFR mutations in advanced lung adenocarcinoma patients. A total of 95 advanced lung adenocarcinoma patients were enrolled in the study. The HPV infection status and presence of EGFR mutations in tumor tissue was evaluated. Patient clinical characteristics were also determined and compared with HPV infection and EGFR mutation status to analyze their impact on progression-free survival. HPV DNA was identified in 27/95 (28.4%) lung adenocarcinoma tumors and was most common in patients with lymph node metastasis (P=0.016). A total of 44/95 (46.3%) cases exhibited EGFR mutations, which were predominantly observed in female patients and non-smokers. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) and multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma. Patients with both HPV infections and EGFR mutations exhibit a marked decrease in the risk of lung cancer progression when compared with those without HPV infection or EGFR mutations (adjusted HR=0.640; 95% confidence interval: 0.488–0.840; P=0.001). HPV infection was significantly associated with EGFR mutations in advanced lung adenocarcinoma patients. Furthermore, patients with HPV infections exhibited the longest progression-free survival times, which may be due to good response to tyrosine kinase inhibitor- or platinum-based-adjuvant therapy in these patients. Patients with EGFR mutations exhibited a better prognosis when compared with those exhibiting wild-type EGFR, regardless of HPV status. PMID:27602120

  2. Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma

    PubMed Central

    Li, Ming; Deng, Fang; Qian, Li-Ting; Meng, Shui-Ping; Zhang, Yang; Shan, Wu-Lin; Zhang, Xiao-Lei; Wang, Bao-Long

    2016-01-01

    Previous studies have demonstrated an association between human papillomavirus (HPV) and mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer patients; however, few studies have investigated this association in advanced lung adenocarcinoma patients undergoing gefitinib treatment. The present study investigated the association between HPV and EGFR mutations in advanced lung adenocarcinoma patients. A total of 95 advanced lung adenocarcinoma patients were enrolled in the study. The HPV infection status and presence of EGFR mutations in tumor tissue was evaluated. Patient clinical characteristics were also determined and compared with HPV infection and EGFR mutation status to analyze their impact on progression-free survival. HPV DNA was identified in 27/95 (28.4%) lung adenocarcinoma tumors and was most common in patients with lymph node metastasis (P=0.016). A total of 44/95 (46.3%) cases exhibited EGFR mutations, which were predominantly observed in female patients and non-smokers. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) and multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma. Patients with both HPV infections and EGFR mutations exhibit a marked decrease in the risk of lung cancer progression when compared with those without HPV infection or EGFR mutations (adjusted HR=0.640; 95% confidence interval: 0.488–0.840; P=0.001). HPV infection was significantly associated with EGFR mutations in advanced lung adenocarcinoma patients. Furthermore, patients with HPV infections exhibited the longest progression-free survival times, which may be due to good response to tyrosine kinase inhibitor- or platinum-based-adjuvant therapy in these patients. Patients with EGFR mutations exhibited a better prognosis when compared with those exhibiting wild-type EGFR, regardless of HPV status.

  3. [Efficacy of first-line afatinib versus chemotherapy in EGFR mutation positive pulmonary adenocarcinoma].

    PubMed

    Sárosi, Veronika; Balikó, Zoltán

    2014-12-01

    Therapy of patients with advanced NSCLC has lately changed due to the algorithm based on the presence or absence of oncogenic mutations. There is an agreement nowadays that in the presence of activating EGFR mutations, the administration of EGFR TKI (gefitinib, erlotinib, afatinib) is the most efficacious initial treatment. Unlike the first-generation TKIs, afatinib is a new, irreversible ErbB blocker, selectively and effectively blocking signals from the ErbB family receptors. Afatinib's marketing authorization is based on a large, randomized, phase III clinical trial, LUX-Lung 3, where patients in the control arm were treated with the best available chemotherapy (pemetrexed/cisplatin combination). Primary endpoint was progression-free survival (PFS). Patients with common EGFR mutations showed a PFS of 13.6 months when treated with afatinib, while treatment in the control arm resulted in a PFS of 6.9 months. Overall survival (OS) was 31.6 and 28.2 months, respectively. LUX-Lung 3 has been followed by the LUX-Lung 6 trial, comparing afatinib treatment to traditional chemotherapy (gemcitabine/cisplatin) in Asian patients with NSCLC harboring EGFR mutations. This clinical trial has also proved benefit of afatinib: PFS was 11.0 months in the afatinib arm and 5.6 months in the control arm by independent reviewer, while OS was 23.6 months and 23.5 months, respectively. Similarity of the OS values in both trials is explained by the cross-over treatment. When further analyzing OS data, a statistically significant difference between the afatinib and the control arm was seen in the EGFR exon 19 del subgroup (LUX-Lung 3: 33.3 vs. 21.1 months, LUX-Lung 6: 31.4 vs. 18.4 months, respectively). PMID:25517450

  4. Immunostaining with EGFR mutation-specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples.

    PubMed

    Fan, Xiangshan; Liu, Biao; Xu, Haodong; Yu, Bo; Shi, Shanshan; Zhang, Jin; Wang, Xuan; Wang, Jiandong; Lu, Zhenfeng; Ma, Henghui; Zhou, Xiaojun

    2013-08-01

    Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas.

  5. 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

  6. Src inhibitors act through different mechanisms in Non-Small Cell Lung Cancer models depending on EGFR and RAS mutational status

    PubMed Central

    Formisano, Luigi; D'Amato, Valentina; Servetto, Alberto; Brillante, Simona; Raimondo, Lucia; Di Mauro, Concetta; Marciano, Roberta; Orsini, Roberta Clara; Cosconati, Sandro; Randazzo, Antonio; Parsons, Sarah J.; Montuori, Nunzia; Veneziani, Bianca Maria; De Placido, Sabino

    2015-01-01

    Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs. PMID:26325669

  7. Radiotherapy of non-small-cell lung cancer in the era of EGFR gene mutations and EGF receptor tyrosine kinase inhibitors.

    PubMed

    Moschini, Ilaria; Dell'Anna, Cristina; Losardo, Pier Luigi; Bordi, Paola; D'Abbiero, Nunziata; Tiseo, Marcello

    2015-01-01

    Non-small-cell lung cancer (NSCLC) occurs, approximately, in 80-85% of all cases of lung cancer. The majority of patients present locally advanced or metastatic disease when diagnosed, with poor prognosis. The discovery of activating mutations in the EGFR gene has started a new era of personalized treatment for NSCLC patients. To improve the treatment outcome in patients with unresectable NSCLC and, in particular, EGFR mutated, a combined strategy of radiotherapy and medical treatment can be undertaken. In this review we will discuss preclinical data regarding EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) and radiotherapy, available clinical trials investigating efficacy and toxicity of combined treatment (thoracic or whole brain radiotherapy and EGFR-TKIs) and, also, the role of local radiation in mutated EGFR patients who developed EGFR-TKI resistance.

  8. Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation.

    PubMed

    Zhang, Shirong; Zheng, Xiaoliang; Huang, Haixiu; Wu, Kan; Wang, Bing; Chen, Xufeng; Ma, Shenglin

    2015-03-20

    Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.

  9. NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

    PubMed

    Mozzi, Alessandra; Forcella, Matilde; Riva, Alice; Difrancesco, Carlotta; Molinari, Francesca; Martin, Vittoria; Papini, Nadia; Bernasconi, Barbara; Nonnis, Simona; Tedeschi, Gabriella; Mazzucchelli, Luca; Monti, Eugenio; Fusi, Paola; Frattini, Milo

    2015-08-01

    Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation. PMID:25922362

  10. Micropapillary: A component more likely to harbour heterogeneous EGFR mutations in lung adenocarcinomas

    PubMed Central

    Cai, Yi-Ran; Dong, Yu-Jie; Wu, Hong-Bo; Liu, Zi-Chen; Zhou, Li-Juan; Su, Dan; Chen, Xue-Jing; Zhang, Li; Zhao, Ying-Li

    2016-01-01

    The micropapillary (MP) subtype has recently been established to be a distinct marker of poor prognosis in lung adenocarcinomas (LACs). According to the 2015 WHO classification system, LAC constituents are required to be precisely reported. T790M mutation and an insertion in exon 20 (E20ins) are associated with EGFR-TKI resistance. A total of 211 LAC patients were involved in this study, and EGFR mutations were determined using an amplification refractory mutation system (ARMS). Sex, smoking history, lymph node status, and clinical stage differed significantly between the EGFR wild type and mutant groups (p < 0.05). The EGFR mutation occurred more frequently in female, non-smokers, ACs with papillary (85.7%) or MP components (91.4%) (p < 0.001). Twenty ACs with naïve T790M or E20ins were microdissected. The AC constituents metastasizing to lymph nodes exhibited a phenotype and EGFR status that was consistent with the primary loci constituents. Glomerulus-like solid components exhibited the same EGFR status as the surrounding T790M-mutated MP components. The MP and glomerulus-like portions in AC tumours exhibited a congenial EGFR status, but the acinar cells with papillary cells were heterogeneous. The naïve T790M mutants, although minor in the MP component, dramatically increased after EGFR-TKI therapy and indicate that the MP components feature intrinsic heterogeneity. PMID:27046167

  11. Afatinib in Treatment-Naive Patients With EGFR-Mutated Lung Adenocarcinoma With Brain Metastasis: A Case Series.

    PubMed

    Li, Shih-Hong; Hsieh, Meng-Heng; Fang, Yueh-Fu

    2015-10-01

    Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) were previously the standard first-line treatments for lung cancers with activating EGFR mutations. The first-generation reversible EGFR TKIs, gefitinib and erlotinib, demonstrated substantial efficacy in the treatment of brain metastases from EGFR-mutated lung adenocarcinoma. However, the efficacy of afatinib, the second-generation irreversible EGFR TKI, as the first-line treatment in lung adenocarcinoma patients with brain metastasis has yet to be evaluated.Here, we report cases of 3 patients who received afatinib alone as the first-line treatment in combination with whole-brain radiotherapy or following surgical resection of brain metastases. All 3 patients had EGFR L858R mutation. The first patient had lung adenocarcinoma with brain metastasis and no neurologic symptoms. After consultation, she received afatinib as a first-line treatment. Chest computed tomography and brain magnetic resonance imaging (MRI) showed partial response. The second patient had lung adenocarcinoma accompanied with a metastatic brain lesion associated with seizures. This patient received whole-brain radiotherapy and afatinib treatment following brain MRI and subsequently showed significant regression of the brain metastasis. The third patient had strabismus of the right eye, and brain MRI showed a single tumor at the cerebellar pontine angle. This patient underwent surgical resection of the tumor followed by afatinib treatment. He refused adjuvant radiotherapy after surgery for brain metastasis. The brain MRI showed no recurrent brain metastasis, and the patient had relatively less neurologic deficiency.This series of 3 cases indicate that afatinib may be an appropriate first-line treatment alternative in patients having lung adenocarcinoma with EGFR mutations. Further retrospective analyses and prospective clinical trials are required to substantiate the efficacy of afatinib in the treatment of brain

  12. Autocrine epiregulin activates EGFR pathway for lung metastasis via EMT in salivary adenoid cystic carcinoma

    PubMed Central

    Xu, Dongliang; Liao, Yueling; Zhang, Ling; Liu, Liu; Yu, Wenwen; Wang, Yanan; He, Yue; Hu, Jingzhou; Guo, Wenzheng; Wang, Tong; Sun, Beibei; Song, Hongyong; Yin, Huijing; Liu, Jingyi; Wu, Yadi; Zhu, Hanguang; Zhou, Binhua P.; Deng, Jiong; Zhang, Zhiyuan

    2016-01-01

    Salivary adenoid cystic carcinoma (SACC) is characterized by invasive local growth and a high incidence of lung metastasis. Patients with lung metastasis have a poor prognosis. Treatment of metastatic SACC has been unsuccessful, largely due to a lack of specific targets for the metastatic cells. In this study, we showed that epidermal growth factor receptors (EGFR) were constitutively activated in metastatic lung subtypes of SACC cells, and that this activation was induced by autocrine expression of epiregulin (EREG), a ligand of EGFR. Autocrine EREG expression was increased in metastatic SACC-LM cells compared to that in non-metastatic parental SACC cells. Importantly, EREG-neutralizing antibody, but not normal IgG, blocked the autocrine EREG-induced EGFR phosphorylation and the migration of SACC cells, suggesting that EREG-induced EGFR activation is essential for induction of cell migration and invasion by SACC cells. Moreover, EREG-activated EGFR stabilized Snail and Slug, which promoted EMT and metastatic features in SACC cells. Of note, targeting EGFR with inhibitors significantly suppressed both the motility of SACC cells in vitro and lung metastasis in vivo. Finally, elevated EREG expression showed a strong correlation with poor prognosis in head and neck cancer. Thus, targeting the EREG-EGFR-Snail/Slug axis represents a novel strategy for the treatment of metastatic SACC even no genetic EGFR mutation. PMID:26958807

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

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    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 of 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.

  14. The correlation between EGFR mutation status and the risk of brain metastasis in patients with lung adenocarcinoma.

    PubMed

    Li, Bo; Sun, Suo-Zhu; Yang, Ming; Shi, Jian-Ling; Xu, Wei; Wang, Xi-Fan; Song, Mao-Min; Chen, Huo-Ming

    2015-08-01

    To explore the correlation between epidermal growth factor receptor (EGFR) mutation status and the risk of brain metastasis (BM) in patients with lung adenocarcinoma, the clinical data of 100 patients with pathologically confirmed lung adenocarcinoma and known EGFR mutation status at exon 18, 19, 20, or 21 were analyzed retrospectively. The incidence of BM was similar between patients with wild-type EGFR and those with EGFR mutations (p = 0.48). However, among patients with EGFR mutations, the incidence of BM was significantly higher in patients with mutation at exon 19 than in patients with mutation at other sites (p = 0.007). Besides, among patients with heterochronous BM, 66.7 % had EGFR mutations. Regarding brain-metastasis-free survival (BMFS), patients with EGFR sensitive mutations (mutation at exon 19/21/and dual mutation) had significantly shorter BMFS compared with patients with wild-type EGFR (p = 0.018). For patients treated only with chemotherapy, BM was an unfavorable prognostic factor. Patients with BM had worse overall survival compared with those without BM (p = 0.035). However, in patients with BM and EGFR sensitive mutations, those treated with tyrosine kinase inhibitors (TKIs) had significantly longer overall survival compared with those treated with chemotherapy only (p = 0.0081). In conclusion, among patients with EGFR mutations, those mutated at exon 19 had the highest incidence of BM. Furthermore, patients with EGFR mutations are more likely to develop heterochronous BM. The BMFS was significantly shorter in patients with EGFR sensitive mutations. TKIs improved the survival of patients with lung adenocarcinoma and BM who harbored EGFR sensitive mutations.

  15. Evaluation of EGFR mutation status in cytology specimens: an institutional experience.

    PubMed

    Aisner, D L; Deshpande, C; Baloch, Z; Watt, C D; Litzky, L A; Malhotra, B; Sepulveda, A R; Langer, C; Evans, T; Van Deerlin, V M

    2013-04-01

    Epidermal growth factor receptor (EGFR) mutation status has been shown to predict response to anti-EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). In patients with advanced-stage NSCLC, evaluation of mutational status is increasingly requested on biopsy or fine-needle aspiration specimens, which often have limited material. There are limited data on the suitability of cytology cell blocks (CB) for EGFR mutation testing. In this study, we report our institutional experience with cytology cell block material for EGFR mutation testing. We retrospectively reviewed EGFR mutation analyses performed on 234 surgical (SP) and cytology (CB) from October 2007 to May 2010. One hundred ninety-two SP specimens and 42 CB specimens were evaluated for EGFR mutation. CB specimens were evaluated for overall specimen size based on aggregate cellularity in comparison to small biopsy specimens, and percent tumor. Of the 192 SP and 42 CB specimens, 31 (16.1%) and 11 (26.2%) were positive for EGFR mutation, respectively; there does not appear to be an association between mutation detection rate and the source of the specimen (P = 0.124). Limited DNA was obtained from 70.0% (29/42), including 81.8% (9/11) of those which were mutation positive. Additionally, 45.4% (5/11) of mutation positive specimens had extremely low DNA yields. Although 16.6% (7/42) of CB specimens had <10% tumor, all 11 mutation positive CB cases had >10% tumor. These data indicate that CB specimens provide an alternative source for molecular evaluation of NSCLC, and that tumor percentage may be more important than specimen size and/or DNA yield in determining the suitability of these specimens for testing.

  16. Acquired Resistance to EGFR Kinase Inhibitors Associated with a Novel T854A Mutation in a Patient with EGFR-Mutant Lung Adenocarcinoma

    PubMed Central

    Bean, James; Riely, Gregory J.; Balak, Marissa; Marks, Jenifer L.; Ladanyi, Marc; Miller, Vincent A.; Pao, William

    2008-01-01

    Purpose Somatic mutations in the tyrosine kinase domain of EGFR are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance. Experimental Design Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then performed to characterize biological properties of a novel mutant EGFR allele. Results A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R-mutant lung adenocarcinoma after long-term treatment with TKIs. The T854A mutation was not detected in a pretreatment tumor sample. Crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays demonstrate that the EGFR T854A mutation abrogates inhibition of tyrosine phosphorylation by erlotinib. Such resistance appears to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992. Conclusions The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket. PMID:19010870

  17. Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR

    PubMed Central

    Campbell, Patrick; Morton, Penny; Takeichi, Takuya; Salam, Amr; Roberts, Nerys; Proudfoot, Laura E.; Mellerio, Jemima E.; Aminu, Kingi; Wellington, Cheryl; Patil, Sachin N.; Akiyama, Masashi; Liu, Lu; McMillan, James R.; Aristodemou, Sophia; Ishida-Yamamoto, Akemi; Abdul-Wahab, Alya; Petrof, Gabriela; Fong, Kenneth; Harnchoowong, Sarawin; Stone, Kristina; Harper, John I.; McLean, W. H. Irwin; Simpson, Michael A.; Parsons, Maddy; McGrath, John A.

    2014-01-01

    Epidermal growth factor receptor (EGFR) signaling is fundamentally important for tissue homeostasis through EGFR/ligand interactions that stimulate numerous signal transduction pathways. Aberrant EGFR signaling has been reported in inflammatory and malignant diseases but thus far no primary inherited defects in EGFR have been recorded. Using whole-exome sequencing, we identified a homozygous loss-of-function missense mutation in EGFR (c.1283G>A; p.Gly428Asp) in a male infant with life-long inflammation affecting the skin, bowel and lungs. During the first year of life, his skin showed erosions, dry scale, and alopecia. Subsequently, there were numerous papules and pustules – similar to the rash seen in patients receiving EGFR inhibitor drugs. Skin biopsy demonstrated an altered cellular distribution of EGFR in the epidermis with reduced cell membrane labeling, and in vitro analysis of the mutant receptor revealed abrogated EGFR phosphorylation and EGF-stimulated downstream signaling. Microarray analysis on the patient’s skin highlighted disturbed differentiation/premature terminal differentiation of keratinocytes and upregulation of several inflammatory/innate immune response networks. The boy died aged 2.5 years from extensive skin and chest infections as well as electrolyte imbalance. This case highlights the major mechanism of epithelial dysfunction following EGFR signaling ablation and illustrates the broader impact of EGFR inhibition on other tissues. PMID:24691054

  18. EGFR regulates macrophage activation and function in bacterial infection.

    PubMed

    Hardbower, Dana M; Singh, Kshipra; Asim, Mohammad; Verriere, Thomas G; Olivares-Villagómez, Danyvid; Barry, Daniel P; Allaman, Margaret M; Washington, M Kay; Peek, Richard M; Piazuelo, M Blanca; Wilson, Keith T

    2016-09-01

    EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

  19. EGFR regulates macrophage activation and function in bacterial infection.

    PubMed

    Hardbower, Dana M; Singh, Kshipra; Asim, Mohammad; Verriere, Thomas G; Olivares-Villagómez, Danyvid; Barry, Daniel P; Allaman, Margaret M; Washington, M Kay; Peek, Richard M; Piazuelo, M Blanca; Wilson, Keith T

    2016-09-01

    EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis. PMID:27482886

  20. 001. EGFR mutations in Greek patients with lung adenocarcinoma: a multi-center study (preliminary results)

    PubMed Central

    Zarogoulidis, Pavlos; Kallianos, Anastasios; Kerasiotis, Haralampos; Marosis, Konstantinos; Vlastos, Fotios; Veldekis, Ioannis; Aggelidou, Maria; Zarogoulidis, Konstantinos; Rapti, Aggeliki

    2015-01-01

    Background Integration of mutational epidermal growth factor receptor (EGFR) profiling to identify driver alterations in a clinical setting, is necessary to facilitate personalized lung cancer medicine. A tumor genotyping EGFR panel was developed and the Greek Mutation Study was initiated as a prospective tumor genotyping study. This study reports the frequency of driver genetic alterations in Greek lung adenocarcinoma patients, and epidemiology correlations with EGFR genotype. Methods Between November 2012 and June 2014, 211 lung adenocarcinoma patients were included in this study. EGFR mutations were tested in lung cancer tissues and histologic specimen. All patients completed informed consent. The method was high resolution melting analysis (HRMA) about mutations 18-21 and further analysis with method sequencing-ABI prism 3130 sequencer (DNA). Eighteen Surgically resected tissues (8.5%), 174 tumor biopsies (82%) and 19 (9%) body cavity fluids were collected and tested for EGFR mutations. We describe clinical and epidemiological profile of EGFR positive adenocarcinoma patients. Results Genetic EGFR positive mutations were detected in 10.8% (24 of 221) of all patients, fifteen women and nine men. All women were nonsmokers (100%) and seven of nine (78%) men were smokers. Mean age of this group was 64.3 years old. Eighteen patients were with stage IV and rest of them IIIa. None of them was operable. Conclusions This is one report of tumor EGFR positive Greek patients with adenocarcinoma. These data suggest that patients profiling data using a mutational testing platform would be valuable for observation for each group of patients helping clinical doctor for further molecular-targeted therapeutics in lung cancer.

  1. MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells.

    PubMed

    Presutti, Dario; Santini, Simonetta; Cardinali, Beatrice; Papoff, Giuliana; Lalli, Cristiana; Samperna, Simone; Fustaino, Valentina; Giannini, Giuseppe; Ruberti, Giovina

    2015-01-01

    Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation

  2. MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

    PubMed Central

    Presutti, Dario; Santini, Simonetta; Cardinali, Beatrice; Papoff, Giuliana; Lalli, Cristiana; Samperna, Simone; Fustaino, Valentina; Giannini, Giuseppe; Ruberti, Giovina

    2015-01-01

    Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation

  3. A cautionary lesson on the use of targeted methods for EGFR mutation analysis: a case report.

    PubMed

    Walsh, K; Wallace, W A; Butler, R; Mackean, M J; Harrison, D J; Stirling, D; Oniscu, A

    2014-08-01

    Epidermal growth factor receptor (EGFR) mutation analysis is recommended for lung cancer patients prior to the prescription of first-line EGFR tyrosine kinase inhibitors in order to predict response to treatment. There are many methods available to identify mutations in the EGFR gene; a large number of clinical laboratories use the therascreen EGFR RGQ PCR kit (Qiagen). We report a case where this kit detected an exon 19 deletion, predicting sensitivity to tyrosine kinase inhibitors (TKIs), which on further analysis was found to be a 2 bp indel (c.2239_2240delinsCC, p.(Leu747Pro)). Two of four published cases with this mutation were found to be associated with resistance to EGFR TKI. The sample was also tested using two other commercial kits, one of which indicated a deletion. This is a rare mutation making the erroneous detection of a deletion unlikely; however, it is important that clinical laboratories are aware of the potential failings of two commercial kits for EGFR mutation analysis. PMID:24811487

  4. EGFR-tyrosine kinase inhibitor treatment in a patient with advanced non-small cell lung cancer and concurrent exon 19 and 21 EGFR mutations: A case report and review of the literature

    PubMed Central

    YANG, YANG; ZHANG, BIAO; LI, RUTIAN; LIU, BAORUI; WANG, LIFENG

    2016-01-01

    Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered to be effective treatments for advanced non-small cell lung cancer (NSCLC) patients with sensitizing EGFR mutations, including exon 19 deletion and exon 21 L858R mutations. However, with the development of EGFR mutation detection assays, patients with complex EGFR mutations are emerging, and their response to EGFR-TKIs remains unclear. The present study reports a case of a 62-year-old, non-smoking female patient with advanced NSCLC, presenting with concurrent EGFR 19+21 sensitizing mutations, who had a poor response to the first-line EGFR-TKI erlotinib and succumbed 5 months subsequent to diagnosis. Furthermore, the present study performed a literature review, and 18 patients with complex EGFR 19+21 mutations that had received EGFR-TKIs were identified. The majority of these patients responded well to EGFR-TKIs. To the best of our knowledge, the present case is the first to report a patient with lung adenocarcinoma with complex EGFR 19+21 sensitizing mutations that had a poor clinical response to a first-line EGFR-TKI. Despite the 70% response rate of sensitizing EGFR-mutant NSCLCs to EGFR-TKIs, there is still a proportion of patients that experience de novo resistance, and heterogeneity is likely to be important in this resistance mechanism. Therefore, comprehensive genomic detection assays and multi-targeted therapies for patients with NSCLC with complex EGFR mutations require additional investigation. PMID:27123149

  5. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer.

    PubMed

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders; Skov, Birgit G

    2014-12-01

    Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR PCR kit, Qiagen, UK; reference method). For immunohistochemistry, antibodies against exon19 deletions (clone 6B6), exon21 mutations (clone 43B2) from Cell Signaling Technology (Boston, USA) and EGFR variantIII (clone 218C9) from Dako (Copenhagen, DK) were applied. Protein expression was evaluated, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94.4-99.4%). Sensitivity of exon19 antibody was 63.2% (95% confidence interval=38.4-83.7%) and of exon21 antibody was 80.0% (95% confidence interval=44.4-97.5%). Seven exon19 and four exon21 mutations were false negatives (immunohistochemistry negative, RT-PCR positive). Two exon19 and three exon21 mutations were false positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC

  6. Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer

    PubMed Central

    2016-01-01

    EGFR and KRAS mutations are two of the most common mutations that are present in lung cancer. Screening and detecting these mutations are of issue these days, and many different methods and tissue samples are currently used to effectively detect these two mutations. In this study, we aimed to evaluate the testing for EGFR and KRAS mutations by pyrosequencing method, and compared the yield of cytology versus histology specimens in a consecutive series of patients with lung cancer. We retrospectively reviewed EGFR and KRAS mutation results of 399 (patients with EGFR mutation test) and 323 patients (patients with KRAS mutation test) diagnosed with lung cancer in Konkuk University Medical Center from 2008 to 2014. Among them, 60 patients had received both EGFR and KRAS mutation studies. We compared the detection rate of EGFR and KRAS tests in cytology, biopsy, and resection specimens. EGFR and KRAS mutations were detected in 29.8% and 8.7% of total patients, and the positive mutation results of EGFR and KRAS were mutually exclusive. The detection rate of EGFR mutation in cytology was higher than non-cytology (biopsy or resection) materials (cytology: 48.5%, non-cytology: 26.1%), and the detection rate of KRAS mutation in cytology specimens was comparable to non-cytology specimens (cytology: 8.3%, non-cytology: 8.7%). We suggest that cytology specimens are good alternatives that can readily substitute tissue samples for testing both EGFR and KRAS mutations. Moreover, pyrosequencing method is highly sensitive in detecting EGFR and KRAS mutations in lung cancer patients. PMID:27478332

  7. Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer.

    PubMed

    Lee, Seung Eun; Lee, So-Young; Park, Hyung-Kyu; Oh, Seo-Young; Kim, Hee-Joung; Lee, Kye-Young; Kim, Wan-Seop

    2016-08-01

    EGFR and KRAS mutations are two of the most common mutations that are present in lung cancer. Screening and detecting these mutations are of issue these days, and many different methods and tissue samples are currently used to effectively detect these two mutations. In this study, we aimed to evaluate the testing for EGFR and KRAS mutations by pyrosequencing method, and compared the yield of cytology versus histology specimens in a consecutive series of patients with lung cancer. We retrospectively reviewed EGFR and KRAS mutation results of 399 (patients with EGFR mutation test) and 323 patients (patients with KRAS mutation test) diagnosed with lung cancer in Konkuk University Medical Center from 2008 to 2014. Among them, 60 patients had received both EGFR and KRAS mutation studies. We compared the detection rate of EGFR and KRAS tests in cytology, biopsy, and resection specimens. EGFR and KRAS mutations were detected in 29.8% and 8.7% of total patients, and the positive mutation results of EGFR and KRAS were mutually exclusive. The detection rate of EGFR mutation in cytology was higher than non-cytology (biopsy or resection) materials (cytology: 48.5%, non-cytology: 26.1%), and the detection rate of KRAS mutation in cytology specimens was comparable to non-cytology specimens (cytology: 8.3%, non-cytology: 8.7%). We suggest that cytology specimens are good alternatives that can readily substitute tissue samples for testing both EGFR and KRAS mutations. Moreover, pyrosequencing method is highly sensitive in detecting EGFR and KRAS mutations in lung cancer patients.

  8. [Histology-based algorithm in the molecular diagnosis of mutations of the Epidernal Growth Factor Receptor (EGFR) in non-small cell lung cancer].

    PubMed

    Popper, Helmut; Wrba, Fritz; Gruber-Mösenbacher, Ulrike; Hulla, Wolfgang; Pirker, Robert; Hilbe, Wolfgang; Studnicka, Michael; Mohn-Staudner, Andrea; Ploner, Ferdinand

    2011-05-01

    Patients with stage IIIB and IV non-small cell lung carcinoma (NSCLC) harboring an activating mutation of the Epidermal Growth Factor Receptor (EGFR) Gene should be treated first-line with Gefitinib, an EGFR tyrosine kinase inhibitor (TKI). EGF receptor mutations are most common in adenocarcinomas, especially non-mucinous type, rare in squamous cell carcinomas and sarcomatoid carcinomas, and do not occur in neuroendocrine carcinomas. Therefore, the Pulmonary Pathology Working Group of the Austrian Society of Pathology, after intense discussions and in consensus with Oncologists and Pulmonologists, recommends a priori EGFR mutation analysis for all cases of adenocarcinoma, and for all other NSCLC upon clinical request. This will markedly reduce waiting time for those patients, which most likely will have the greatest benefit from EGFR TKI therapy.

  9. The Frequency of EGFR Mutation in Lung Adenocarcinoma and the Efficacy of Tyrosine Kinase Inhibitor Therapy in a Hungarian Cohort of Patients.

    PubMed

    Sárosi, Veronika; Balikó, Zoltán; Smuk, Gábor; László, Terézia; Szabó, Mariann; Ruzsics, István; Mezősi, Emese

    2016-10-01

    In the last decades new therapeutic drugs have been developed for the treatment of non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs) significantly increase the progression free survival (PFS) of patients with NSCLC carrying epidermal growth factor receptor (EGFR) mutations. This type of lung cancer occurs mainly among non-smoking women and Asian origin. However, the new ESMO guideline recommends EGFR mutation analysis in every patient with NSCLC, because in patients with activating EGFR mutation, TKIs should be considered as first line therapy. In our recent work, we analyzed data of patients with EGFR-mutant adenocarcinoma from January 2009. The number of patients investigated was 446, among them 44 cases were positive for EGFR mutation. The ratio of positive cases was 9.86 % that is lower than the average mutation rate in Europe and much lower than that found in Asia. The exon 19 deletion was detected in 61.4 % of the patients, while L858R point mutation in exon 21 was observed in 34.1 % of them. In one subject, both exon 19 and 21 mutations were present simultaneously. A rare mutation located in exon 21 was found in another patient. TKI therapy was conducted in 38 patients. The disease control rate by TKI therapy was 85.7 %; primary resistance was documented in five subjects. Non-smoking patients with EGFR mutant adenocarcinoma had the highest benefit from TKI treatment. Our data support the recommendation that EGFR mutation status should be defined in all cases of locally advanced or metastatic lung adenocarcinoma.

  10. A comparison of methods for EGFR mutation testing in non-small cell lung cancer.

    PubMed

    Young, Elizabeth C; Owens, Martina M; Adebiyi, Idowu; Bedenham, Tina; Butler, Rachel; Callaway, Jonathan; Cranston, Treena; Crosby, Charlene; Cree, Ian A; Dutton, Laura; Faulkes, Catherine; Faulkner, Claire; Howard, Emma; Knight, Julia; Huang, Yuanxue; Lavender, Louise; Lazarou, Lazarus P; Liu, Hongxiang; Mair, Debbie; Milano, Antonio; Sandell, Stacey; Skinner, Alison; Wallace, Andrew; Williams, Maggie; Spivey, Vicky; Goodall, John; Frampton, Jonathan; Ellard, Sian

    2013-12-01

    EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

  11. Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

    PubMed

    Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

    2013-09-01

    A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed. PMID:23828210

  12. Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

    PubMed Central

    McFadden, David G.; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K.; Song, Xiaoling; Pirun, Mono; Santiago, Philip M.; Kim-Kiselak, Caroline; Platt, James T.; Lee, Emily; Hodges, Emily; Rosebrock, Adam P.; Bronson, Roderick T.; Socci, Nicholas D.; Hannon, Gregory J.; Jacks, Tyler; Varmus, Harold

    2016-01-01

    Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. PMID:27702896

  13. Functional EGFR germline polymorphisms may confer risk for EGFR somatic mutations in non-small cell lung cancer, with a predominant effect on exon 19 microdeletions

    PubMed Central

    Liu, Wanqing; He, Lijun; Ramírez, Jacqueline; Krishnaswamy, Soundararajan; Kanteti, Rajani; Wang, Yi-Ching; Salgia, Ravi; Ratain, Mark J

    2011-01-01

    Somatic mutations in the EGFR tyrosine kinase (TK) domain play a critical role in the development and treatment of non-small cell lung cancer (NSCLC). Strong genetic influence on susceptibility to these mutations has been suggested. To identify the genetic factors conferring risk for the EGFR TK mutations in NSCLC, a case-control study was conducted in 141 Taiwanese NSCLC patients by focusing on three functional polymorphisms in the EGFR gene [-216G/T, intron 1(CA)n and R497K]. Allelic imbalance (AI) of the EGFR -216G/T polymorphism was also tested in the heterozygous patients as well as in the NCI-60 cancer cell lines to further verify its function. We found that the frequencies of the alleles -216T and CA-19 are significantly higher in the patients with any mutation (p=0.032 and 0.01, respectively), in particular in those with exon 19 microdeletions (p=0.006 and 0.033, respectively), but not in the patients with L858R mutation. The -216T allele is favored to be amplified in both tumor DNA of lung cancer patients and cancer cell lines. We conclude that the local haplotype structures across the EGFR gene may favor the development of cellular malignancies and thus significantly confer risk to the occurrence of EGFR mutations in NSCLC, particularly the exon 19 microdeletions. PMID:21292812

  14. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases

    PubMed Central

    Lee, Taebum; Lee, Boram; Choi, Yoon-La; Han, Joungho; Ahn, Myung-Ju; Um, Sang-Won

    2016-01-01

    Background: Although epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and anaplastic lymphoma kinase (ALK) mutations in non-small cell lung cancer (NSCLC) were thought to be mutually exclusive, some tumors harbor concomitant mutations. Discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. Methods: In 6,637 patients with NSCLC, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. Clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease-free survival. Histologic features included dominant patterns, nuclear and cytoplasmic features. Results: All patients were diagnosed with adenocarcinoma and had an EGFR mutation. Six patients had concomitant KRAS mutations and the other six had KRAS mutations. Five of six EGFR-KRAS mutation patients showed papillary and acinar histologic patterns with hobnail cells. Three of six received EGFR tyrosine kinase inhibitor (TKI) and showed partial response for 7–29 months. All six EGFR-ALK mutation patients showed solid or cribriform patterns and three had signet ring cells. Five of six EGFR-ALK mutation patients received EGFR TKI and/or ALK inhibitor and four showed partial response or stable disease, except for one patient who had acquired an EGFR mutation. Conclusions: EGFR and ALK mutations play an important role as driver mutations in double mutated NSCLC, and morphologic analysis can be used to predict treatment response. PMID:27086595

  15. EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

    PubMed

    Giannini, Riccardo; Lupi, Cristiana; Sensi, Elisa; Alì, Greta; Proietti, Agnese; Boldrini, Laura; Servadio, Adele; Giordano, Mirella; Macerola, Elisabetta; Bruno, Rossella; Borrelli, Nicla; Chella, Antonio; Melfi, Franca; Lucchi, Marco; Ribechini, Alessandro; Vasile, Enrico; Cappuzzo, Federico; Mussi, Alfredo; Fontanini, Gabriella

    2016-08-01

    Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large‑scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix‑assisted laser desorption ionization‑time of flight (MALDI‑TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI‑TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI‑TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI‑TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time. PMID:27373829

  16. Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers

    PubMed Central

    Nomura, Masaharu; Shigematsu, Hisayuki; Li, Lin; Suzuki, Makoto; Takahashi, Takao; Estess, Pila; Siegelman, Mark; Feng, Ziding; Kato, Harubumi; Marchetti, Antonio; Shay, Jerry W; Spitz, Margaret R; Wistuba, Ignacio I; Minna, John D; Gazdar, Adi F

    2007-01-01

    Background The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers. Methods and Findings We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were

  17. Proteomic profiling of patient-derived glioblastoma xenografts identifies a subset with activated EGFR: implications for drug development.

    PubMed

    Brown, Kristine E; Chagoya, Gustavo; Kwatra, Shawn G; Yen, Timothy; Keir, Stephen T; Cooter, Mary; Hoadley, Katherine A; Rasheed, Ahmed; Lipp, Eric S; Mclendon, Roger; Ali-Osman, Francis; Bigner, Darell D; Sampson, John H; Kwatra, Madan M

    2015-06-01

    The development of drugs to inhibit glioblastoma (GBM) growth requires reliable pre-clinical models. To date, proteomic level validation of widely used patient-derived glioblastoma xenografts (PDGX) has not been performed. In the present study, we characterized 20 PDGX models according to subtype classification based on The Cancer Genome Atlas criteria, TP53, PTEN, IDH 1/2, and TERT promoter genetic analysis, EGFR amplification status, and examined their proteomic profiles against those of their parent tumors. The 20 PDGXs belonged to three of four The Cancer Genome Atlas subtypes: eight classical, eight mesenchymal, and four proneural; none neural. Amplification of EGFR gene was observed in 9 of 20 xenografts, and of these, 3 harbored the EGFRvIII mutation. We then performed proteomic profiling of PDGX, analyzing expression/activity of several proteins including EGFR. Levels of EGFR phosphorylated at Y1068 vary considerably between PDGX samples, and this pattern was also seen in primary GBM. Partitioning of 20 PDGX into high (n = 5) and low (n = 15) groups identified a panel of proteins associated with high EGFR activity. Thus, PDGX with high EGFR activity represent an excellent pre-clinical model to develop therapies for a subset of GBM patients whose tumors are characterized by high EGFR activity. Further, the proteins found to be associated with high EGFR activity can be monitored to assess the effectiveness of targeting EGFR. The development of drugs to inhibit glioblastoma (GBM) growth requires reliable pre-clinical models. We validated proteomic profiles using patient-derived glioblastoma xenografts (PDGX), characterizing 20 PDGX models according to subtype classification based on The Cancer Genome Atlas (TCGA) criteria, TP53, PTEN, IDH 1/2, and TERT promoter genetic analysis, EGFR amplification status, and examined their proteomic profiles against those of their parent tumors. Proteins found to be associated with high EGFR activity represent potential

  18. EGFR Somatic Mutations in Lung Tumors: Radon Exposure and Passive-smoking in Former- and Never-smoking U.S. Women

    PubMed Central

    Taga, Masataka; Mechanic, Leah E.; Hagiwara, Nobutoshi; Vähäkangas, Kirsi H.; Bennett, William P.; Alavanja, Michael C. R.; Welsh, Judith A.; Khan, Mohammed A.; Lee, Adam; Diasio, Robert; Edell, Eric; Bungum, Aaron; Jang, Jin Sung; Yang, Ping; Jen, Jin; Harris, Curtis C.

