Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: Activation of MET as one mechanism for drug resistance
Introduction
The human epidermal growth factor receptor (EGFR) is a well established drug target in colon, head and neck as well as non-small cell lung cancer (NSCLC). However, the population of patients responding to EGFR-targeted therapies is smaller than expected from the high proportion of patient tumours expressing EGFR, due to primary resistance.1, 2 In addition, most patients only respond transiently to therapy with EGFR-targeted drugs because of the development of acquired (secondary) resistance. Therefore, it is of importance to gain knowledge of the molecular mechanisms of drug resistance in order to identify patients with tumours that are fully addicted to an active EGFR, and also to guide the selection of combination treatments or alternative targeted therapeutics.
Cetuximab (Erbitux, C225) is a chimeric mouse-human monoclonal antibody of the IgG1 subclass that binds to the EGFR with ligand-competitive properties.3 Besides inhibiting ligand binding, cetuximab disables the activated EGFR conformation required for receptor dimerization and phosphorylation,4 inducing receptor downregulation. As a consequence, EGFR downstream signalling through the PI3K/Akt and Ras/Raf/Erk pathways is antagonized.5, 6 The roles of antibody-dependent cell-mediated cytotoxicity7 and of autophagy8 have not been extensively studied, but are likely to contribute to clinical efficacy.
Cetuximab is approved for the treatment of advanced colorectal cancer as well as for squamous cell carcinoma of the head and neck.2, 9 However, the response rate of cetuximab monotherapy with 10.8% in advanced colorectal cancer is low, and median time-to-progression is still only 1.5 months.10 The low response rate is the result of primary (intrinsic) resistance, as described below, and includes mechanisms identified in acquired drug resistance. EGFR amplification and high expression of the ligands amphiregulin and epiregulin have been described for susceptibility to cetuximab.2, 11, 12 Mutations in downstream components of EGFR signalling, most notably KRAS, NRAS and BRAF, are related to drug resistance. In metastatic colorectal cancer, KRAS mutations were found in about 33% of non-responding patients, but only in up to 10% of patients with disease control.2 Also NRAS mutations in cetuximab treated colorectal cancer had lower response rates than wild-type tumours.13 Activating BRAF mutations were identified in about 10% of patients and correlated to cetuximab resistance in some studies11 but not others.2 Similarly, phosphoinositol-3-kinase (PI3K) mutations possibly affect the cetuximab response, but conflicting data has been reported.14 Acquired resistance to cetuximab has been experimentally addressed by continuous treatment of cell lines in vitro, which attained resistance by (i) upregulation of alternative growth factor receptors, namely HER2 (ErbB2) and HER3 (ErbB3), (ii) MET activation,15 (iii) obstruction of EGFR interaction with c-Cbl, leading to failure of EGFR downregulation16 and finally (iv) high activity of Src family kinases.17
The EGFR tyrosine kinase inhibitors (TKI) gefitinib (Iressa®) and erlotinib (Tarceva®) are approved for the treatment of advanced NSCLC and erlotinib for pancreatic cancer. Acquired resistance in patients treated with these drugs is mainly due to mutations in the kinase domain.18, 19 Primary resistance mechanisms by activation of alternative receptors have been described. The HER2 variant HER2YVMA is expressed in 4% of NSCLC20 and confers resistance by ligand-independent EGFR activation.21 Expression of the insulin-like growth factor-1 receptor (IGF-1R) has been implicated in weakened response of NSCLC cell lines to TKIs, but not to cetuximab.22, 23 A combination of gefitinib with an IGF-1R targeted antibody prevented recurrence of the A431 xenograft.24
MET contributes to primary and acquired resistance to TKIs. MET is the receptor for hepatocyte growth factor (HGF, also called scatter factor) and its constitutive activation in cancer leads to invasive growth and metastasis.25 MET amplification and signalling via HER3 have been found in preclinical models of acquired resistance and in NSCLC patients.26 High HGF expression has been described in 40% of patients progressing under gefitinib therapy, but not in the original lung tumours.27 Further, MET was highly expressed in an EGFR/HER2 inhibitor resistant NSCLC cell line and interacted with EGFR and HER2.28 In accordance with this, 6% of NSCLC patients had high levels of phosphorylated MET, associated with resistance to gefitinib.29 Nevertheless, a role of MET in primary resistance to cetuximab has not been established so far.
