Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines

PurposeThe therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line.MethodsEGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods.ResultsWe assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance.ConclusionThese data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

[1]  Steven P Gygi,et al.  Signaling networks assembled by oncogenic EGFR and c-Met , 2008, Proceedings of the National Academy of Sciences.

[2]  I. Bahar,et al.  [Epidermal growth factor receptor]. , 1994, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[3]  J. Engelman,et al.  Acquired resistance to tyrosine kinase inhibitors during cancer therapy. , 2008, Current opinion in genetics & development.

[4]  P. Andrews,et al.  Mechanisms of acquired resistance to cisplatin. , 1994, Cancer treatment and research.

[5]  Douglas R Lowy,et al.  E‐cadherin‐mediated adhesion inhibits ligand‐dependent activation of diverse receptor tyrosine kinases , 2004, The EMBO journal.

[6]  P. Jänne,et al.  Mechanisms of Acquired Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non–Small Cell Lung Cancer , 2008, Clinical Cancer Research.

[7]  R. Seruca,et al.  EGFR regulates RhoA-GTP dependent cell motility in E-cadherin mutant cells. , 2007, Human molecular genetics.

[8]  F. Lordick,et al.  Cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric cancer: a phase II study of the Arbeitsgemeinschaft Internistische Onkologie (AIO) , 2010, British Journal of Cancer.

[9]  A. Bardelli,et al.  Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[10]  P. Elvin,et al.  Inhibition of Src Impairs the Growth of Met-Addicted Gastric Tumors , 2010, Clinical Cancer Research.

[11]  J. Haerting,et al.  Chemotherapy in advanced gastric cancer: a systematic review and meta-analysis based on aggregate data. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[12]  R. Langer,et al.  Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie (AIO) , 2011, BMC Cancer.

[13]  H. Yokozaki,et al.  Molecular characteristics of eight gastric cancer cell lines established in Japan , 2000, Pathology international.

[14]  Sanjay Goel,et al.  PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. , 2008, Cancer research.

[15]  Keith L. Ligon,et al.  Coactivation of Receptor Tyrosine Kinases Affects the Response of Tumor Cells to Targeted Therapies , 2007, Science.

[16]  H. Höfler,et al.  Germline mutations of the E-cadherin(CDH1) and TP53 genes, rather than of RUNX3 and HPP1, contribute to genetic predisposition in German gastric cancer patients , 2004, Journal of Medical Genetics.

[17]  S. Kubicka,et al.  Cetuximab with irinotecan, folinic acid and 5-fluorouracil as first-line treatment in advanced gastroesophageal cancer: a prospective multi-center biomarker-oriented phase II study. , 2011, Annals of oncology : official journal of the European Society for Medical Oncology.

[18]  P. Jänne,et al.  Primary resistance to cetuximab therapy in EGFR FISH-positive colorectal cancer patients , 2008, British Journal of Cancer.

[19]  Xin Liu,et al.  Cetuximab enhances the activities of irinotecan on gastric cancer cell lines through downregulating the EGFR pathway upregulated by irinotecan , 2011, Cancer Chemotherapy and Pharmacology.

[20]  T. Cenci,et al.  Is there a role for IGF1R and c-MET pathways in resistance to cetuximab in metastatic colorectal cancer? , 2011, Clinical colorectal cancer.

[21]  J. Minna,et al.  Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. , 2006, Cancer research.

[22]  L. Norton,et al.  Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[23]  C. Mathers,et al.  Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 , 2010, International journal of cancer.

[24]  F. Boyd Some Practical Considerations , 1924 .

[25]  Heinz Höfler,et al.  Mutations of the human E‐cadherin (CDH1) gene , 1998, Human mutation.

[26]  H. Höfler,et al.  E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. , 1994, Cancer research.

[27]  Y. Bang,et al.  Phase II study and biomarker analysis of cetuximab combined with modified FOLFOX6 in advanced gastric cancer , 2009, British Journal of Cancer.

[28]  T. Beckers,et al.  Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance. , 2011, European journal of cancer.

[29]  F. Luo,et al.  Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels , 2006, Molecular Cancer Therapeutics.

[30]  F. Hirsch,et al.  ErbB-3 expression is associated with E-cadherin and their coexpression restores response to gefitinib in non-small-cell lung cancer (NSCLC). , 2009, Annals of oncology : official journal of the European Society for Medical Oncology.