    2012-01-01

    Background Lung cancer patients with mutations in EGFR tyrosine kinase have improved prognosis when treated with EGFR inhibitors. We hypothesized that EGFR mutations may be related to residential radon or passive tobacco smoke. Methods This hypothesis was investigated by analyzing EGFR mutations in seventy lung tumors from a population of never and long-term former female smokers from Missouri with detailed exposure assessments. The relationship with passive-smoking was also examined in never-smoking female lung cancer cases from the Mayo clinic. Results Overall, the frequency of EGFR mutation was 41% [95% Confidence Interval (CI): 32-49%]. Neither radon nor passive-smoking exposure was consistently associated with EGFR mutations in lung tumors. Conclusions The results suggest that EGFR mutations are common in female, never-smoking, lung cancer cases from the U.S, and EGFR mutations are unlikely due to exposure to radon or passive-smoking. PMID:22523180

  19. Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond

    PubMed Central

    Rogers, Toni-Maree; Fellowes, Andrew; Bell, Anthony; Fox, Stephen

    2015-01-01

    Somatic mutational profiling in cancer has revolutionized the practice of clinical oncology. The discovery of driver mutations in non-small cell lung cancer (NSCLC) is an example of this. Molecular testing of lung adenocarcinoma is now considered standard of care and part of the diagnostic algorithm. This article provides an overview of the workflow of molecular testing in a clinical diagnostic laboratory discussing in particular novel assays that are currently in use for somatic mutation detection in NSCLC focussing on epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK), ROS1 and RET rearrangements. PMID:25870795

  20. EGFR kinase domain mutation positive lung cancers are sensitive to intrapleural perfusion with hyperthermic chemotherapy (IPHC) complete treatment

    PubMed Central

    Zhang, Hongjuan; Zhan, Cheng; Ke, Ji; Xue, Zhiqiang; Zhang, Aiqun; Xu, Kaifeng; Shen, Zhirong; Yu, Lei; Chen, Liang

    2016-01-01

    Lung cancer is the global leading cause of cancer-related deaths. A significant portion of lung cancer patients harbor kinase domain mutations in the epidermal growth factor receptor (EGFR). While EGFR tyrosine kinase inhibitors (TKI) effectively shrink tumors harboring mutant EGFR, clinical efficacy is limited by the development of TKI resistance. Effective alternatives are desperately needed in clinic for treating EGFR kinase domain mutation positive lung cancer. In our clinic in treating M1a lung cancer patients through intrapleural perfusion with hyperthermic chemotherapy (IPHC) followed by cycles of systemic chemotherapy (we termed this procedure IPHC complete treatment, IPHC-CT), we found dramatic tumor shrinkage in mutant EGFR-positive patients. We further confirmed the sensitivity of EGFR mutation-positive lung cancer cell lines derived from patients to HC (hyperthermic chemotherapy) treatment. We found that hyperthermia promoted accumulation of cisplatin in lung cancer cells. Hyperthermia and cisplatin synergistically downregulated the EGFR protein level, leading to quenching of signal from EGFR and induction of apoptosis. Our work therefore showed IPHC-CT is an effective treatment for EGFR kinase domain mutation positive lung cancer patients. PMID:26654941

  1. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

    SciTech Connect

    Yun, C.H.; Mengwasser, K.E.; Toms, A.V.; Woo, M.S.; Greulich, H.; Wong, K.K.; Meyerson, M.; Eck, M.J.

    2008-07-15

    Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the 'gatekeeper' residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a 'generic' resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

  2. [Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer].

    PubMed

    Shuai, Sun; Yuliang, Deng

    2015-12-01

    Circulating tumor cells (CTCs) are cells that shed from a primary tumor and enter the peripheral blood circulation. The CTCs are closely associated with tumor development and metastasis because of its high heterogeneity. However, there are still no effective methods to detect single-cell heterogeneity of the CTCs. To this end, we developed a method to detect gene mutation in CTCs at the single-cell level and applied it to the detection of EGFR gene mutation in single lung cancer CTC. Specifically, the rare CTCs were captured from blood using an integrated microfluidic system, and then were released into a microchip with thousands of nanoliter wells to isolate single CTC. The single CTC was then transferred into a PCR tube under the microscope for single-cell genome amplification and detection of EGFR gene mutation. We firstly modified chip and capillary and optimized PCR conditions (annealing temperature, number of cycles) using non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H1650 and NCI-H1975 as samples, which showed maximal amplification after 30 cycles with an annealing temperature at 59℃. We then successfully detected blood samples from NSCLC patients using this method. 5 CTCs were obtained from 2 mL patient's blood and the sequencing of EGFR exons 18, 19, 20 and 21 showed no mutations. Our results demonstrated that this method is sensitive enough to detect gene mutation in single CTC and has guiding significance in clinic research. PMID:26704950

  3. Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion.

    PubMed

    Whitsett, Timothy G; Cheng, Emily; Inge, Landon; Asrani, Kaushal; Jameson, Nathan M; Hostetter, Galen; Weiss, Glen J; Kingsley, Christopher B; Loftus, Joseph C; Bremner, Ross; Tran, Nhan L; Winkles, Jeffrey A

    2012-07-01

    Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs. PMID:22634180

  4. IGF-1R/epithelial-to-mesenchymal transition (EMT) crosstalk suppresses the erlotinib-sensitizing effect of EGFR exon 19 deletion mutations

    PubMed Central

    Vazquez-Martin, Alejandro; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Torres-Garcia, Violeta Zenobia; Corominas-Faja, Bruna; Cuyàs, Elisabet; Bonavia, Rosa; Visa, Joana; Martin-Castillo, Begoña; Barrajón-Catalán, Enrique; Micol, Vicente; Bosch-Barrera, Joaquim; Menendez, Javier A.

    2013-01-01

    Using non-small cell lung carcinoma (NSCLC) cells harboring the erlotinib-sensitizing Epidermal Growth Factor Receptor (EGFR) exon 19 mutation delE746-A750, we developed erlotinib-refractory derivatives in which hyperactive Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling associated with enrichment in epithelial-to-mesenchymal transition (EMT)-related morphological and transcriptional features. We then explored whether an IGF-1R/EMT crosstalk was sufficient to promote erlotinib refractoriness in the absence of second-site EGFR mutations, MET and AXL hyperactivation. Transforming Growth Factor-beta1 (TGFβ1)-induced mesenchymal trans-differentiation was sufficient to impede erlotinib functioning in the presence of drug-sensitive delE746-A750 EGFR mutation. Pharmacological blockade of IGF-1R fully prevented the TGFβ1's ability to activate an EMT protein signature [E-cadherin low/vimentin high]. The sole presence of erlotinib was capable of rapidly activate an IGF-1R-dependent, vimentin-enriched mesenchymal-like phenotype in delE746-A750-mutated epithelial cells. Even if transient, NSCLC cells' intrinsic plasticity to undergo crosstalk between IGF-1R and EMT signaling pathways can sufficiently eliminate the erlotinib-sensitizing effect of highly prevalent EGFR mutations and suggests the urgent need for dual IGF-1R/EMT-targeting strategies to circumvent erlotinib resistance. PMID:23994953

  5. Detection of the EGFR mutation in exhaled breath condensate from a heavy smoker with squamous cell carcinoma of the lung.

    PubMed

    Zhang, Dan; Takigawa, Nagio; Ochi, Nobuaki; Tanimoto, Yasushi; Noujima, Daisuke; Chen, Yan Yan; Tanimoto, Mitsune; Kiura, Katsuyuki

    2011-09-01

    A 61-year-old male smoker (40 pack-years) presented with right chest pain. Computed tomography of the chest revealed a cavitary mass in the right lower lobe. A transbronchial biopsy showed squamous cell carcinoma. We examined epidermal growth factor receptor (EGFR) mutations in exhaled breath condensate (EBC). The DNA extracted from his EBC showed a deletion mutation in exon 19. Subsequently, the del E746-A750 mutation in exon 19 in a transbronchial tissue specimen was confirmed. Although he underwent whole-brain irradiation against multiple brain metastases, he had paralysis of the left side of the body and his performance status was 3. The patient was treated with gefitinib. He had marked tumor regression and no symptoms. Although only a small percentage of heavy smokers with squamous cell carcinoma harbor EGFR mutations, they probably benefit from EGFR-tyrosine kinase inhibitors. EGFR mutation status in the patients having such clinical features might be examined. PMID:21684624

  6. Immunohistochemical staining with EGFR mutation-specific antibodies: high specificity as a diagnostic marker for lung adenocarcinoma.

    PubMed

    Wen, Yong Hannah; Brogi, Edi; Hasanovic, Adnan; Ladanyi, Marc; Soslow, Robert A; Chitale, Dhananjay; Shia, Jinru; Moreira, Andre L

    2013-09-01

    We previously demonstrated a high specificity of immunohistochemistry using epidermal growth factor receptor (EGFR) mutation-specific antibodies in lung adenocarcinoma and correlation with EGFR mutation analysis. In this study, we assessed EGFR mutation status by immunohistochemistry in a variety of extrapulmonary malignancies, especially those that frequently show EGFR overexpression. Tissue microarrays containing triplicate cores of breast carcinomas (n=300), colorectal carcinomas (n=65), pancreatic adenocarcinoma (n=145), and uterine carcinosarcoma or malignant mixed müllerian tumors (n=25) were included in the study. Tissue microarray of lung adenocarcinoma with known EGFR mutation status was used as reference. Immunohistochemistry was performed using antibodies specific for the E746-A750del and L858R mutations. In pulmonary adenocarcinoma, a staining intensity of 2+ or 3+ correlates with mutation status and is therefore considered as positive. Out of 300 breast carcinomas, 293 (98%) scored 0, 5 (2%) had 1+ staining, 2 (1%) were 2+ for the L858R antibody. All breast carcinomas scored 0 with the E746-A750 antibody. All the colorectal, pancreatic carcinomas and malignant mixed müllerian tumors were negative (0) for both antibodies. Molecular analysis of the breast carcinomas that scored 2+ for L858R showed no mutation. Our results show that EGFR mutation-specific antibodies could be an additional tool distinguishing primary versus metastatic carcinomas in the lung. False-positivity can be seen in breast carcinoma but is extremely rare (1%).

  7. Celecoxib-erlotinib combination delays growth and inhibits angiogenesis in EGFR-mutated lung cancer

    PubMed Central

    Li, Yi Xiao; Wang, Jia Le; Gao, Meng; Tang, Hao; Gui, Rong; Fu, Yun Feng

    2016-01-01

    Combination treatment for non-small cell lung cancer (NSCLC) is becoming more popular due to the anticipation that it may be more effective than single drug treatment. In addition, there are efforts to genetically screen patients for specific mutations in light of attempting to administer specific anticancer agents that are most effective. In this study, we evaluate the anticancer and anti-angiogenic effects of low dose celecoxib-erlotinib combination in NSCLC in vitro and in vivo. In NSCLC cells harboring epidermal growth factor receptor (EGFR) mutations, combination celecoxib-erlotinib treatment led to synergistic cell death, but there was minimal efficacy in NSCLC cells with wild-type EGFR. In xenograft models, combination treatment also demonstrated greater inhibition of tumor growth compared to individual treatment. The anti-tumor effect observed was secondary to the targeting of angiogenesis, evidenced by decreased vascular endothelial growth factor A (VEGFA) levels and decreased levels of CD31 and microvessel density. Combination treatment targets angiogenesis through the modulation of of the PI3K/AKT and ERK/Raf1-1 pathway in NSCLC with EGFR exon 19 deletions. These findings may have significant clinical implications in patients with tumors harboring EGFR exon 19 deletions as they may be particularly sensitive to this regimen. PMID:27508092

  8. Celecoxib-erlotinib combination delays growth and inhibits angiogenesis in EGFR-mutated lung cancer.

    PubMed

    Li, Yi Xiao; Wang, Jia Le; Gao, Meng; Tang, Hao; Gui, Rong; Fu, Yun Feng

    2016-01-01

    Combination treatment for non-small cell lung cancer (NSCLC) is becoming more popular due to the anticipation that it may be more effective than single drug treatment. In addition, there are efforts to genetically screen patients for specific mutations in light of attempting to administer specific anticancer agents that are most effective. In this study, we evaluate the anticancer and anti-angiogenic effects of low dose celecoxib-erlotinib combination in NSCLC in vitro and in vivo. In NSCLC cells harboring epidermal growth factor receptor (EGFR) mutations, combination celecoxib-erlotinib treatment led to synergistic cell death, but there was minimal efficacy in NSCLC cells with wild-type EGFR. In xenograft models, combination treatment also demonstrated greater inhibition of tumor growth compared to individual treatment. The anti-tumor effect observed was secondary to the targeting of angiogenesis, evidenced by decreased vascular endothelial growth factor A (VEGFA) levels and decreased levels of CD31 and microvessel density. Combination treatment targets angiogenesis through the modulation of of the PI3K/AKT and ERK/Raf1-1 pathway in NSCLC with EGFR exon 19 deletions. These findings may have significant clinical implications in patients with tumors harboring EGFR exon 19 deletions as they may be particularly sensitive to this regimen. PMID:27508092

  9. Structural signature of the G719S-T790M double mutation in the EGFR kinase domain and its response to inhibitors

    NASA Astrophysics Data System (ADS)

    George Priya Doss, C.; Rajith, B.; Chakraborty, Chiranjib; Nagasundaram, N.; Ali, Shabana Kouser; Zhu, Hailong

    2014-08-01

    Some individuals with non-small-cell lung cancer (NSCLC) benefit from therapies targeting epidermal growth factor receptor (EGFR), and the characterization of a new mechanism of resistance to the EGFR-specific antibody gefitinib will provide valuable insight into how therapeutic strategies might be designed to overcome this particular resistance mechanism. The G719S and T790M mutations and their combination were involved in causing different conformational redistribution of EGFR. In the present computational study, we analyzed the impact and structural influence of G719S/T790M double mutation (DM) in EGFR with ligand (gefitinib) through molecular dynamic simulation (50 ns) and docking analysis. We observed the escalation in distance between the functional loop and activation loop with respect to T790M mutation compared to the G719S mutation. Furthermore, we confirmed that the G719S mutation causes the ligand to move closer to the hinge region, whereas T790M makes the ligand escape from the binding pocket. Obtained results provide with an explanation for the resistance induced by T790M and a vital clue for the design of drugs to combat gefitinib resistance.

  10. BIM Gene Polymorphism Lowers the Efficacy of EGFR-TKIs in Advanced Nonsmall Cell Lung Cancer With Sensitive EGFR Mutations: A Systematic Review and Meta-Analysis.

    PubMed

    Huang, Wu Feng; Liu, Ai Hua; Zhao, Hai Jin; Dong, Hang Ming; Liu, Lai Yu; Cai, Shao Xi

    2015-08-01

    The strong association between bcl-2-like 11 (BIM) triggered apoptosis and the presence of epidermal growth factor receptor (EGFR) mutations has been proven in nonsmall cell lung cancer (NSCLC). However, the relationship between EGFR-tyrosine kinase inhibitor's (TKI's) efficacy and BIM polymorphism in NSCLC EGFR is still unclear.Electronic databases were searched for eligible literatures. Data on objective response rates (ORRs), disease control rates (DCRs), and progression-free survival (PFS) stratified by BIM polymorphism status were extracted and synthesized based on random-effect model. Subgroup and sensitivity analyses were conducted.A total of 6 studies that involved a total of 773 EGFR mutant advanced NSCLC patients after EGFR-TKI treatment were included. In overall, non-BIM polymorphism patients were associated with significant prolonged PFS (hazard ratio 0.63, 0.47-0.83, P = 0.001) compared to patients with BIM polymorphism. However, only marginal improvements without statistical significance in ORR (odds ratio [OR] 1.71, 0.91-3.24, P = 0.097) and DCR (OR 1.56, 0.85-2.89, P = 0.153) were observed. Subgroup analyses showed that the benefits of PFS in non-BIM polymorphism group were predominantly presented in pooled results of studies involving chemotherapy-naive and the others, and retrospective studies. Additionally, we failed to observe any significant benefit from patients without BIM polymorphism in every subgroup for ORR and DCR.For advanced NSCLC EGFR mutant patients, non-BIM polymorphism ones are associated with longer PFS than those with BIM polymorphism after EGFR-TKIs treatment. BIM polymorphism status should be considered an essential factor in studies regarding EGFR-targeted agents toward EGFR mutant patients.

  11. Sea-urchin-like Au nanocluster with surface-enhanced raman scattering in detecting epidermal growth factor receptor (EGFR) mutation status of malignant pleural effusion.

    PubMed

    Wang, Lei; Guo, Ting; Lu, Qiang; Yan, Xiaolong; Zhong, Daixing; Zhang, Zhipei; Ni, Yunfeng; Han, Yong; Cui, Daxiang; Li, Xiaofei; Huang, Lijun

    2015-01-14

    Somatic mutations in the epidermal growth factor receptor (EGFR) gene are common in patients with lung adenocarcinomas and are associated with sensitivity to the small-molecule tyrosine kinase inhibitors (TKIs). For 10%-50% of the patients who experienced malignant pleural effusion (MPE), pathological diagnosis might rely exclusively on finding lung cancer cells in the MPE. Current methods based on polymerase chain reaction were utilized to test EGFR mutation status of MPE samples, but the accuracy of the test data was very low, resulting in many patients losing the chance of TKIs treatment. Herein, we synthesized the sea-urchin-like Au nanocluster (AuNC) with an average diameter of 92.4 nm, composed of 15-nm nanopricks. By introducing abundant sharp nanopricks, the enhancement factor of AuNC reached at 1.97 × 10(7). After capped with crystal violet (CV), polyethylene glycol, and EGFR mutation specific antibody, the AuNC-EGFR had excellent surface-enhanced Raman scattering (SERS) activity and EGFR mutation targeted recognition capability in lung cancer cells. Characteristic SERS signal at 1617 cm(-1) of CV was linear correlation with the number of H1650 cells, demonstrating the minimum detection limit as 25 cells in a 1-mL suspension. The gold mass in single H1650 cells exposed to AuNC-E746_750 for 2 h ranged from 208.6 pg to 231.4 pg, which approximately corresponded to 56-62 AuNCs per cell. Furthermore, SERS was preclinically utilized to test EGFR mutation status in MPE samples from 35 patients with lung adenocarcinoma. Principal component analysis (PCA) and the support vector machine (SVM) algorithm were constructed for EGFR mutation diagnostic analysis, yielding an overall accuracy of 90.7%. SERS measurement based on sea-urchin-like AuNC was an efficient method for EGFR mutation detection in MPE, and it might show great potential in applications such as predicting gene typing of clinical lung cancer in the near future.

  12. EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation.

    PubMed

    Yamada, Teppei; Azuma, Koichi; Muta, Emi; Kim, Jintaek; Sugawara, Shunichi; Zhang, Guang Lan; Matsueda, Satoko; Kasama-Kawaguchi, Yuri; Yamashita, Yuichi; Yamashita, Takuto; Nishio, Kazuto; Itoh, Kyogo; Hoshino, Tomoaki; Sasada, Tetsuro

    2013-01-01

    Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non-small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR

  13. Different clinical effects upon separate inhibition of coexisting EGFR and PI3KCA mutations in a lung adenocarcinoma patient.

    PubMed

    Tiseo, Marcello; Bersanelli, Melissa; Perrone, Federica; Tamborini, Elena; Settanni, Giulio; Busico, Adele; Rossi, Giulio; Ardizzoni, Andrea; Pelosi, Giuseppe

    2015-02-01

    We present a very interesting case of lung adenocarcinoma carrying an uncommon EGFR exon 19 insertion with concomitant PIK3CA mutation showing dramatic and persisting improvement upon erlotinib therapy, after progression during PI3K inhibitor. PMID:25555368

  14. Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation

    PubMed Central

    Kim, Nayoung; Cho, Ahye; Watanabe, Hideo; Choi, Yoon-La; Aziz, Meraj; Kassner, Michelle; Joung, Je-Gun; Park, Angela KJ; Francis, Joshua M.; Bae, Joon Seol; Ahn, Soo-min; Kim, Kyoung-Mee; Park, Joon Oh; Park, Woong-Yang; Ahn, Myung-Ju; Park, Keunchil; Koo, Jaehyung; Yin, Hongwei Holly; Cho, Jeonghee

    2016-01-01

    Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients. PMID:26883194

  15. Determination of HER2 and p53 Mutations by Sequence Analysis Method and EGFR/Chromosome 7 Gene Status by Fluorescence in Situ Hybridization for the Predilection of Targeted Therapy Modalities in Immunohistochemically Triple Negative Breast Carcinomas in Turkish Population.

    PubMed

    Pala, Emel Ebru; Bayol, Umit; Keskin, Elif Usturali; Ozguzer, Alp; Kucuk, Ulku; Ozer, Ozge; Koc, Altug

    2015-09-01

    Triple negative breast cancer (TNBC), an agressive subtype accounts nearly 15 % of all breast carcinomas. Conventional chemotherapy is the only treatment modality thus new, effective targeted therapy methods have been investigated. Epidermal growth factor receptor (EGFR) inhibitors give hope according to the recent studies results. Also therapeutic agents have been tried against aberrant p53 signal activity as TNBC show high p53 mutation rates. Our aim was to detect the incidence of mutations/amplifications identified in TNBC in our population. Here we used sequence analysis to detect HER2 (exon 18-23), p53 (exon 5-8) mutations; fluorescence in situ hybridization (FISH) method to analyse EGFR/chromosome 7 centromere gene status in 82 immunohistochemically TNBC. Basaloid phenotype was identified in 49 (59.8 %) patients. EGFR amplification was noted in 5 cases (6.1 %). All EGFR amplified cases showed EGFR overexpression by immunohistochemistry (IHC). p53 mutations were identified in 33 (40.2 %) cases. Almost 60 % of the basal like breast cancer cases showed p53 mutation. Only one case showed HER2 mutation (exon 20:g.36830_3). Our results showed that gene amplification is not the unique mechanism in EGFR overexpression. IHC might be used in the decision of anti-EGFR therapy in routine practice. p53 mutation rate was lower than the rates reported in the literature probably due to ethnic differences and low sensitivity of sanger sequences in general mutation screening. We also established the rarity of HER2 mutation in TNBC. In conclusion EGFR and p53 are the major targets in TNBC also for our population.

  16. EGFR mutation status in brain metastases of non-small cell lung carcinoma.

    PubMed

    Burel-Vandenbos, Fanny; Ambrosetti, Damien; Coutts, Michael; Pedeutour, Florence

    2013-01-01

    Brain metastases are a frequent and grave complication of non-small cell lung carcinoma (NSCLC). The prognosis is generally poor, despite standard therapy based on surgery and radiotherapy. A degree of understanding of the molecular basis of tumors has led to the development of targeted agents with promising initial findings for the treatment of NSCLC. EGFR mutations have been identified which are associated with significant sensitivity to EGFR tyrosine kinase inhibitors (TKI) and correlate with improved outcome in patients with NSCLC who are treated with these agents. The adoption of treatment tailored to the genetic make-up of individual tumors could lead to substantial therapeutic improvements, and such targeted therapy might be considered as a therapeutic option for brain metastases in the future. We review current knowledge about EGFR mutation status in the specific context of brain metastasis: its association with the response of brain metastases to TKI, its prevalence in brain metastases, and the correlation between mutation status in metastases as compared to the corresponding primary lung carcinoma. PMID:23086434

  17. EGFR Mutation Positive Stage IV Non-Small-Cell Lung Cancer: Treatment Beyond Progression

    PubMed Central

    Van Assche, Katrijn; Ferdinande, Liesbeth; Lievens, Yolande; Vandecasteele, Katrien; Surmont, Veerle

    2014-01-01

    Non-small-cell lung cancer (NSCLC) is the leading cause of death from cancer for both men and women. Chemotherapy is the mainstay of treatment in advanced disease, but is only marginally effective. In about 30% of patients with advanced NSCLC in East Asia and in 10–15% in Western countries, epidermal growth factor receptor (EGFR) mutations are found. In this population, first-line treatment with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib, or afatinib is recommended. The treatment beyond progression is less well-defined. In this paper, we present three patients, EGFR mutation positive, with local progression after an initial treatment with TKI. These patients were treated with local radiotherapy. TKI was temporarily stopped and restarted after radiotherapy. We give an overview of the literature and discuss the different treatment options in case of progression after TKI: TKI continuation with or without chemotherapy, TKI continuation with local therapy, alternative dosing or switch to next-generation TKI or combination therapy. There are different options for treatment beyond progression in EGFR mutation positive metastatic NSCLC, but the optimal strategy is still to be defined. Further research on this topic is ongoing. PMID:25538894

  18. EGFR mediates hyperlipidemia-induced renal injury via regulating inflammation and oxidative stress: the detrimental role and mechanism of EGFR activation

    PubMed Central

    Fang, Qilu; Zou, Chunpeng; Zhong, Peng; Lin, Feng; Li, Weixin; Wang, Lintao; Zhang, Yali; Zheng, Chao; Wang, Yi; Li, Xiaokun; Liang, Guang

    2016-01-01

    Previous studies have implicated inflammation, oxidative stress, and fibrosis as key factors in the development of obesity-induced kidney diseases. Epidermal growth factor receptor (EGFR) plays an important role in cancer development. Recently, the EGFR pathway has been increasingly implicated in chronic cardiovascular diseases via regulating inflammation and oxidative stress. However, it is unclear if EGFR is involved in obesity-related kidney injury. Using ApoE−/− and C57BL/6 mice models and two specific EGFR inhibitors, we investigated the potential effects of EGFR inhibition in the treatment of obesity-related nephropathy and found that EGFR inhibition alleviates renal inflammation, oxidative stress and fibrosis. In NRK-52E cells, we also elucidated the mechanism behind hyperlipidemia-induced EGFR activation. We observed that c-Src and EGFR forms a complex, and following PA stimulation, it is the successive phosphorylation, not formation, of the c-Src/EGFR complex that results in the subsequent cascade activation. Second, we found that TLR4 regulates the activation EGFR pathway mainly through the phosphorylation of the c-Src/EGFR complex. These results demonstrate the detrimental role of EGFR in the pathogenesis of obesity-related nephropathy, provide a new understanding of the mechanism behind hyperlipidemia/FFA-induced EGFR activation, and support the use of EGFR inhibitors in the treatment of obesity-induced kidney diseases. PMID:27014908

  19. Can EGFR mutation status be reliably determined in pre-operative needle biopsies from adenocarcinomas of the lung?

    PubMed

    Lindahl, Kim Hein; Sørensen, Flemming Brandt; Jonstrup, Søren Peter; Olsen, Karen Ege; Loeschke, Siegfried

    2015-04-01

    The identification of EGFR mutations in non-small-cell lung cancer is important for selecting patients, who may benefit from treatment with EGFR tyrosine kinase inhibitors. The analysis is usually performed on cytological aspirates and/or histological needle biopsies, representing a small fraction of the tumour volume. The aim of the present investigation was to evaluate the diagnostic performance of this molecular test. We retrospectively included 201 patients with primary adenocarcinoma of the lung. EGFR mutation status (exon 19 deletions and exon 21 L858R point mutation) was evaluated on both pre-operative biopsies (131 histological and 70 cytological) and on the surgical specimens, using PCR. Samples with low tumour cell fraction were assigned to laser micro-dissection (LMD). We found nine (4.5%) patients with EGFR mutation in the lung tumour resections, but failed to identify mutation in one of the corresponding pre-operative, cytological specimens. Several (18.4%) analyses of the pre-operative biopsies were inconclusive, especially in case of biopsies undergoing LMD and regarding exon 21 analysis. Discrepancy of mutation status in one patient may reflect intra-tumoural heterogeneity or technical issues. Moreover, several inconclusive results in the diagnostic biopsies reveal that attention must be paid on the suitability of pre-operative biopsies for EGFR mutation analysis.

  20. The Role of PIK3CA Mutations among Lung Adenocarcinoma Patients with Primary and Acquired Resistance to EGFR Tyrosine Kinase Inhibition

    PubMed Central

    Wu, Shang-Gin; Chang, Yih-Leong; Yu, Chong-Jen; Yang, Pan-Chyr; Shih, Jin-Yuan

    2016-01-01

    To understand the impact of PIK3CA mutations on clinical characteristics and treatment response to epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs) of lung adenocarcinoma, we examined PIK3CA and EGFR mutations in lung adenocarcinoma patients, and analyzed their clinical outcomes. Surgically excised tumor, bronchoscopy biopsy/brushing specimens and pleural effusions were prospectively collected from 1029 patients. PIK3CA and EGFR mutations were analyzed by RT-PCR and direct sequencing. In EGFR TKI-nave specimens, PIK3CA mutation rate was 1.8% (14/760). Twelve patients had coexisting PIK3CA and EGFR mutations. Among the 344 EGFR TKI-treated EGFR mutant patients, there was no significant difference in treatment response (p = 0.476) and progression-free survival (p = 0.401) of EGFR TKI between PIK3CA mutation-positive and negative patients. The PIK3CA mutation rate in lung adenocarcinoma with acquired resistance to EGFR TKI is not higher than that in EGFR TKI-naïve tissue specimens (2.9% (6/207) vs. 1.8%; p = 0.344). Of the 74 patients with paired specimens (TKI-naïve and acquired resistance to TKIs) only one patient (1.4%) developed acquired PIK3CA (E545K) mutation, and he also had acquired EGFR (T790M) mutation. In conclusion, PIK3CA mutation may not be associated with primary resistance to EGFR TKI among lung adenocarcinoma patients. Acquired PIK3CA mutation related to EGFR TKI treatment is rare. PMID:27734950

  1. Impact of genetic profiles on the efficacy of anti-EGFR antibodies in metastatic colorectal cancer with KRAS mutation.

    PubMed

    Kishiki, Tomokazu; Ohnishi, Hiroaki; Masaki, Tadahiko; Ohtsuka, Kouki; Ohkura, Yasuo; Furuse, Jyunji; Sugiyama, Masanori; Watanabe, Takashi

    2014-07-01

    Reports indicate that, even in KRAS-mutated colon cancer, there are subsets of patients who benefit from anti-EGFR monoclonal antibody (MoAb) treatment. The aim of the present study was to identify genetic profiles that contribute to the responsiveness of metastatic colorectal cancer (mCRC) to anti-EGFR MoAb. We retrospectively evaluated the efficacy of anti-EGFR MoAb in mCRC patients with KRAS mutations according to KRAS mutational subtypes, BRAF and PIK3CA mutational status and PTEN and MET expression. Among 21 patients with KRAS-mutant tumors, 8 (38%) harbored p.G13D, 7 (33%) harbored p.G12V, 5 (24%) harbored p.G12D, and 1 (5%) harbored p.G12C mutation. Patients with the p.G13D mutation exhibited a significantly higher disease control rate than patients with other KRAS mutations (P=0.042), and tended to show a longer progression-free survival (PFS) than patients with other KRAS mutations with marginal significance (P=0.074). Patients with loss of PTEN had significantly shorter PFS than those with normal PTEN expression in patients with KRAS mutations (P=0.044). MET overexpression was significantly associated with shorter PFS compared to normal MET expression in patients with KRAS mutations (P=0.016). Our data demonstrated the potential utility of alterations in PTEN and MET expression as predictive markers for response to anti-EGFR MoAbs in mCRC patients with KRAS mutations. In addition, we confirmed the predictive value of the KRAS p.G13D mutation for better response to anti-EGFR therapies in comparison with other KRAS mutations. PMID:24839940

  2. Relationship between epidermal growth factor receptor (EGFR) mutation and serum cyclooxygenase-2 Level, and the synergistic effect of celecoxib and gefitinib on EGFR expression in non-small cell lung cancer cells.

    PubMed

    Li, Na; Li, Huanhuan; Su, Fan; Li, Jing; Ma, Xiaoping; Gong, Ping

    2015-01-01

    Epidermal growth factor receptor (EGFR) mutations occur mostly in patients with lung adenocarcinoma; such patients are also more likely to express cyclooxygenase-2 (COX-2), indicating a possible relationship between EGFR mutation and COX-2. The COX-2 and EGFR pathways mutually enhance their procarcinogenic effects in different tumor types. Therefore, simultaneous EGFR and COX-2 inhibition may be a promising therapeutic approach for patients with lung adenocarcinoma. We obtained tissue and serum samples from patients with non-small cell lung cancer (NSCLC) to detect the relationship between EGFR mutation and serum COX-2 level. Subsequently, gefitinib was combined with celecoxib to investigate the efficacy of inhibition in vitro in two NSCLC cell lines: HCC827 (del E746-A750) and A549 (wild-type EGFR). The cells were treated with gefitinib or celecoxib alone or with gefitinib plus celecoxib. Cell proliferation and apoptosis were assessed and correlated with expression of COX-2 and phosphorylated (p)-EGFR. The EGFR mutation rate of the high-COX-2 patients was significantly higher than that in the low-COX-2 patients. Multivariate analysis showed that high COX-2 levels were independently associated with EGFR mutation. Celecoxib and gefitinib inhibited cell growth in both cell lines. At sufficiently high concentrations, celecoxib plus gefitinib significantly mutually enhanced their anti-proliferative and apoptotic effects in both cell lines. At low concentrations, the combination had no additional effects on A549 cells. There was increased down regulation of COX-2 and p-EGFR when both cell lines were treated with high-concentration celecoxib plus gefitinib compared to either agent alone. This study demonstrates that high serum COX-2 levels may indicate EGFR mutations and that the efficacy of combined celecoxib and gefitinib is significantly greater in NSCLC cells with EGFR mutations; at high concentrations, the combination is efficacious in wild-type NSCLC cells.

  3. Utilization of Structure-Based Design to Identify Novel, Irreversible Inhibitors of EGFR Harboring the T790M Mutation.

    PubMed

    Hennessy, Edward J; Chuaqui, Claudio; Ashton, Susan; Colclough, Nicola; Cross, Darren A E; Debreczeni, Judit É; Eberlein, Cath; Gingipalli, Lakshmaiah; Klinowska, Teresa C M; Orme, Jonathan P; Sha, Li; Wu, Xiaoyun

    2016-05-12

    A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

  4. Selective Antitumor Activity of Ibrutinib in EGFR-Mutant Non–Small Cell Lung Cancer Cells

    PubMed Central

    Gao, Wen; Wang, Michael; Wang, Li; Lu, Haibo; Wu, Shuhong; Dai, Bingbing; Ou, Zhishuo; Zhang, Liang; Heymach, John V.; Gold, Kathryn A.; Minna, John; Roth, Jack A.; Hofstetter, Wayne L.; Swisher, Stephen G.

    2014-01-01

    Ibrutinib, which irreversibly inhibits Bruton tyrosine kinase, was evaluated for antitumor activity in a panel of non–small cell lung cancer (NSCLC) cell lines and found to selectively inhibit growth of NSCLC cells carrying mutations in the epidermal growth factor receptor (EGFR) gene, including T790M mutant and erlotinib-resistant H1975 cells. Ibrutinib induced dose-dependent inhibition of phosphor-EGFR at both Y1068 and Y1173 sites, suggesting ibrutinib functions as an EGFR inhibitor. Survival was analyzed by Kaplan–Meier estimation and log-rank test. All statistical tests were two-sided. In vivo study showed that ibrutinib statistically significantly suppressed H1975 tumor growth and prolonged survival of the tumor bearing mice (n = 5 per group). The mean survival times for solvent- and erlotinib-treated mice were both 17.8 days (95% confidence interval [CI] = 14.3 to 21.3 days), while the mean survival time for ibrutinib-treated mice was 29.8 days (95% CI = 26.0 to 33.6 days, P = .008). Our results indicate that ibrutinib could be a candidate drug for treatment of EGFR-mutant NSCLC, including erlotinib-resistant tumors. PMID:25214559

  5. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor.

    PubMed

    Finlay, M Raymond V; Anderton, Mark; Ashton, Susan; Ballard, Peter; Bethel, Paul A; Box, Matthew R; Bradbury, Robert H; Brown, Simon J; Butterworth, Sam; Campbell, Andrew; Chorley, Christopher; Colclough, Nicola; Cross, Darren A E; Currie, Gordon S; Grist, Matthew; Hassall, Lorraine; Hill, George B; James, Daniel; James, Michael; Kemmitt, Paul; Klinowska, Teresa; Lamont, Gillian; Lamont, Scott G; Martin, Nathaniel; McFarland, Heather L; Mellor, Martine J; Orme, Jonathon P; Perkins, David; Perkins, Paula; Richmond, Graham; Smith, Peter; Ward, Richard A; Waring, Michael J; Whittaker, David; Wells, Stuart; Wrigley, Gail L

    2014-10-23

    Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.

  6. 18F-FDG uptake for prediction EGFR mutation status in non-small cell lung cancer.

    PubMed

    Guan, Jian; Xiao, Nan J; Chen, Min; Zhou, Wen L; Zhang, Yao W; Wang, Shuang; Dai, Yong M; Li, Lu; Zhang, Yue; Li, Qin Y; Li, Xiang Z; Yang, Mi; Wu, Hu B; Chen, Long H; Liu, Lai Y

    2016-07-01

    Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) are a response to EGFR-tyrosine kinase inhibitor. However, a lack of sufficient tumor tissue has been a limitation for determining EGFR mutation status in clinical practice. The objective of this study was to predict EGFR mutation status in NSCLC patients based on a model including maximum standardized uptake value (SUVmax) and clinical features.We retrospectively reviewed NSCLC patients undergoing EGFR mutation testing and pretreatment positron emission tomography/computed tomography between March 2009 and December 2013. The relationships of EGFR mutations with both SUVmax and patient characteristics were evaluated, and a multivariate logistic regression analysis was performed. The model was assessed by area under the receiver-operating characteristic curve (AUC) and was prospectively validated during January to June 2014.Three hundred and sixteen patients meeting the criteria were enrolled for model construction. The SUVmax values were significantly lower for EGFR mutations (mean, 9.5 ± 5.74) than for EGFR wild-type (mean, 12.7 ± 6.43; P < 0.001). ROC curve analysis showed that the SUVmax cutoff point was 8.1, for which the AUC was 0.65 (95% confidence interval [CI], 0.60-0.72). In addition, multivariate analysis also showed that low SUVmax (≤8.1) was a predictor of EGFR mutations, for which the AUC was 0.77, combining nonsmoking history and primary tumor size (≤5 cm). Eighty-five patients were enrolled to validate the predictive model, and the overall accuracy, sensitivity, and specificity were 77.6%, 64.6% (95% CI 40.7-82.8), and 82.5% (95% CI 70.9-91.0), respectively.The specific FDG uptake value could be considered to effectively predict EGFR mutation status of NSCLC patients by considering smoking history and primary tumor size when genetic tests are not available. PMID:27472739

  7. Detection of EML4-ALK fusion gene and features associated with EGFR mutations in Chinese patients with non-small-cell lung cancer

    PubMed Central

    Wen, Miaomiao; Wang, Xuejiao; Sun, Ying; Xia, Jinghua; Fan, Liangbo; Xing, Hao; Zhang, Zhipei; Li, Xiaofei

    2016-01-01

    Purpose Echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). Methods We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. Results Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ2 test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. Conclusion EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent. PMID:27103824

  8. EGFR phosphorylates FAM129B to promote Ras activation

    PubMed Central

    Ji, Haitao; Lee, Jong-Ho; Wang, Yugang; Pang, Yilin; Zhang, Tao; Xia, Yan; Zhong, Lianjin; Lyu, Jianxin; Lu, Zhimin

    2016-01-01

    Ras GTPase-activating proteins (GAPs) are important regulators for Ras activation, which is instrumental in tumor development. However, the mechanism underlying this regulation remains elusive. We demonstrate here that activated EGFR phosphorylates the Y593 residue of the protein known as family with sequence similarity 129, member B (FAM129B), which is overexpressed in many types of human cancer. FAM129B phosphorylation increased the interaction between FAM129B and Ras, resulting in reduced binding of p120-RasGAP to Ras. FAM129B phosphorylation promoted Ras activation, increasing ERK1/2- and PKM2-dependent β-catenin transactivation and leading to the enhanced glycolytic gene expression and the Warburg effect; promoting tumor cell proliferation and invasion; and supporting brain tumorigenesis. Our studies unearthed a novel and important mechanism underlying EGFR-mediated Ras activation in tumor development. PMID:26721396

  9. Insufficiency of peripheral blood as a substitute tissue for detecting EGFR mutations in lung cancer: a meta-analysis.