[31]  R. Berardi,et al.  Phase II study of cetuximab in combination with FOLFIRI in patients with untreated advanced gastric or gastroesophageal junction adenocarcinoma (FOLCETUX study). , 2007, Annals of oncology : official journal of the European Society for Medical Oncology.

[32]  J. Nährig,et al.  Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: a multivariate analysis using a standardized immunohistochemical detection system , 2004, Modern Pathology.

[33]  L. Chin,et al.  Frequent met oncogene amplification in a Brca1/Trp53 mouse model of mammary tumorigenesis. , 2006, Cancer research.

[34]  R H Wheeler,et al.  Phase I study of anti--epidermal growth factor receptor antibody cetuximab in combination with radiation therapy in patients with advanced head and neck cancer. , 2001, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[35]  M. Boyd,et al.  Some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen , 1995 .

[36]  T. Fujii,et al.  Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. , 2008, Cancer research.

[37]  J. Christensen,et al.  HGF Rescues Colorectal Cancer Cells from EGFR Inhibition via MET Activation , 2009, Clinical Cancer Research.

[38]  G. Tortora,et al.  EGFR antagonists in cancer treatment. , 2008, The New England journal of medicine.

[39]  A. Balmain,et al.  Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7 , 2010, Oncogene.

[40]  U. McDermott,et al.  Acquired resistance of non-small cell lung cancer cells to MET kinase inhibition is mediated by a switch to epidermal growth factor receptor dependency. , 2010, Cancer research.

[41]  中川 誠人 Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites , 2002 .

[42]  S. Hirohashi,et al.  E-cadherin gene mutations in human gastric carcinoma cell lines. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Langer,et al.  DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer. , 2011, European journal of cancer.

[44]  G. Mills,et al.  Cetuximab attenuates metastasis and u-PAR expression in non-small cell lung cancer: u-PAR and E-cadherin are novel biomarkers of cetuximab sensitivity. , 2009, Cancer research.

[45]  D A Scudiero,et al.  Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. , 1989, Journal of the National Cancer Institute.

[46]  L. Schwartz,et al.  Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[47]  S. Agarwal,et al.  Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells , 2009, British Journal of Cancer.

[48]  Jin Won Kim,et al.  The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines. , 2008, Cancer letters.

[49]  F. Lee,et al.  Correlation of pharmacokinetics with the antitumor activity of Cetuximab in nude mice bearing the GEO human colon carcinoma xenograft , 2005, Cancer Chemotherapy and Pharmacology.

[50]  P. Harari,et al.  Establishment and Characterization of a Model of Acquired Resistance to Epidermal Growth Factor Receptor Targeting Agents in Human Cancer Cells , 2009, Clinical Cancer Research.

[51]  A. Bardelli,et al.  Phase II study of cetuximab in combination with cisplatin and docetaxel in patients with untreated advanced gastric or gastro-oesophageal junction adenocarcinoma (DOCETUX study) , 2009, British Journal of Cancer.

[52]  G. Adams,et al.  Monoclonal antibody therapy of cancer , 1999, Nature Biotechnology.

[53]  C. Caldas,et al.  Genetic screening for hereditary diffuse gastric cancer , 2003, Expert review of molecular diagnostics.

[54]  W. Birchmeier,et al.  E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells , 1991, The Journal of cell biology.

[55]  K. Muenchen Cetuximab plus oxaliplatin- based chemotherapy in the treatment of colorectal cancer , 2008 .

[56]  R. Langer,et al.  Enhanced activation of epidermal growth factor receptor caused by tumor-derived E-cadherin mutations. , 2008, Cancer research.

[57]  N. Rosen,et al.  HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancer cells , 2008, Molecular Cancer Therapeutics.

[58]  Xifeng Wu,et al.  Sensitivity to Epidermal Growth Factor Receptor Inhibitor Requires E-Cadherin Expression in Urothelial Carcinoma Cells , 2008, Clinical Cancer Research.

[59]  M. Dietel,et al.  In vitro prediction of cytostatic drug resistance in primary cell cultures of solid malignant tumours. , 1993, European journal of cancer.

[60]  Hayao Nakanishi,et al.  Interleukin‐2 potentiation of cetuximab antitumor activity for epidermal growth factor receptor‐overexpressing gastric cancer xenografts through antibody‐dependent cellular cytotoxicity , 2008, Cancer science.

[61]  Joon-Oh Park,et al.  MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling , 2007, Science.