    PubMed

    Li, Zhijun; Zhang, Yongjun; Bao, Wenlong; Jiang, Chuming

    2014-12-01

    The detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer tissues is necessary for effective treatment with EGFR tyrosine kinase inhibitors. However, tumor tissues may not be available in all situations. Studies have evaluated the potential use of serum or plasma for detecting the EGFR mutation status, but the results have been inconclusive. Here, a meta-analysis was performed to determine whether blood samples could serve as substitutes for tissue specimens in detecting the EGFR mutation status. Databases, including PubMed and Embase, were searched for relevant studies published from 2005 to 2013 that included true-positive, false-positive, true-negative, and false-negative values of the EGFR mutation status of the blood compared with tissue specimens. Summary receiver operating characteristic curves were developed to explore the threshold effect. Spearman's correlation coefficient was calculated to analyze the heterogeneity between studies. Pooled sensitivity and specificity were evaluated using Meta-DiSc version 1.4. Thirteen articles involving 1,591 cases were enrolled, with a pooled sensitivity and specificity of 64.5 % (95 % CI = 0.605-0.683) and 88.5 % (95 % CI = 0.863-0.904), respectively. Heterogeneity among the studies was caused by factors other than threshold effect. The findings were influenced by test method (p = 0.0354). Blood samples had a high specificity and relatively low sensitivity for detecting EGFR mutations compared to tumor tissues. The results of this meta-analysis suggest that peripheral blood is insufficient as a substitute for tumor tissues in detecting EGFR mutations in clinical practice.

  10. Association of variations in HLA class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma

    PubMed Central

    Shiraishi, Kouya; Okada, Yukinori; Takahashi, Atsushi; Kamatani, Yoichiro; Momozawa, Yukihide; Ashikawa, Kyota; Kunitoh, Hideo; Matsumoto, Shingo; Takano, Atsushi; Shimizu, Kimihiro; Goto, Akiteru; Tsuta, Koji; Watanabe, Shun-ichi; Ohe, Yuichiro; Watanabe, Yukio; Goto, Yasushi; Nokihara, Hiroshi; Furuta, Koh; Yoshida, Akihiko; Goto, Koichi; Hishida, Tomoyuki; Tsuboi, Masahiro; Tsuchihara, Katsuya; Miyagi, Yohei; Nakayama, Haruhiko; Yokose, Tomoyuki; Tanaka, Kazumi; Nagashima, Toshiteru; Ohtaki, Yoichi; Maeda, Daichi; Imai, Kazuhiro; Minamiya, Yoshihiro; Sakamoto, Hiromi; Saito, Akira; Shimada, Yoko; Sunami, Kuniko; Saito, Motonobu; Inazawa, Johji; Nakamura, Yusuke; Yoshida, Teruhiko; Yokota, Jun; Matsuda, Fumihiko; Matsuo, Keitaro; Daigo, Yataro; Kubo, Michiaki; Kohno, Takashi

    2016-01-01

    Lung adenocarcinoma driven by somatic EGFR mutations is more prevalent in East Asians (30–50%) than in European/Americans (10–20%). Here we investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA class II at 6p21.32 (rs2179920; P =5.1 × 10−17, per-allele OR=1.36) and 6p21.1 (FOXP4) (rs2495239; P=3.9 × 10−9, per-allele OR=1.19) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma. This study indicates that multiple genetic factors underlie the risk of lung adenocarcinomas with EGFR mutations. PMID:27501781

  11. Association of variations in HLA class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma.

    PubMed

    Shiraishi, Kouya; Okada, Yukinori; Takahashi, Atsushi; Kamatani, Yoichiro; Momozawa, Yukihide; Ashikawa, Kyota; Kunitoh, Hideo; Matsumoto, Shingo; Takano, Atsushi; Shimizu, Kimihiro; Goto, Akiteru; Tsuta, Koji; Watanabe, Shun-Ichi; Ohe, Yuichiro; Watanabe, Yukio; Goto, Yasushi; Nokihara, Hiroshi; Furuta, Koh; Yoshida, Akihiko; Goto, Koichi; Hishida, Tomoyuki; Tsuboi, Masahiro; Tsuchihara, Katsuya; Miyagi, Yohei; Nakayama, Haruhiko; Yokose, Tomoyuki; Tanaka, Kazumi; Nagashima, Toshiteru; Ohtaki, Yoichi; Maeda, Daichi; Imai, Kazuhiro; Minamiya, Yoshihiro; Sakamoto, Hiromi; Saito, Akira; Shimada, Yoko; Sunami, Kuniko; Saito, Motonobu; Inazawa, Johji; Nakamura, Yusuke; Yoshida, Teruhiko; Yokota, Jun; Matsuda, Fumihiko; Matsuo, Keitaro; Daigo, Yataro; Kubo, Michiaki; Kohno, Takashi

    2016-01-01

    Lung adenocarcinoma driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). Here we investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA class II at 6p21.32 (rs2179920; P =5.1 × 10(-17), per-allele OR=1.36) and 6p21.1 (FOXP4) (rs2495239; P=3.9 × 10(-9), per-allele OR=1.19) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma. This study indicates that multiple genetic factors underlie the risk of lung adenocarcinomas with EGFR mutations. PMID:27501781

  12. Prognostic factors for brain metastases from non-small cell lung cancer with EGFR mutation: influence of stable extracranial disease and erlotinib therapy.

    PubMed

    Sekine, Akimasa; Satoh, Hiroaki; Iwasawa, Tae; Tamura, Katsumi; Hayashihara, Kenji; Saito, Takefumi; Kato, Terufumi; Arai, Mito; Okudela, Koji; Ohashi, Kenichi; Ogura, Takashi

    2014-10-01

    The aim of this study was to explore prognostic factors for non-small cell lung cancer (NSCLC) patients with brain metastases (BM) on the basis of EGFR mutation status. Among 779 consecutive NSCLC patients who underwent EGFR mutation screening, all 197 patients with BM were divided according to the EGFR mutation status. The prognostic factors, including patient characteristics at the time of BM diagnosis, treatment history, and radiologic features, were analyzed. Of 197 patients with BM, 108 had wild-type EGFR and 89 had EGFR mutation. The patients with EGFR mutation presented longer overall survival after BM diagnosis (OS) than those with wild-type EGFR, regardless of whether BM was synchronous or metachronous. For the patients with EGFR mutation, favorable prognostic factors in multivariate analysis were age<65 (p=0.037), good performance status (PS) (p<0.0001), cranial radiotherapy (p=0.020), previous chemotherapy≤1 regimen (p=0.009), stable extracranial disease at BM diagnosis (p=0.022), and erlotinib therapy after BM diagnosis (p=0.0015). On the other hand, favorable prognostic factors for the patients with wild-type EGFR were only good PS (p=0.0037) and cranial radiotherapy (p=0.0005). Among patients treated with erlotinib after BM diagnosis, the patients with exon 19 deletion showed longer OS than those with exon 21 point mutation (p=0.019). The prognostic factors for NSCLC patients with BM were different according to the EGFR mutation status. Particularly in NSCLC patients with EGFR mutation and stable extracranial disease, regular cranial evaluation for detecting asymptomatic BM would lead to good prognosis. In addition, erlotinib therapy would be preferable in NSCLC patients with BM and EGFR mutation, especially those with exon 19 deletion.

  13. A new generation of companion diagnostics: cobas BRAF, KRAS and EGFR mutation detection tests.

    PubMed

    Angulo, Barbara; Lopez-Rios, Fernando; Gonzalez, David

    2014-06-01

    The cobas(®) (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an 'end-to-end' solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use. PMID:24844134

  14. Standardization of Epidermal Growth Factor Receptor (EGFR) Measurement by Quantitative Immunofluorescence and Impact on Antibody-Based Mutation Detection in Non–Small Cell Lung Cancer

    PubMed Central

    Dimou, Anastasios; Agarwal, Seema; Anagnostou, Valsamo; Viray, Hollis; Christensen, Stephen; Gould Rothberg, Bonnie; Zolota, Vassiliki; Syrigos, Konstantinos; Rimm, David L.

    2011-01-01

    Challenges in measurement of epidermal growth factor receptor (EGFR) protein expression have led to conflicting data on its prognostic value and discontinuation of its use for prediction of response. Herein is described a quantitative standardized assay for EGFR and its use in a series of retrospective cohorts of patients with non–small cell lung cancer (NSCLC). The AQUA technology of quantitative immunofluorescence was used in conjunction with Western blot analysis to calculate the absolute concentration of EGFR in two independent NSCLC cohorts (170 from Yale New Haven Hospital and 335 from Sotiria and Patras Hospitals in Greece). EGFR and mutated EGFR were measured using D38B1 antibody and two mutation-specific antibodies. All patients positive or borderline for mutation-specific antibody were genotyped. A threshold for reproducible detection of EGFR was defined as 0.85 ng/μg total protein. EGFR expression demonstrated no prognostic value in either cohort. The mutation rate was 1.79% in the Yale cohort, and 1.52% in the Sotiria/Patras cohort, with no antibody detection–based false-positive cases. No mutations were detected for EGFR concentrations <1.46 ng/μg total protein. In summary, accurate measurement of EGFR still shows no prognostic value in NSCLC. In these two population-based cohorts, the antibody-based EGFR mutation rate was lower than has been frequently reported. PMID:21722621

  15. EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line

    PubMed Central

    Popeda, Marta; Ksiazkiewicz, Magdalena; Grzela, Dawid P.; Walczak, Maciej P.; Banaszczyk, Mateusz; Peciak, Joanna; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant–EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis. PMID:27153109

  16. Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

    PubMed Central

    Schäfer, A.; Pahnke, A.; Schaffert, D.; van Weerden, W.M.; de Ridder, C.M.A.; Rödl, W.; Vetter, A.; Spitzweg, C.; Kraaij, R.; Wagner, E.

    2011-01-01

    Abstract The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes. PMID:21644815

  17. Volumetric tumor growth in advanced NSCLC patients with EGFR mutations during EGFR-TKI therapy: Developing criteria to continue therapy beyond RECIST progression

    PubMed Central

    Nishino, Mizuki; Dahlberg, Suzanne E.; Cardarella, Stephanie; Jackman, David M.; Rabin, Michael S.; Ramaiya, Nikhil H.; Hatabu, Hiroto; Jänne, Pasi A.; Johnson, Bruce E.

    2013-01-01

    Purpose Define volumetric tumor growth rate in advanced NSCLC patients with sensitizing EGFR mutations initially treated with EGFR-TKI therapy beyond progression. Methods The study included 58 advanced NSCLC patients with sensitizing EGFR mutations treated with first-line gefitinib or erlotinib, who had baseline CT showing measurable lung lesion and at least two follow-up CTs while on TKI and experienced volumetric tumor growth. Tumor volume (mm3) of the dominant lung lesion was measured on baseline and follow-up CT scans during therapy. A total of 405 volume measurements were analyzed in a linear mixed effects model, fitting time as a random effect, to define the growth rate of the logarithm of tumor volume (logeV). Results A linear mixed effects model was fitted to predict growth of logeV, adjusting for time in months from baseline. LogeV was estimated as a function of time in months, in patients whose tumors have started growing after nadir: logeV=0.12*time+7.68 In this formula, the regression coefficient for time, 0.12/month, represents the growth rate of logeV (SE: 0.015; p<0.001). When adjusted for baseline volume, logeV0, the growth rate was also 0.12/month (SE: 0.015; p<0.001; logeV =0.12*months+0.72 logeV0+0.61). Conclusion Tumor volume models defined volumetric tumor growth after the nadir in EGFR-mutant advanced NSCLC patients receiving TKI, providing a reference value for the tumor growth rate in patients progressing after the nadir on TKI. The results can be further studied in additional cohorts to develop practical criteria which help to identify patients who are slowly progressing and can safely remain on EGFR-TKIs. PMID:23922022

  18. The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells.

    PubMed

    Amornwichet, Napapat; Oike, Takahiro; Shibata, Atsushi; Nirodi, Chaitanya S; Ogiwara, Hideaki; Makino, Haruhiko; Kimura, Yuka; Hirota, Yuka; Isono, Mayu; Yoshida, Yukari; Ohno, Tatsuya; Kohno, Takashi; Nakano, Takashi

    2015-06-11

    Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

  19. The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells.

    PubMed

    Amornwichet, Napapat; Oike, Takahiro; Shibata, Atsushi; Nirodi, Chaitanya S; Ogiwara, Hideaki; Makino, Haruhiko; Kimura, Yuka; Hirota, Yuka; Isono, Mayu; Yoshida, Yukari; Ohno, Tatsuya; Kohno, Takashi; Nakano, Takashi

    2015-01-01

    Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC. PMID:26065573

  20. PCR-sequencing is a complementary method to amplification refractory mutation system for EGFR gene mutation analysis in FFPE samples.

    PubMed

    Jiang, Junchang; Wang, Chunhua; Yu, Xiaoli; Sheng, Danli; Zuo, Chen; Ren, Minpu; Wu, Yaqin; Shen, Jie; Jin, Mei; Xu, Songxiao

    2015-12-01

    Amplification Refractory Mutation System (ARMS) is the most popular technology for EGFR gene mutation analysis in China. Cutoff Ct or ΔCt values were used to differentiate low mutation abundance cases from no mutation cases. In this study, all of 359 NSCLC samples were tested by ARMS. Seventeen samples with larger Ct or ΔCt than cutoff values were retested by PCR-sequencing. TKI treatment responses were monitored on the cases with ARMS negative and PCR-sequencing positive results. One exon 18 G719X case, 67 exon 19 deletion cases, 2 exon 20 insertion cases, 1 exon 20 T790M case, 60 exon 21 L858R cases, 5 exon 21 L861Q cases and 201 wild type cases were identified by ARMS. Another 22 cases were evaluated as wild type but had later amplification fluorescent curves. Seventeen out of these 22 cases were retested by PCR-sequencing. It turns out that 3 out of 3 cases with exon 19 deletion later amplifications, 2 out of 2 cases with L858R later amplifications and 4 out of 12 cases with T790M later amplifications were identified as mutation positive. Two cases with exon 19 deletion and L858R respectively were treated by TKI and got responses. Our study indicated that PCR-sequencing might be a complementary way to confirm ARMS results with later amplifications.

  1. PCR-sequencing is a complementary method to amplification refractory mutation system for EGFR gene mutation analysis in FFPE samples.

    PubMed

    Jiang, Junchang; Wang, Chunhua; Yu, Xiaoli; Sheng, Danli; Zuo, Chen; Ren, Minpu; Wu, Yaqin; Shen, Jie; Jin, Mei; Xu, Songxiao

    2015-12-01

    Amplification Refractory Mutation System (ARMS) is the most popular technology for EGFR gene mutation analysis in China. Cutoff Ct or ΔCt values were used to differentiate low mutation abundance cases from no mutation cases. In this study, all of 359 NSCLC samples were tested by ARMS. Seventeen samples with larger Ct or ΔCt than cutoff values were retested by PCR-sequencing. TKI treatment responses were monitored on the cases with ARMS negative and PCR-sequencing positive results. One exon 18 G719X case, 67 exon 19 deletion cases, 2 exon 20 insertion cases, 1 exon 20 T790M case, 60 exon 21 L858R cases, 5 exon 21 L861Q cases and 201 wild type cases were identified by ARMS. Another 22 cases were evaluated as wild type but had later amplification fluorescent curves. Seventeen out of these 22 cases were retested by PCR-sequencing. It turns out that 3 out of 3 cases with exon 19 deletion later amplifications, 2 out of 2 cases with L858R later amplifications and 4 out of 12 cases with T790M later amplifications were identified as mutation positive. Two cases with exon 19 deletion and L858R respectively were treated by TKI and got responses. Our study indicated that PCR-sequencing might be a complementary way to confirm ARMS results with later amplifications. PMID:26477713

  2. Dominant Enhancers of Egfr in Drosophila Melanogaster: Genetic Links between the Notch and Egfr Signaling Pathways

    PubMed Central

    Price, J. V.; Savenye, E. D.; Lum, D.; Breitkreutz, A.

    1997-01-01

    The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F(1) screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context. PMID:9383058

  3. Comprehensive detection of diverse exon 19 deletion mutations of EGFR in lung Cancer by a single probe set.

    PubMed

    Bae, Jin Ho; Jo, Seong-Min; Kim, Hak-Sung

    2015-12-15

    Detection of exon 19 deletion mutation of EGFR, one of the most frequently occurring mutations in lung cancer, provides the crucial information for diagnosis and treatment guideline in non-small-cell lung cancer (NSCLC). Here, we demonstrate a simple and efficient method to detect various exon 19 deletion mutations of EGFR using a single probe set comprising of an oligo-quencher (oligo-Q) and a molecular beacon (MB). While the MB hybridizes to both the wild and mutant target DNA, the oligo-Q only binds to the wild target DNA, leading to a fluorescent signal in case of deletion mutation. This enables the comprehensive detection of the diverse exon 19 deletion mutations using a single probe set. We demonstrated the utility and efficiency of the approach by detecting the frequent exon 19 deletion mutations of EGFR through a real-time PCR and in situ fluorescence imaging. Our approach enabled the detection of genomic DNA as low as 0.02 ng, showing a detection limit of 2% in a heterogeneous DNA mixture, and could be used for detecting mutations in a single cell level. The present MB and oligo-Q dual probe system can be used for diagnosis and treatment guideline in NSCLC.

  4. Malignant Pleural Effusion Supernatants Are Substitutes for Metastatic Pleural Tumor Tissues in EGFR Mutation Test in Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Wu, Ning; Nie, Xiaomeng; Xia, Yang; Han, Yiping; Li, Qiang; Zhu, Guanshan; Bai, Chong

    2014-01-01

    Background Though the possibility of using malignant pleural effusions (MPEs) as alternatives for metastatic pleural tumor tissues (MPTTs) in epidermal growth factor receptor (EGFR) mutation test has been examined, due to the lack of studies comparing the results in matching MPEs and MPTTs, the clinical value of MPEs for advanced adenocarcinoma patients with pleural effusions is not confirmed. Methods EGFR mutation statuses in matching MPTTs, MPE supernatants and cell blocks, of 41 patients with advanced lung adenocarcinoma as diagnosed by thoracoscopy were analyzed using amplification refractory mutation system (ARMS). Results EGFR mutations were detected in 46.3% (19/41) of MPTTs, 43.9% (18/41) of MPE supernatants and 56.3% (18/32) of MPE cell blocks by ARMS analysis. Generally, the same EGFR statuses were identified in both MPTTs and matching MPE cell blocks of 81.3% patients (26/32), whereas MPTTs and matching MPE supernatants of 87.8% (36/41) patients shared the same EGFR status. Compared with EGFR mutation detection in MPTTs, the sensitivity of EGFR mutation detection in MPE-cell blocks was 87.5% (14/16), specificity was 75.0% (12/16), while the sensitivity of EGFR mutation detection in MPE-supernatants was 84.2% (16/19), specificity was 90.9% (20/22). Conclusions The high concordance of EGFR mutation statuses between MPEs and MPTTs in lung adenocarcinoma patients with pleural metastasis as determined by ARMS analysis suggests that MPEs, particularly MPE supernatants, may be substitutes for MPTTs in EGFR mutation test. PMID:24587142

  5. EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production.

    PubMed

    Kalinowski, April; Ueki, Iris; Min-Oo, Gundula; Ballon-Landa, Eric; Knoff, David; Galen, Benjamin; Lanier, Lewis L; Nadel, Jay A; Koff, Jonathan L

    2014-07-15

    Airway epithelial cells are the primary cell type involved in respiratory viral infection. Upon infection, airway epithelium plays a critical role in host defense against viral infection by contributing to innate and adaptive immune responses. Influenza A virus, rhinovirus, and respiratory syncytial virus (RSV) represent a broad range of human viral pathogens that cause viral pneumonia and induce exacerbations of asthma and chronic obstructive pulmonary disease. These respiratory viruses induce airway epithelial production of IL-8, which involves epidermal growth factor receptor (EGFR) activation. EGFR activation involves an integrated signaling pathway that includes NADPH oxidase activation of metalloproteinase, and EGFR proligand release that activates EGFR. Because respiratory viruses have been shown to activate EGFR via this signaling pathway in airway epithelium, we investigated the effect of virus-induced EGFR activation on airway epithelial antiviral responses. CXCL10, a chemokine produced by airway epithelial cells in response to respiratory viral infection, contributes to the recruitment of lymphocytes to target and kill virus-infected cells. While respiratory viruses activate EGFR, the interaction between CXCL10 and EGFR signaling pathways is unclear, and the potential for EGFR signaling to suppress CXCL10 has not been explored. Here, we report that respiratory virus-induced EGFR activation suppresses CXCL10 production. We found that influenza virus-, rhinovirus-, and RSV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-dependent CXCL10 production. In addition, inhibition of EGFR during viral infection augmented IRF1 and CXCL10. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

  6. EGFR mutations are associated with favorable intracranial response and progression-free survival following brain irradiation in non-small cell lung cancer patients with brain metastases

    PubMed Central

    2012-01-01

    Background The presence of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) is associated with increased radiosensitivity in vitro. However, the results from clinical studies regarding the radiosensitivity in NSCLC with mutant EGFR are inconclusive. We retrospectively analyzed our NSCLC patients who had been regularly followed up by imaging studies after irradiation for brain metastases, and investigated the impact of EGFR mutations on radiotherapy (RT). Methods Forty-three patients with brain metastases treated with RT, together with EGFR mutation status, demographics, smoking history, performance status, recursive partitioning analysis (RPA) class, tumor characteristics, and treatment modalities, were included. Radiological images were taken at 1 to 3 months after RT, and 3 to 6 months thereafter. Radiographic response was evaluated by RECIST criteria version 1.1 according to the intracranial images before and after RT. Log-rank test and Cox regression model were used to correlate EGFR mutation status and other clinical features with intracranial radiological progression-free survival (RPFS) and overall survival (OS). Results The median follow-up duration was 15 months. Patients with mutant EGFR had higher response rates to brain RT than those with wild-type EGFR (80% vs. 46%; p = 0.037). Logistic regression analysis showed that EGFR mutation status is the only predictor for treatment response (p = 0.032). The median intracranial RPFS was 18 months (95% CI = 8.33-27.68 months). In Cox regression analysis, mutant EGFR (p = 0.025) and lower RPA class (p = 0.026) were associated with longer intracranial RPFS. EGFR mutation status (p = 0.061) and performance status (p = 0.076) had a trend to predict OS. Conclusions Mutant EGFR in NSCLC patients is an independent prognostic factor for better treatment response and longer intracranial RPFS following RT for brain metastases. PMID:23110940

  7. Molecular spectrum of somatic EGFR and KRAS gene mutations in non small cell lung carcinoma: determination of frequency, distribution pattern and identification of novel variations in Indian patients.

    PubMed

    Das, Bibhu Ranjan; Bhaumik, Sangeet; Ahmad, Firoz; Mandsaurwala, Aziz; Satam, Heena

    2015-07-01

    Somatic mutations of EGFR and KRAS gene represent the most common alterations currently known in NSCLC patients. This study explored the frequency, distribution pattern of EGFR and KRAS mutations in Indian patients. The frequencies of EGFR and KRAS mutations were 29 % (116/400) and 4.5 % (6/132) respectively. Both EGFR and KRAS mutations were prevalent in females, and a trend towards higher mutation frequency was seen in patients under ≥ 60 years age. The presence of EGFR and KRAS mutations were higher in adenocarcinomas in comparison to other histological subtype. Sequencing analysis of EGFR exon 18 revealed Inframe deletion (G709_T710 > A) and missense mutation (K713R). Among exon 19 positive cases, 49.3 % (37/75) were in-frame deletions, of which E746_A750del was frequent. Similarly, ~47 % (35/75) cases showed complex mutation involving indel. Among mutations in exon 20 (N = 9), 8 were substitutions, one showed duplication, while all exon 21 mutations were of the missense types with L858R as the most recurrent type. Sequencing analysis of KRAS exon 1 revealed three different types codon 12 substitutions resulting in c34G > T (G12C) (n = 4), c.35G > A (G12D) (n = 1), and c.35G > T (G12V) (n = 1). In conclusion, the present study is an example of molecular diversity of EGFR and KRAS gene in Indian patients and further confirms that the frequency of EGFR and KRAS mutations varies considerably globally. To the best of our knowledge, this is the first Indian study to evaluate KRAS mutation. The current study also served to identify novel variations that added new insights into the genetic heterogeneity of NSCLC.

  8. Features and prognostic impact of distant metastasis in patients with stage IV lung adenocarcinoma harboring EGFR mutations: importance of bone metastasis.

    PubMed

    Fujimoto, Daichi; Ueda, Hiroyuki; Shimizu, Ryoko; Kato, Ryoji; Otoshi, Takehiro; Kawamura, Takahisa; Tamai, Koji; Shibata, Yumi; Matsumoto, Takeshi; Nagata, Kazuma; Otsuka, Kyoko; Nakagawa, Atsushi; Otsuka, Kojiro; Katakami, Nobuyuki; Tomii, Keisuke

    2014-06-01

    Mutated epidermal growth factor receptor (EGFR) and signaling pathways were associated with multiple brain and intra-pulmonary metastases, oncogenic progression and metastasis. However, features of metastasis to other organs and the independent prognostic influence of metastatic lesions were not elucidated in patients with lung cancer harboring EGFR mutations. Between January 2007 and April 2012, we treated 277 patients diagnosed with stage IV lung adenocarcinoma. Studied were 246 patients with available tumor EGFR mutation data who also underwent radiographic evaluation of lung, abdominal, brain, and bone metastases. The EGFR mutated group (N = 98) had significantly more metastatic lesions in the brain and bone than the wild-type group (N = 148): brain, 3 (1-93) versus 2 (1-32) median (range), P = 0.023; bone, 3 (1-43) versus 2 (1-27), P = 0.035, respectively. In addition, EGFR mutations were significantly more frequent in patients with multiple than non-multiple lung metastases (24/40 vs. 12/42, P = 0.004). Multivariate analysis showed that bone metastasis was a significant independent negative predictive factor of overall survival (OS) in patients with mutated [hazard ratio (HR) 2.04; 95 % confidence interval (CI) 1.17-3.64; P = 0.011] and wild-type EGFR (HR 2.09; 95 % CI 1.37-3.20; P < 0.001). In conclusion, patients with mutated EGFR had more lung, brain, and bone metastases, and bone metastasis was an independent negative predictor of OS.

  9. Lung cancer with concurrent EGFR mutation and ROS1 rearrangement: a case report and review of the literature.

    PubMed

    Zhu, You-Cai; Xu, Chun-Wei; Ye, Xiao-Qian; Yin, Man-Xiang; Zhang, Jin-Xian; Du, Kai-Qi; Zhang, Zhi-Hao; Hu, Jian

    2016-01-01

    ROS1 rearrangement has recently emerged as a new molecular subtype in non-small cell lung cancer, and is predominantly found in lung adenocarcinomas compared with other oncogenes such as EGFR, KRAS, or ALK. Patients who have both mutations are extremely rare. Here we report a 50-year-old female diagnosed with adenocarcinoma with sarcomatoid differentiation, who was shown to have EGFR and ROS1 mutations. The patient was treated surgically and received three cycles of adjuvant postoperative chemotherapy. In addition, we reviewed the previously reported cases and related literature. This presentation will provide further understanding of the underlying molecular biology and optimal treatment for non-small cell lung cancer patients with more than one driver mutation. PMID:27486332

  10. Lung cancer with concurrent EGFR mutation and ROS1 rearrangement: a case report and review of the literature

    PubMed Central

    Zhu, You-cai; Xu, Chun-wei; Ye, Xiao-qian; Yin, Man-xiang; Zhang, Jin-xian; Du, Kai-qi; Zhang, Zhi-hao; Hu, Jian

    2016-01-01

    ROS1 rearrangement has recently emerged as a new molecular subtype in non-small cell lung cancer, and is predominantly found in lung adenocarcinomas compared with other oncogenes such as EGFR, KRAS, or ALK. Patients who have both mutations are extremely rare. Here we report a 50-year-old female diagnosed with adenocarcinoma with sarcomatoid differentiation, who was shown to have EGFR and ROS1 mutations. The patient was treated surgically and received three cycles of adjuvant postoperative chemotherapy. In addition, we reviewed the previously reported cases and related literature. This presentation will provide further understanding of the underlying molecular biology and optimal treatment for non-small cell lung cancer patients with more than one driver mutation. PMID:27486332

  11. EGFR mutation detection on routine cytological smears of non-small cell lung cancer by digital PCR: a validation study.

    PubMed

    Malapelle, Umberto; de Luca, Caterina; Vigliar, Elena; Ambrosio, Francesca; Rocco, Danilo; Pisapia, Pasquale; Bellevicine, Claudio; Troncone, Giancarlo

    2016-05-01

    Highly sensitive genotyping techniques are useful to detect epidermal growth factor receptor (EGFR) mutations on lung cancer cytological samples, when these specimens feature only few neoplastic cells. This study aimed to validate digital PCR (dPCR) methodology on cytological material. In plasmid model system, dPCR allowed for the detection of a minimal percentage (1%) of EGFR mutant alleles. Cytological samples (n = 30), with neoplastic cell percentage ranging from 10% to 80% and yielding a quantity of extracted DNA ranging from 1.75 to 60 ng/µL were selected. Results previously generated by fragment length and TaqMan assays (n = 8 exon 19 deletions, n = 2 L858R mutations and n = 20 wild-type DNA) were compared with those obtained by dPCR. Data were highly concordant (96.6%). However, dPCR detected an additional L858R mutation that had been missed by TaqMan assay on a paucicellular smear. This mutation was confirmed by cloning PCR products and sequencing. Thus, dPCR can reliably be used to increase EGFR mutation detection rate on scarcely cellular lung cancer smears. PMID:26893402

  12. The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling.

    PubMed

    Asbagh, Layka Abbasi; Vazquez, Iria; Vecchione, Loredana; Budinska, Eva; De Vriendt, Veerle; Baietti, Maria Francesca; Steklov, Mikhail; Jacobs, Bart; Hoe, Nicholas; Singh, Sharat; Imjeti, Naga-Sailaja; Zimmermann, Pascale; Sablina, Anna; Tejpar, Sabine

    2014-10-30

    Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies. PMID:25301722

  13. Development of Resistance to EGFR-Targeted Therapy in Malignant Glioma Can Occur through EGFR-Dependent and -Independent Mechanisms.

    PubMed

    Klingler, Stefan; Guo, Baofeng; Yao, Jun; Yan, Haiyan; Zhang, Ling; Vaseva, Angelina V; Chen, Sida; Canoll, Peter; Horner, James W; Wang, Y Alan; Paik, Ji-Hye; Ying, Haoqiang; Zheng, Hongwu

    2015-05-15

    Epidermal growth factor receptor (EGFR) is highly amplified, mutated, and overexpressed in human malignant gliomas. Despite its prevalence and growth-promoting functions, therapeutic strategies to inhibit EGFR kinase activity have not been translated into profound beneficial effects in glioma clinical trials. To determine the roles of oncogenic EGFR signaling in gliomagenesis and tumor maintenance, we generated a novel glioma mouse model driven by inducible expression of a mutant EGFR (EGFR*). Using combined genetic and pharmacologic interventions, we revealed that EGFR*-driven gliomas were insensitive to EGFR tyrosine kinase inhibitors, although they could efficiently inhibit EGFR* autophosphorylation in vitro and in vivo. This is in contrast with the genetic suppression of EGFR* induction that led to significant tumor regression and prolonged animal survival. However, despite their initial response to genetic EGFR* extinction, all tumors would relapse and propagate independent of EGFR*. We further showed that EGFR*-independent tumor cells existed prior to treatment and were responsible for relapse following genetic EGFR* suppression. And, the addition of a PI3K/mTOR inhibitor could significantly delay relapse and prolong animal survival. Our findings shed mechanistic insight into EGFR drug resistance in glioma and provide a platform to test therapies targeting aberrant EGFR signaling in this setting.

  14. Decoding the EGFR mutation-induced drug resistance in lung cancer treatment by local surface geometric properties.

    PubMed

    Ma, Lichun; Wang, Debby D; Huang, Yiqing; Wong, Maria P; Lee, Victor H F; Yan, Hong

    2015-08-01

    Epidermal growth factor receptor (EGFR) mutation-induced drug resistance leads to a limited efficacy of tyrosine kinase inhibitors during lung cancer treatments. In this study, we explore the correlations between the local surface geometric properties of EGFR mutants and the progression-free survival (PFS). The geometric properties include local surface changes (four types) of the EGFR mutants compared with the wild-type EGFR, and the convex degrees of these local surfaces. Our analysis results show that the Spearman׳s rank correlation coefficients between the PFS and three types of local surface properties are all greater than 0.6 with small P-values, implying a high significance. Moreover, the number of atoms with solid angles in the ranges of [0.71, 1], [0.61, 1] or [0.5, 1], indicating the convex degree of a local EGFR surface, also shows a strong correlation with the PFS. Overall, these characteristics can be efficiently applied to the prediction of drug resistance in lung cancer treatments, and easily extended to other cancer treatments.

  15. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  16. DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

    PubMed Central

    Habibovic, Aida; Hristova, Milena; Heppner, David E.; Danyal, Karamatullah; Ather, Jennifer L.; Janssen-Heininger, Yvonne M.W.; Irvin, Charles G.; Poynter, Matthew E.; Lundblad, Lennart K.; Dixon, Anne E.; Geiszt, Miklos

    2016-01-01

    Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management. PMID:27812543

  17. Impact of physical size on gefitinib efficacy in patients with non-small cell lung cancer harboring EGFR mutations.

    PubMed

    Ichihara, Eiki; Hotta, Katsuyuki; Takigawa, Nagio; Kudo, Kenichiro; Kato, Yuka; Honda, Yoshihiro; Hayakawa, Hiromi; Minami, Daisuke; Sato, Akiko; Tabata, Masahiro; Tanimoto, Mitsune; Kiura, Katsuyuki

    2013-09-01

    Gefitinib is an essential drug for the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) gene mutations. The approved dosage is 250 mg/body/day without adjustment for physical size such as body surface area (BSA), and the impact of physical size on the efficacy of gefitinib has not been evaluated. Here, we sought to clarify this issue using a retrospective cohort. We reviewed the medical records of patients with consecutive advanced NSCLC harboring EGFR mutations who underwent gefitinib monotherapy at Okayama University Hospital. In total, 101 patients were included in this study, and the median BSA in this cohort was 1.5 m(2). The median progression-free survival (PFS) of the patients with higher BSA (≥1.5 m(2)) was significantly worse than that of those with lower BSA (< 1.5 m(2)) (10.4 vs. 18.0 months; p = 0.019, log-rank test). Multivariate analysis also showed a significant impact of BSA on PFS (hazards ratio, 2.34; 95% confidence interval, 1.78-2.89; p = 0.002). By contrast, no significant association between BSA and PFS was observed in those undergoing cytotoxic chemotherapy (4.0 vs. 5.1 months; p = 0.989, log-rank test), suggesting that BSA is a predictive, rather than a prognostic, marker for gefitinib therapy in EGFR-mutated NSCLC. In conclusion, BSA affected PFS in patients with EGFR-mutated NSCLC who underwent gefitinib monotherapy, suggesting the need for appraisal of BSA-based dose adjustment, even for this molecular target-based therapy.

  18. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients.

    PubMed

    Riediger, Anja Lisa; Dietz, Steffen; Schirmer, Uwe; Meister, Michael; Heinzmann-Groth, Ingrid; Schneider, Marc; Muley, Thomas; Thomas, Michael; Sültmann, Holger

    2016-01-01

    Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods. PMID:27640882

  19. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients

    PubMed Central

    Riediger, Anja Lisa; Dietz, Steffen; Schirmer, Uwe; Meister, Michael; Heinzmann-Groth, Ingrid; Schneider, Marc; Muley, Thomas; Thomas, Michael; Sültmann, Holger

    2016-01-01

    Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods. PMID:27640882

  20. A large-scale cross-sectional study of ALK rearrangements and EGFR mutations in non-small-cell lung cancer in Chinese Han population.

    PubMed

    Hong, Shaodong; Fang, Wenfeng; Hu, Zhihuang; Zhou, Ting; Yan, Yue; Qin, Tao; Tang, Yanna; Ma, Yuxiang; Zhao, Yuanyuan; Xue, Cong; Huang, Yan; Zhao, Hongyun; Zhang, Li

    2014-01-01

    The predictive power of age at diagnosis and smoking history for ALK rearrangements and EGFR mutations in non-small-cell lung cancer (NSCLC) remains not fully understood. In this cross-sectional study, 1160 NSCLC patients were prospectively enrolled and genotyped for EML4-ALK rearrangements and EGFR mutations. Multivariate logistic regression analysis was performed to explore the association between clinicopathological features and these two genetic aberrations. Receiver operating characteristic (ROC) curves methodology was applied to evaluate the predictive value. We showed that younger age at diagnosis was the only independent variable associated with EML4-ALK rearrangements (odds ratio (OR) per 5 years' increment, 0.68; p < 0.001), while lower tobacco exposure (OR per 5 pack-years' increment, 0.88; p < 0.001), adenocarcinoma (OR, 6.61; p < 0.001), and moderate to high differentiation (OR, 2.05; p < 0.001) were independently associated with EGFR mutations. Age at diagnosis was a very strong predictor of ALK rearrangements but poorly predicted EGFR mutations, while smoking pack-years may predict the presence of EGFR mutations and ALK rearrangements but with rather limited power. These findings should assist clinicians in assessing the likelihood of EML4-ALK rearrangements and EGFR mutations and understanding their biological implications in NSCLC.

  1. Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

    PubMed

    Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

    2012-12-01

    Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

  2. Predictive and prognostic value of preoperative serum tumor markers is EGFR mutation-specific in resectable non-small-cell lung cancer

    PubMed Central

    Jiang, Richeng; Wang, Xinyue; Li, Kai

    2016-01-01

    Background The predictive and prognostic value of carcinoembryonic antigen (CEA), cytokeratin-19 fragments (Cyfra21-1), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE) has been investigated in non-small-cell lung cancer (NSCLC) patients. However, few studies have directly focused on the association between these markers and epidermal growth factor receptor (EGFR) mutation status or mutation subtypes. Patients and methods We retrospectively analyzed 1016 patients with stage I-IIIA NSCLC who underwent complete resection between 2008 and 2012. Correlations between serum tumor marker levels and EGFR mutations and survival parameters were analyzed and prognostic factors were identified. Results Cyfra21-1 levels (P = 0.032 for disease-free survival [DFS]; P < 0.001 for overall survival [OS]) and clinical stage were identified as independent predictive and prognostic factors in EGFR-mutated adenocarcinoma patients. CEA levels (P < 0.001 for DFS; P = 0.002 for OS) and clinical stage were independently predictive and prognostic in EGFR wild-type adenocarcinoma patients. Further stratification analysis revealed that in EGFR exon 19 deletion adenocarcinomas, elevated Cyfra21-1 was an independent prognostic factor (P = 0.002). Within the Leu858Arg substitution subgroup, increased CEA (P = 0.005) and clinical stage were predictive factors of DFS, while elevated CEA (P = 0.005) and Cyfra21-1 (P = 0.027) were independent prognostic factors. Conclusion Cyfra21-1 and CEA exhibit different predictive and prognostic values between EGFR-mutated and wild-type adenocarcinomas, as well as between EGFR mutation subtypes. The prognostic impact of preoperative serum tumor markers should be evaluated together with EGFR mutation status. PMID:27072585

  3. The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

    PubMed Central

    Chang, Xiaofei; Izumchenko, Eugene; Solis, Luisa M.; Kim, Myoung Sook; Chatterjee, Aditi; Ling, Shizhang; Monitto, Constance L.; Harari, Paul M.; Hidalgo, Manuel; Goodman, Steve N.; Wistuba, Ignacio I.; Bedi, Atul; Sidransky, David

    2013-01-01

    The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01). PMID:23935914

  4. Expression of Tenascin C, EGFR, E-Cadherin, and TTF-1 in Medullary Thyroid Carcinoma and the Correlation with RET Mutation Status.

    PubMed

    Steiner, Florian; Hauser-Kronberger, Cornelia; Rendl, Gundula; Rodrigues, Margarida; Pirich, Christian

    2016-01-01

    Tenascin C expression correlates with tumor grade and indicates worse prognosis in several tumors. Epidermal growth factor receptor (EGFR) plays an important role in driving proliferation in many tumors. Loss of E-cadherin function is associated with tumor invasion and metastasis. Thyroid transcription factor-1 (TTF-1) is involved in rearranged during transfection (RET) transcription in Hirschsprung's disease. Tenascin C, EGFR, E-cadherin, TTF-1-expression, and their correlations with RET mutation status were investigated in 30 patients with medullary thyroid carcinoma (MTC) (n = 26) or C-cell hyperplasia (n = 4). Tenascin C was found in all, EGFR in 4/26, E-cadherin in 23/26, and TTF-1 in 25/26 MTC. Tenascin C correlated significantly with tumor proliferation (overall, r = 0.61, p < 0.005; RET-mutated, r = 0.81, p < 0.01). E-cadherin showed weak correlation, whereas EGFR and TTF-1 showed no significant correlation with tumor proliferation. EGFR, E-cadherin, and TTF-1 showed weak correlation with proliferation of RET-mutated tumors. Correlation between TTF-1 and tenascin C, E-cadherin, and EGFR was r = -0.10, 0.37, and 0.21, respectively. In conclusion, MTC express tenascin C, E-cadherin, and TTF-1. Tenascin C correlates significantly with tumor proliferation, especially in RET-mutated tumors. EGFR is low, and tumors expressing EGFR do not exhibit higher proliferation. TTF-1 does not correlate with RET mutation status and has a weak correlation with tenascin C, E-cadherin, and EGFR expression. PMID:27409604

  5. Expression of Tenascin C, EGFR, E-Cadherin, and TTF-1 in Medullary Thyroid Carcinoma and the Correlation with RET Mutation Status.

    PubMed

    Steiner, Florian; Hauser-Kronberger, Cornelia; Rendl, Gundula; Rodrigues, Margarida; Pirich, Christian

    2016-07-09

    Tenascin C expression correlates with tumor grade and indicates worse prognosis in several tumors. Epidermal growth factor receptor (EGFR) plays an important role in driving proliferation in many tumors. Loss of E-cadherin function is associated with tumor invasion and metastasis. Thyroid transcription factor-1 (TTF-1) is involved in rearranged during transfection (RET) transcription in Hirschsprung's disease. Tenascin C, EGFR, E-cadherin, TTF-1-expression, and their correlations with RET mutation status were investigated in 30 patients with medullary thyroid carcinoma (MTC) (n = 26) or C-cell hyperplasia (n = 4). Tenascin C was found in all, EGFR in 4/26, E-cadherin in 23/26, and TTF-1 in 25/26 MTC. Tenascin C correlated significantly with tumor proliferation (overall, r = 0.61, p < 0.005; RET-mutated, r = 0.81, p < 0.01). E-cadherin showed weak correlation, whereas EGFR and TTF-1 showed no significant correlation with tumor proliferation. EGFR, E-cadherin, and TTF-1 showed weak correlation with proliferation of RET-mutated tumors. Correlation between TTF-1 and tenascin C, E-cadherin, and EGFR was r = -0.10, 0.37, and 0.21, respectively. In conclusion, MTC express tenascin C, E-cadherin, and TTF-1. Tenascin C correlates significantly with tumor proliferation, especially in RET-mutated tumors. EGFR is low, and tumors expressing EGFR do not exhibit higher proliferation. TTF-1 does not correlate with RET mutation status and has a weak correlation with tenascin C, E-cadherin, and EGFR expression.

  6. Integrated cell-based platform to study EGFR activation and transactivation.

    PubMed

    Caruso, Marie-Elaine; Clément, Paule; Parent, Stéphane; Dupriez, Vincent; Bossé, Roger; Rouleau, Nathalie

    2013-09-01

    The epidermal growth factor receptor (EGFR) pathway is one of the most deregulated molecular pathways in human epithelial cancers. Many approved drugs were optimized to directly target EGFR but yielded only modest clinical improvement in cancer patients due to low efficacy and drug resistance. Transactivation of EGFR by other cell surface receptors such as G-protein-coupled receptors (GPCRs) was proposed to explain this lack of efficacy. Even if direct EGFR activation and transactivation by GPCR contribute to the activation of the same signaling pathways, they are often studied as independent events resulting in partial investigation of a drug's mechanism of action. We present a novel high-throughput approach that integrates interrogation of direct activation of EGFR and its transactivation via GPCR activation. Using distinct technology platforms, three readouts were used to measure (1) direct activation of GPCR via cyclic adenosine monophosphate (cAMP) detection, (2) direct activation of EGFR through the release of intracellular Ca(2+), and (3) EGFR transactivation by GPCR using the detection of p-extracellular-signal-regulated kinases 1/2 (p-ERK1/2). In addition to being simple, quick, and homogenous, our methods were shown to be more sensitive than those in current use. These enabling tools should improve the knowledge pertaining to GPCRs and receptor tyrosine kinases trans-regulation and facilitate the design of more potent and better targeted new therapeutic strategies.

  7. EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function.

    PubMed

    Phuchareon, Janyaporn; McCormick, Frank; Eisele, David W; Tetsu, Osamu

    2015-07-21

    Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

  8. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Park, Jangchul; Kobayashi, Yoshihisa; Urayama, Kevin Y.; Yamaura, Hidekazu; Yatabe, Yasushi; Hida, Toyoaki

    2016-01-01

    This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB–IV) and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups’ characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159), KRAS mutations (n = 55), or ALK rearrangements (n = 51). Among the three groups, we evaluated only patients with stage IIIB–IV lung adenocarcinoma who had EGFR mutations (n = 126), KRAS mutations (n = 35), or ALK rearrangements (n = 47). We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009). Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003). Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively). Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049). Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively). Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively). In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  9. Mechanisms of resistance to EGFR tyrosine kinase inhibitors.

    PubMed

    Huang, Lihua; Fu, Liwu

    2015-09-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies.

  10. Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase

    PubMed Central

    Au, A E-L; Sashindranath, M; Borg, R J; Kleifeld, O; Andrews, R K; Gardiner, E E; Medcalf, R L; Samson, A L

    2014-01-01

    Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade. PMID:25210793

  11. Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients.

    PubMed

    Roengvoraphoj, Monic; Tsongalis, Gregory J; Dragnev, Konstantin H; Rigas, James R

    2013-12-01

    Activation of the epidermal growth factor receptor (EGFR) pathway has been implicated in tumorigenesis in non-small cell lung cancer (NSCLC), the most common type of lung cancer. As a result, EGFR has become a key focus for the development of personalized therapy, with several molecular biomarkers having been investigated as potential predictors of response with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC (e.g., EGFR expression, EGFR gene copy gain, and EGFR mutations). Of these, activating mutations in EGFR have thus far given the most consistent results based on the available evidence from preclinical studies and clinical trials. In an attempt to identify patients who are most likely to benefit from treatment with EGFR TKIs, EGFR mutation testing is being increasingly utilized in clinical practice. Currently in the United States, no EGFR TKI or accompanying mutational test is approved for the identification and first-line treatment of patients with advanced NSCLC. However, the first-generation EGFR TKIs, erlotinib and gefitinib, as well as investigational ErbB family TKIs and EGFR mutation testing methods are being evaluated in this setting. This review will discuss EGFR mutation testing as a biomarker of response to EGFR TKIs and the evolution of EGFR mutational analysis in NSCLC. Completed and ongoing clinical trials evaluating currently available or investigational EGFR TKIs as first-line therapy in molecularly and clinically selected patients with NSCLC, with a focus on trials in patients whose tumors have EGFR mutations, will also be reviewed.

  12. Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

    PubMed Central

    Fritsch, Cornelia; Klaver, Arjen; Kanger, Johannes S.; Jovin, Thomas M.; Arndt-Jovin, Donna J.

    2013-01-01

    Background Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. Methodology/Principal Findings The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. Conclusions/Significance We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor

  13. HER2 mutated breast cancer responds to treatment with single agent neratinib, a second generation HER2/EGFR tyrosine kinase inhibitor

    PubMed Central

    Ben–Baruch, Noa Efrat; Bose, Ron; Kavuri, Shyam M.; Ma, Cynthia X.; Ellis, Matthew J.

    2015-01-01

    Activating mutations in the HER2 tyrosine kinase have been identified in human breast cancers that lack HER2 gene amplification. These patients are not candidates for HER2 targeted drugs under current standards of care, but preclinical data strongly suggest that these patients will benefit from anti-HER2 drugs. In this case report, we describe a young woman with metastatic breast cancer whose tumor was found to carry a HER2 L755S mutation, which is in the kinase domain of HER2. Treatment with the second generation HER2/EGFR tyrosine kinase inhibitor, neratinib, resulted in partial response and dramatic improvement in the patient’s function status. This partial response lasted 11 months and when the patient’s cancer progressed, she was treated with neratinib plus capecitabine and her cancer again responded. This second response parallels the benefit seen with continuing trastuzumab in HER2 amplified breast cancer after disease progression. This case is the first report, to our knowledge, of successful single agent treatment of HER2 mutated breast cancer. Two clinical trials of neratinib for HER2 mutated, metastatic breast cancer are currently enrolling patients. Further, data from The Cancer Genome Atlas project have identified HER2 mutations in a wide range of solid tumors, including bladder, colorectal, and non-small cell lung cancer, suggesting that clinical trials of neratinib or neratinib-based combinations for HER2 mutated solid tumors is warranted. PMID:26358790

  14. 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.

  15. EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib

    PubMed Central

    Gallant, Jean-Nicolas; Sheehan, Jonathan H.; Shaver, Timothy M.; Bailey, Mark; Lipson, Doron; Chandramohan, Raghu; Brewer, Monica Red; York, Sally J.; Kris, Mark G.; Pietenpol, Jennifer A.; Ladanyi, Marc; Miller, Vincent A.; Ali, Siraj M.; Meiler, Jens; Lovly, Christine M.

    2015-01-01

    Oncogenic EGFR mutations are found in 10-35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). In analyzing the tumor from a 33-year-old male never smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18-25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD-transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI, afatinib. The patient eventually progressed, at which time, re-sequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target. PMID:26286086

  16. Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network

    PubMed Central

    Beau-Faller, M.; Prim, N.; Ruppert, A.-M.; Nanni-Metéllus, I.; Lacave, R.; Lacroix, L.; Escande, F.; Lizard, S.; Pretet, J.-L.; Rouquette, I.; de Crémoux, P.; Solassol, J.; de Fraipont, F.; Bièche, I.; Cayre, A.; Favre-Guillevin, E.; Tomasini, P.; Wislez, M.; Besse, B.; Legrain, M.; Voegeli, A.-C.; Baudrin, L.; Morin, F.; Zalcman, G.; Quoix, E.; Blons, H.; Cadranel, J.

    2014-01-01

    Background There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. Patients and methods EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. Results Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12–24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08–0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6–21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). Conclusions Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment. PMID:24285021

  17. A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

    PubMed

    Moores, Sheri L; Chiu, Mark L; Bushey, Barbara S; Chevalier, Kristen; Luistro, Leopoldo; Dorn, Keri; Brezski, Randall J; Haytko, Peter; Kelly, Thomas; Wu, Sheng-Jiun; Martin, Pauline L; Neijssen, Joost; Parren, Paul W H I; Schuurman, Janine; Attar, Ricardo M; Laquerre, Sylvie; Lorenzi, Matthew V; Anderson, G Mark

    2016-07-01

    Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.

  18. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study

    PubMed Central

    Douillard, J-Y; Ostoros, G; Cobo, M; Ciuleanu, T; McCormack, R; Webster, A; Milenkova, T

    2014-01-01

    Background: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). Methods: Treatment: gefitinib 250 mg day−1 until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. Results: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8% adenocarcinoma 97.2% never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5–77.7), DCR 90.6% (95% CI 83.5–94.8), median PFS 9.7 months (95% CI 8.5–11.0), median OS 19.2 months (95% CI 17.0–NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15% SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8–74.7). Conclusion: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable. PMID:24263064

  19. Assessment of MAGE-A expression in resected non-small cell lung cancer in relation to clinicopathologic features and mutational status of EGFR and KRAS.

    PubMed

    Ayyoub, Maha; Memeo, Lorenzo; Alvarez-Fernández, Emilio; Colarossi, Cristina; Costanzo, Rosario; Aiello, Eleonora; Martinetti, Daniela; Valmori, Danila

    2014-10-01

    Non-small cell lung cancer (NSCLC) is a major public health problem, accounting for more cancer-related deaths than any other cancer. Both immunotherapy, based on the expression of tumor-specific antigens, and targeted therapy, based on the presence of oncogenic mutations, are under development for NSCLC. In this study, we analyzed the expression of MAGE-A, a cancer-testis antigen, in tumors from a cohort of patients with resected NSCLC with respect to their clinicopathologic characteristics and their mutational status for the EGFR and KRAS genes. We found MAGE-A expression by IHC in 43% of the tumors. MAGE-A expression was significantly more frequent in squamous tumors than in adenocarcinomas, did not correlate with disease stage, but was correlated significantly with high tumor grade and worse survival. EGFR and KRAS mutations were present in adenocarcinomas, but not in squamous tumors. Whereas the presence of EGFR mutations did not seem to affect survival, the presence of KRAS mutations was associated with early-stage disease and better survival. MAGE-A expression was absent from adenocarcinomas with KRAS mutations, but not significantly different in tumors with or without EGFR mutations. Together, the reported results provide guidance for the design of combination therapies in patients with NSCLC.

  20. 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.

  1. Extracellular PKM2 induces cancer proliferation by activating the EGFR signaling pathway

    PubMed Central

    Hsu, Ming-Chuan; Hung, Wen-Chun; Yamaguchi, Hirohito; Lim, Seung-Oe; Liao, Hsin-Wei; Tsai, Chia-Hua; Hung, Mien-Chie

    2016-01-01

    Pyruvate kinase is a key enzyme in the glycolytic pathway that converts phosphoenolpyruvate to pyruvate, and the M2 isoform of pyruvate kinase (PKM2) is associated with cancer. PKM2 has been reported to function independently of its pyruvate kinase activity, which is crucial for cancer cell proliferation. Moreover, there is growing evidence indicating that dimeric PKM2 is released from tumor cells into the circulation of cancer patients. However, the role of secreted PKM2 in cancer is not well understood. Here, we found that the phosphorylation level of epidermal growth factor receptor (EGFR) significantly increased upon the exposure of cells to the recombinant PKM2 protein. In addition, secreted PKM2 induces EGFR phosphorylation and activates the EGFR downstream signaling in triple-negative breast cancer cells. In contrast, knocking down PKM2 decreased EGFR phosphorylation. Moreover, expression of R399E mutant PKM2, which has been reported to preferentially form a dimer, enhanced EGFR phosphorylation, cellular transformation, and cell proliferation more strongly than the wild-type PKM2. Thus, our study revealed a novel function of extracellular PKM2 in the promoting cancer cell proliferation through EGFR activation. PMID:27152240

  2. Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

    PubMed

    Clapéron, A; Guedj, N; Mergey, M; Vignjevic, D; Desbois-Mouthon, C; Boissan, M; Saubaméa, B; Paradis, V; Housset, C; Fouassier, L

    2012-03-15

    Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis

  3. Anti-EGFR therapeutic efficacy correlates directly with inhibition of STAT3 activity.

    PubMed

    Ung, Nelson; Putoczki, Tracy L; Stylli, Stanley S; Ng, Irvin; Mariadason, John M; Chan, Timothy A; Zhu, Hong-Jian; Luwor, Rodney B

    2014-05-01

    Several agents targeting the epidermal growth factor receptor (EGFR) have been FDA-approved to treat cancer patients with varying tumor types including metastatic colorectal cancer. Many patients treated with anti-EGFR therapy however do not respond and those that do initially respond often acquire resistance. Here we show a clear correlation between the efficacy of anti-EGFR inhibitors with their ability to inhibit STAT3 activity in A431 epidermoid carcinoma cells and in a series of wt K-RAS expressing human colon cancer cell lines. Furthermore, the ability of cetuximab to inhibit growth also correlated with its ability to inhibit STAT3 activity in tumor xenograft animal studies. In addition, stable knockdown of the STAT3 phosphatase, protein tyrosine phosphatase receptor delta (PTPRD) resulted in enhanced STAT3 activity and subsequent resistance to cetuximab in DIFI colon carcinoma cells. This resistance could be reversed by STAT3 inhibition. Finally, HN5 cells with acquired resistance to the EGFR tyrosine kinase inhibitor, AG1478 displayed greater STAT3 activity than the HN5 control cell line. These AG1478-refractory HN5 cells were re-sensitized to AG1478, cetuximab and erlotinib when co-treated with a STAT3 inhibitor. Taken together, our current data indicates a key role of STAT3 activity in promoting resistance to anti-EGFR therapy and suggests that anti-EGFR therapy in combination with inhibitors that block STAT3 may provide therapeutic benefit for patients with mCRC and other EGFR driven tumor types.

  4. Genomic activation of the EGFR and HER2-neu genes in a significant proportion of invasive epithelial ovarian cancers

    PubMed Central

    Vermeij, Joanna; Teugels, Erik; Bourgain, Claire; Xiangming, Ji; in 't Veld, Peter; Ghislain, Vanessa; Neyns, Bart; De Grève, Jacques

    2008-01-01

    Background The status of the EGFR and HER2-neu genes has not been fully defined in ovarian cancer. An integrated analysis of both genes could help define the proportion of patients that would potentially benefit from targeted therapies. Methods We determined the tumour mutation status of the entire tyrosine kinase (TK) domain of the EGFR and HER2-neu genes in a cohort of 52 patients with invasive epithelial ovarian cancer as well as the gene copy number and protein expression of both genes in 31 of these patients by DGGE and direct sequecing, immunohistochemistry and Fluorescent in Situ Hybridisation (FISH). Results The EGFR was expressed in 59% of the cases, with a 2+/3+ staining intensity in 38%. HER2-neu expression was found in 35%, with a 2/3+ staining in 18%. No mutations were found in exons 18–24 of the TK domains of EGFR and HER2-neu. High polysomy of the EGFR gene was observed in 13% of the invasive epthelial cancers and amplification of the HER2-neu gene was found in 10% and correlated with a high expression level by immunohistochemistry. Mutations within the tyrosine kinase domain were not found in the entire TK domain of both genes, but have been found in very rare cases by others. Conclusion Genomic alteration of the HER2-neu and EGFR genes is frequent (25%) in ovarian cancer. EGFR/HER2-neu targeted therapies should be investigated prospectively and specifically in that subset of patients. PMID:18182111

  5. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    PubMed

    Cai, Xin; Sheng, Jianhui; Tang, Chuanning; Nandakumar, Vijayalakshmi; Ye, Hua; Ji, Hong; Tang, Haiying; Qin, Yu; Guan, Hongwei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Wu, Taihua; Lin, Hongli

    2014-01-01

    Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations. PMID:24760004

  6. 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

  7. EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing

    PubMed Central

    Francis, Joshua M.; Zhang, Cheng-Zhong; Maire, Cecile L.; Jung, Joonil; Manzo, Veronica E.; Adalsteinsson, Viktor A.; Homer, Heather; Haidar, Sam; Blumenstiel, Brendan; Pedamallu, Chandra Sekhar; Ligon, Azra H.; Love, J. Christopher; Meyerson, Matthew; Ligon, Keith L.

    2014-01-01

    Glioblastomas with EGFR amplification represent approximately 50% of newly diagnosed cases and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor with implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here we applied a population-based single-cell whole genome sequencing methodology to characterize genomic heterogeneity in EGFR amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal loss-of-heterozygosity and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR Carboxyl-terminal deletions) identified in the bulk tumor segregate into non-overlapping subclonal populations. In vitro and in vivo functional studies show EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment. PMID:24893890

  8. [Response of Erlotinib in Lung Adenocarcinoma Harboring EGFR Sensitive Mutation in Cerebrospinal Fluid: Case Report].

    PubMed

    Li, Xiaoyan; Yang, Hui; Xu, Huayan; Wang, Shasha; Gao, Hongjun

    2016-01-01

    背景与目的 脑是非小细胞肺癌(non-small cell lung cancer, NSCLC)常见的转移部位。有研究显示表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors, EGFR-TKI)可透过血脑屏障,发挥抗肿瘤作用。本例报道采用突变扩增阻滞系统(amplification refractory mutation system, ARMS)检测脑脊液EGFR突变指导临床治疗的可行性,并分析TKI治疗肺癌脑转移的疗效和安全性。方法 腰穿取得脑脊液标本,检测颅内压力,检验常规、生化及肿瘤标志物,查找脱落细胞,采用ARMS法检测EGFR基因突变,得到阳性结果后给予盐酸厄洛替尼(erlotinib,tarceva,特罗凯)治疗,150 mg,每天1次。按照实体瘤疗效评价标准1.1版(Response Evaluation Criteriation in Solid Tumours, RECIST v1.1)评价客观疗效,按照不良反应通用术语标准4.0版(Common Terminology Criteria for Adverse Events v4.0, CTC AE v4.0)评估用药期间发生的不良事件。结果 该患者多线治疗后,颅内病灶控制欠佳,脑脊液中发现异型细胞,EGFR基因19外显子缺失突变,服用厄洛替尼4周后,颅内客观疗效为部分缓解(partial response, PR),颅外客观疗效为疾病稳定(stable disease, SD),颅内无进展生存期(progression-free survival, PFS)10.5个月,总生存期(overall survival, OS)11个月。主要不良反应为皮疹(1级)。结论 脑脊液检测EGFR突变可为制定治疗策略提供理论支持,根据不同的突变状态给予小分子靶向药物联合化疗,可分别控制颅内及颅外病灶。.

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

    PubMed Central

    2014-01-01

    Background 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. Methods 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. Results 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. Conclusions miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy. PMID:24650032

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

    SciTech Connect

    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 can 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.

  11. 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

  12. Lung Adenocarcinoma with EGFR Amplification has Distinct Clinicopathologic and Molecular Features in Never-Smokers

    PubMed Central

    Sholl, Lynette M.; Yeap, Beow Y.; Iafrate, A. John; Holmes-Tisch, Alison J.; Chou, Yi-Ping; Wu, Ming-Tsang; Goan, Yih-Gang; Su, Li; Benedittini, Elisa; Yu, Jian; Loda, Massimo; Jänne, Pasi A.; Christiani, David C.; Chirieac, Lucian R.

    2009-01-01

    In a subset of lung adenocarcinomas the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. We selected to study 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion-mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, low and high polysomy (100% v 54%, P=0.009). EGFR amplification occurred invariably on the mutated and not the wildtype allele (median mutated:wildtype ratios 14.0 v .33, P=0.003), was associated with solid histology (P=0.008), and advanced clinical stage (P=0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival 16 v 31 months, P=0.01) and when adjusted for stage (P=0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and demonstrate distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology. PMID:19826035

  13. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

    PubMed

    Zheng, Junfang; Dai, Yuanping; Yang, Zhiyu; Yang, Longyan; Peng, Zhiqiang; Meng, Ran; Xiong, Ying; He, Junqi

    2016-01-01

    Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular

  14. AXL mediates resistance to PI3Kα inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas

    PubMed Central

    Elkabets, Moshe; Pazarentzos, Evangelos; Juric, Dejan; Sheng, Qing; Pelossof, Raphael A.; Brook, Samuel; Benzaken, Ana Oaknin; Rodon, Jordi; Morse, Natasha; Yan, Jenny Jiacheng; Liu, Manway; Das, Rita; Chen, Yan; Tam, Angela; Wang, Huiqin; Liang, Jinsheng; Gurski, Joseph M.; Kerr, Darcy A.; Rosell, Rafael; Teixidó, Cristina; Huang, Alan; Ghossein, Ronald A.; Rosen, Neal; Bivona, Trever G.; Scaltriti, Maurizio; Baselga, José

    2015-01-01

    Summary Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous carcinoma (SCC) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance we have observed that SCCs cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)- protein kinase C (PKC), which in turn activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance. PMID:25873175

  15. Economic Evaluation of Companion Diagnostic Testing for EGFR Mutations and First-Line Targeted Therapy in Advanced Non-Small Cell Lung Cancer Patients in South Korea

    PubMed Central

    Lim, Eun-A; Bae, Eunmi; Lim, Jaeok; Shin, Young Kee; Choi, Sang-Eun

    2016-01-01

    Background As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR) mutation status compared with a no-testing strategy that is the current clinical practice in South Korea. Methods A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer’s perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles). Results In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs) were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs) increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib. Conclusion Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy. PMID:27483001

  16. Dose-Dependent Mutation Rates Determine Optimum Erlotinib Dosing Strategies for EGFR Mutant Non-Small Cell Lung Cancer Patients

    PubMed Central

    Liu, Lin L.; Li, Fei; Pao, William; Michor, Franziska

    2015-01-01

    Background The advent of targeted therapy for cancer treatment has brought about a paradigm shift in the clinical management of human malignancies. Agents such as erlotinib used for EGFR-mutant non-small cell lung cancer or imatinib for chronic myeloid leukemia, for instance, lead to rapid tumor responses. Unfortunately, however, resistance often emerges and renders these agents ineffective after a variable amount of time. The FDA-approved dosing schedules for these drugs were not designed to optimally prevent the emergence of resistance. To this end, we have previously utilized evolutionary mathematical modeling of treatment responses to elucidate the dosing schedules best able to prevent or delay the onset of resistance. Here we expand on our approaches by taking into account dose-dependent mutation rates at which resistant cells emerge. The relationship between the serum drug concentration and the rate at which resistance mutations arise can lead to non-intuitive results about the best dose administration strategies to prevent or delay the emergence of resistance. Methods We used mathematical modeling, available clinical trial data, and different considerations of the relationship between mutation rate and drug concentration to predict the effectiveness of different dosing strategies. Results We designed several distinct measures to interrogate the effects of different treatment dosing strategies and found that a low-dose continuous strategy coupled with high-dose pulses leads to the maximal delay until clinically observable resistance. Furthermore, the response to treatment is robust against different assumptions of the mutation rate as a function of drug concentration. Conclusions For new and existing targeted drugs, our methodology can be employed to compare the effectiveness of different dose administration schedules and investigate the influence of changing mutation rates on outcomes. PMID:26536620

  17. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    SciTech Connect

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  18. Successful treatment with afatinib after grade 3 hepatotoxicity induced by both gefitinib and erlotinib in EGFR mutation-positive non-small cell lung cancer.

    PubMed

    Zenke, Yoshitaka; Umemura, Shigeki; Sugiyama, Eri; Kirita, Keisuke; Matsumoto, Shingo; Yoh, Kiyotaka; Niho, Seiji; Ohmatsu, Hironobu; Goto, Koichi

    2016-09-01

    Hepatotoxicity is a major cause of the withdrawal of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) when treating EGFR mutation-positive non-small cell lung cancer (NSCLC). We report a case in which gefitinib- and elrotinib-induced severe hepatotoxicity arose in a patient with the uridine diphosphate glucuronosyltransferase isoform 1A1 (UGT1A1) and cytochrome p450 3A5 (CYP3A5) poor metabolizer phenotypes. Afatinib is not significantly metabolized by cytochrome p450-mediated pathways. We describe successful management of the patient's tumor by switching to afatinib. Evaluation of single nucleotide polymorphisms (SNPs) in metabolic enzymes might be useful to predict severe hepatotoxicity induced by EGFR-TKIs. PMID:27565905

  19. Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.

    PubMed

    Sun, Sheng; Zhou, Xi; Zhang, Wei; Gallick, Gary E; Kuang, Jian

    2015-03-15

    Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.

  20. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells.

    PubMed

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients' outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCMTGF, FCMPDGF) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCMB). FCMTGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCMTGF≫FCMPDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCMTGF>FCMPDGF) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. PMID:24394543

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

    SciTech Connect

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

    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 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 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 cycle of

  2. 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

  3. Alpha6beta4 integrin crosslinking induces EGFR clustering and promotes EGF-mediated Rho activation in breast cancer

    PubMed Central

    Gilcrease, Michael Z; Zhou, Xiao; Lu, Xiaolin; Woodward, Wendy A; Hall, Brian E; Morrissey, Phillip J

    2009-01-01

    Background The α6β4 integrin is overexpressed in the basal subtype of breast cancer and plays an important role in tumor cell motility and invasion. EGFR is also overexpressed in the basal subtype of breast cancer, and crosstalk between α6β4 integrin and EGFR appears to be important in tumor progression. Methods We evaluated the effects of α6β4 crosslinking on the distribution and function of EGFR in breast carcinoma cell line MDA-MB-231. Receptor distribution was evaluated by fluorescence microscopy and multispectral imaging flow cytometry, and ligand-mediated EGFR signaling was evaluated using Western blots and a Rho pull-down assay. Results Antibody-mediated crosslinking of α6β4 integrin was sufficient to induce cell-surface clustering of not only α6β4 but also EGFR in nonadherent cells. The induced clustering of EGFR was observed minimally after 5 min of integrin crosslinking but was more prominent after 15 min. EGFR clustering had minimal effect on the phosphorylation of Akt or Erk1,2 in response to EGF in suspended cells or in response to HB-EGF in adherent cells. However, EGFR clustering induced by crosslinking α6β4 had a marked effect on Rho activation in response to EGF. Conclusion Crosslinking α6β4 integrin in breast carcinoma cells induces EGFR clustering and preferentially promotes Rho activation in response to EGF. We hypothesize that this integrin-EGFR crosstalk may facilitate tumor cell cytoskeletal rearrangements important for tumor progression. PMID:19470173

  4. [Value of immunohistochemical staining with mutation-specific antibodies in detecting EGFR mutations: a meta-analysis].

    PubMed

    Ma, Qing; Wang, Jing; Zhong, Diansheng; Ning, Chao; Liu, Chang; Xiao, Ping

    2014-06-20

    背景与目的 已有的研究表明:表皮生长因子受体(epidermal growth factor receptor, EGFR)基因突变是非小细胞肺癌(non-small cell lung cancer, NSCLC)患者应用表皮生长因子受体酪氨酸激酶抑制剂(EGFR tyrosine kinase inhibitor, EGFR-TKI)治疗疗效的最重要的预测因子。EGFR基因突变的患者对于使用TKIs分子靶向药物治疗疗效更敏感。其突变检测对肺癌一线靶向治疗选择尤为关键。研究分析特异性抗体免疫组化法(immunohistochemistry, IHC)检测EGFR突变与DNA测序法比较的敏感度与特异度,明确该方法准确性及临床应用价值。 方法 通过Pubmed数据库检索所有符合检索条件的文献,末次检索日期2013年3月26日,根据纳入和排除标准进行进一步筛选,采用诊断试验meta分析方法,分析特异性抗体免疫组化方法与DNA直接测序法对比的敏感度与特异度。结果 10篇文献纳入meta分析,L858R 1,679例,E746-A750del 1,041例,诊断比值比(diagnositic odds ratio, DOR)分别为225.17(95%CI: 55.67-910.69)和267.16(95%CI: 132.45-538.88);SROC曲线AUC分别为0.948,4(SEAUC=0.014,4)和0.981,3(SEAUC=0.009,9),Q*统计量分别为0.888,3(SEQ*=0.019,2)和0.9397(SEQ*=0.019,1)。结论 以上两种特异性抗体IHC鉴别EGFR突变的特异度高,灵敏度较高,作为筛查突变方法可行性高,具有一定的临床应用价值。

  5. EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells.

    PubMed

    Martínez-Palacián, A; del Castillo, G; Herrera, B; Fernández, M; Roncero, C; Fabregat, I; Sánchez, A

    2012-02-01

    Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in

  6. Activating HER2 mutations in HER2 gene amplification negative breast cancer

    PubMed Central

    Bose, Ron; Kavuri, Shyam M.; Searleman, Adam C.; Shen, Wei; Shen, Dong; Koboldt, Daniel C.; Monsey, John; Goel, Nicholas; Aronson, Adam B.; Li, Shunqiang; Ma, Cynthia X.; Ding, Li; Mardis, Elaine R.; Ellis, Matthew J.

    2012-01-01

    Data from eight breast cancer genome sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized thirteen HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGFR exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings demonstrate that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. PMID:23220880

  7. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  8. Ibrutinib selectively and irreversibly targets EGFR (L858R, Del19) mutant but is moderately resistant to EGFR (T790M) mutant NSCLC Cells

    PubMed Central

    Wang, Wenchao; Hu, Chen; Ye, Zi; Zhao, Zheng; Wang, Li; Li, Xixiang; Yu, Kailin; Liu, Juan; Wu, Jiaxin; Yan, Xiao-E; Zhao, Peng; Wang, Jinhua; Wang, Chu; Weisberg, Ellen L.; Gray, Nathanael S.; Yun, Cai-Hong; Liu, Jing; Chen, Liang; Liu, Qingsong

    2015-01-01

    Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M ‘gatekeeper’ mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib's effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo. These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC. PMID:26375053

  9. MITF Modulates Therapeutic Resistance through EGFR Signaling.

    PubMed

    Ji, Zhenyu; Erin Chen, Yiyin; Kumar, Raj; Taylor, Michael; Jenny Njauw, Ching-Ni; Miao, Benchun; Frederick, Dennie T; Wargo, Jennifer A; Flaherty, Keith T; Jönsson, Göran; Tsao, Hensin

    2015-07-01

    Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF (V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs), although the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR-induced vemurafenib resistance is ligand dependent. We employed whole-genome expression analysis and discovered that vemurafenib resistance correlated with the loss of microphthalmia-associated transcription factor (MITF), along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance, and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients' tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an "autocrine drug resistance loop" is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition. PMID:25789707

  10. NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor.

    PubMed

    Galvani, Elena; Sun, Jing; Leon, Leticia G; Sciarrillo, Rocco; Narayan, Ravi S; Sjin, Robert Tjin Tham; Lee, Kwangho; Ohashi, Kadoaki; Heideman, Daniëlle A M; Alfieri, Roberta R; Heynen, Guus J; Bernards, René; Smit, Egbert F; Pao, William; Peters, Godefridus J; Giovannetti, Elisa

    2015-12-15

    The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

  11. NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor

    PubMed Central

    Galvani, Elena; Sun, Jing; Leon, Leticia G.; Sciarrillo, Rocco; Narayan, Ravi S.; Tjin Tham Sjin, Robert; Lee, Kwangho; Ohashi, Kadoaki; Heideman, Daniëlle A.M.; Alfieri, Roberta R.; Heynen, Guus J.; Bernards, René; Smit, Egbert F.; Pao, William; Peters, Godefridus J.; Giovannetti, Elisa

    2015-01-01

    The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs. PMID:26015408

  12. [Detection and evaluation of EGFR mutation status in serum of patients with advanced non-small cell lung cancer treated with EGFR-TKIs].

    PubMed

    Ma, Ling; Liu, Li; Zhang, Tao; Shan, Li

    2013-06-01

    背景与目的 小分子酪氨酸激酶抑制剂(tyrosine kinase inhibitors, TKIs)对于表皮生长因子受体(epidermal growth factor receptor, EGFR)基因突变的肺癌患者显示出良好的治疗效果。本研究旨在探讨晚期非小细胞肺癌(non-small cell lung cancer, NSCLC)患者血清EGFR基因突变状态与EGFR-TKIs疗效的关系。 方法 检测80例一线口服EGFR-TKIs晚期NSCLC患者血清EGFR基因的突变状态,对患者进行长期随访并评价治疗效果。结果 80例患者血清EGFR基因突变27例(33.8%),其中外显子19缺失突变12例(44.4%),外显子21点突变15例(55.6%);血清EGFR基因突变患者的有效率(55.6%, 15/27)高于野生型患者(17.0%, 9/53),差异具有统计学意义(χ2=0.370, P<0.001);血清EGFR基因突变患者中位无进展生存时间(progress free survival, PFS)明显长于野生型患者(9.8个月 vs 5.7个月,P=0.014)。结论 血清EGFR基因突变患者一线口服EGFR-TKIs的疗效优于野生型患者,血清EGFR基因状态可为EGFR-TKIs的一线治疗提供有效依据。

  13. EGF receptor mutations in lung cancer: from humans to mice and maybe back to humans.

    PubMed

    Arteaga, Carlos L

    2006-06-01

    Deletions in exon 19 and nucleotide substitutions in exon 21 are the most common mutations of the EGFR (ErbB1) in NSCLC. These mutations endow the receptor with constitutive kinase activity. Most tumors expressing these mutants respond well to EGFR tyrosine kinase inhibitors, suggesting that they are dependent on mutant EGFR signaling. Two groups developed transgenic mice in which expression of these mutants is temporally induced in mouse lung. Mice expressing EGFR mutants develop bronchioloalveolar cancer and lung adenocarcinoma, which are highly sensitive to EGFR inhibitors. These mouse models provide important opportunities for studying the biology of NSCLC and the refinement of anti-EGFR therapies.

  14. BRAF V600E mutation and resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer: a meta-analysis.

    PubMed

    Mao, Chen; Liao, Ru-Yan; Qiu, Li-Xin; Wang, Xi-Wen; Ding, Hong; Chen, Qing

    2011-04-01

    Epidemiologic studies have evaluated the association between BRAF mutations and resistance to the treatment of anti-EGFR monoclonal antibodies (MoAb) in patients with metastatic colorectal cancer (mCRC). However, the results are still inconclusive. To derive a more precise estimation of the relationship, we performed this meta-analysis. A total of 11 studies were included in the final meta-analysis. There were seven studies for unselected mCRC patients and four studies for patients with wild type KRAS mCRC. Among unselected mCRC patients, BRAF V600E mutation was detected in 48 of 546 primary tumors (8.8%). The objective response rate (ORR) of patients with mutant BRAF was 29.2% (14/48), whereas the ORR of patients with wild-type BRAF was 33.5% (158/472).The overall RR for ORR of mutant BRAF patients over wild-type BRAF patients was 0.86 (95% CI=0.57-1.30; P=0.48). For patients with KRAS wild-type mCRC, BRAF V600E mutation was detected in 40 of 376 primary tumors (10.6%). The ORR of patients with mutant BRAF was 0.0% (0/40), whereas the ORR of patients with wild-type BRAF was 36.3% (122/336). The pooled RR of mutant BRAF patients over wild-type BRAF patients was 0.14 (95% CI=0.04-0.53; P=0.004). In conclusion, this meta-analysis provides evidence that BRAF V600E mutation is associated with lack of response in wild-type KRAS mCRC treated with anti-EGFR MoAbs. BRAF mutation may be used as an additional biomarker for the selection of mCRC patients who might benefit from anti-EGFR MoAbs therapy.

  15. Mutation profiling in chinese patients with metastatic colorectal cancer and its correlation with clinicopathological features and anti-EGFR treatment response

    PubMed Central

    Wang, Fang; Zhao, Qi; Zhang, Dong-Sheng; Wang, Feng-Hua; Wang, Zhi-Qiang; Luo, Hui-Yan; He, Ming-Ming; Wang, De-Shen; Jin, Ying; Ren, Chao; Qiu, Miao-Zhen; Ren, Jian; Pan, Zhi-Zhong; Li, Yu-Hong; Shao, Jiao-Yong; Xu, Rui-Hua

    2016-01-01

    An increasing number of studies reveal the significance of genetic markers in guiding target treatment and refining prognosis. This retrospective observational study aims to assess the mutation profile of metastatic colorectal cancer (mCRC) in Chinese population with the help of MassARRAY® technique platform and OncoCarta™ Panel. 322 Chinese patients with mCRC who received clinical molecular testing as part of their standard care were investigated. 80 patients received cetuximab palliative treatment. 238 common hot-spot mutations of 19 cancer related genes in the OncoCarta™ Panel were tested. 44 mutations in 11 genes were detected in 156 cases (48.4%). At least one mutation was identified in 38.5% (124/322) of all tested cases, two concomitant mutations in 9.0% (29/322) and three mutations in 3 cases (<1%). KRAS was the most frequently mutated gene (34.8%), followed by PIK3CA (9.6%), NRAS (4.3%), BRAF (3.4%), EGFR (2.5%) and HRAS (1.2%). Less frequent mutations were detected in PDGFRA, RET, AKT1, FGFR1, and ERBB2. Co-mutation of RAS family subtypes was observed in 5 patients, and KRAS and BRAF concurrent mutation in 1 patient. KRAS, NRAS, BRAF and PIK3CA mutations had association with some clinicopathological features statistically. Patients identified as wild-type in all 19 genes had better objective response rate when treated with cetuximab. The clinical molecular testing with OncoCarta™ Panel supplemented the limited data of mCRC in Chinese population, and offered a clearer landscape of multiple gene mutational profile in not only clinically prognostic KRAS, NRAS, BRAF and PIK3CA genes, but also less frequent mutated genes. Knowledge of these multiple gene mutation patterns may give clues in exploring interesting accompanying co-occurrence relationship or mutually exclusive relationship between mutated genes, as well as in predicting benefit of all-wild-type patients from anti-EGFR treatment. PMID:27050078

  16. Mutation profiling in chinese patients with metastatic colorectal cancer and its correlation with clinicopathological features and anti-EGFR treatment response.

    PubMed

    Li, Zhe-Zhen; Wang, Feng; Zhang, Zi-Chen; Wang, Fang; Zhao, Qi; Zhang, Dong-Sheng; Wang, Feng-Hua; Wang, Zhi-Qiang; Luo, Hui-Yan; He, Ming-Ming; Wang, De-Shen; Jin, Ying; Ren, Chao; Qiu, Miao-Zhen; Ren, Jian; Pan, Zhi-Zhong; Li, Yu-Hong; Shao, Jiao-Yong; Xu, Rui-Hua

    2016-05-10

    An increasing number of studies reveal the significance of genetic markers in guiding target treatment and refining prognosis. This retrospective observational study aims to assess the mutation profile of metastatic colorectal cancer (mCRC) in Chinese population with the help of MassARRAY® technique platform and OncoCarta™ Panel.322 Chinese patients with mCRC who received clinical molecular testing as part of their standard care were investigated. 80 patients received cetuximab palliative treatment. 238 common hot-spot mutations of 19 cancer related genes in the OncoCarta™ Panel were tested.44 mutations in 11 genes were detected in 156 cases (48.4%). At least one mutation was identified in 38.5% (124/322) of all tested cases, two concomitant mutations in 9.0% (29/322) and three mutations in 3 cases (<1%). KRAS was the most frequently mutated gene (34.8%), followed by PIK3CA (9.6%), NRAS (4.3%), BRAF (3.4%), EGFR (2.5%) and HRAS (1.2%). Less frequent mutations were detected in PDGFRA, RET, AKT1, FGFR1, and ERBB2. Co-mutation of RAS family subtypes was observed in 5 patients, and KRAS and BRAF concurrent mutation in 1 patient. KRAS, NRAS, BRAF and PIK3CA mutations had association with some clinicopathological features statistically. Patients identified as wild-type in all 19 genes had better objective response rate when treated with cetuximab.The clinical molecular testing with OncoCarta™ Panel supplemented the limited data of mCRC in Chinese population, and offered a clearer landscape of multiple gene mutational profile in not only clinically prognostic KRAS, NRAS, BRAF and PIK3CA genes, but also less frequent mutated genes. Knowledge of these multiple gene mutation patterns may give clues in exploring interesting accompanying co-occurrence relationship or mutually exclusive relationship between mutated genes, as well as in predicting benefit of all-wild-type patients from anti-EGFR treatment. PMID:27050078

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

    SciTech Connect

    Sakuma, Yuji; Yamazaki, Yukiko; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Matsukuma, Shoichi; Koizume, Shiro; Miyagi, Yohei

    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 cells 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.

  18. Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance

    PubMed Central

    Naumov, George N.; Nilsson, Monique B.; Cascone, Tina; Briggs, Alexandra; Straume, Oddbjorn; Akslen, Lars A.; Lifshits, Eugene; Byers, Lauren Averett; Xu, Li; Wu, Hua-kang; Jänne, Pasi; Kobayashi, Susumu; Halmos, Balazs; Tenen, Daniel; Tang, Xi M.; Engelman, Jeffrey; Yeap, Beow; Folkman, Judah; Johnson, Bruce E.; Heymach, John V.

    2010-01-01

    Purpose The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non–small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. Experimental Design We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Results Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. Conclusion These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients. PMID:19447865

  19. Detection of EGFR mutation in supernatant, cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing.

    PubMed

    Lin, Jie; Gu, Ye; Du, Rui; Deng, Min; Lu, Yaodan; Ding, Yanqing

    2014-01-01

    To determine epidermal growth factor receptor (EGFR) mutation in advanced non-small cell lung cancer (NSCLC) patients and compare the detection efficiency between different sample resources, both high resolution melting (HRM) analysis and direct sequencing method were used to analyze 36 pleural effusion samples and 22 matched biopsy tumor tissues collected from NSCLC patients. For each pleural effusion sample, the supernatant and the cell pellets were examined separately. Among all the 36 cases of pleural effusion samples, 18 mutations of EGFR were found in cell-free supernatant while 13 mutations were found in the cell pellets as detected by HRM analysis. In the 22 matched samples, 13 cases of EGFR mutations were identified in paraffin-embedded biopsy tissue samples, 12 cases in the cell-free supernatant and 9 cases in the cell pellets of pleural effusion. EGFR mutations in 15 cases out of the total 36 pleural effusion samples detected by direct sequencing were also identified by HRM analysis, giving 100% efficiency for HRM method. The results established the important role of HRM as a reliable and efficient method to determine EGFR mutation status and indicated the feasibility of using pleural effusion in replacement of biopsy tissues in particular clinical cases. Furthermore, the cell-free supernatant of pleural effusion might be a better resource for mutation detection than cell pellets.

  20. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.

    PubMed

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J; Lipschutz, Joshua H

    2014-12-15

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.

  1. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

    PubMed Central

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M.; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F.; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J.

    2014-01-01

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury. PMID:25298525

  2. Blockade of EGFR and MEK intercepts heterogeneous mechanisms of acquired resistance to anti-EGFR therapies in colorectal cancer.

    PubMed

    Misale, Sandra; Arena, Sabrina; Lamba, Simona; Siravegna, Giulia; Lallo, Alice; Hobor, Sebastijan; Russo, Mariangela; Buscarino, Michela; Lazzari, Luca; Sartore-Bianchi, Andrea; Bencardino, Katia; Amatu, Alessio; Lauricella, Calogero; Valtorta, Emanuele; Siena, Salvatore; Di Nicolantonio, Federica; Bardelli, Alberto

    2014-02-19

    Colorectal cancers (CRCs) that are sensitive to the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.

  3. A comparative analysis of EGFR mutation status in association with the efficacy of TKI in combination with WBRT/SRS/surgery plus chemotherapy in brain metastasis from non-small cell lung cancer.

    PubMed

    Cai, Ling; Zhu, Jian-fei; Zhang, Xue-wen; Lin, Su-xia; Su, Xiao-dong; Lin, Peng; Chen, Kai; Zhang, Lan-jun

    2014-11-01

    We proposed to identify the efficacy of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) using whole brain radiotherapy (WBRT)/stereotactic radiosurgery (SRS)/surgery in brain metastases from patients with non-small cell lung cancer (NSCLC) and clarify the association between treatment outcome and EGFR gene mutation status. A total of 282 patients with NSCLC brain metastases who underwent WBRT/SRS/surgery alone or in combination with TKI were enrolled in our study from 2003-2013. Amplification mutation refractory system technology was used to determine the EGFR mutation status in 109 tissue samples. EGFR mutation detection was performed in 109 patients with tumor tissues. The EGFR positive rate was 50 % (55/109), including 26 exon 19 deletions and 24 L858R mutations. The median follow-up time was 28 months. The median overall survival, median progression-free survival of intracranial disease, and median progression-free survival of extracranial disease was significantly longer for patients with TKI treatment (31.9 vs 17.0 months, P < 0.0001; 19.8 vs 12.0 months, P < 0.0001; and 19.6 vs 12.3 months, P < 0.0001; respectively). In subgroup analysis within the TKI group, patients harboring EGFR mutations had better extracranial disease control (20.4 vs 14.1 months, P = 0.032). Administration of TKI agents with conventional therapy compared with conventional therapy alone might be beneficial for overall survival, progression-free survival of intracranial disease and progression-free survival of extracranial disease in patients with brain metastases from NSCLC independent of EGFR mutations.

  4. Differences among lesions with exon 19, exon 21 EGFR mutations and wild types in surgically resected non-small cell lung cancer

    PubMed Central

    Jin, Ying; Chen, Ming; Yu, Xinmin

    2016-01-01

    The clinical behavior of patients with advanced non-small cell lung cancer (NSCLC) differ between epidermal growth factor receptor (EGFR) exon 19 deletion (Ex19) and EGFR exon 21 L858R mutation (Ex21). This study aimed to evaluate whether these differences exist in surgically resected NSCLC. A total of 198 patients with surgically resected NSCLC harbouring Ex19 (n = 53), Ex21 (n = 51), and EGFR wild-type (Wt) (n = 94) were analyzed. The clinicopathological features, laboratory parameters, recurrent sites and disease-free survival (DFS) were compared according to mutational EGFR status. Ex21 occurred more frequently in female (p < 0.001), never-smokers (p < 0.001), adenocarcinoma (p < 0.001), low grade (p = 0.013) than Wt lesions. Ex19 occurred more frequently in female (p = 0.016), never-smokers (p = 0.008), adenocarcinoma (p < 0.001), low grade (p = 0.025) than Wt lesions. Ex 21 lesions (p = 0.026) had larger lepidic components than Wt lesions. Wt lesions had larger mucinous variant components than Ex21 lesions (p = 0.045) and Ex19 lesions (p = 0.015). Ex21 lesions were associated with lower pretreatment neutrophil: lymphocyte ratio (NLR) than Wt lesions (p = 0.017). The recurrent sites and DFS were similar among patients with Wt, Ex19 and Ex21. PMID:27527915

  5. Dacomitinib in lung cancer: a "lost generation" EGFR tyrosine-kinase inhibitor from a bygone era?

    PubMed

    Ou, Sai-Hong Ignatius; Soo, Ross A

    2015-01-01

    EGFR tyrosine-kinase inhibitors (TKIs) have now been firmly established as the first-line treatment for non-small-cell lung cancer (NSCLC) patients harboring activating EGFR mutations, based on seven prospective randomized Phase III trials. However, despite significantly improved overall response rate and improved median progression-free survival when compared to platinum-doublet chemotherapy, EGFR-mutant NSCLC patients treated with EGFR TKIs invariably progress due to the emergence of acquired resistances, with the gatekeeper T790M mutation accounting for up to 60% of the resistance mechanisms. Second-generation irreversible EGFR TKIs were developed in part to inhibit the T790M mutation, in addition to the common activating EGFR mutations. Dacomitinib is one such second-generation EGFR TKI designed to inhibit both the wild-type (WT) EGFR and EGFR T790M. Afatinib is another second-generation EGR TKI that has been now been approved for the first-line treatment of EGFR-mutant NSCLC patients, while dacomitinib continues to undergo clinical evaluation. We will review the clinical development of dacomitinib from Phase I to Phase III trials, including the two recently published negative large-scale randomized Phase III trials (ARCHER 1009, NCIC-BR-26). Results from another large-scale randomized trial (ARCHER 1050) comparing dacomitinib to gefitinib as first-line treatment of advanced treatment-naïve EGFR-mutant NSCLC patients will soon be available and will serve as the lynchpin trial for the potential approval of dacomitinib in NSCLC. Meanwhile, third-generation EGFR TKIs (eg, CO-1686 [rociletinib], AZ9291, HM61713, EGF816, and ASP8273) that preferentially and potently inhibit EGFR T790M but not WT EGFR are in full-scale clinical development, and some of these EGFR TKIs have received "breakthrough" designation by the US Food and Drug Administration and will likely be approved in late 2015. Given the rapid development of third-generation EGFR TKIs and the approval of

  6. HDAC6 regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells.

    PubMed

    Liu, Wei; Fan, Lucy X; Zhou, Xia; Sweeney, William E; Avner, Ellis D; Li, Xiaogang

    2012-01-01

    We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.

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

    PubMed

    Chen, Zhi-Dong; Xu, Liang; Tang, Kan-Kai; Gong, Fang-Xiao; Liu, Jing-Quan; Ni, Yin; Jiang, Ling-Zhi; Hong, Jun; Han, Fang; Li, Qian; Yang, Xiang-Hong; Sun, Ren-Hua; Mo, Shi-Jing

    2016-09-10

    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 IKKβ 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.

  8. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

    PubMed Central

    Cross, Darren A. E.; Ashton, Susan E.; Ghiorghiu, Serban; Eberlein, Cath; Nebhan, Caroline A.; Spitzler, Paula J.; Orme, Jonathon P.; Finlay, M. Raymond V.; Ward, Richard A.; Mellor, Martine J.; Hughes, Gareth; Rahi, Amar; Jacobs, Vivien N.; Brewer, Monica Red; Ichihara, Eiki; Sun, Jing; Jin, Hailing; Ballard, Peter; Al-Kadhimi, Katherine; Rowlinson, Rachel; Klinowska, Teresa; Richmond, Graham H. P.; Cantarini, Mireille; Kim, Dong-Wan; Ranson, Malcolm R.; Pao, William

    2014-01-01

    First generation EGF receptor tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit in patients with advanced EGFR mutant (EGFRm+) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent and selective third generation irreversible inhibitor of both EGFRm+ sensitizing and T790M resistance mutants that spares wild-type EGFR. This monoanilino-pyrimidine compound is structurally distinct from other third generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Pre-clinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm+ and EGFRm+/T790M mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models. The treatment of two patients with advanced EGFRm T790M+ NSCLC is described as proof of principle. PMID:24893891

  9. Design, synthesis and in vitro anti-proliferative activity of 4,6-quinazolinediamines as potent EGFR-TK inhibitors.

    PubMed

    Mowafy, Samar; Farag, Nahla A; Abouzid, Khaled A M

    2013-03-01

    4-Anilino-6-substituted-quinazolines were designed, synthesized and evaluated for EGFR-TK and tumor growth inhibitory activities. The target compounds were designed with enamine ester or urea moieties appended at the C-6 of quinazoline as additional hydrogen bond acceptor functions. Most of the synthesized compounds displayed potent EGFR-TK inhibitory activity at 10 μM and the 6-ureido-anilinoquinazoline derivative 7a showed IC50 value of 0.061 μM. Moreover, six compounds were tested by National Cancer Institute (NCI), USA for their anti-proliferative activity at 10 μM in full NCI 60 cell panel. Compound 7a was further assayed for five dose molar ranges in full NCI 60 cell panel and exhibited remarkable growth inhibitory activity pattern against Non-Small Cell Lung Cancer EKVX (GI50 = 0.37 μM), NCI-H322M (GI50 = 0.36 μM), Renal Cancer A498 (GI50 = 0.46 μM), TK-10 (GI50 = 0.99 μM) and Breast Cancer MDA-MB-468 (GI50 = 1.096 μM) which are of high EGFR expression. Docking study was performed for the active compounds into ATP binding site of EGFR-TK which showed similar binding mode to gefitinib and additional binding with Cys-773 at the gatekeeper of EGFR-TK enzyme.

  10. Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR

    SciTech Connect

    Garrett, T.; Burgess, A; Gan, H; Luwor, R; Cartwright, G; Walker, F; Orchard, S; Clayton, A; Nice, E; et. al.

    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. 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.

  11. Mapping inhibitor response to the in-frame deletions, insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer.

    PubMed

    Ning, Jingheng; Wu, Qi; Liu, Zhenguo; Wang, Jianhui; Lin, Xianfu

    2016-01-01

    Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase's active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation-π interactions and hydrophobic contacts.

  12. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26079946

  14. Characteristics and overall survival of EGFR mutation-positive non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors: a retrospective analysis for 1660 Japanese patients

    PubMed Central

    Inoue, Akira; Yoshida, Kazushi; Morita, Satoshi; Imamura, Fumio; Seto, Takashi; Okamoto, Isamu; Nakagawa, Kazuhiko; Yamamoto, Nobuyuki; Muto, Satoshi; Fukuoka, Masahiro

    2016-01-01

    Background The Japan Guidelines of Lung Cancer Therapy recommend epidermal growth factor receptor-tyrosine kinase inhibitors as a first-line therapy for advanced/recurrent non-small cell lung cancer patients with epidermal growth factor receptor mutation. Although survival periods in recent reports of epidermal growth factor receptor-tyrosine kinase inhibitor treatment have been getting longer, the reasons why are unclear. We investigated the survival, prognostic factors and real-world treatment of non-small cell lung cancer patients with epidermal growth factor receptor mutation in clinical practice. Methods Non-small cell lung cancer patients (n = 1660) who started first-line treatment from January 2008 to December 2012 were enrolled. Patients were diagnosed with epidermal growth factor receptor mutation-positive advanced/recurrent non-small cell lung cancer by histology or cytology samples. The primary objective was to estimate overall survival. The secondary objectives were to determine prognostic factors, real-world treatment patterns and efficacy of gefitinib treatment. We calculated the treatment exposure rate for each treatment category using the following formula: exposure rate = person-years for the treatment category/total person-years × 100. Results The median overall survival was 30.8 months. Sex, age, histology, epidermal growth factor receptor mutation type, clinical stage and performance status affected overall survival. The exposure rates for all epidermal growth factor receptor-tyrosine kinase inhibitors, gefitinib and platinum-doublet chemotherapy were 62.1, 46.4 and 8.5% respectively. Overall 56.1% of patients were administered gefitinib as first-line therapy, and 39.0% were treated with ≥2 epidermal growth factor receptor-tyrosine kinase inhibitor regimens. The median progression-free survival in the first-line gefitinib group was 11.4 months. Factors affecting prognosis were sex, histology, clinical stage and performance status. Conclusion

  15. Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer

    PubMed Central

    Bardelli, Alberto; Corso, Simona; Bertotti, Andrea; Hobor, Sebastijan; Valtorta, Emanuele; Siravegna, Giulia; Sartore-Bianchi, Andrea; Scala, Elisa; Cassingena, Andrea; Zecchin, Davide; Apicella, Maria; Migliardi, Giorgia; Galimi, Francesco; Lauricella, Calogero; Zanon, Carlo; Perera, Timothy; Veronese, Silvio; Corti, Giorgio; Amatu, Alessio; Gambacorta, Marcello; Diaz, Luis A.; Sausen, Mark; Velculescu, Victor E.; Comoglio, Paolo; Trusolino, Livio; Di Nicolantonio, Federica; Giordano, Silvia; Siena, Salvatore

    2014-01-01

    EGFR targeted monoclonal antibodies are effective in a subset of metastatic colorectal tumors (mCRC). Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in patients who do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies demonstrate that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived CRC xenografts, MET amplification correlated with resistance to EGFR blockade which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in CRC and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification. PMID:23729478

  16. Enhanced gefitinib-induced repression of the epidermal growth factor receptor pathway by ataxia telangiectasia-mutated kinase inhibition in non-small-cell lung cancer cells.

    PubMed

    Misumi, Keizo; Sun, Jiying; Kinomura, Aiko; Miyata, Yoshihiro; Okada, Morihito; Tashiro, Satoshi

    2016-04-01

    The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR-TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non-small-cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR-TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR-TKIs, we examined the cross-talk of the EGFR pathways with ataxia telangiectasia-mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR-TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.

  17. Erlotinib plus bevacizumab as an effective treatment for leptomeningeal metastases from EGFR mutation-positive non-small cell lung cancer.

    PubMed

    Sakata, Yoshihiko; Kawamura, Kodai; Shingu, Naoki; Ichikado, Kazuya

    2016-09-01

    Leptomeningeal metastasis is a severe complication of non-small cell lung cancer. Its prognosis is very poor and conventional treatments have limited efficacy. However, epidermal growth factor receptor-tyrosine kinase inhibitors have exhibited high response rates in EGFR mutation-positive lung cancer patients with central nervous system metastases. It has been postulated that this could be due to the penetration of agents into the central nervous system and a high cerebrospinal fluid concentration is a key consideration in measuring treatment effect. Bevacizumab has also been used as an effective therapeutic agent in patients with central nervous system metastases. However, the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitor doublet therapy for leptomeningeal metastases and the cerebrospinal fluid penetration of epidermal growth factor receptor-tyrosine kinase inhibitors have yet to be determined. Moreover, the safety of this doublet regimen in patients with a poor general condition is not known. Herein, we report on a case treated with erlotinib plus bevacizumab for leptomeningeal metastases from EGFR mutation-positive non-small cell lung cancer. The patient's performance status significantly improved and the cerebrospinal fluid penetration rate of erlotinib plus bevacizumab was equal to or greater than the past reports of erlotinib alone. PMID:27565925

  18. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

  19. Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

    PubMed

    Centuori, Sara M; Gomes, Cecil J; Trujillo, Jesse; Borg, Jamie; Brownlee, Joshua; Putnam, Charles W; Martinez, Jesse D

    2016-07-01

    Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects. PMID:27086143

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

    PubMed Central

    Li, Ting; Yuan, Wanqiong; Mo, Xiaoning; Li, Henan; He, Qihua; Ma, Dalong; Han, Wenling

    2015-01-01

    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. PMID:26528697

  1. The sex-limited effects of mutations in the EGFR and TGF-β signaling pathways on shape and size sexual dimorphism and allometry in the Drosophila wing.

    PubMed

    Testa, Nicholas D; Dworkin, Ian

    2016-06-01

    Much of the morphological diversity in nature-including among sexes within a species-is a direct consequence of variation in size and shape. However, disentangling variation in sexual dimorphism for both shape (SShD), size (SSD), and their relationship with one another remains complex. Understanding how genetic variation influences both size and shape together, and how this in turn influences SSD and SShD, is challenging. In this study, we utilize Drosophila wing size and shape as a model system to investigate how mutations influence size and shape as modulated by sex. Previous work has demonstrated that mutations in epidermal growth factor receptor (EGFR) and transforming growth factor-β (TGF-β) signaling components can influence both wing size and shape. In this study, we re-analyze this data to specifically address how they impact the relationship between size and shape in a sex-specific manner, in turn altering the pattern of sexual dimorphism. While most mutations influence shape overall, only a subset have a genotypic specific effect that influences SShD. Furthermore, while we observe sex-specific patterns of allometric shape variation, the effects of most mutations on allometry tend to be small. We discuss this within the context of using mutational analysis to understand sexual size and shape dimorphism.

  2. The sex-limited effects of mutations in the EGFR and TGF-β signaling pathways on shape and size sexual dimorphism and allometry in the Drosophila wing.

    PubMed

    Testa, Nicholas D; Dworkin, Ian

    2016-06-01

    Much of the morphological diversity in nature-including among sexes within a species-is a direct consequence of variation in size and shape. However, disentangling variation in sexual dimorphism for both shape (SShD), size (SSD), and their relationship with one another remains complex. Understanding how genetic variation influences both size and shape together, and how this in turn influences SSD and SShD, is challenging. In this study, we utilize Drosophila wing size and shape as a model system to investigate how mutations influence size and shape as modulated by sex. Previous work has demonstrated that mutations in epidermal growth factor receptor (EGFR) and transforming growth factor-β (TGF-β) signaling components can influence both wing size and shape. In this study, we re-analyze this data to specifically address how they impact the relationship between size and shape in a sex-specific manner, in turn altering the pattern of sexual dimorphism. While most mutations influence shape overall, only a subset have a genotypic specific effect that influences SShD. Furthermore, while we observe sex-specific patterns of allometric shape variation, the effects of most mutations on allometry tend to be small. We discuss this within the context of using mutational analysis to understand sexual size and shape dimorphism. PMID:27038022

  3. Fyn and Src are Effectors of Oncogenic EGFR Signaling in Glioblastoma Patients

    PubMed Central

    Lu, Kan V.; Zhu, Shaojun; Cvrljevic, Anna; Huang, Tiffany T.; Sarkaria, Shawn; Ahkavan, David; Dang, Julie; Dinca, Eduard B.; Plaisier, Seema B.; Oderberg, Isaac; Lee, Yohan; Chen, Zugen; Caldwell, Jeremy S.; Xie, Yongmin; Loo, Joseph A.; Seligson, David; Chakravari, Arnab; Lee, Francis Y.; Weinmann, Roberto; Cloughesy, Timothy F.; Nelson, Stanley F.; Bergers, Gabriele; Graeber, Thomas; Furnari, Frank B.; James, C. David; Cavenee, Webster K.; Johns, Terrance G.; Mischel, Paul S.

    2009-01-01

    Activating EGFR mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We demonstrate that the Src family kinases (SFKs) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples demonstrated frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. PMID:19690143

  4. Copper improves the anti-angiogenic activity of disulfiram through the EGFR/Src/VEGF pathway in gliomas.

    PubMed

    Li, Yi; Fu, Shi-Yuan; Wang, Li-Hui; Wang, Fang-Yang; Wang, Nan-Nan; Cao, Qi; Wang, Ya-Ting; Yang, Jing-Yu; Wu, Chun-Fu

    2015-12-01

    Disulfiram (DSF) possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. DSF also potently inhibits angiogenesis, but the effect of Cu on this anti-angiogenic activity is unknown. Here we show that DSF inhibits the proliferation, migration, invasion, adhesion and complex tube formation of human umbilical vascular endothelial cells (HUVECs). Aortic ring assays and Matrigel plug assays revealed that DSF significantly inhibited the formation of microvessels. Importantly, Cu improved the anti-angiogenic activity of DSF in all these assays, while copper alone had no effect. DSF/Cu treatment of U87 human glioblastoma cells resulted in suppression of VEGF secretion through the EGFR/c-Src/VEGF pathway. Reduction of EGFR phosphorylation disables recruitment of multiple Src homology 2 (SH2) domains, resulting in transcriptional down-regulation of VEGF. The role of EGFR/c-Src/VEGF pathway was further confirmed by using specific inhibitor, which significantly improved the anti-angiogenic activity of DSF/Cu. DSF/Cu also exerted increased anti-tumor effects on subcutaneous and intracerebral U87 xenograft models by reducing microvessel density (MVD) and VEGF expression. These results indicate that Cu improves the anti-angiogenic activity of DSF by targeting the EGFR/Src/VEGF signaling pathway, thus providing a rationale for the use of DSF/Cu rather than DSF alone as an angiogenesis inhibitor in clinical applications.

  5. Dacomitinib in lung cancer: a “lost generation” EGFR tyrosine-kinase inhibitor from a bygone era?

    PubMed Central

    Ou, Sai-Hong Ignatius; Soo, Ross A

    2015-01-01

    EGFR tyrosine-kinase inhibitors (TKIs) have now been firmly established as the first-line treatment for non-small-cell lung cancer (NSCLC) patients harboring activating EGFR mutations, based on seven prospective randomized Phase III trials. However, despite significantly improved overall response rate and improved median progression-free survival when compared to platinum-doublet chemotherapy, EGFR-mutant NSCLC patients treated with EGFR TKIs invariably progress due to the emergence of acquired resistances, with the gatekeeper T790M mutation accounting for up to 60% of the resistance mechanisms. Second-generation irreversible EGFR TKIs were developed in part to inhibit the T790M mutation, in addition to the common activating EGFR mutations. Dacomitinib is one such second-generation EGFR TKI designed to inhibit both the wild-type (WT) EGFR and EGFR T790M. Afatinib is another second-generation EGR TKI that has been now been approved for the first-line treatment of EGFR-mutant NSCLC patients, while dacomitinib continues to undergo clinical evaluation. We will review the clinical development of dacomitinib from Phase I to Phase III trials, including the two recently published negative large-scale randomized Phase III trials (ARCHER 1009, NCIC-BR-26). Results from another large-scale randomized trial (ARCHER 1050) comparing dacomitinib to gefitinib as first-line treatment of advanced treatment-naïve EGFR-mutant NSCLC patients will soon be available and will serve as the lynchpin trial for the potential approval of dacomitinib in NSCLC. Meanwhile, third-generation EGFR TKIs (eg, CO-1686 [rociletinib], AZ9291, HM61713, EGF816, and ASP8273) that preferentially and potently inhibit EGFR T790M but not WT EGFR are in full-scale clinical development, and some of these EGFR TKIs have received “breakthrough” designation by the US Food and Drug Administration and will likely be approved in late 2015. Given the rapid development of third-generation EGFR TKIs and the approval

  6. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells.

    PubMed

    Feng, Yi; Ying, Hai-Yan; Qu, Ying; Cai, Xiao-Bo; Xu, Ming-Yi; Lu, Lun-Gen

    2016-09-01

    Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.

  7. Epidermal Growth Factor Receptor Mutation Is Associated With Longer Local Control After Definitive Chemoradiotherapy in Patients With Stage III Nonsquamous Non–Small-Cell Lung Cancer

    SciTech Connect

    Yagishita, Shigehiro; Horinouchi, Hidehito; Katsui Taniyama, Tomoko; Nakamichi, Shinji; Kitazono, Satoru; Mizugaki, Hidenori; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide

    2015-01-01

    Purpose: To determine the frequency and clinical significance of epidermal growth factor receptor (EGFR) mutations in patients with potentially curable stage III non–small-cell lung cancer (NSCLC) who are eligible for definitive chemoradiotherapy (CRT). Patients and Methods: Between January 2001 and December 2010, we analyzed the EGFR mutational status in consecutive NSCLC patients who were treated by CRT. The response rate, relapse-free survival, 2-year relapse-free rate, initial relapse sites, and overall survival of the patients were investigated. Results: A total of 528 patients received CRT at our hospital during the study period. Of these, 274 were diagnosed as having nonsquamous NSCLC. Sufficient specimens for mutational analyses could be obtained from 198 of these patients. The proportion of patients with EGFR activating mutations was 17%. In addition to the well-known characteristics of patients carrying EGFR mutations (female, adenocarcinoma, and never/light smoker), the proportion of cases with smaller primary lesions (T1/2) was found to be higher in patients with EGFR mutations than in those with wild-type EGFR. Patients with EGFR mutations showed similar response rate, relapse-free survival, and 2-year relapse-free rates as compared to patients with wild-type EGFR. Local relapses as the site of initial relapse occurred significantly less frequently in patients with EGFR mutation (4% vs 21%; P=.045). Patients with EGFR mutations showed longer local control (adjusted hazard ratio 0.49; P=.043). After disease progression, a majority of the patients with EGFR mutations received EGFR tyrosine kinase inhibitors (62%), and these patients showed longer postprogression survival than those with wild-type EGFR. Conclusions: Our study is the first to show radiosensitive biology of EGFR-mutated tumors in definitive CRT with curative intent. This finding could serve as a credible baseline estimate of EGFR-mutated population in stage III nonsquamous NSCLC.

  8. [Advances in the Research of Autophagy in EGFR-TKI Treatment and Resistance 
in Lung Cancer].

    PubMed

    Zhang, Qicheng; Xu, Ke

    2016-09-20

    Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is a group of targeted-drugs which effectively inhibits the growth of tumor cells with sensitive mutations in EGFR. However, the innate and acquired resistance are major obstacles of the efficiency. Autophagy is a highly conserved self-digesting process in cells, which is considered to be associated with cancer development andchemoresistance. The activation of EGFR may regulate autophagy through multiple signal pathways. EGFR-TKIs can induce autophagy, however, the function of the inducted autophagy remains biphasic. On one hand, autophagy induced by EGFR-TKI acts as a cytoprotective response in cancer cells, and autophagy inhibitors can enhance the cytotoxic effects of EGFR-TKI. On the other hand, a high level of autophagy after treatment of EGFR-TKI can also result in autophagic cell death lacking features of apoptosis, and the combination of EGFR-TKI with autophagy inducer might be beneficial. Thus, autophagy regulation represents a promising approach for improving the efficiency of EGFR-TKI in the treatment of cancer patients. Here we summarized the signaling pathways involved in EGFR-TKI induced autophagy, and reviewed the roles of autophagy in the treatment and chemoresistance of EGFR-TKI treatment in lung cancer. PMID:27666552

  9. 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...

  10. Silibinin meglumine, a water-soluble form of milk thistle silymarin, is an orally active anti-cancer agent that impedes the epithelial-to-mesenchymal transition (EMT) in EGFR-mutant non-small-cell lung carcinoma cells.

    PubMed

    Cufí, Sílvia; Bonavia, Rosa; Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; Martin-Castillo, Begoña; Barrajón-Catalán, Enrique; Visa, Joana; Segura-Carretero, Antonio; Bosch-Barrera, Joaquim; Joven, Jorge; Micol, Vicente; Menendez, Javier A

    2013-10-01

    Silibinin is the primary active constituent of a crude extract (silymarin) from milk thistle plant (Silybum marianum) seeds. We explored the ability of an oral milk thistle extract formulation that was enriched with a water-soluble form of silibinin complexed with the amino-sugar meglumine to inhibit the growth of non-small-cell lung carcinoma (NSCLC) mouse xenografts. As a single agent, oral silibinin meglumine notably decreased the overall volumes of NSCLC tumors as efficiently as did the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Concurrent treatment with silibinin meglumine impeded the regrowth of gefitinib-unresponsive tumors, resulting in drastic tumor growth prevention. Because the epithelial-to-mesenchymal transition (EMT) is required by a multiplicity of mechanisms of resistance to EGFR TKIs, we evaluated the ability of silibinin meglumine to impede the EMT in vitro and in vivo. Silibinin-meglumine efficiently prevented the loss of markers associated with a polarized epithelial phenotype as well as the de novo synthesis of proteins associated with the mesenchymal morphology of transitioning cells. Our current findings with this non-toxic, orally active, and water-soluble silibinin formulation might facilitate the design of clinical trials to test the administration of silibinin meglumine-containing injections, granules, or beverages in combination with EGFR TKIs in patients with EGFR-mutated NSCLC.

  11. Effect of BRAF V600E mutation on tumor response of anti-EGFR monoclonal antibodies for first-line metastatic colorectal cancer treatment: a meta-analysis of randomized studies.

    PubMed

    Cui, Dandan; Cao, Dan; Yang, Yu; Qiu, Meng; Huang, Ying; Yi, Cheng

    2014-03-01

    Anti-EGFR monoclonal antibodies (anti-EGFR MoAbs) in metastatic colorectal cancer (mCRC) treatment are still not effective in all patients. This study aimed to evaluate the relationship between BRAF V600E mutation and the tumor response of anti-EGFR MoAbs for first-line treatment in mCRC patients. We searched the MEDLINE and EMBASE databases, using the key words that included colorectal cancer, cetuximab, panitumumab, and BRAF mutation and retrieved 445 articles. Among them four were included in the systematic review. Relative risks (RRs) with 95% confidence intervals (CI) for response rate were calculated. BRAF mutation carriers had worse ORR than non-carriers in mCRC patients with KRAS wild-type in first-line treatment whether adding anti-EGFR MoAb to chemotherapy or not (RR = 0.43, [95% CI 0.16-0.75]; RR = 0.38, [95% CI 0.20-0.73]). But in the unselected patients whose KRAS mutation were unknown, BRAF mutation carriers had similar ORR whether adding cetuximab to chemotherapy or not (RR = 0.45, [95% CI 0.18-1.09]; RR = 0.57, [95% CI 0.15-2.23]). In BRAF mutation carriers adding anti-EGFR MoAb to chemotherapy was similar to chemotherapy alone whether in patients with wild-type KRAS or unselected patients (RR = 1.61, [95% CI 0.57-4.47]; RR = 0.71, [95% CI 0.18-2.77]). But in the BRAF mutation non-carriers, adding anti-EGFR MoAb produced a clear benefit in response rate than chemotherapy alone and this advantage was restricted to KRAS wild-type patients (RR = 1.48, [95% CI 1.28-1.71]). BRAF mutation decreases tumor response in first-line treatment whether cetuximab was given or not in patients with KRAS wild-type, and anti-EGFR MoAb produces a clear benefit in response rate in patients with BRAF and KRAS wild-type.

  12. TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun

    PubMed Central

    Wang, Tao; Wang, Ting; Niu, Mengjie; Zhang, Shengli; Jia, Lintao; Li, Shengqing

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers. PMID:26745678

  13. 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

  14. 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.

  15. Design, synthesis and molecular docking of α,β-unsaturated cyclohexanone analogous of curcumin as potent EGFR inhibitors with antiproliferative activity.

    PubMed

    Xu, Yun-Yun; Cao, Yi; Ma, Hailkuo; Li, Huan-Qiu; Ao, Gui-Zhen

    2013-01-15

    A type of novel α,β-unsaturated cyclohexanone analogous, which designed based on the curcumin core structure, have been discovered as potential EGFR inhibitors. These compounds exhibit potent antiproliferative activity in two human tumor cell lines (Hep G2 and B16-F10). Among them, compounds I(3) and I(12) displayed the most potent EGFR inhibitory activity (IC(50) = 0.43 μM and 1.54 μM, respectively). Molecular docking of I(12) into EGFR TK active site was also performed. This inhibitor nicely fitting the active site might well explain its excellent inhibitory activity.

  16. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  17. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

    PubMed Central

    Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

    2015-01-01

    Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies. PMID:26540293

  18. Successful treatment of a patient with Li–Fraumeni syndrome and metastatic lung adenocarcinoma harboring synchronous EGFR L858R and ERBB2 extracellular domain S310F mutations with the pan-HER inhibitor afatinib

    PubMed Central

    Jia, Yuxia; Ali, Siraj M; Saad, Shumaila; Chan, Courtney A; Miller, Vincent A; Halmos, Balazs

    2014-01-01

    We report the case of a young, never-smoker woman with Li–Fraumeni syndrome and advanced lung adenocarcinoma refractory to multiple lines of conventional chemotherapy and negative for actionable alterations by routine testing. Comprehensive genomic profiling by clinical-grade next generation sequencing was performed on 3320 exons of 184 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer. The tumor was found to harbor both EGFR L858R and ERBB2 S310F alterations and also tested positive for a known TP53 germline mutation. The presence of the EGFR mutation was further validated by direct sequencing. Based on these results, a dual EGFR/ERBB2 inhibitor, afatinib, was chosen for treatment. The patient achieved a rapid, complete, and durable response to afatinib monotherapy, both clinically and radiographically. The treatment was very well tolerated. This unique case raises practical questions as to the challenges of molecular testing and highlights the potential association of p53 mutations with concurrent EGFR and ERBB2 aberrations. As this case powerfully illustrates, the combination of broad genomic profiling and targeted therapy guided by mutational analysis offers the possibility of precision management of refractory advanced adenocarcinoma in the background of neoplastic syndromes. PMID:24835218

  19. 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

  20. Anti-EGFR function of EFEMP1 in glioma cells and patient prognosis

    PubMed Central

    Hu, Yuanjie; Gao, Hengjun; Vo, Christopher; Ke, Chao; Pan, Francine; Yu, Liping; Siegel, Eric; Hess, Kenneth R.; Linskey, Mark E.; Zhou, Yi-Hong

    2014-01-01

    EGFR is one of the key oncogenes subjected to targeted therapy for several cancers, as it is known to be amplified and/or mutated in up to 40% of malignant gliomas. EFEMP1, a fibulin-like extracellular protein, exerts both tumor suppressive and oncogenic effects in various cancers and glioma cell models. Although EFEMP1's anti-cancer activity has most commonly been attributed to its anti-angiogenic effects, we showed for gliomas that EFEMP1's binding to EGFR accounts for its suppression of the intracranial tumorigenicity of glioma cells expressing high levels of EGFR. In gliomas where EFEMP1 expression, and thus the anti-EGFR effect of EFEMP1, was suppressed, heightened levels of EGFR expression were associated with unfavorable patient outcomes in prognostic models. Results from the current study clearly demonstrate the impact that the anti-EGFR function of EFEMP1 has on the expression of EGFR and patient prognosis. A glioma prognostic model also suggests EFEMP1's context-dependent oncogenic function in gliomas expressing low levels of EGFR. Hence the level of EFEMP1 expression may have a predictive value for choosing patients for anti-EGFR therapy. PMID:25594013

  1. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

    SciTech Connect

    Zhou, Wenjun; Ercan, Dalia; Chen, Liang; Yun, Cai-Hong; Li, Danan; Capelletti, Marzia; Cortot, Alexis B.; Chirieac, Lucian; Iacob, Roxana E.; Padera, Robert; Engen, John R.; Wong, Kwok-Kin; Eck, Michael J.; Gray, Nathanael S.; Jänne, Pasi A.

    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 potent 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.

  2. Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

    PubMed Central

    Schrumpf, Melanie; Talebian Yazdi, Mehrdad; Ruano, Dina; Forte, Giusi I.; Nederlof, Petra M.; Veselic, Maud; Rabe, Klaus F.; Annema, Jouke T.; Smit, Vincent; Morreau, Hans; van Wezel, Tom

    2011-01-01

    Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients. PMID:21408138

  3. Rational bases for the development of EGFR inhibitors for cancer treatment.

    PubMed

    Bianco, Roberto; Gelardi, Teresa; Damiano, Vincenzo; Ciardiello, Fortunato; Tortora, Giampaolo

    2007-01-01

    Growth factor receptors and their ligands not only regulate normal cell processes but have been also identified as key regulators of human cancer formation. The epidermal growth factor receptor (EGFR/ErbB1/HER1) belongs to the ErbB/HER-family of tyrosine kinase receptors (RTKs). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Several evidences suggest that cooperation of multiple ErbB receptors and ligands is required for the induction of cell transformation. In this respect, EGFR, upon activation, sustains a complex and redundant network of signal transduction pathways with the contribution of other trans-membrane receptors. EGFR has been found to be expressed and altered in a variety of malignancies and clearly it plays a significant role in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Moreover, amplification of the EGFR gene and mutations in the EGFR tyrosine kinase domain have been recently reported in human carcinomas. As a result, investigators have developed approaches to inhibit the effects of EGFR activation, with the aim of blocking tumor growth and invasion. A number of agents targeting EGFR, including specific antibodies directed against its ligand-binding domain and small molecules inhibiting its tyrosine kinase activity are either in clinical trials or are already approved for clinical treatment. This article reviews the EGFR role in carcinogenesis and tumor progression as rational bases for the development of specific therapeutic inhibitors.

  4. [Epidermal growth factor receptor (EGFR): therapeutic target in the treatment of lung adenocarcinoma].

    PubMed

    Schwab, Richárd; Peták, István; Pintér, Ferenc; Szabó, Edit; Kánya, Melinda; Tamási, Anna; Várkondi, Edit; Almási, Andrea; Szokolóczi, Orsolya; Pápay, Judit; Moldvay, Judit; Kéri, György; Kopper, László

    2005-11-13

    Revolution in biotechnology made possible to identify those gene errors, which via their encoded proteins (mostly kinase enzymes) are key players in tumor development, growth and progression, and could be considered as molecular targets in tumor diagnosis and therapy. Activity of EGFR (epidermal growth factor receptor), an outstanding representative of the regulatory cell surface receptors, can be inhibited by drugs proved for clinical use. In the past year many groups observed that those lung adenocarcinoma cells, which contain activating mutation in the tyrosine kinase domain of EGFR show remarkable sensitivity to anti-EGFR compounds. The basis of the effective therapy is the identification of the mutations. The clinical advantage of EGFR is an example from the coming age of tumor chemotherapy, when the presence of molecular targets will guide the therapeutic choice.

  5. A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer

    PubMed Central

    OFUJI, KAZUYA; TADA, YOSHITAKA; YOSHIKAWA, TOSHIAKI; SHIMOMURA, MANAMI; YOSHIMURA, MAYUKO; SAITO, KEIGO; NAKAMOTO, YASUNARI; NAKATSURA, TETSUYA

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated marked clinical activity against non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations. However, in most cases, patients develop acquired resistance to EGFR-TKI therapy. The threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation is the most common acquired resistance mutation, and is present in ~50% cases of TKI resistance. New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are required. We evaluated the immunogenicity of an antigen derived from EGFR with the T790M mutation. Using BIMAS we selected several EGFR T790M-derived peptides bound to human leukocyte antigen (HLA)-A*02:01. T790M-A peptide (789–797) (IMQLMPFGC)-specific cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors. An established T790M-A-specific CTL line showed reactivity against the NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2−, T790M+), and the corresponding wild-type peptide (ITQLMPFGC)-pulsed T2 cells using an interferon-γ (IFN-γ) enzyme-linked immuno spot (ELISPOT) assay. This CTL line also demonstrated peptide-specific cytotoxicity against H1975-A2 cells. This finding suggests that the EGFR T790M mutation-derived antigen could be a new target for cancer immunotherapy. PMID:25532027

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

    PubMed

    Gu, Da-Min; Lu, Pei-Hua; Zhang, Ke; Wang, Xiang; Sun, Min; Chen, Guo-Qian; Wang, Qiong

    2015-02-13

    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 through 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. PMID:25582778

  7. 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

  8. EGF‑stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib‑resistant human lung cancer cell line.

    PubMed

    Nishimura, Yukio; Takiguchi, Soichi; Ito, Shigeru; Itoh, Kazuyuki

    2015-04-01

    The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of

  9. Unexpected responses to EGFR inhibition in NSCLC

    PubMed Central

    Stella, Giulia M.; Valizia, Claudio; Zorzetto, Michele; Inghilleri, Simona; Valentini, Adele; Dore, Roberto; Colombo, Sara; Valentino, Francesco; Orlandoni, Giulio; Morbini, Patrizia

    2015-01-01

    The presence of activating mutations of the epidermal growth factor receptor (EGFR)-gene identifies a distinct and clinically relevant molecular subset of non-small-cell lung cancer. It is now well demonstrated that EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are superior to standard chemotherapy in this subset of tumors. Nevertheless, in many cases, responses are not durable and last for 6–12 months due to the occurrence of secondary or acquired resistance. Here we present three cases of EGFR-mutant lung adenocarcinomas (ADC), that showed an unexpected response to anti-EGFR small molecules. The first patient presented a continued 89 month-long response to erlotinib in a tumor recurred after surgery and conventional chemotherapy. In the other cases, subclinically persistent tumor in the lung tissue was documented histologically in lung resections performed after partial response to TKI treatment. The persistence of interstitial and endolymphatic tumor cells after TKI treatment might explain the common observation of tumor relapse after TKI discontinuation, and sustain the decision to continue treatment in responsive patients as in our first case. PMID:26744648

  10. Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial

    PubMed Central

    Kapp, Joshua R; Diss, Tim; Spicer, James; Gandy, Michael; Schrijver, Iris; Jennings, Lawrence J; Li, Marilyn M; Tsongalis, Gregory J; de Castro, David Gonzalez; Bridge, Julia A; Wallace, Andrew; Deignan, Joshua L; Hing, Sandra; Butler, Rachel; Verghese, Eldo; Latham, Gary J; Hamoudi, Rifat A

    2015-01-01

    Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation. PMID:25430497

  11. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  12. Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

    PubMed Central

    Bertrand, T; Lesept, F; Chevilley, A; Lenoir, S; Aimable, M; Briens, A; Hommet, Y; Bardou, I; Parcq, J; Vivien, D

    2015-01-01

    Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects. PMID:26469972

  13. EGFR Signaling in Liver Diseases

    PubMed Central

    Komposch, Karin; Sibilia, Maria

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that is activated by several ligands leading to the activation of diverse signaling pathways controlling mainly proliferation, differentiation, and survival. The EGFR signaling axis has been shown to play a key role during liver regeneration following acute and chronic liver damage, as well as in cirrhosis and hepatocellular carcinoma (HCC) highlighting the importance of the EGFR in the development of liver diseases. Despite the frequent overexpression of EGFR in human HCC, clinical studies with EGFR inhibitors have so far shown only modest results. Interestingly, a recent study has shown that in human HCC and in mouse HCC models the EGFR is upregulated in liver macrophages where it plays a tumor-promoting function. Thus, the role of EGFR in liver diseases appears to be more complex than what anticipated. Further studies are needed to improve the molecular understanding of the cell-specific signaling pathways that control disease development and progression to be able to develop better therapies targeting major components of the EGFR signaling network in selected cell types. In this review, we compiled the current knowledge of EGFR signaling in different models of liver damage and diseases, mainly derived from the analysis of HCC cell lines and genetically engineered mouse models (GEMMs). PMID:26729094

  14. Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab

    PubMed Central

    2014-01-01

    Background Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. Findings We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. Conclusion The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy. PMID:24894453

  15. Somatic mutation analysis of KRAS, BRAF, HER2 and PTEN in EGFR mutation-negative non-small cell lung carcinoma: determination of frequency, distribution pattern and identification of novel deletion in HER2 gene from Indian patients.

    PubMed

    Bhaumik, Sangeet; Ahmad, Firoz; Das, Bibhu Ranjan

    2016-10-01

    Somatic mutations of KRAS, BRAF, HER2, PTEN genes are the most important molecular markers after the EGFR gene mutation. The current study evaluated the frequency and distribution pattern of KRAS, BRAF, HER2, PTEN mutation in Indian non-small cell lung carcinoma patients. The frequency of KRAS, BRAF, HER2, PTEN mutations was 6.4 % (14/204), 1.5 % (3/204), 1.5 % (3/204), 0 % (0/204), respectively. KRAS, BRAF, HER2 mutations were more prevalent in males than in females. KRAS and HER2 showed a trend of a higher frequency of mutation in the age group of <60 years, whereas BRAF mutations were more frequent in the age group of ≥60 years. Sequencing analysis of KRAS gene revealed c.34G>T (G12C) (n = 8), c.35G>A (G12D) (n = 3), c.35G>T (G12 V) (n = 1) and c.34G>T (G12C)/c.41T>C (V14A) (n = 2) mutations. Three different BRAF mutations (L584P: n = 1, V600E: n = 1, K601E: n = 1) were detected. Two cases harboured c.2324_2325ins12 (ATACGTGATGGC duplication) in HER2 gene, and one case was positive for NG_007503.2 (NM_001005862.2):c.2218-4del. It is less certain, but still quite possible that this mutation will affect splicing as the deletion of one C actually brings in one additional purine into the region. In conclusion, the present study demonstrates an instance of diverse nature of KRAS, BRAF, HER2 and PTEN gene in Indian patients and confirms that the frequency of these gene mutations varies globally. To the best of our knowledge, this is the first Indian study to evaluate KRAS, BRAF, HER2 and PTEN gene mutations.

  16. Somatic mutation analysis of KRAS, BRAF, HER2 and PTEN in EGFR mutation-negative non-small cell lung carcinoma: determination of frequency, distribution pattern and identification of novel deletion in HER2 gene from Indian patients.

    PubMed

    Bhaumik, Sangeet; Ahmad, Firoz; Das, Bibhu Ranjan

    2016-10-01

    Somatic mutations of KRAS, BRAF, HER2, PTEN genes are the most important molecular markers after the EGFR gene mutation. The current study evaluated the frequency and distribution pattern of KRAS, BRAF, HER2, PTEN mutation in Indian non-small cell lung carcinoma patients. The frequency of KRAS, BRAF, HER2, PTEN mutations was 6.4 % (14/204), 1.5 % (3/204), 1.5 % (3/204), 0 % (0/204), respectively. KRAS, BRAF, HER2 mutations were more prevalent in males than in females. KRAS and HER2 showed a trend of a higher frequency of mutation in the age group of <60 years, whereas BRAF mutations were more frequent in the age group of ≥60 years. Sequencing analysis of KRAS gene revealed c.34G>T (G12C) (n = 8), c.35G>A (G12D) (n = 3), c.35G>T (G12 V) (n = 1) and c.34G>T (G12C)/c.41T>C (V14A) (n = 2) mutations. Three different BRAF mutations (L584P: n = 1, V600E: n = 1, K601E: n = 1) were detected. Two cases harboured c.2324_2325ins12 (ATACGTGATGGC duplication) in HER2 gene, and one case was positive for NG_007503.2 (NM_001005862.2):c.2218-4del. It is less certain, but still quite possible that this mutation will affect splicing as the deletion of one C actually brings in one additional purine into the region. In conclusion, the present study demonstrates an instance of diverse nature of KRAS, BRAF, HER2 and PTEN gene in Indian patients and confirms that the frequency of these gene mutations varies globally. To the best of our knowledge, this is the first Indian study to evaluate KRAS, BRAF, HER2 and PTEN gene mutations. PMID:27637917

  17. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR

    PubMed Central

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Javier Carmona, F; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; van den Oord, Joost; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2016-01-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors. PMID:26030178

  18. Pinin facilitated proliferation and metastasis of colorectal cancer through activating EGFR/ERK signaling pathway

    PubMed Central

    Zhu, Xianjun; Wang, Yutian; Xu, Zhuoluo; Luo, Jun; Wang, Da; Guo, Weihong; Li, Xiaomei; Xin, Sainan; Yu, Jiang; Li, Guoxin

    2016-01-01

    Increasing emphasis has been put on the influence of desmosome related proteins on progress of colorectal cancer (CRC). Pinin (PNN) is a desmosome-associated molecule that has been reported its overexpression could increase desmoglein 2 (DSG2) and E-cadherin (E-ca) levels. However, it was documented that DSG2 and E-ca had opposite functions in CRC. Thus, we attempted to elucidate function and mechanism of PNN in CRC. Herein, we revealed that overexpression of PNN was significantly correlated with the aggressive characteristics and indicated poor overall survival of CRC patients. In addition, the proliferation, invasion in vitro, and tumorigenic growth, metastasis in vivo were also promoted by the up-regulation of PNN. It was also verified that up-regulation of PNN increased the expression of DSG2 and activated the EGFR/ERK signaling pathway. Our findings suggested that PNN, as a valuable marker of prognosis, has important influence on the progression of CRC. PMID:27107420

  19. Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR.

    PubMed

    Vizoso, Miguel; Ferreira, Humberto J; Lopez-Serra, Paula; Carmona, F Javier; Martínez-Cardús, Anna; Girotti, Maria Romina; Villanueva, Alberto; Guil, Sonia; Moutinho, Catia; Liz, Julia; Portela, Anna; Heyn, Holger; Moran, Sebastian; Vidal, August; Martinez-Iniesta, Maria; Manzano, Jose L; Fernandez-Figueras, Maria Teresa; Elez, Elena; Muñoz-Couselo, Eva; Botella-Estrada, Rafael; Berrocal, Alfonso; Pontén, Fredrik; Oord, Joost van den; Gallagher, William M; Frederick, Dennie T; Flaherty, Keith T; McDermott, Ultan; Lorigan, Paul; Marais, Richard; Esteller, Manel

    2015-07-01

    Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.

  20. The scaffolding protein NHERF1 sensitizes EGFR-dependent tumor growth, motility and invadopodia function to gefitinib treatment in breast cancer cells.

    PubMed

    Bellizzi, Antonia; Greco, Maria Raffaella; Rubino, Rosa; Paradiso, Angelo; Forciniti, Stefania; Zeeberg, Katrine; Cardone, Rosa Angela; Reshkin, Stephan Joel

    2015-03-01

    Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

  1. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer: a systematic review and cost-effectiveness analysis.

    PubMed Central

    Westwood, Marie; Joore, Manuela; Whiting, Penny; van Asselt, Thea; Ramaekers, Bram; Armstrong, Nigel; Misso, Kate; Severens, Johan; Kleijnen, Jos

    2014-01-01

    BACKGROUND Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment with standard chemotherapy. Patients with NSCLC are therefore tested for EGFR-TK tumour gene mutations to inform treatment decisions. There are a variety of tests available to detect these mutations. The different tests vary in the specific mutations that they attempt to detect, the amount of tumour cells needed for the test to work, the time that it takes to give a result, the error rate of the test, and the cost of the test. OBJECTIVE To compare the performance and cost-effectiveness of EGFR-TK mutation tests used to identify previously untreated adults with locally advanced or metastatic NSCLC, who may benefit from first-line treatment with TKIs. DATA SOURCES Twelve databases to August 2012 [including MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations and Daily Update (OvidSP), EMBASE, Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment database (HTA), Science Citation Index (SCI), Latin American and Caribbean Health Sciences Literature (LILACS), BIOSIS Previews, NIHR Health Technology Assessment programme, PROSPERO (International Prospective Register of Systematic Reviews)], research registers and conference proceedings. A web-based survey gathered data on technical performance of EGFR-TK mutation tests. METHODS Randomised controlled trials were assessed for methodological quality using the Cochrane risk of bias tool. Diagnostic accuracy studies were assessed using QUADAS-2. There were insufficient data for meta-analysis. For accuracy studies, we calculated sensitivity and specificity together with 95% confidence intervals (CIs). Survival data were summarised

  2. Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

    PubMed

    Li, Minghui; Kales, Stephen C; Ma, Ke; Shoemaker, Benjamin A; Crespo-Barreto, Juan; Cangelosi, Andrew L; Lipkowitz, Stanley; Panchenko, Anna R

    2016-02-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  3. Epidermal growth factor receptor kinase domain mutations are rare in salivary gland carcinomas

    PubMed Central

    Dahse, R; Driemel, O; Schwarz, S; Dahse, J; Kromeyer-Hauschild, K; Berndt, A; Kosmehl, H

    2009-01-01

    Activating mutations within the epidermal growth factor (EGFR) tyrosine kinase domain identify non-small cell lung cancer patients with improved clinical response to tyrosine kinase inhibitor therapy. Recently, we identified two EGFR mutations in a cohort of 25 salivary gland carcinomas (SGCs) by screening the tumour samples for the both most common hotspot mutations in exons 19 and 21 by allele-specific PCR. Here, we present a comprehensive sequencing analysis of the entire critical EGFR tyrosine kinase domain in 65 SGC of the main histopathological types. We found EGFR mutations in the tyrosine kinase domain to be a rare event in SGCs. No additional mutations other than the two known exon 19 deletions (c.2235_2249del15) in a mucoepidermoid carcinoma and an adenoid cystic carcinoma have been detected. Other putative predictive markers for EGFR-targeted therapy in SGCs might be relevant and should be investigated. PMID:19174819

  4. Epithelial Cell-Derived a Disintegrin and Metalloproteinase-17 Confers Resistance to Colonic Inflammation Through EGFR Activation

    PubMed Central

    Shimoda, Masayuki; Horiuchi, Keisuke; Sasaki, Aya; Tsukamoto, Tetsuya; Okabayashi, Koji; Hasegawa, Hirotoshi; Kitagawa, Yuko; Okada, Yasunori

    2016-01-01

    Epithelial regeneration is a key process for the recovery from ulcerative colitis (UC). Here we demonstrate that a disintegrin and metalloproteinase-17 (ADAM17), a main sheddase for tumor necrosis factor (TNF)-α, is essential for defensive epithelial properties against UC by promoting epithelial cell growth and goblet cell differentiation in mouse and human. Mice with systemic deletion of Adam17 developed severe dextran sulfate sodium-induced colitis when compared to mice with myeloid cell Adam17 deletion or control littermates. ADAM17 was predominantly expressed by regenerating epithelia in control mice, and its loss or inhibition attenuated epidermal growth factor receptor (EGFR) activation, epithelial proliferation, mucus production and barrier functions. Conversely, ectopic EGFR stimulation promoted epithelial regeneration thereby partially rescuing the severe colitis caused by ADAM17 deficiency. In UC patients, epithelial ADAM17 expression positively correlated with both cell proliferation and goblet cell number. These findings suggest that maintaining ADAM17–EGFR epithelial signaling is necessary for the recovery from UC and would be beneficial to therapeutic strategies targeting ADAM17-mediated TNF-α shedding. PMID:27077118

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

    SciTech Connect

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    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 PDAC 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.

  6. Dynamics of circulating tumor DNA represented by the activating and resistant mutations in epidermal growth factor receptor tyrosine kinase inhibitor treatment.

    PubMed

    Uchida, Junji; Imamura, Fumio; Kukita, Yoji; Oba, Shigeyuki; Kumagai, Toru; Nishino, Kazumi; Inoue, Takako; Kimura, Madoka; Kato, Kikuya

    2016-03-01

    Circulating tumor DNA (ctDNA) is an emerging field of cancer research. For lung cancer, non-invasive genotyping of EGFR is the foremost application. The activating mutations represent the ctDNA from all cancer cells, and the T790M-resistant mutation represents that from resistant cells. We examined the ctDNA dynamics of EGFR mutations by using deep sequencing with a massively parallel DNA sequencer. We obtained 190 plasma samples from 57 patients at various times during the treatment course and classified them according to treatment status. The mutation detection rate of exon 19 deletion/L858R in plasma was high at the initiation of treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI; P = 0.001), suppressed during EGFR-TKI treatment before disease progression, and elevated after the onset of disease progression (P = 0.023). The mutation detection rate of T790M was low until the onset of disease progression and elevated thereafter (P = 0.01). Samples across the development of disease progression were obtained from 10 patients and showed a correlation between increased ctDNA level and disease progression. Decreased ctDNA level in response to the initiation of EGFR-TKI was observed in 4 of 6 eligible patients. In two patients, the ctDNA dynamics suggested the presence of cancer cell populations only with the T790M mutation. In another patient, the T790M ctDNA represented cell subpopulations that respond to cytotoxic agents differently from the major population. Considering the high incidence, ctDNA could be a clinical parameter to complement information from image analyses.

  7. [Clinical Research of EGFR and KRAS Mutation in Fine Needle Aspiration Cytology Specimens of Non-small Cell Lung Carcinoma].

    PubMed

    Zhang, Zhihui; Wu, Xilan; Ying, Jianming; Li, Junling; Qiu, Tian; Guo, Huiqin; Zhao, Huan; Shan, Ling; Ling, Yun

    2015-04-01

    背景与目的 肺癌患者靶向药物治疗前,需要检测表皮生长因子受体(epidermal growth factor receptor, EGFR)和KRAS基因是否突变。本研究旨在探讨细针吸取悬浮液标本检测非小细胞肺癌EGFR、KRAS基因突变的意义。方法 细胞学悬浮液标本,Real-time PCR法检测EGFR 18-21号外显子,KRAS 2号外显子12、13密码子突变。结果 检测85例肺癌转移淋巴结针吸标本,EGFR突变率37.3%,KRAS突变率7.2%。19例组织切片标本,与细胞学结果一致(kappa=1.0)。13例有EGFR突变,临床分期IV期患者, 使用酪氨酸激酶抑制剂治疗,2例完全缓解(complete remission, CR)(16.7%);8例部分缓解(partial remission, PR)(66.7%);3例最佳稳定疾病(stable disease, SD)(25.0%)。结论 细针吸取标本检测EGFR、KRAS基因突变,标本取材容易、简单、方便,具有较高的临床实用性。.

  8. Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal

    PubMed Central

    Hong, Hue-Hua L.; Hoenerhoff, Mark J.; Ton, Thai-Vu; Herbert, Ronald A.; Kissling, Grace E.; Hooth, Michelle J.; Behl, Mamta; Witt, Kristine L.; Smith-Roe, Stephanie L.; Sills, Robert C.; Pandiri, Arun R.

    2015-01-01

    Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kras, Egfr and Tp53 mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD) induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors, and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors, and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominantly in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assays indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents. PMID:26059825

  9. Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells

    PubMed Central

    Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

    2016-01-01

    Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future. PMID:27362942

  10. Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells.

    PubMed

    Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

    2016-01-01

    Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future.

  11. Model-based analysis of HER activation in cells co-expressing EGFR, HER2 and HER3.

    PubMed

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    2013-01-01

    The HER/ErbB family of receptor tyrosine kinases drives 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 panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, 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 human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 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. PMID:23990774

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

    PubMed Central

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    2013-01-01

    The HER/ErbB family of receptor tyrosine kinases drives 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 panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, 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 human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 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. PMID:23990774

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

    SciTech Connect

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    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 panel 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.

  14. EGFR trans-activation by urotensin II receptor is mediated by β-arrestin recruitment and confers cardioprotection in pressure overload-induced cardiac hypertrophy.

    PubMed

    Esposito, Giovanni; Perrino, Cinzia; Cannavo, Alessandro; Schiattarella, Gabriele G; Borgia, Francesco; Sannino, Anna; Pironti, Gianluigi; Gargiulo, Giuseppe; Di Serafino, Luigi; Franzone, Anna; Scudiero, Laura; Grieco, Paolo; Indolfi, Ciro; Chiariello, Massimo

    2011-06-01

    Urotensin II (UTII) and its seven trans-membrane receptor (UTR) are up-regulated in the heart under pathological conditions. Previous in vitro studies have shown that UTII trans-activates the epidermal growth factor receptor (EGFR), however, the role of such novel signalling pathway stimulated by UTII is currently unknown. In this study, we hypothesized that EGFR trans-activation by UTII might exert a protective effect in the overloaded heart. To test this hypothesis, we induced cardiac hypertrophy by transverse aortic constriction (TAC) in wild-type mice, and tested the effects of the UTII antagonist Urantide (UR) on cardiac function, structure, and EGFR trans-activation. After 7 days of pressure overload, UR treatment induced a rapid and significant impairment of cardiac function compared to vehicle. In UR-treated TAC mice, cardiac dysfunction was associated with reduced phosphorylation levels of the EGFR and extracellular-regulated kinase (ERK), increased apoptotic cell death and fibrosis. In vitro UTR stimulation induced membrane translocation of β-arrestin 1/2, EGFR phosphorylation/internalization, and ERK activation in HEK293 cells. Furthermore, UTII administration lowered apoptotic cell death induced by serum deprivation, as shown by reduced TUNEL/Annexin V staining and caspase 3 activation. Interestingly, UTII-mediated EGFR trans-activation could be prevented by UR treatment or knockdown of β-arrestin 1/2. Our data show, for the first time in vivo, a new UTR signalling pathway which is mediated by EGFR trans-activation, dependent by β-arrestin 1/2, promoting cell survival and cardioprotection.

  15. EGFR blockade prevents glioma escape from BRAFV600E targeted therapy

    PubMed Central

    Prados, Michael; Weiss, William A.; James, C. David; Nicolaides, Theodore

    2015-01-01

    Mutational activation of BRAF (BRAFV600E) occurs in pediatric glioma and drives aberrant MAPK signaling independently of upstream cues. Targeted monotherapy against BRAFV600E displays efficacy in pre-clinical models of glioma, however xenograft tumors adapt rapidly and escape from the growth-inhibitory effects of BRAF-targeted therapy. Here, we show that intrinsic resistance to a BRAFV600E specific inhibitor stems, in part, from feedback activation of EGFR and downstream signaling pathways. BRAFV600E inhibition suppresses MAPK signaling, which in turn downregulates the EGFR phosphatase PTPN9, resulting in sustained EGFR phosphorylation and enhanced EGFR activity. We demonstrated that overexpression of PTPN9 reduces EGFR phosphorylation and cooperates with BRAFV600E inhibitor PLX4720 to reduce MAPK and Akt signaling, resulting in decreased glioma cell viability. Moreover, pharmacologic inhibition of EGFR combined with inhibition of BRAFV600E to reduce growth of glioma cell lines and orthotopic glioma xenograft by decreasing tumor cell proliferation while increasing apoptosis, with resultant significant extension of animal subject survival. Our data support clinical evaluation of BRAFV600E and EGFR targeted therapy in treating BRAFV600E glioma. PMID:26023796

  16. TARGETING THE ONCOGENIC MUC1-C PROTEIN INHIBITS MUTANT EGFR-MEDIATED SIGNALING AND SURVIVAL IN NON-SMALL CELL LUNG CANCER CELLS

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Tchaicha, Jeremy; Kikuchi, Eiki; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Purpose Non-small cell lung cancers (NSCLC) that express the EGF receptor (EGFR) with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. Experimental Design Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival and tumorigenicity. Results Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC→AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK→ERK activation, colony formation and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT and MEK→ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. Conclusions These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors. PMID:25189483

  17. 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

    SciTech Connect

    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 1–3 μM 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. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  18. 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

  19. EGFR gene deregulation mechanisms in lung adenocarcinoma: A molecular review.

    PubMed

    Tsiambas, Evangelos; Lefas, Alicia Y; Georgiannos, Stavros N; Ragos, Vasileios; Fotiades, Panagiotis P; Grapsa, Dimitra; Stamatelopoulos, Athanasios; Kavantzas, Nikolaos; Patsouris, Efstratios; Syrigos, Konstantinos

    2016-08-01

    For the last two decades, evolution in molecular biology has expanded our knowledge in decoding a broad spectrum of genomic imbalances that progressively lead normal cells to a neoplastic state and finally to complete malignant transformation. Concerning oncogenes and signaling transduction pathways mediated by them, identification of specific gene alterations remains a critical process for handling patients by applying targeted therapeutic regimens. The epidermal growth factor receptor (EGFR) signaling pathway plays a crucial role in regulating cell proliferation, differentiation and apoptosis in normal cells. EGFR mutations and amplification represent the gene's main deregulation mechanisms in cancers of different histo-genetic origin. Furthermore, intra-cancer molecular heterogeneity due to clonal rise and expansion mainly explains the variable resistance to novel anti-EGFR monoclonal antibody (mAb), and also tyrosine kinase inhibitors (TKIs). According to recently published 2015 WHO new classification, lung cancer is the leading cause of death related to cancer and its incidence is still on the increase worldwide. The majority of patients suffering from lung cancer are diagnosed with epithelial tumors (adenocarcinoma predominantly and squamous cell carcinoma represent ∼85% of all pathologically defined lung cancer cases). In those patients, EGFR-activating somatic mutations in exons 18/19/20/21 modify patients' sensitivity (i.e. exon 21 L858R, exon 19 LREA deletion) or resistance (ie exon 20 T790M and/or insertion) to TKI mediated targeted therapeutic strategies. Additionally, the role of specific micro-RNAs that affect EGFR regulation is under investigation. In the current review, we focused on EGFR gene/protein structural and functional aspects and the corresponding alterations that occur mainly in lung adenocarcinoma to critically modify its molecular landscape. PMID:27461822

  20. A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery.

    PubMed

    Antczak, Christophe; Mahida, Jeni P; Bhinder, Bhavneet; Calder, Paul A; Djaballah, Hakim

    2012-08-01

    Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell-permeable modulators for development. For this purpose, we have used our recently optimized epidermal growth factor receptor (EGFR) biosensor assay to screen for modulators of EGFR activity. Here, we report on its validation under high-throughput screening (HTS) conditions displaying a signal-to-noise ratio of 21 and a Z' value of 0.56-attributes of a robust cell-based assay. We performed a pilot screen against a library of 6912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95%, respectively. Follow-up dose-response studies revealed that 12 of the 13 known EGFR inhibitors in the library were confirmed as hits. ZM-306416, a vascular endothelial growth factor receptor (VEGFR) antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone, and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.

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

    SciTech Connect

    Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.; Yang, J.-L.

    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 of 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.

  2. 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

  3. 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. PMID:26229444

  4. Genome wide analysis of transcript levels after perturbation of the EGFR pathway in the Drosophila ovary.

    PubMed

    Jordan, Katherine C; Hatfield, Steven D; Tworoger, Michael; Ward, Ellen J; Fischer, Karin A; Bowers, Stuart; Ruohola-Baker, Hannele

    2005-03-01

    Defects in the epidermal growth factor receptor (EGFR) pathway can lead to aggressive tumor formation. Activation of this pathway during normal development produces multiple outcomes at the cellular level, leading to cellular differentiation and cell cycle activation. To elucidate the downstream events induced by this pathway, we used genome-wide cDNA microarray technology to identify potential EGFR targets in Drosophila oogenesis. We focused on genes for which the transcriptional responses due to EGFR pathway activation and inactivation were in opposite directions, as this is expected for genes that are directly regulated by the pathway in this tissue type. We perturbed the EGFR pathway in epithelial follicle cells using seven different genetic backgrounds. To activate the pathway, we overexpressed an activated form of the EGFR (UAS-caEGFR), and an activated form of the signal transducer Raf (UAS-caRaf); we also over- or ectopically expressed the downstream homeobox transcription factor Mirror (UAS-mirr) and the ligand-activating serine protease Rhomboid (UAS-rho). To reduce pathway activity we used loss-of-function mutations in the ligand (gurken) and receptor (torpedo). From microarrays containing 6,255 genes, we found 454 genes that responded in an opposite manner in gain-of-function and loss-of-function conditions among which are many Wingless signaling pathway components. Further analysis of two such components, sugarless and pangolin, revealed a function for these genes in late follicle cell patterning. Of interest, components of other signaling pathways were also enriched in the EGFR target group, suggesting that one reason for the pleiotropic effects seen with EGFR activity in cancer progression and development may be its ability to regulate many other signaling pathways. PMID:15704171

  5. Genome wide analysis of transcript levels after perturbation of the EGFR pathway in the Drosophila ovary.

    PubMed

    Jordan, Katherine C; Hatfield, Steven D; Tworoger, Michael; Ward, Ellen J; Fischer, Karin A; Bowers, Stuart; Ruohola-Baker, Hannele

    2005-03-01

    Defects in the epidermal growth factor receptor (EGFR) pathway can lead to aggressive tumor formation. Activation of this pathway during normal development produces multiple outcomes at the cellular level, leading to cellular differentiation and cell cycle activation. To elucidate the downstream events induced by this pathway, we used genome-wide cDNA microarray technology to identify potential EGFR targets in Drosophila oogenesis. We focused on genes for which the transcriptional responses due to EGFR pathway activation and inactivation were in opposite directions, as this is expected for genes that are directly regulated by the pathway in this tissue type. We perturbed the EGFR pathway in epithelial follicle cells using seven different genetic backgrounds. To activate the pathway, we overexpressed an activated form of the EGFR (UAS-caEGFR), and an activated form of the signal transducer Raf (UAS-caRaf); we also over- or ectopically expressed the downstream homeobox transcription factor Mirror (UAS-mirr) and the ligand-activating serine protease Rhomboid (UAS-rho). To reduce pathway activity we used loss-of-function mutations in the ligand (gurken) and receptor (torpedo). From microarrays containing 6,255 genes, we found 454 genes that responded in an opposite manner in gain-of-function and loss-of-function conditions among which are many Wingless signaling pathway components. Further analysis of two such components, sugarless and pangolin, revealed a function for these genes in late follicle cell patterning. Of interest, components of other signaling pathways were also enriched in the EGFR target group, suggesting that one reason for the pleiotropic effects seen with EGFR activity in cancer progression and development may be its ability to regulate many other signaling pathways.

  6. Five-year survival in EGFR-mutant metastatic lung adenocarcinoma treated with EGFR-TKIs

    PubMed Central

    Lin, Jessica J.; Cardarella, Stephanie; Lydon, Christine A.; Dahlberg, Suzanne E.; Jackman, David M.; Jänne, Pasi A.; Johnson, Bruce E.

    2016-01-01

    Introduction Activating mutations in the epidermal growth factor receptor (EGFR) predict for prolonged progression-free survival in patients with advanced non-small cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) versus chemotherapy. Long-term survival outcomes, however, remain undefined. The objective of this study was to determine the 5-year survival in these patients, and identify clinical factors associated with overall survival (OS). Methods Patients with EGFR-mutant metastatic lung adenocarcinoma treated with erlotinib or gefitinib at Dana-Farber Cancer Institute between 2002 and 2009 were included. OS was analyzed. Results Among 137 patients, median PFS and OS were 12.1 months (95% CI, 10.2-13.5 months) and 30.9 months (95% CI, 28.2-35.7 months), respectively. Twenty patients (14.6%) were 5-year survivors. In multivariate analysis, exon 19 deletions (hazard ratio [HR], 0.63; 95% CI, 0.44-0.91; P = 0.01), absence of extrathoracic (HR 0.62; 95% CI, 0.41-0.93; P = 0.02) or brain metastasis (HR 0.48; 95% CI, 0.30-0.77, P = 0.002), and not a current smoker (HR 0.23; 95% CI, 0.09-0.59; P = 0.002) were associated with prolonged OS. Age, gender, stage at diagnosis, liver or bone or adrenal metastasis, specific TKI, and line of TKI therapy were not associated with OS. Conclusions Our data suggest that the prevalence of 5-year survival among EGFR-mutant metastatic lung adenocarcinoma patients treated with erlotinib or gefitinib is 14.6%. Exon 19 deletions and absence of extrathoracic or brain metastasis are associated with prolonged survival. Based on our findings, clinicians can gain an enhanced estimation of long-term outcomes in this population. PMID:26724471

  7. 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

  8. ABT-414, an Antibody-Drug Conjugate Targeting a Tumor-Selective EGFR Epitope.

    PubMed

    Phillips, Andrew C; Boghaert, Erwin R; Vaidya, Kedar S; Mitten, Michael J; Norvell, Suzanne; Falls, Hugh D; DeVries, Peter J; Cheng, Dong; Meulbroek, Jonathan A; Buchanan, Fritz G; McKay, Laura M; Goodwin, Neal C; Reilly, Edward B

    2016-04-01

    Targeting tumor-overexpressed EGFR with an antibody-drug conjugate (ADC) is an attractive therapeutic strategy; however, normal tissue expression represents a significant toxicity risk. The anti-EGFR antibody ABT-806 targets a unique tumor-specific epitope and exhibits minimal reactivity to EGFR in normal tissue, suggesting its suitability for the development of an ADC. We describe the binding properties and preclinical activity of ABT-414, an ABT-806 monomethyl auristatin F conjugate. In vitro, ABT-414 selectively kills tumor cells overexpressing wild-type or mutant forms of EGFR. ABT-414 inhibits the growth of xenograft tumors with high EGFR expression and causes complete regressions and cures in the most sensitive models. Tumor growth inhibition is also observed in tumor models with EGFR mutations, including activating mutations and those with the exon 2-7 deletion [EGFR variant III (EGFRvIII)], commonly found in glioblastoma multiforme. ABT-414 exhibits potent cytotoxicity against glioblastoma multiforme patient-derived xenograft models expressing either wild-type EGFR or EGFRvIII, with sustained regressions and cures observed at clinically relevant doses. ABT-414 also combines with standard-of-care treatment of radiation and temozolomide, providing significant therapeutic benefit in a glioblastoma multiforme xenograft model. On the basis of these results, ABT-414 has advanced to phase I/II clinical trials, and objective responses have been observed in patients with both amplified wild-type and EGFRvIII-expressing tumors. Mol Cancer Ther; 15(4); 661-9. ©2016 AACR. PMID:26846818

  9. Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer: a new era begins.

    PubMed

    Remon, J; Morán, T; Majem, M; Reguart, N; Dalmau, E; Márquez-Medina, D; Lianes, P

    2014-02-01

    The discovery of mutated oncogenes has opened up a new era for the development of more effective treatments for non-small cell lung cancer patients (NSCLC) harbouring EGFR mutations. However, patients with EGFR-activating mutation ultimately develop acquired resistance (AR). Several studies have identified some of the mechanisms involved in the development of AR to EGFR tyrosine kinase inhibitors (TKI) that can be potential therapeutic strategies, although in up to 30% of cases, the underlying mechanism of AR are still unexplained. In this review we aim to summarize the main mechanisms of AR to EGFR TKI and some clinical strategies that can be used in the daily clinical practice to overcome this resistance and try to prolong the outcomes in this subgroup of patients.

  10. Overexpression of wildtype EGFR is tumorigenic and denotes a therapeutic target in non-small cell lung cancer

    PubMed Central

    Mu, Libing; Tang, Yanna; Gao, Lei; Ren, Shengxiang; Cao, Dengfeng; Zhou, Lixin; Zhang, Aiqun; Liu, Deruo; Zhou, Caicun; Wong, Kwok-Kin; Yu, Lei; Zhang, Li; Chen, Liang

    2016-01-01

    Current guidelines for lung cancer treatment with EGFR tyrosine kinase inhibitors (TKI) include only patients with mutated EGFR, although some patients with wildtype EGFR (wt-EGFR) have exhibited positive responses to this therapy as well. Biomarkers predicting the benefit from EGFR-TKIs treatment remain to be determined for patients with wild-type EGFR. Here, we report that wt-EGFR overexpression transformed cells in vitro and induced tumorigenesis in vivo in transgenic mouse models. Wt-EGFR driven lung cancer was hypersensitive to TKI treatment in mouse model. Lung cancer patients with high-expression of wt-EGFR showed longer Overall Survival in comparison to low-expression patients after TKI treatment. Our data therefore suggest that treatment with EGFR inhibitors should be extended to include not only patients with mutated EGFR but also a subset of patients with overexpression of wt-EGFR. PMID:26646697

  11. 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...

  12. Comparison of the efficacy of icotinib in patients with non-small-cell lung cancer according to the type of epidermal growth factor receptor mutation

    PubMed Central

    Xue, Zhang Xiao; Wen, Wang Xiu; Zhuang, Yu; Hua, Zang Jian; Xia, Yang Ni

    2016-01-01

    Icotinib hydrochloride is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with preclinical and clinical activity in non-small-cell lung cancer (NSCLC). Exon 19 deletion and L858R point mutation are the most commonly encountered EGFR mutations in NSCLC, and they predict improved clinical outcomes following treatment with icotinib. The objective of this study was to evaluate the differential clinical efficacy of icotinib in patients with exon 19 deletion or L858R point mutation of the EGFR gene. A total of 104 patients with advanced NSCLC, who harbored exon 19 deletion or L858R point mutation of EGFR and were treated with icotinib, were enrolled in this study. The tumor response and progression-free survival were evaluated. There were no significant differences between patients with EGFR exon 19 deletion and those with L858R point mutation who received treatment with icotinib. PMID:27588191

  13. TGF-β and Hypoxia/Reoxygenation Promote Radioresistance of A549 Lung Cancer Cells through Activation of Nrf2 and EGFR

    PubMed Central

    Lee, Sae-lo-oom; Ryu, Hwani; Son, A-rang; Seo, Bitna; Kim, Jooyoung; Jung, Seung-Youn; Song, Jie-Young; Hwang, Sang-Gu; Ahn, Jiyeon

    2016-01-01

    Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF-) β separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-β on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-β under hypoxia/reoxygenation conditions. Combined treatment with TGF-β and hypoxia activated epidermal growth factor receptor (EGFR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-β, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-β and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS), while treatment with N-acetyl-l-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-β under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR), and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-β and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR. PMID:26904167

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

    SciTech Connect

    Gu, Da-min; Lu, Pei-Hua; Zhang, Ke; Wang, Xiang; Sun, Min; Chen, Guo-Qian; Wang, Qiong

    2015-02-13

    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 through 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.

  15. Panaxydol, a component of Panax ginseng, induces apoptosis in cancer cells through EGFR activation and ER stress and inhibits tumor growth in mouse models.

    PubMed

    Kim, Hee Suk; Lim, Jang Mi; Kim, Joo Young; Kim, Yongjin; Park, Serkin; Sohn, Jeongwon

    2016-03-15

    We reported previously that panaxydol, a component of Panax ginseng roots, induced mitochondria-mediated apoptosis preferentially in transformed cells. This study demonstrates that EGFR activation and the resulting ER stress mediate panaxydol-induced apoptosis, and that panaxydol suppresses in vivo tumor growth in syngeneic and xenogeneic mouse tumor models. In addition, we elucidated that CaMKII and TGF-β-activated kinase (TAK1) participate in p38/JNK activation by elevated cytoplasmic Ca(2+) concentration ([Ca(2+)]c). In MCF-7 cells, EGFR was activated immediately after exposure to panaxydol, and this activation was necessary for induction of apoptosis, suggesting that panaxydol might be a promising anticancer candidate, especially for EGFR-addicted cancer. Activation of PLCγ followed EGFR activation, resulting in Ca(2+) release from the endoplasmic reticulum (ER) via inositol triphosphate and ryanodine receptors. ER Ca(2+) release triggered mitochondrial Ca(2+) uptake indirectly through oxidative stress and ensuing ER stress. Elevated [Ca(2+)]c triggered sequential activation of calmodulin/CaMKII, TAK1 and p38/JNK. As shown previously, p38 and JNK activate NADPH oxidase. Here, it was shown that the resulting oxidative stress triggered ER stress. Among the three signaling branches of the unfolded protein response, protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 or activating transcription factor 6, played a role in transmitting the apoptosis signal. PERK induced C/EBP homologous protein (CHOP), and CHOP elevated Bim expression, initiating mitochondrial Ca(2+) uptake and apoptosis. In summary, we identified roles of EGFR, the CAMKII-TAK1-p38/JNK pathway, and ER stress in panaxydol-induced apoptosis and demonstrated the in vivo anticancer effect of panaxydol.

  16. Panaxydol, a component of Panax ginseng, induces apoptosis in cancer cells through EGFR activation and ER stress and inhibits tumor growth in mouse models.

    PubMed

    Kim, Hee Suk; Lim, Jang Mi; Kim, Joo Young; Kim, Yongjin; Park, Serkin; Sohn, Jeongwon

    2016-03-15

    We reported previously that panaxydol, a component of Panax ginseng roots, induced mitochondria-mediated apoptosis preferentially in transformed cells. This study demonstrates that EGFR activation and the resulting ER stress mediate panaxydol-induced apoptosis, and that panaxydol suppresses in vivo tumor growth in syngeneic and xenogeneic mouse tumor models. In addition, we elucidated that CaMKII and TGF-β-activated kinase (TAK1) participate in p38/JNK activation by elevated cytoplasmic Ca(2+) concentration ([Ca(2+)]c). In MCF-7 cells, EGFR was activated immediately after exposure to panaxydol, and this activation was necessary for induction of apoptosis, suggesting that panaxydol might be a promising anticancer candidate, especially for EGFR-addicted cancer. Activation of PLCγ followed EGFR activation, resulting in Ca(2+) release from the endoplasmic reticulum (ER) via inositol triphosphate and ryanodine receptors. ER Ca(2+) release triggered mitochondrial Ca(2+) uptake indirectly through oxidative stress and ensuing ER stress. Elevated [Ca(2+)]c triggered sequential activation of calmodulin/CaMKII, TAK1 and p38/JNK. As shown previously, p38 and JNK activate NADPH oxidase. Here, it was shown that the resulting oxidative stress triggered ER stress. Among the three signaling branches of the unfolded protein response, protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 or activating transcription factor 6, played a role in transmitting the apoptosis signal. PERK induced C/EBP homologous protein (CHOP), and CHOP elevated Bim expression, initiating mitochondrial Ca(2+) uptake and apoptosis. In summary, we identified roles of EGFR, the CAMKII-TAK1-p38/JNK pathway, and ER stress in panaxydol-induced apoptosis and demonstrated the in vivo anticancer effect of panaxydol. PMID:26421996

  17. Molecular Dynamics Analysis of Binding of Kinase Inhibitors to WT EGFR and the T790M Mutant.

    PubMed

    Park, Jiyong; McDonald, Joseph J; Petter, Russell C; Houk, K N

    2016-04-12

    Epidermal growth factor receptor (EGFR) inhibitors interrupt EGFR-dependent cellular signaling pathways that lead to accelerated tumor growth and proliferation. Mutation of a threonine in the inhibitor binding pocket, known as the "gatekeeper", to methionine (T790M) confers acquired resistance to several EGFR-selective inhibitors. We studied interactions between EGFR inhibitors and the gatekeeper residues of the target protein. Thermodynamic integration (TI) with Amber14 indicates that the binding energies of gefitinib and AEE788 to the active state of the T790M mutant EGFR is 3 kcal/mol higher than to the wild type (WT), whereas ATP binding energy to the mutant is similar to the WT. Using metadynamics MD simulations with NAMD v2.9, the conformational equilibrium between the inactive resting state and the catalytically competent activate state was determined for the WT EGFR. When combined with the results obtained by Sutto and Gervasio, our simulations showed that the T790M point mutation lowers the free energy of the active state by 5 kcal/mol relative to the inactive state of the enzyme. Relative to the WT, the T790M mutant binds gefitinib more strongly. The T790M mutation is nevertheless resistant due to its increased binding of ATP. By contrast, the binding of AEE788 to the active state causes a conformational change in the αC-helix adjacent to the inhibitor binding pocket, that results in a 2 kcal/mol energy penalty. The energy penalty explains why the binding of AEE788 to the T790M mutant is unfavorable relative to binding to WT EGFR. These results establish the role of the gatekeeper mutation on inhibitor selectivity. Additional molecular dynamics (MD) simulations, TI, and metadynamics MD simulations reveal the origins of the changes in binding energy of WT and mutants. PMID:27010480

  18. Activating STAT6 mutations in follicular lymphoma

    PubMed Central

    Yildiz, Mehmet; Li, Hongxiu; Bernard, Denzil; Amin, Nisar A.; Ouillette, Peter; Jones, Siân; Saiya-Cork, Kamlai; Parkin, Brian; Jacobi, Kathryn; Shedden, Kerby; Wang, Shaomeng; Chang, Alfred E.; Kaminski, Mark S.

    2015-01-01

    Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell–based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) –induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4–induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis. PMID:25428220

  19. Epidermal growth factor receptor exon 20 p.S768I mutation in non-small cell lung carcinoma: A case report combined with a review of the literature and investigation of clinical significance

    PubMed Central

    IMPROTA, GIUSEPPINA; PETTINATO, ANGELA; GIERI, STEFANIA; SCANDURRA, GIUSEPPA; SKOVRIDER-RUMINSKI, WOJCIECH; HØGDALL, ESTRID; FRAGGETTA, FILIPPO

    2016-01-01

    Epidermal growth factor receptor (EGFR) plays a significant role in non-small cell lung cancer (NSCLC), the most prevalent form of lung cancer worldwide. Therefore, EGFR may be a useful molecular target for personalized therapy utilizing tyrosine kinase inhibitors (TKIs). Somatic activating EGFR mutations may be used to identify tumors sensitive to the effects of small-molecule EGFR-TKIs (gefitinib and erlotinib), and alternative, less frequently observed mutations, including the majority of mutations identified within exon 20, may be associated with a lack of response to TKIs. However, due to the comparative rarity of EGFR exon 20 mutations, clinical information concerning the association between EGFR exon 20 mutations and responsiveness to TKIs has been limited within the relevant literature, particularly for certain rare mutations, including p.S768I. The current study reports the case of a patient with NSCLC harboring a p.S768I mutation in the EGFR gene [a substitution at codon 768 of exon 20 (c.2303G>T, p.S768I)], as well as a mutation at codon 719, exon 18 (p.G719A). The relevant literature concerning this rare EGFR somatic mutation is also reviewed. PMID:26870223

  20. Enhanced Depolarization-Induced Pulmonary Vasoconstriction Following Chronic Hypoxia Requires EGFR-Dependent Activation of NAD(P)H Oxidase 2

    PubMed Central

    Norton, Charles E.; Broughton, Brad R.S.; Jernigan, Nikki L.; Walker, Benjimen R.

    2013-01-01

    Abstract Aims: Chronic hypoxia (CH) enhances depolarization-induced myofilament Ca2+ sensitization and resultant pulmonary arterial constriction through superoxide (O2−)-dependent stimulation of RhoA. Because NAD(P)H oxidase (NOX) has been implicated in the development of pulmonary hypertension, we hypothesized that vascular smooth muscle (VSM) depolarization increases NOX-derived O2− production leading to myofilament Ca2+ sensitization and augmented vasoconstrictor reactivity following CH. As epidermal growth factor receptor (EGFR) mediates Rac1-dependent NOX activation in renal mesangial cells, we further sought to examine the role EGFR plays in this response. Results: Vasoconstrictor responses to depolarizing concentrations of KCl were greater in lungs isolated from CH (4 wk, 0.5 atm) rats compared to normoxic controls, and this effect of CH was abolished by the general NOX inhibitor, apocynin. CH similarly augmented KCl-induced vasoconstriction and O2− generation (assessed using the fluorescent indicator, dihydroethidium) in Ca2+-permeabilized, pressurized small pulmonary arteries. These latter responses to CH were prevented by general inhibition of NOX isoforms (apocynin, diphenylene iodonium), and by selective inhibition of NOX 2 (gp91ds-tat), Rac1 (NSC 23766), and EGFR (AG 1478). Consistent with these observations, CH increased KCl-induced EGFR phosphorylation, and augmented depolarization-induced Rac1 activation in an EGFR-dependent manner. Innovation: This study establishes a novel signaling axis in VSM linking membrane depolarization to contraction that is independent of Ca2+ influx, and which mediates myofilament Ca2+ sensitization in the hypertensive pulmonary circulation. Conclusion: CH augments membrane depolarization-induced pulmonary VSM Ca2+ sensitization and vasoconstriction through EGFR-dependent stimulation of Rac1 and NOX 2. Antioxid. Redox Signal. 18, 1777–1788. PMID:22966991

  1. Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

    PubMed

    Tuononen, Katja; Mäki-Nevala, Satu; Sarhadi, Virinder Kaur; Wirtanen, Aino; Rönty, Mikko; Salmenkivi, Kaisa; Andrews, Jenny M; Telaranta-Keerie, Aino I; Hannula, Sari; Lagström, Sonja; Ellonen, Pekka; Knuuttila, Aija; Knuutila, Sakari

    2013-05-01

    The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

  2. CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling

    PubMed Central

    Rodríguez-Nieves, José A.; Patalano, Susan C.; Almanza, Diego; Gharaee-Kermani, Mehrnaz; Macoska, Jill A.

    2016-01-01

    Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis. PMID:27434301

  3. CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling.

    PubMed

    Rodríguez-Nieves, José A; Patalano, Susan C; Almanza, Diego; Gharaee-Kermani, Mehrnaz; Macoska, Jill A

    2016-01-01

    Benign prostate hyperplasia (BPH), an enlargement of the prostate common in aging in men, is associated with urinary voiding dysfunction manifest as Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal smooth muscle contractions are known to play key roles in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the unregulated differentiation of fibroblasts or other precursor cell types into myofibroblasts, which is usually accomplished by activation of the TGFβ/TGFβR axis. Previously we reported that the CXC-type chemokines, CXCL5, CXCL8 and CXCL12, which are up-regulated in the aging in the prostate, can drive this differentiation process as well in the absence of TGFβ. Based on this data we sought to elucidate the molecular mechanisms employed by CXCL12, and its receptor CXCR4, during prostate myofibroblast phenoconversion. The results of these studies suggest that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGFβ/TGFβR axis activation or Smad signaling. Here we report that CXCL12/CXCR4 axis activation promotes signaling through the EGFR and downstream MEK/ERK and PI3K/Akt pathways during myofibroblast phenoconversion, but not through TGFβ/TGFβR and downstream Smad signaling, in prostate fibroblasts undergoing myofibroblast phenoconversion. We document that EGFR transactivation is required for CXCL12-mediated signaling and expression of genes associate with myofibroblast phenoconversion (α-SMA, COL1a1). Our study successfully identified TGFβ/TGFβR-independent molecular mechanisms that promote CXCL12/CXCR4-induced myofibroblast phenoconversion. This information may be crucial for the development of novel therapies and potential biomarkers for prostatic fibrosis. PMID:27434301

  4. Deletion Mutations Keep Kinase Inhibitors in the Loop

    PubMed Central

    Freed, Daniel M.; Park, Jin H.; Radhakrishnan, Ravi; Lemmon, Mark A.

    2016-01-01

    Effective clinical application of conformationally selective kinase inhibitors requires tailoring drug choice to the tumor's activating mutation(s). In this issue of Cancer Cell, Foster et al. (2016) describe how activating deletions in BRAF, EGFR, and HER2 cause primary resistance to common inhibitors, suggesting strategies for improved inhibitor selection. PMID:27070691

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

    PubMed

    Gao, Zhan; Bu, Yongjun; Liu, Xiaozhuan; Wang, Xugang; Zhang, Guofu; Wang, Erhui; Ding, Shibin; Liu, Yongfeng; Shi, Ruling; Li, Qiaoyun; Fu, Jianhong; Yu, Zengli

    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 cell 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. PMID:26971374

  6. T11TS inhibits Angiopoietin-1/Tie-2 signaling, EGFR activation and Raf/MEK/ERK pathway in brain endothelial cells restraining angiogenesis in glioma model.

    PubMed

    Bhattacharya, Debanjan; Chaudhuri, Suhnrita; Singh, Manoj Kumar; Chaudhuri, Swapna

    2015-06-01

    Malignant gliomas represent one of the most aggressive and hypervascular primary brain tumors. Angiopoietin-1, the peptide growth factor activates endothelial Tie-2 receptor promoting vessel maturation and vascular stabilization steps of angiogenesis in glioma. Epidermal growth factor receptor (EGFR) and Tie-2 receptor on endothelial cells once activated transmits signals through downstream Raf/MEK/ERK pathway promoting endothelial cell proliferation and migration which are essential for angiogenesis induction. The in vivo effect of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) on angiopoietin-1/Tie-2 axis, EGFR signaling and Raf/MEK/ERK pathway in glioma associated endothelial cells has not been investigated previously. The present study performed with rodent glioma model aims to investigate the effect of T11TS treatment on angiopoietin-1/Tie-2 signaling, EGFR activity and Raf/MEK/ERK pathway in glioma associated endothelial cells within glioma milieu. T11TS administration in rodent glioma model inhibited angiopoietin-1 expression and attenuated Tie-2 expression and activation in glioma associated brain endothelial cells. T11TS treatment also downregulated total and phosphorylated EGFR expression in glioma associated endothelial cells. Additionally T11TS treatment inhibited Raf-1 expression, MEK-1 and ERK-1/2 expression and phosphorylation in glioma associated brain endothelial cells. Thus T11TS therapy remarkably inhibits endothelial angiopoietin-1/Tie-2 signaling associated with vessel maturation and simultaneously antagonizes endothelial cell proliferation signaling by blocking EGFR activation and components of Raf/MEK/ERK pathway. Collectively, the findings demonstrate a multi-targeted anti-angiogenic activity of T11TS which augments the potential for clinical translation of T11TS as an effective angiogenesis inhibitor for glioma treatment.

  7. Correlations between EGFR gene polymorphisms and pleural metastasis of lung adenocarcinoma

    PubMed Central

    Guo, Haisheng; Xing, Yunhui; Mu, Ailan; Li, Xia; Li, Tingshan; Bian, Xia; Yang, Chunmei; Zhang, Xiaolei; Liu, Yuefen; Wang, Xunguo

    2016-01-01

    Proliferation, growth, and differentiation of cells are strictly controlled by the signal system of epidermal growth factor receptor (EGFR). If any link of the EGFR signals system is interfered with or damaged, the proliferation, growth, and differentiation of cells would become uncontrolled. EGFR is overexpressed in a variety of malignant tumors, such as non-small-cell lung cancer, colorectal cancer and breast cancer. Results of the study have proved that EGFR overexpression is closely associated with mutations and variants of the EGFR genes, whose mutations and variants are associated with occurrence, metastasis, and prognosis of different types of tumors, including lung cancer. This study is aimed at investigating whether the polymorphisms of CA simple sequence repeat in intron 1 (CA-SSR1), -216G/T, and R497K in the EGFR are able to induce EGFR activation and whether overexpression is associated with pleural metastasis of lung adenocarcinoma. A total of 432 lung adenocarcinoma patients with pleural metastasis (metastasis group) and 424 patients with lung adenocarcinoma but without pleural metastasis (nonmetastasis group) were enrolled in this study. For all patients, the CA-SSR1 genotypes were determined by capillary electrophoresis, polymerase chain reaction amplification, and direct DNA sequencing, and the R497K and -216G/T genotypes were determined by polymerase chain reaction amplification and direct DNA sequencing. EGFR expression was evaluated by immunohistochemical staining in primary tumor tissues with different -216G/T, R497K, and CA-SSR1 genotypes. Our results showed significant differences between pleural metastasis and nonmetastasis groups in the genotype and allele distribution of -216G/T, R497K, and CA-SSR1 polymorphisms of the EGFR gene. The -216T allele, Arg allele, and shorter CA-SSR1 (<17) had significantly increased risks of pleural metastasis compared with the -216G allele, Lys allele, and longer CA-SSR1 (≥17), respectively. The expression

  8. Correlations between EGFR gene polymorphisms and pleural metastasis of lung adenocarcinoma

    PubMed Central

    Guo, Haisheng; Xing, Yunhui; Mu, Ailan; Li, Xia; Li, Tingshan; Bian, Xia; Yang, Chunmei; Zhang, Xiaolei; Liu, Yuefen; Wang, Xunguo

    2016-01-01

    Proliferation, growth, and differentiation of cells are strictly controlled by the signal system of epidermal growth factor receptor (EGFR). If any link of the EGFR signals system is interfered with or damaged, the proliferation, growth, and differentiation of cells would become uncontrolled. EGFR is overexpressed in a variety of malignant tumors, such as non-small-cell lung cancer, colorectal cancer and breast cancer. Results of the study have proved that EGFR overexpression is closely associated with mutations and variants of the EGFR genes, whose mutations and variants are associated with occurrence, metastasis, and prognosis of different types of tumors, including lung cancer. This study is aimed at investigating whether the polymorphisms of CA simple sequence repeat in intron 1 (CA-SSR1), -216G/T, and R497K in the EGFR are able to induce EGFR activation and whether overexpression is associated with pleural metastasis of lung adenocarcinoma. A total of 432 lung adenocarcinoma patients with pleural metastasis (metastasis group) and 424 patients with lung adenocarcinoma but without pleural metastasis (nonmetastasis group) were enrolled in this study. For all patients, the CA-SSR1 genotypes were determined by capillary electrophoresis, polymerase chain reaction amplification, and direct DNA sequencing, and the R497K and -216G/T genotypes were determined by polymerase chain reaction amplification and direct DNA sequencing. EGFR expression was evaluated by immunohistochemical staining in primary tumor tissues with different -216G/T, R497K, and CA-SSR1 genotypes. Our results showed significant differences between pleural metastasis and nonmetastasis groups in the genotype and allele distribution of -216G/T, R497K, and CA-SSR1 polymorphisms of the EGFR gene. The -216T allele, Arg allele, and shorter CA-SSR1 (<17) had significantly increased risks of pleural metastasis compared with the -216G allele, Lys allele, and longer CA-SSR1 (≥17), respectively. The expression

  9. Correlations between EGFR gene polymorphisms and pleural metastasis of lung adenocarcinoma.

    PubMed

    Guo, Haisheng; Xing, Yunhui; Mu, Ailan; Li, Xia; Li, Tingshan; Bian, Xia; Yang, Chunmei; Zhang, Xiaolei; Liu, Yuefen; Wang, Xunguo

    2016-01-01

    Proliferation, growth, and differentiation of cells are strictly controlled by the signal system of epidermal growth factor receptor (EGFR). If any link of the EGFR signals system is interfered with or damaged, the proliferation, growth, and differentiation of cells would become uncontrolled. EGFR is overexpressed in a variety of malignant tumors, such as non-small-cell lung cancer, colorectal cancer and breast cancer. Results of the study have proved that EGFR overexpression is closely associated with mutations and variants of the EGFR genes, whose mutations and variants are associated with occurrence, metastasis, and prognosis of different types of tumors, including lung cancer. This study is aimed at investigating whether the polymorphisms of CA simple sequence repeat in intron 1 (CA-SSR1), -216G/T, and R497K in the EGFR are able to induce EGFR activation and whether overexpression is associated with pleural metastasis of lung adenocarcinoma. A total of 432 lung adenocarcinoma patients with pleural metastasis (metastasis group) and 424 patients with lung adenocarcinoma but without pleural metastasis (nonmetastasis group) were enrolled in this study. For all patients, the CA-SSR1 genotypes were determined by capillary electrophoresis, polymerase chain reaction amplification, and direct DNA sequencing, and the R497K and -216G/T genotypes were determined by polymerase chain reaction amplification and direct DNA sequencing. EGFR expression was evaluated by immunohistochemical staining in primary tumor tissues with different -216G/T, R497K, and CA-SSR1 genotypes. Our results showed significant differences between pleural metastasis and nonmetastasis groups in the genotype and allele distribution of -216G/T, R497K, and CA-SSR1 polymorphisms of the EGFR gene. The -216T allele, Arg allele, and shorter CA-SSR1 (<17) had significantly increased risks of pleural metastasis compared with the -216G allele, Lys allele, and longer CA-SSR1 (≥17), respectively. The expression

  10. Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer.

    PubMed

    Brave, Sandra R; Odedra, Rajesh; James, Neil H; Smith, Neil R; Marshall, Gayle B; Acheson, Kerry L; Baker, Dawn; Howard, Zoe; Jackson, Lynsay; Ratcliffe, Kirsty; Wainwright, Anna; Lovick, Susan C; Hickinson, D Mark; Wilkinson, Robert W; Barry, Simon T; Speake, Georgina; Ryan, Anderson J

    2011-07-01

    Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response. PMID:21537841

  11. Delay of treatment change following objective progression on first-line erlotinib in EGFR-mutant lung cancer

    PubMed Central

    Lo, Peter C.; Dahlberg, Suzanne E.; Nishino, Mizuki; Johnson, Bruce E.; Sequist, Lecia V.; Jackman, David M.; Jänne, Pasi A; Oxnard, Geoffrey R.

    2015-01-01

    Background Erlotinib is a highly active EGFR kinase inhibitor approved for first-line use in lung cancers harboring EGFR mutations. Anecdotal experience suggests this drug may provide continued disease control following objective progression of disease (PD), however this has not been systematically studied. Methods Patients with RECIST-defined PD on three prospective trials of first-line erlotinib in advanced lung cancer were studied retrospectively, comparing progression characteristics of cases with and without EGFR sensitizing mutations. Factors influencing time to treatment change (TTC), defined as the time from PD until start of a new systemic therapy or death, were studied. Rate of tumor progression was assessed by comparing tumor measurements between the PD scan and the preceding scan. Results 92 eligible patients were studied: 42 with an EGFR sensitizing mutation and 50 without. The EGFR-mutant cohort had a slower rate of progression (p = 0.003) and a longer TTC (p < 0.001). Among EGFR-mutant cancers, 28 (66%) continued single-agent erlotinib following PD and 21 (50%) were able to delay change in systemic therapy for >3 months; only 2 received local debulking therapy during that period. Multivariate analysis of EGFR-mutant cases demonstrated that longer time to progression, slower rate of progression, and lack of new extrathoracic metastases were associated with a longer TTC. Conclusions A change in systemic therapy can commonly be delayed in patients with EGFR-mutant lung cancer objectively progressing on first-line erlotinib, particularly in those with a longer time to progression, a slow rate of progression, and lack of new extrathoracic metastases. PMID:25876525

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

    PubMed

    Gao, Gongming; Shen, Nan; Jiang, Xuefeng; Sun, Huiqing; Xu, Nanwei; Zhou, Dong; Nong, Luming; Ren, Kewei

    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 proliferation (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.

  13. Tankyrase and the canonical Wnt pathway protect lung cancer cells from EGFR inhibition

    PubMed Central

    Casás-Selves, Matias; Kim, Jihye; Zhang, Zhiyong; Helfrich, Barbara A.; Gao, Dexiang; Porter, Christopher C.; Scarborough, Hannah A.; Bunn, Paul A.; Chan, Daniel C.; Tan, Aik Choon; DeGregori, James

    2013-01-01

    Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histological subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome shRNA screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC. PMID:22738915

  14. Synergistic anti-proliferative and pro-apoptotic activity of combined therapy with bortezomib, a proteasome inhibitor, with anti-epidermal growth factor receptor (EGFR) drugs in human cancer cells.

    PubMed

    Cascone, Tina; Morelli, Maria Pia; Morgillo, Floriana; Kim, Woo-Young; Rodolico, Gabriella; Pepe, Stefano; Tortora, Giampaolo; Berrino, Liberato; Lee, Ho-Young; Heymach, John V; Ciardiello, Fortunato

    2008-09-01

    The proteasome plays a pivotal role in the turnover of regulatory transduction proteins induced by activated cell membrane growth factor receptors. The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. Proteasome inhibition may sensitize human cancer cell lines to EGFR inhibitors. We investigated the growth inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42 nM). A significant synergistic growth inhibitory effect was observed with the combination of bortezomib and each EGFR inhibitor in all cell lines (combination index, CI, range 0.10-0.55), which was accompanied by a significant induction in apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib. In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR inhibitor treatment induced a more effective inhibition of EGFR-activated down-stream signals, including a marked suppression in activated, phosphorylated Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt protected A549 cells by cell growth inhibition and apoptosis following treatment with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity in different human cancer cells which possess a functional EGFR-dependent autocrine growth pathway through to a more efficient and sustained inhibition of Akt.

  15. [Research Progress of EGFR-TKI Therapy for Patients with Central Nervous System 
Metastases from Non-small Cell Lung Cancer].

    PubMed

    Jin, Yinghua; Xin, Tao

    2016-08-20

    Approximately half of all patients with non-small cell lung cancer (NSCLC) develop central nervous system metastases during the course of their disease which indicate poor prognosis. A part of NSCLC patients demonstrates activating epidermal growth factor receptor gene (EGFR) mutations who represent effectiveness and well tolerance of EGFR-specific tyrosine kinase inhibitors (TKIs) therapy. Although the systemic efficacy of targeted agents is established, the efficacy of central nervous system (CNS) metastases is not as well characterized. In this article, we review recent data on the use of EGFR inhibitors for treatment of patients with NSCLC and CNS metastases. PMID:27561797

  16. Second-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs): a better mousetrap? A review of the clinical evidence.

    PubMed

    Ou, Sai-Hong Ignatius

    2012-09-01

    The discovery of activating epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) in 2004 heralded the era of molecular targeted therapy in NSCLC. First-generation small molecule, reversible tyrosine kinase inhibitors (TKIs) of EGFR, gefitinib and erlotinib, had been approved for second- or third-line treatment of NSCLC prior to the knowledge of these mutations. However, resistance to gefitinib and erlotinib invariably develops after prolonged clinical use. Two second-generation irreversible EGFR TKIs, afatinib (BIBW 2992) and dacomitinib (PF-00299804), that can potentially overcome the majority of these resistances are in late stage clinical development. Here I will review the clinical data of EGFR TKIs and discuss the appropriate future role of afatinib and dacomitinib in NSCLC: whether as replacement of erlotinib or gefitinib or only after erlotinib or gefitinib failure and whether different subgroups would benefit from different approaches.

  17. Renal cortical hexokinase and pentose phosphate pathway activation through the EGFR/Akt signaling pathway in endotoxin-induced acute kidney injury.

    PubMed

    Smith, Joshua A; Stallons, L Jay; Schnellmann, Rick G

    2014-08-15

    While disruption of energy production is an important contributor to renal injury, metabolic alterations in sepsis-induced AKI remain understudied. We assessed changes in renal cortical glycolytic metabolism in a mouse model of sepsis-induced AKI. A specific and rapid increase in hexokinase (HK) activity (∼2-fold) was observed 3 h after LPS exposure and maintained up to 18 h, in association with a decline in renal function as measured by blood urea nitrogen (BUN). LPS-induced HK activation occurred independently of HK isoform expression or mitochondrial localization. No other changes in glycolytic enzymes were observed. LPS-mediated HK activation was not sufficient to increase glycolytic flux as indicated by reduced or unchanged pyruvate and lactate levels in the renal cortex. LPS-induced HK activation was associated with increased glucose-6-phosphate dehydrogenase activity but not glycogen production. Mechanistically, LPS-induced HK activation was attenuated by pharmacological inhibitors of the EGF receptor (EGFR) and Akt, indicating that EGFR/phosphatidylinositol 3-kinase/Akt signaling is responsible. Our findings reveal LPS rapidly increases renal cortical HK activity in an EGFR- and Akt-dependent manner and that HK activation is linked to increased pentose phosphate pathway activity.

  18. Discovery of Selective and Noncovalent Diaminopyrimidine-Based Inhibitors of Epidermal Growth Factor Receptor Containing the T790M Resistance Mutation

    PubMed Central

    2015-01-01

    Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties. PMID:25383627

  19. A multicenter blinded study evaluating EGFR and KRAS mutation testing methods in the clinical non-small cell lung cancer setting--IFCT/ERMETIC2 Project Part 1: Comparison of testing methods in 20 French molecular genetic National Cancer Institute platforms.

    PubMed

    Beau-Faller, Michèle; Blons, Hélène; Domerg, Caroline; Gajda, Dorota; Richard, Nicolas; Escande, Fabienne; Solassol, Jérôme; Denis, Marc G; Cayre, Anne; Nanni-Metellus, Isabelle; Olschwang, Sylviane; Lizard, Sarab; Piard, Fabienne; Pretet, Jean-Luc; de Fraipont, Florence; Bièche, Ivan; de Cremoux, Patricia; Rouquette, Isabelle; Bringuier, Pierre-Paul; Mosser, Jean; Legrain, Michèle; Voegeli, Anne-Claire; Saulnier, Patrick; Morin, Franck; Pignon, Jean-Pierre; Zalcman, Gérard; Cadranel, Jacques

    2014-01-01

    Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non-small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%-16% versus 40%; 95% CI, 39%-42%; P < 0.001]. Mutated cases increased from 42% (95% CI, 31%-54%) to 53% (95% CI, 41%-64%), with three supplementary EGFR mutations (p.G179A at exon 18 and p.L858R and p.L861Q at exon 21) and five supplementary KRAS mutations, when using alternative testing instead of direct sequencing. False-positive results were observed when using a PCR-based sizing assay, high-resolution melting, or pyrosequencing. Concordance analysis returned good kappa test scores for EGFR exon 19 and KRAS analysis when comparing sequencing with alternative methods and revealed no difference between alternative techniques themselves.

  20. Long-Term Treatment with Erlotinib for EGFR Wild-Type Non-Small Cell Lung Cancer: A Case Report.

    PubMed

    Polychronidou, Genovefa; Papakotoulas, Pavlos

    2013-01-01

    The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are known to have greater efficacy in EGFR mutation-positive non-small cell lung cancer (NSCLC), although erlotinib also has activity in wild-type disease. We report the successful long-term maintenance treatment of a patient with EGFR wild-type NSCLC with gefitinib and later erlotinib. The patient (male; 44 years old; smoker) was diagnosed with EGFR wild-type NSCLC after computer tomography had revealed a mediastinal mass, and histology and mutation testing had identified the tumor as an EGFR wild-type grade 3 adenocarcinoma. The patient received multiple rounds of chemotherapy, followed by gefitinib maintenance (3 years). Later on, he received erlotinib maintenance and developed a persistent rash (grade 1/2) that lasted throughout the treatment. The patient's condition has remained stable on erlotinib for more than 5 years, with no evidence of progression. We describe the patient's disease course and treatment in the context of EGFR TKI therapy and the prognostic factors for long-term clinical outcomes of NSCLC, including the development of erlotinib-induced rash.

  1. Molecular docking based screening of novel designed chalcone series of compounds for their anti-cancer activity targeting EGFR kinase domain

    PubMed Central

    Rao, Chennu Maruthi Malya Prasada; Yejella, Rajendra Prasad; Rehman, Rehman Shaik Abdul; Basha, Syed Hussain

    2015-01-01

    Epidermal growth factor receptors (EGFR) are critical for the growth of many tumors and expressed at high levels in about one third of epithelial cancers. Hence, blockade of the binding sites for EGFR has been hypothesized as an effective anti-cancer therapy. Chalcone derivative compounds have been shown to be highly effective anti-cancer agents, however there are still so many novel derivatives possible, one of which might get us the best targeted EGFR inhibitor. In this effort directed towards the discovery of novel, potent anti-tumor agents for the treatment of cancer, in the present study a library of novel chalcone series of compounds has been designed and evaluated for their anti-cancer activity targeting EGFR kinase domain using various computational approaches. Among the twenty five novel designed chalcone series of compounds, all of them have found to be successfully docking inside the active binding domain of EGFR receptor target with a binding energy in a range of -6.10 to -9.25 Kcal/mol with predicted IC50 value range of 33.50 micor molar to 164.66 nano molar respectively. On the other hand, calculated 2DQSAR molecular descriptor properties of the compounds showed promising ADME parameters and found to be well in compliance with Lipinski׳s rule of five. Among all the twenty five compounds tested, compound 21 ((2E)-3-(anthracen-9-yl)-1-phenylprop-2-2n-1- one) was found to be the best lead like molecule with a binding energy of -9.25 kcal/mol with predicted IC50 value of 164.66 nano molar. Conclusively, novel designed compound 21 of the present study have shown promising anti-cancer potential worth considering for further evaluations. PMID:26339147

  2. The non-receptor tyrosine kinase Ack1 regulates the fate of activated EGFR by inducing trafficking to the p62/NBR1 pre-autophagosome.

    PubMed

    Jones, Sylwia; Cunningham, Debbie L; Rappoport, Joshua Z; Heath, John K

    2014-03-01

    Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.

  3. eGFR

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? Estimated Glomerular Filtration Rate (eGFR) Share this page: Was ... page helpful? Also known as: Glomerular Filtration Rate, Estimated; GFR; eGFR; Calculated Glomerular Filtration Rate; cGFR Formal ...

  4. ROLE OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN THE ACTIVATION OF MEK INDUCED BY ZN EXPOSURE

    EPA Science Inventory

    Zn is a ubiquitous ambient air pollutant typically found associated with particulate matter. Divalent Zn inhibits tyrosine phosphatases and induces EGFR- and MAPK- dependent signaling in human airway epithelial cells. To further characterize Zn-induced intracellular signaling, ...

  5. Combined targeting of EGFR/HER promotes anti-tumor efficacy in subsets of KRAS mutant lung cancer resistant to single EGFR blockade

    PubMed Central

    Umelo, Ijeoma Adaku; De Wever, Olivier; Kronenberger, Peter; Van Deun, Jan; Noor, Alfiah; Singh, Kshitiz; Teugels, Erik; Chen, Gang; Bracke, Marc; De Grève, Jacques

    2015-01-01

    KRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers. PMID:25992771

  6. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

    PubMed

    Zuo, Jian-Hong; Zhu, Wei; Li, Mao-Yu; Li, Xin-Hui; Yi, Hong; Zeng, Gu-Qing; Wan, Xun-Xun; He, Qiu-Yan; Li, Jian-Huang; Qu, Jia-Quan; Chen, Yu; Xiao, Zhi-Qiang

    2011-09-01

    EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

  7. PTEN mutation and epidermal growth factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression in human glioblastoma cells by transactivating the proximal VEGF promoter.

    PubMed

    Pore, Nabendu; Liu, Shuang; Haas-Kogan, Daphne A; O'Rourke, Donald M; Maity, Amit

    2003-01-01

    Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.

  8. ETS1 inactivation causes innate drug resistance to EGFR inhibitors.

    PubMed

    Tetsu, Osamu; Phuchareon, Janyaporn; Eisele, David W; McCormick, Frank

    2016-03-01

    Mutations in epidermal growth factor receptor (EGFR) are found in approximately 10% of lung cancers. Treatment with EGFR inhibitors, although promising, has surprisingly resulted in greater than 90% tumor reduction in only 5% of cases, prompting us to investigate the mechanism of innate drug resistance. PMID:27308601

  9. Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

    PubMed Central

    Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

    2016-01-01

    Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity. PMID:27279498

  10. Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

    NASA Astrophysics Data System (ADS)

    Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

    2016-06-01

    Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity.

  11. Cathepsin S attenuates endosomal EGFR signalling: A mechanical rationale for the combination of cathepsin S and EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Chien-Chang; Lee, Cheng-Che; Lin, Hsiao-Han; Chang, Jang-Yang

    2016-01-01

    EGF-mediated EGFR endocytosis plays a crucial role in the attenuation of EGFR activation by sorting from early endosomes to late endosomes and transporting them into lysosomes for the final proteolytic degradation. We previously observed that cathepsin S (CTSS) inhibition induces tumour cell autophagy through the EGFR-mediated signalling pathway. In this study, we further clarified the relationship between CTSS activities and EGFR signalling regulation. Our results revealed that CTSS can regulate EGFR signalling by facilitating EGF-mediated EGFR degradation. CTSS inhibition delayed the EGFR degradation process and caused EGFR accumulation in the late endosomes at the perinuclear region, which provides spatial compartments for prolonged EGFR and sustained downstream signal transducer and activator of transcription 3 and AKT signalling. Notably, cellular apoptosis was markedly enhanced by combining treatment with the EGFR inhibitor Iressa and CTSS inhibitor 6r. The data not only reveal a biological role of CTSS in EGFR signalling regulation but also evidence a rationale for its clinical evaluation in the combination of CTSS and EGFR tyrosine kinase inhibitors. PMID:27387133

  12. Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer.

    PubMed

    Saber, Ali; van der Wekken, Anthonie J; Kerner, Gerald S M A; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T Jeroen N; Groen, Harry J M

    2016-01-01

    Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14-14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04-0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.

  13. Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer.

    PubMed

    Saber, Ali; van der Wekken, Anthonie J; Kerner, Gerald S M A; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T Jeroen N; Groen, Harry J M

    2016-01-01

    Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14-14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04-0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients. PMID:27008036

  14. Multiple myeloma acquires resistance to EGFR inhibitor via induction of pentose phosphate pathway.

    PubMed

    Chen, Yan; Huang, Ruibin; Ding, Jianghua; Ji, Dexiang; Song, Bing; Yuan, Liya; Chang, Hong; Chen, Guoan

    2015-04-20

    Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM.

  15. EGFR- and AKT-mediated reduction in PTEN expression contributes to tyrphostin resistance and is reversed by mTOR inhibition in endometrial cancer cells.

    PubMed

    Li, Tian; Yang, Yuebo; Li, Xiaomao; Xu, Chengfang; Meng, Lirong

    2012-02-01

    Loss or mutation of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene is associated with resistance to epidermal growth factor receptor (EGFR) inhibitors. However, the mechanism underlying remains elusive. In this study, we aimed to explore whether sensitivity to the EGFR tyrosine kinase inhibitor (TKI) is affected by PTEN status in endometrial cancer cells. PTEN siRNA and the PTEN gene were transfected into HEC-1A and Ishikawa endometrial cancer cells using lentiviral vectors. Cells were treated under various concentrations of RG14620 and rapamycin, which are EGFR and mammalian target of rapamycin (mTOR) inhibitors, respectively. The IC(50) of RG16420 was determined by using the MTT method. Cell apoptosis and the cell cycle were studied, and activation of EGFR, AKT, and p70S6 were detected by Western blot analysis. Loss of PTEN promoted cell proliferation and led to significant increases in the levels of EGFR, phospho-EGFR, AKT, phospho-AKT, and phospho-mTOR proteins. Ishikawa and HEC-1A(PTENkd) cells that displayed loss and inactivation of PTEN function were resistant to RG14620. HEC-1A and Ishikawa(PTEN) cells with intact PTEN were sensitive to RG14620. The combination of two inhibitors was more effective than both monotherapies, particularly in carcinoma cells with PTEN dysfunction. Decreased phospho-EGFR protein expression was observed in all cell lines that were sensitive to RG14620. Decreased phospho-AKT and phospho-p70S6 protein expression was observed in PTEN-intact cells that were sensitive to RG14620. PTEN loss results in resistance to EGFR TKI, which was reversed by PTEN reintroduction or mTOR inhibitor treatment. The combined treatment of EGFR TKI and the mTOR inhibitor provided a synergistic effect by promoting cell death in PTEN-deficient and PTEN-intact endometrial cancer cells, particularly in PTEN-deficient carcinoma cells with up-regulated EGFR activation.

  16. Epidermal growth factor receptor (EGFR) signaling requires a specific endoplasmic reticulum thioredoxin for the post-translational control of receptor presentation to the cell surface.

    PubMed

    Dong, Aiwen; Wodziak, Dariusz; Lowe, Anson W

    2015-03-27

    The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.

  17. Oxopyrido[2,3-d]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors

    PubMed Central

    2015-01-01

    In nonsmall cell lung cancer (NSCLC), the threonine790–methionine790 (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFRL858R,T790M with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFRL858R,T790M driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR. PMID:26396685

  18. Rational combination of targeted therapies as a strategy to overcome the mechanisms of resistance to inhibitors of EGFR signaling.

    PubMed

    Bianco, Roberto; Damiano, Vincenzo; Gelardi, Teresa; Daniele, Gennaro; Ciardiello, Fortunato; Tortora, Giampaolo

    2007-01-01

    The epidermal growth factor receptor (EGFR) has been widely used as a target for novel anticancer agents, such as blocking antibodies and small molecular weight tyrosine kinase compounds. In spite of recent advances in cancer cell biology, leading to the introduction of clinically active new drugs, such as cetuximab, panitumumab and erlotinib, unfortunately disease control remains unsuccessful due to the presence of constitutive resistance to EGFR inhibitors in most patients and the development of acquired resistance in the responders. A large number of molecular abnormalities in tumor cells seem to partly contribute to their resistance to anti-EGFR therapy: increased angiogenesis, constitutive activation of downstream mediators, overexpression of other tyrosine kinase receptors. Moreover, some mutations in the EGFR receptor kinase domain seem to play a crucial role in determining the sensitivity of cancer cells to specific inhibitors by altering the conformation of the receptor and its activity. The development of rational combinations of anticancer agents and EGFR inhibitors, able to exert synergistic cytotoxic interactions, has been widely accepted and used in both preclinical and clinical studies. Although the failure of large clinical trial based on empirical combination of anti-EGFR and classic chemotherapeutic agents, several preclinical data seems to support the hypothesis that combining EGFR inhibitors and other novel agents could efficiently inhibit tumor growth and overcome intrinsic resistance to a single-agent based therapy. This review focuses on the role of complementary signalling pathways in the development of resistance to EGFR targeting agents and the rationale to combine novel inhibitors as anticancer therapy.

  19. Clinicopathologic characteristics of EGFR, KRAS, and ALK alterations in 6,595 lung cancers

    PubMed Central

    Lee, Boram; Lee, Taebum; Lee, Se-Hoon; Choi, Yoon-La; Han, Joungho

    2016-01-01

    Background EGFR, KRAS, and ALK alterations are major genetic changes found in non-small cell lung cancers (NSCLCs). Testing advanced lung adenocarcinoma tumors for these three genes is now standard care. The purpose of this study was to investigate the clinicopathologic expression pattern of these three genes in East Asian NSCLC patients. Patients and methods We conducted a retrospective study of all patients tested for mutations of these three genes at a single institute in Korea between 2006 and 2014. Study data were extracted from electronic medical records. Univariate and multivariate logistic regression analyses were used to measure associations between clinicopathologic features and alterations of EGFR, KRAS, and ALK. Results We detected 12 EGFR-mutated tumors with additional mutations in KRAS (N=6, 0.1%) or ALK (N=6, 0.1%). General clinicopathologic characteristics of tumors with EGFR, KRAS, or ALK mutations were similar to previous reports. Patients having EGFR L858R point mutations were older than patients having EGFR exon 19 deletions. EGFR G719X point mutations were more common in men and smokers than exon 19 deletions or L858R point mutations. Tumors having KRAS G12C mutations were less often of mucinous type than those with G12D or G12V, mutations. Conclusions This is the largest three gene molecular epidemiology study in East Asian NSCLC patients. Each genetic alteration was associated with distinct clinicopathologic characteristics. Furthermore, different age and sex are associated with different subtypes of EGFR and KRAS mutations. PMID:26992209

  20. Combined treatment with silibinin and epidermal growth factor receptor tyrosine kinase inhibitors overcomes drug resistance caused by T790M mutation.

    PubMed

    Rho, Jin Kyung; Choi, Yun Jung; Jeon, Byung-Suk; Choi, Su Jin; Cheon, Gi Jeong; Woo, Sang-Keun; Kim, Hye-Ryoun; Kim, Cheol Hyeon; Choi, Chang-Min; Lee, Jae Cheol

    2010-12-01

    Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) produce an initially dramatic response in lung cancer patients harboring a mutation in the EGFR gene, development of acquired resistance is almost inevitable. A secondary mutation of threonine 790 (T790M) is associated with approximately half of the cases of acquired resistance. This study investigated whether the addition of silibinin to therapy with gefitinib or erlotinib could overcome T790M-mediated drug resistance considering that silibinin has various antitumor effects, including EGFR modulation. Silibinin selectively reduced the activity of the EGFR family (EGFR, ErbB2, and ErbB3) through the inhibition of receptor dimerization in lung cancer cells with EGFR mutations, but not in those harboring the wild type. In primary and acquired resistant cells with T790M, addition of silibinin enhanced the ability of EGFR-TKIs to downregulate EGFR signals and to inhibit cell growth. Similarly, the combination of silibinin and erlotinib effectively suppressed tumor growth in erlotinib resistance-bearing PC-9 xenografts. The results indicate that the addition of silibinin to EGFR-TKIs is a promising strategy to overcome T790M-mediated drug resistance.

  1. The autophagy inhibitor chloroquine overcomes the innate resistance to erlotinib of non-small cell lung cancer cells with wild-type EGFR

    PubMed Central

    Zou, Yiyu; Ling, Yi-He; Sironi, Juan; Schwartz, Edward L.; Perez-Soler, Roman; Piperdi, Bilal

    2013-01-01

    Introduction The EGFR inhibitor erlotinib is much less effective in NSCLC tumors with wild-type EGFR than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process that may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. Methods We used wild-type EGFR erlotinib-sensitive and -resistant NSCLC cell lines to determine if inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. Results Erlotinib at a clinically relevant concentration (2 μM) induced autophagy in NSCLC cells with wild type EGFR, and the degree of induction was greater in resistant than in sensitive cells, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5 and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not due to the potentiation of erlotinib’s effects on autophagy, cell cycle arrest, inhibition of EGF receptor, or the inhibition of signaling downstream of the EGFR. Rather CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. Conclusions The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant of resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors. PMID:23575415

  2. C/EBPα Short-Activating RNA Suppresses Metastasis of Hepatocellular Carcinoma through Inhibiting EGFR/β-Catenin Signaling Mediated EMT

    PubMed Central

    Chen, Xuejiao; Wu, Lili; Liu, Weihui; Habib, Nagy A.; Bie, Ping; Xia, Feng

    2016-01-01

    Hepatocellular carcinoma is associated with high mortality, and tumor metastasis is an important reason for poor prognosis. However, metastasis has not been effectively prevented in clinical therapy and the mechanisms underlying metastasis have not been fully characterized. CCAAT/enhancer-binding protein-α (C/EBPα) is a transcriptional regulator with an essential role in tumor metastasis. We used short-activating RNAs (saRNA) to enhance expression of C/EBPα. Intravenous injection of C/EBPα-saRNA in a nude mouse liver orthotopic xenograft tumor model inhibited intrahepatic and distant metastasis. C/EBPα-saRNA-treated mice showed increased serum levels of albumin and decreased alanine aminotransferase (ALT), glutamic-oxalacetic transaminase (AST), indicating a role of C/EBPα in improving liver function. Migration and invasion were inhibited in hepatoma cell lines transfected with C/EBPα-saRNA. We also observed an inhibition of epithelial-mesenchymal transition (EMT) and suppression of epidermal growth factor receptor (EGFR), EGFR phosphorylation, and β-catenin in C/EBPa-saRNA-transfected cells. Our results suggested that C/EBPα-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/β-catenin signaling pathway mediated EMT in vitro and in vivo. PMID:27050434

  3. EGFR — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. Mutations in this gene are associated with lung cancer. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2010

  4. Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking

    SciTech Connect

    Shankaran, Harish; Zhang, Yi; Opresko, Lee; Resat, Haluk

    2008-06-27

    The integration of experimental measurements and computational predictions is a powerful means to understand the molecular mechanisms of complex biological systems. The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing different levels of HER1 and HER2. A mathematical model that includes the fundamental biochemical/biophysical processes involved in receptor activation was used to fit the experimental data, and 19 independent parameters including receptor dimerization affinities, trafficking rates and relative phosphorylation levels were estimated. The parameter values quantitatively support existing ideas on the effect of HER2 on HER1 phosphorylation dynamics, and enable us to predict receptor dimerization patterns in the cell lines. The integrated approach described here can be applied to obtain a predictive understanding of other biomolecular systems.

  5. Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury.

    PubMed

    Akhtar, Saghir; Yousif, Mariam H M; Chandrasekhar, Bindu; Benter, Ibrahim F

    2012-01-01

    This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.

  6. JNK Pathway Activation Modulates Acquired Resistance to EGFR/HER2-Targeted Therapies.

    PubMed

    Manole, Simin; Richards, Edward J; Meyer, Aaron S

    2016-09-15

    Resistance limits the effectiveness of receptor tyrosine kinase (RTK)-targeted therapies. Combination therapies targeting resistance mechanisms can considerably improve response, but will require an improved understanding of when particular combinations will be effective. One common form of resistance is bypass signaling, wherein RTKs not targeted by an inhibitor can direct reactivation of pathways essential for survival. Although this mechanism of resistance is well appreciated, it is unclear which downstream signaling events are responsible. Here, we apply a combined experimental- and statistical modeling-based approach to identify a set of pathway reactivation essential for RTK-mediated bypass resistance. Differences in the downstream pathway activation provided by particular RTKs lead to qualitative differences in the capacity of each receptor to drive therapeutic resistance. We identify and validate that the JNK pathway is activated during and strongly modulates bypass resistance. These results identify effective therapeutic combinations that block bypass-mediated resistance and provide a basic understanding of this network-level change in kinase dependence that will inform the design of prognostic assays for identifying effective therapeutic combinations in individual patients. Cancer Res; 76(18); 5219-28. ©2016 AACR. PMID:27450453

  7. Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment.

    PubMed

    Shi, Puyu; Oh, You-Take; Zhang, Guojing; Yao, Weilong; Yue, Ping; Li, Yikun; Kanteti, Rajani; Riehm, Jacob; Salgia, Ravi; Owonikoko, Taofeek K; Ramalingam, Suresh S; Chen, Mingwei; Sun, Shi-Yong

    2016-10-01

    The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. The current study has demonstrated that Met amplification and hyperactivation is a resistance mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed amplified Met gene and hyperactivated Met, and were cross-resistant to AZD9291 or erlotinib. Met inhibition overcame the resistance of these cell lines to AZD9291 both in vitro and in vivo, including enhancement of apoptosis or G1 cell cycle arrest. Hence, we suggest that Met inhibition is also an effective strategy to overcome resistance of certain EGFR-mutated NSCLCs with Met amplification to AZD9291, warranting the further clinical validation of our findings. PMID:27450722

  8. Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells.

    PubMed

    Rubio, Ignacio; Rennert, Knut; Wittig, Ute; Beer, Katrin; Dürst, Matthias; Stang, Stacey L; Stone, Jim; Wetzker, Reinhard

    2006-09-01

    Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Höhn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system

  9. Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction

    PubMed Central

    Turk, Harmony F.; Barhoumi, Rola; Chapkin, Robert S.

    2012-01-01

    The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA. PMID:22761867

  10. Activation of EGFR, HER2 and HER3 by neurotensin/neurotensin receptor 1 renders breast tumors aggressive yet highly responsive to lapatinib and metformin in mice

    PubMed Central

    Mourra, Najat; Liu, Jin; De Wever, Olivier; Llorca, Frédérique Penault; Cayre, Anne; Kouchkar, Amal; Gompel, Anne; Forgez, Patricia

    2014-01-01

    A present challenge in breast oncology research is to identify therapeutical targets which could impact tumor progression. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 20% of breast cancers, and NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in invasive breast carcinomas. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here, we depict the cellular mechanisms activated by NTS, and contributing to breast cancer cell aggressiveness. We show that neurotensin (NTS) and its high affinity receptor (NTSR1) contribute to the enhancement of experimental tumor growth and metastasis emergence in an experimental mice model. This effect ensued following EGFR, HER2, and HER3 over-expression and autocrine activation and was associated with an increase of metalloproteinase MMP9, HB-EGF and Neuregulin 2 in the culture media. EGFR over expression ensued in a more intense response to EGF on cellular migration and invasion. Accordingly, lapatinib, an EGFR/HER2 tyrosine kinase inhibitor, as well as metformin, reduced the tumor growth of cells overexpressing NTS and NTSR1. All cellular effects, such as adherence, migration, invasion, altered by NTS/NTSR1 were abolished by a specific NTSR1 antagonist. A strong statistical correlation between NTS-NTSR1-and HER3 (p< 0.0001) as well as NTS-NTSR1-and HER3- HER2 (p< 0.001) expression was found in human breast tumors. Expression of NTS/NTSR1 on breast tumoral cells creates a cellular context associated with cancer aggressiveness by enhancing epidermal growth factor receptor activity. We propose the use of labeled NTS/NTSR1 complexes to enlarge the population eligible for therapy targeting HERs tyrosine kinase inhibitor or HER2 overexpression. PMID:25249538

  11. Detection of epidermal growth factor receptor mutations in plasma by mutant-enriched PCR assay for prediction of the response to gefitinib in patients with non-small-cell lung cancer.

    PubMed

    He, Chen; Liu, Ming; Zhou, Chengzhi; Zhang, Jiexia; Ouyang, Ming; Zhong, Nanshan; Xu, Jun

    2009-11-15

    The high frequency of epidermal growth factor receptor (EGFR) mutations in tyrosine kinase inhibitor-responsive non-small-cell lung cancer (NSCLC) cases is now well established, highlighting the predictive value of activating EGFR mutations in guiding the clinical use of EGFR-targeted therapies. However, specimen source and methods for EGFR mutation analysis are limited by tissue availability and technical feasibility in clinical application. Therefore, the current study is designed to establish a blood-based approach for the assessment of EGFR mutations in NSCLC patients, in particular the advanced stage, and to test its clinical application. Plasma samples were obtained from the enrolled 134 NSCLC patients. The detection rate of the EGFR exon19 deletions and exon21 L858R was 49.3% (66/134) by the blood-based, mutant-enriched polymerase chain reaction. In the paired tumor and plasma samples, the detected mutant types of each pair respectively by direct sequencing and mutant-enriched polymerase chain reaction were concordant in 17 of 18 (94.4%). In the patients treated with gefitinib as a second-line therapy, those with plasma EGFR mutation have a prolonged median progression-free survival compared with those with EGFR wild type (7.609 vs. 2.877 months, p = 0.002). On comparing the efficacy of gefitinib with that of docetaxel, it was found that the median progression-free survival was significantly longer for patients treated with gefitinib than those with docetaxel in those harboring plasma EGFR mutation (7.609 vs. 3.192 months, p = 0.006). These results suggest that the blood-based EGFR mutations test has the ability to provide a reliable guidance for clinical decision making for the treatment of the advanced NSCLC patients.

  12. Activating mutations in CTNNB1 in aldosterone producing adenomas

    PubMed Central

    Åkerström, Tobias; Maharjan, Rajani; Sven Willenberg, Holger; Cupisti, Kenko; Ip, Julian; Moser, Ana; Stålberg, Peter; Robinson, Bruce; Alexander Iwen, K.; Dralle, Henning; Walz, Martin K.; Lehnert, Hendrik; Sidhu, Stan; Gomez-Sanchez, Celso; Hellman, Per; Björklund, Peyman

    2016-01-01

    Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5–10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for β-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3β binding domain in exon 3. The mutations were associated with stabilized β-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation. PMID:26815163

  13. Somatic Activating PIK3CA Mutations Cause Venous Malformation.

    PubMed

    Limaye, Nisha; Kangas, Jaakko; Mendola, Antonella; Godfraind, Catherine; Schlögel, Matthieu J; Helaers, Raphael; Eklund, Lauri; Boon, Laurence M; Vikkula, Miikka

    2015-12-01

    Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects. PMID:26637981

  14. HER2 activating mutations are targets for colorectal cancer treatment

    PubMed Central

    Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

    2015-01-01

    The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

  15. Bi-Directional SIFT Predicts a Subset of Activating Mutations

    PubMed Central

    Lee, William; Lazarus, Robert A.; Zhang, Zemin

    2009-01-01

    Advancements in sequencing technologies have empowered recent efforts to identify polymorphisms and mutations on a global scale. The large number of variations and mutations found in these projects requires high-throughput tools to identify those that are most likely to have an impact on function. Numerous computational tools exist for predicting which mutations are likely to be functional, but none that specifically attempt to identify mutations that result in hyperactivation or gain-of-function. Here we present a modified version of the SIFT (Sorting Intolerant from Tolerant) algorithm that utilizes protein sequence alignments with homologous sequences to identify functional mutations based on evolutionary fitness. We show that this bi-directional SIFT (B-SIFT) is capable of identifying experimentally verified activating mutants from multiple datasets. B-SIFT analysis of large-scale cancer genotyping data identified potential activating mutations, some of which we have provided detailed structural evidence to support. B-SIFT could prove to be a valuable tool for efforts in protein engineering as well as in identification of functional mutations in cancer. PMID:20011534

  16. TERT promoter mutations and monoallelic activation of TERT in cancer

    PubMed Central

    Huang, F W; Bielski, C M; Rinne, M L; Hahn, W C; Sellers, W R; Stegmeier, F; Garraway, L A; Kryukov, G V

    2015-01-01

    Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer. PMID:26657580

  17. Insight into the Inhibition of Drug-Resistant Mutants of the Receptor Tyrosine Kinase EGFR.

    PubMed

    Engel, Julian; Becker, Christian; Lategahn, Jonas; Keul, Marina; Ketzer, Julia; Mühlenberg, Thomas; Kollipara, Laxmikanth; Schultz-Fademrecht, Carsten; Zahedi, René P; Bauer, Sebastian; Rauh, Daniel

    2016-08-26

    Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.

  18. Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway.

    PubMed

    Suriyo, Tawit; Tachachartvanich, Phum; Visitnonthachai, Daranee; Watcharasit, Piyajit; Satayavivad, Jutamaad

    2015-12-01

    Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth.

  19. Desmocollin 3 mediates follicle stimulating hormone-induced ovarian epithelial cancer cell proliferation by activating the EGFR/Akt signaling pathway.

    PubMed

    Yang, Xiao; Wang, Jing; Li, Wen-Ping; Jin, Zhi-Jun; Liu, Xiao-Jun

    2015-01-01

    Follicle-stimulating hormone (FSH) is associated with the pathogenesis of ovarian cancer. We sought to explore whether desmocollin 3 (Dsc3) mediates FSH-induced ovarian epithelial cancer cell proliferation and whether the EGFR/Akt signaling pathway may be involved in this process. Dsc3 positivity in ovarian tissue specimens from 72 patients was assessed by immunohistochemistry. The positive expression rates of Dsc3 were similar in ovarian cancer tissues (24/31:77.4%) and borderline ovarian tumor tissues (18/22:81.8%) (P>0.05), but were significantly higher in these cancerous tissues than in benign ovarian cyst tissues (3/19:15.8%) (P<0.05). Consistently, the expression of Dsc3 in four out of five ovarian cancer cells (HO8910, Skov3ip, Skov and Hey cells, but not ES-2 and in borderline ovarian MCV152 tumor cells was higher than in the immortalized ovarian epithelial cell line, Moody. FSH up-regulated the expression of Dsc3 and EGFR in a dose- and time-dependent manner. Furthermore, a converse relationship between the expression of Dsc3, EFGR and PI3K/Akt signaling was elucidated using RNA interference and PI3K/Akt inhibitor in the absence and presence of FSH. A role for these proteins in FSH-induced cell proliferation was verified, highlighting their interdependence in mediating ovarian cancer cell function. These results suggest that Dsc3 can mediate FSH-induced ovarian cancer cell proliferation by activating the EGFR/Akt signaling pathway.

  20. Niacin activates the PI3K/Akt cascade via PKC- and EGFR-transactivation-dependent pathways through hydroxyl-carboxylic acid receptor 2.

    PubMed

    Sun, Huawang; Li, Guo; Zhang, Wenjuan; Zhou, Qi; Yu, Yena; Shi, Ying; Offermanns, Stefan; Lu, Jianxin; Zhou, Naiming

    2014-01-01

    Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.

  1. Network quantification of EGFR signaling unveils potential for targeted combination therapy

    PubMed Central

    Klinger, Bertram; Sieber, Anja; Fritsche-Guenther, Raphaela; Witzel, Franziska; Berry, Leanne; Schumacher, Dirk; Yan, Yibing; Durek, Pawel; Merchant, Mark; Schäfer, Reinhold; Sers, Christine; Blüthgen, Nils

    2013-01-01

    The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model. PMID:23752269

  2. Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II.

    PubMed

    Peng, Kesong; Tian, Xinqiao; Qian, Yuanyuan; Skibba, Melissa; Zou, Chunpeng; Liu, Zhiguo; Wang, Jingying; Xu, Zheng; Li, Xiaokun; Liang, Guang

    2016-03-01

    Cardiac hypertrophy is an important risk factor for heart failure. Epidermal growth factor receptor (EGFR) has been found to play a role in the pathogenesis of various cardiovascular diseases. The aim of this current study was to examine the role of EGFR in angiotensin II (Ang II)-induced cardiac hypertrophy and identify the underlying molecular mechanisms. In this study, we observed that both Ang II and EGF could increase the phospohorylation of EGFR and protein kinase B (AKT)/extracellular signal-regulated kinase (ERK), and then induce cell hypertrophy in H9c2 cells. Both pharmacological inhibitors and genetic silencing significantly reduced Ang II-induced EGFR signalling pathway activation, hypertrophic marker overexpression, and cell hypertrophy. In addition, our results showed that Ang II-induced EGFR activation is mediated by c-Src phosphorylation. In vivo, Ang II treatment significantly led to cardiac remodelling including cardiac hypertrophy, disorganization and fibrosis, accompanied by the activation of EGFR signalling pathway in the heart tissues, while all these molecular and pathological alterations were attenuated by the oral administration with EGFR inhibitors. In conclusion, the c-Src-dependent EGFR activation may play an important role in Ang II-induced cardiac hypertrophy, and inhibition of EGFR by specific molecules may be an effective strategy for the treatment of Ang II-associated cardiac diseases. PMID:26762600

  3. Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations.

    PubMed

    Alcalay, Roy N; Levy, Oren A; Waters, Cheryl C; Fahn, Stanley; Ford, Blair; Kuo, Sheng-Han; Mazzoni, Pietro; Pauciulo, Michael W; Nichols, William C; Gan-Or, Ziv; Rouleau, Guy A; Chung, Wendy K; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Marder, Karen; Zhang, Xiaokui

    2015-09-01

    Glucocerebrosidase (GBA) mutations have been associated with Parkinson's disease in numerous studies. However, it is unknown whether the increased risk of Parkinson's disease in GBA carriers is due to a loss of glucocerebrosidase enzymatic activity. We measured glucocerebrosidase enzymatic activity in dried blood spots in patients with Parkinson's disease (n = 517) and controls (n = 252) with and without GBA mutations. Participants were recruited from Columbia University, New York, and fully sequenced for GBA mutations and genotyped for the LRRK2 G2019S mutation, the most common autosomal dominant mutation in the Ashkenazi Jewish population. Glucocerebrosidase enzymatic activity in dried blood spots was measured by a mass spectrometry-based assay and compared among participants categorized by GBA mutation status and Parkinson's disease diagnosis. Parkinson's disease patients were more likely than controls to carry the LRRK2 G2019S mutation (n = 39, 7.5% versus n = 2, 0.8%, P < 0.001) and GBA mutations or variants (seven homozygotes and compound heterozygotes and 81 heterozygotes, 17.0% versus 17 heterozygotes, 6.7%, P < 0.001). GBA homozygotes/compound heterozygotes had lower enzymatic activity than GBA heterozygotes (0.85 µmol/l/h versus 7.88 µmol/l/h, P < 0.001), and GBA heterozygotes had lower enzymatic activity than GBA and LRRK2 non-carriers (7.88 µmol/l/h versus 11.93 µmol/l/h, P < 0.001). Glucocerebrosidase activity was reduced in heterozygotes compared to non-carriers when each mutation was compared independently (N370S, P < 0.001; L444P, P < 0.001; 84GG, P = 0.003; R496H, P = 0.018) and also reduced in GBA variants associated with Parkinson's risk but not with Gaucher disease (E326K, P = 0.009; T369M, P < 0.001). When all patients with Parkinson's disease were considered, they had lower mean glucocerebrosidase enzymatic activity than controls (11.14 µmol/l/h versus 11.85 µmol/l/h, P = 0.011). Difference compared to controls persisted in patients with

  4. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  5. Inequalities in lung cancer: a world of EGFR.

    PubMed

    Carbonnaux, Mélodie; Souquet, Pierre-Jean; Meert, Anne-Pascale; Scherpereel, Arnaud; Peters, Matthew; Couraud, Sébastien

    2016-05-01

    Epidermal growth factor receptor gene (EGFR) mutation status has emerged as a crucial issue in lung cancer management. Availability and cost of tests and tyrosine kinase inhibitors (TKIs) may vary as a function of country development.We conducted a prospective specialist opinion survey to map EGFR test and EGFR-TKI availability and detect associations with the Human Development Index (HDI). A questionnaire was sent to specialists in thoracic oncology in all United Nations Member States.We obtained responses from 74 countries, comprising 78% of the worldwide population. Nonresponding countries had significantly lower HDI rank than responding countries. EGFR mutation analysis was routinely available in 57 countries (70% of the worldwide population). The cost of the test was EGFR mutation testing and EGFR-TKIs are widely accessible in routine practice worldwide. However, there are large discrepancies in access to this innovative treatment path and in its cost for patients as a function of country development. PMID:27030679

  6. Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer.

    PubMed

    Aponte, Margarita; Jiang, Wei; Lakkis, Montaha; Li, Ming-Jiang; Edwards, Dale; Albitar, Lina; Vitonis, Allison; Mok, Samuel C; Cramer, Daniel W; Ye, Bin

    2008-07-15

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

  7. IGFBP2 potentiates nuclear EGFR-STAT3 signaling.

    PubMed

    Chua, C Y; Liu, Y; Granberg, K J; Hu, L; Haapasalo, H; Annala, M J; Cogdell, D E; Verploegen, M; Moore, L M; Fuller, G N; Nykter, M; Cavenee, W K; Zhang, W

    2016-02-11

    Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

  8. Cadmium at nanomolar concentrations activates Raf-MEK-ERK1/2 MAPKs signaling via EGFR in human cancer cell lines.

    PubMed

    Ali, Imran; Damdimopoulou, Pauliina; Stenius, Ulla; Halldin, Krister

    2015-04-25

    Cadmium (Cd) is an environmental contaminant classified as carcinogenic to humans by the International Agency for Research on Cancer, supported by data from occupational exposure. Environmentally relevant dietary exposure to Cd has recently been associated with osteoporosis and cancers of the prostate, endometrium, and breast in the general population. The low exposure effects have been proposed to result from endocrine modulative properties of Cd, which mimic the physiological actions of estrogen and androgen. However, the mechanism of action of Cd is an unanswered question. We have shown previously, using mouse models, that canonical estrogen receptor signaling is not involved in estrogen mimicry effects of Cd. Instead, low-level Cd exposure stimulated the mitogen-activated protein kinases (MAPKs) ERK1/2 in these mice. Here we investigate further the ERK1/2 MAPK signaling activation by Cd in vitro by using nanomolar concentrations of cadmium chloride (CdCl2) in three different human carcinoma cell lines: HepG2, MCF-7, and ECC-1. The findings also were confirmed in previously collected mouse tissue samples. We show that 10(-8)M levels of CdCl2 activate ERK1/2 (Tyr 204) and the p53 specific ubiquitin ligase Mdm2 (Ser 166) via Raf and MEK by acting through the epidermal growth factor receptor (EGFR). Furthermore, our results suggest that the CdCl2-induced activation of ERK1/2 and Mdm2 may interfere with the p53 response to genotoxic compounds in cancer cell lines. Our data collectively suggest that nanomolar levels of CdCl2 activate Raf-MEK-ERK1/2 via EGFR. We hypothesize that this signaling cascade may be involved in observed low exposure effects of Cd in certain human populations.

  9. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.

    PubMed

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke

    2015-05-22

    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist.

  10. EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells.

    PubMed

    Sugita, Shohei; Ito, Kentaro; Yamashiro, Yutaro; Moriya, Shota; Che, Xiao-Fang; Yokoyama, Tomohisa; Hiramoto, Masaki; Miyazawa, Keisuke

    2015-05-22

    Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOP/GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist. PMID:25858318

  11. IGFBP2 potentiates nuclear EGFR-STAT3 signaling

    PubMed Central

    Chua, Corrine Yingxuan; Liu, Yuexin; Granberg, Kirsi J.; Hu, Limei; Haapasalo, Hannu; Annala, Matti J.; Cogdell, David E.; Verploegen, Maartje; Moore, Lynette M.; Fuller, Gregory N.; Nykter, Matti; Cavenee, Webster K.; Zhang, Wei

    2015-01-01

    Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of EGFR, which subsequently activates STAT3 signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from the TCGA database for human glioma. A high level of all 3 proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient dataset. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by 2 distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target. PMID:25893308

  12. SKLB-287, a novel oral multikinase inhibitor of EGFR and VEGFR2, exhibits potent antitumor activity in LoVo colorectal tumor model.

    PubMed

    Chen, X; Liu, Y; Yang, H-W; Zhou, S; Cheng, C; Zheng, M-W; Zhong, L; Fu, X-Y; Pan, Y-L; Ma, S; Tang, Y; Chen, Y-Z; Li, L-L; Yang, S-Y

    2014-01-01

    Colorectal cancer (CRC) is the third common cancer and most of the chemotherapies of CRC currently used often suffer limited efficacy and large side effects. Targeted small-molecule by anti-tumor drugs are thought a promising strategy for improving the efficacy and reducing the side effects. In this investigation, we report a novel multikinase inhibitor, termed SKLB-287, which was discovered by us recently. SKLB-287 could efficiently inhibit the activation of endothelial growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2). It displayed very good anti-proliferative activity against LoVo CRC cells and considerable antiangiogenic potency in transgenic zebrafish embryos. Oral administration of SKLB-287 resulted in dose-dependent suppression of tumor growth in LoVo xenograft mouse model. Immunohistochemistry was adopted to examine the in vivo anti-tumor mechanism of action of SKLB-287.

  13. Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots.

    PubMed

    Kawashima, Nagako; Nakayama, Kenichi; Itoh, Kohji; Itoh, Tamitake; Ishikawa, Mitsuru; Biju, Vasudevanpillai

    2010-01-25

    The current work explores intermolecular interactions involved in the lateral propagation of cell-signaling by epidermal growth factor receptors (EGFRs). Activation of EGFRs by binding an EGF ligand in the extracellular domain of the EGFR and subsequent dimerization of the EGFR initiates cell-signaling. We investigated interactions between EGFRs in living cells by using single-molecule microscopy, Förster resonance energy transfer (FRET), and atomic force microscopy. By analyzing time-correlated intensity and propagation trajectories of quantum dot (QD)-labeled EGFR single molecules, we found that signaling dimers of EGFR [(EGF-EGFR)(2)] are continuously formed in cell membrane through reversible association of heterodimers [EGF(EGFR)(2)]. Also, we found that the lateral propagation of EGFR activation takes place through transient association of a heterodimer with predimers [(EGFR)(2)]. We varified the transient association between activated EGFR and predimers using FRET from QD-labeled heterodimers to Cy5-labeled predimers and correlated topography and fluorescence imaging. Without extended single-molecule fluorescence imaging and by using bio-conjugated QDs, reversible receptor dimerization in the lateral activation of EGFR remained obscured.

  14. Constitutive asymmetric dimerization drives oncogenic activation of epidermal growth factor receptor carboxyl-terminal deletion mutants

    PubMed Central

    Park, Angela K.J.; Francis, Joshua M.; Park, Woong-Yang; Park, Joon-Oh; Cho, Jeonghee

    2015-01-01

    Genomic alterations targeting the Epidermal Growth Factor Receptor (EGFR) gene have been strongly associated with cancer pathogenesis. The clinical effectiveness of EGFR targeted therapies, including small molecules directed against the kinase domain such as gefitinib, erlotinib and afatinib, have been proven successful in treating non-small cell lung cancer patients with tumors harboring EGFR kinase domain mutations. Recent large-scale genomic studies in glioblastoma and lung cancer have identified an additional class of oncogenic mutations caused by the intragenic deletion of carboxy-terminal coding regions. Here, we report that combinations of exonic deletions of exon 25 to 28 lead to the oncogenic activation of EGF receptor in the absence of ligand and consequent cellular transformation, indicating a significant role of C-terminal domain in modulating EGFR activation. Furthermore, we show that the oncogenic activity of the resulting C-terminal deletion mutants are efficiently inhibited by EGFR-targeted drugs including erlotinib, afatinib, dacomitinib as well as cetuximab, expanding the therapeutic rationale of cancer genome-based EGFR targeted approaches. Finally, in vivo and in vitro preclinical studies demonstrate that constitutive asymmetric dimerization in mutant EGFR is a key mechanism for oncogenic activation and tumorigenesis by C-terminal deletion mutants. Therefore, our data provide compelling evidence for oncogenic activation of C-terminal deletion mutants at the molecular level and we propose that C-terminal deletion status of EGFR can be considered as a potential genomic marker for EGFR-targeted therapy. PMID:25826094

  15. 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

  16. Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling

    SciTech Connect

    Skuland, Tonje Øvrevik, Johan; Låg, Marit; Schwarze, Per; Refsnes, Magne

    2014-08-15

    Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and—epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50 nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles. - Highlights: • Silica nanoparticles induce IL-6 and CXCL8 via NFκB and MAPKinase p38 in BEAS-2B • Silica nanoparticles induce release of the EGF-receptor ligand TGF-α • TGF-α release contributes to the IL-6 and CXCL8 release • Phosphorylation of p38 is involved in release of TGF-α.

  17. AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells

    PubMed Central

    Abdelhamed, Sherif; Ogura, Keisuke; Yokoyama, Satoru; Saiki, Ikuo; Hayakawa, Yoshihiro

    2016-01-01

    While cancer development and progression can be controlled by cytotoxic T cells, it is also known that tumor-specific CD8+T cells become functionally impaired by acquiring a group of inhibitory receptors known as immune checkpoints. Amongst those, programmed death-1 (PD-1) is one of the most recognized negative regulators of T cell function. In non-small lung cancers (NSCLCs), the aberrant activation of epidermal growth factor receptor (EGFR) is known to induce PD-L1 expression and further the treatment with gefitinib, a tyrosine kinase inhibitor (TKI) for EGFR, decrease the expression of PD-L1 on NSCLC. Given the acquired resistance to gefitinib treatment frequently observed by developing secondary-site mutations limiting its efficacy, it is important to understand the downstream mechanism of activated-EGFR signaling for regulating PD-L1 in NSCLC. In this study, we demonstrated that AKT-STAT3 pathway could be a potential target for regulating the surface expression of PD-L1 on NSCLCs with aberrant EGFR activity and, further, the inhibition of AKT or STAT3 activity could down-regulate the expression of PD-L1 even in gefitinib-resistant NSCLCs. These results highlight an importance of AKT-STAT3 pathway as a promising target for potentiating anti-tumor immune responses by regulating PD-L1 expression on cancer cells with aberrant EGFR activity.

  18. EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors

    PubMed Central

    Ohashi, Shinya; Natsuizaka, Mitsuteru; Wong, Gabrielle S.; Michaylira, Carmen Z.; Grugan, Katharine D.; Stairs, Douglas B.; Kalabis, Jiri; Vega, Maria E.; Kalman, Ross A.; Nakagawa, Momo; Klein-Szanto, Andres J; Herlyn, Meenhard; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2010-01-01

    Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis. PMID:20424117

  19. DNA methylation down-regulates EGFR expression in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor.

    PubMed

    Zhang, Fu-quan; Yang, Wen-tao; Duan, Shan-zhou; Xia, Ying-chen; Zhu, Rong-ying; Chen, Yong-bing

    2015-06-10

    Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.