In the present study we investigated the response towards cetuximab of 79 different patient-derived tumour xenografts, generated at Oncotest through implantation of primary patient material and shown to be of high clinical relevance.30 [XF, Xenograft Freiburg (cancer histotypes: CXF, colon, GXF, gastric, HNXF, head and neck, LXF, lung (LXFA, adenocarcinoma, LXFE, epidermoid, LXFL, large cell), MAXF, mammary).] In addition to cetuximab activity in vivo, KRAS, BRAF and NRAS mutational status, EGFR ligand expression and pathway activation, as well as MET, HER2 and HER3 activation were investigated. In a representative subgroup changes in EGFR and downstream pathway activation upon cetuximab treatment were analysed. The cetuximab-resistant lung adenocarcinoma LXFA 526 and LXFA 1647 were studied in more detail. Both tumours expressed very high levels of activated MET concurrent with cetuximab resistance.
Section snippets
Reagents and antibodies
Erbitux was from Merck-Serono, EGF from R&D systems, 5FU from Sigma, PF-04217903 from Selleck Chemicals LLC, XTT assay from Roche, bead suspension assays of total and phospho Erk1/2, Akt, and phospho EGFR, p70S6K from Bio-Rad and total EGFR, p70S6K from Millipore. Antibodies were: MET (25H5), pMET (3D7) (Cell Signalling Technology), EGFR (Millipore), β-actin and GAPDH (Abcam), mouse IgG (Santa Cruz Biotechnology), and phospho ErbB3 ELISA was from R&D.
Cell lines and xenograft models and cetuximab treatment
All xenografts (except HT29 (NCI), DiFi
Anti-tumour activity profile of cetuximab in a panel of patient-derived tumour xenografts
Anti-tumour activity of cetuximab was tested in a panel of 79 patient-derived xenografts at a dose of 30 mg/kg, which is equivalent to the clinical maintenance dose in humans, given i.p. once weekly for 3 weeks (qd7x3). Growth curves of four representative experiments are shown in Fig. 1. The NSCLC adenocarcinoma LXFA 629 and the head and neck cancer HNXF 1853 are examples of cetuximab sensitive and the NSCLC adenocarcinoma LXFA 526 and LXFA 1647 for resistant tumours. The cetuximab response of
Discussion
The clinical use of cetuximab and other targeted cancer therapeutics is ultimately linked to an understanding of the factors, which render tumours susceptible or resistant to treatment. In the present study we determined the response to cetuximab therapy in 79 patient-derived tumour xenografts. The overall rate of tumour response was 13%.
The prediction of cetuximab response remains elusive. Activating mutations of KRAS, BRAF and NRAS are common in human malignancies, like colorectal and NSCL
Conflict of interest statement
None declared.
Acknowledgements
The authors are grateful to Bio-Rad for the supply of Bio-Plex assay kits for the Erbitux proteomic screen, Hendrikje Weidmann and Eva-Maria Egenter for performance of clonogenic assays, Claudia Rappl for performance of the phospho ErbB3 ELISA and Dr. Jennifer Yang of Eli Lilly and Company for valuable discussion and proof reading of the manuscript.
References (48)
- et al.
The EGF receptor family: spearheading a merger of signaling and therapeutics
Curr Opin Cell Biol
(2007) - et al.
Effector mechanisms of therapeutic antibodies against ErbB receptors
Curr Opin Immunol
(2008) - et al.
Structural basis for inhibition of the epidermal growth factor receptor by cetuximab
Cancer Cell
(2005) - et al.
Survival of cancer cells is maintained by EGFR independent of its kinase activity
Cancer Cell
(2008) - et al.
Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis
Lancet Oncol
(2010) - et al.
Clinical relevance of EGFR- and KRAS-status in colorectal cancer patients treated with monoclonal antibodies directed against the EGFR
Cancer Treat Rev
(2009) - et al.
HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors
Cancer Cell
(2006) - et al.
Resistance to epidermal growth factor receptor-targeted therapy
Drug Resist Updat
(2005) - et al.
Role of cMET expression in non-small-cell lung cancer patients treated with EGFR tyrosine kinase inhibitors
Ann Oncol
(2008) - et al.
Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery
Eur J Cancer
(2004)
Cetuximab, its clinical use and future perspectives
Anticancer Drugs
Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways
Clin Cancer Res
Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer
Cancer Res
Impact of FcγRIIa-FcγRIIIa polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan
J Clin Oncol
Cetuximab plus oxaliplatin-based chemotherapy in the treatment of colorectal cancer
Expert Rev Anticancer Ther
Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer
N Engl J Med
Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer
J Clin Oncol
Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab
J Clin Oncol
Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members
Oncogene
Epidermal growth factor receptor (EGFR) ubiquitination as a mechanism of acquired resistance escaping treatment by the anti-EGFR monoclonal antibody cetuximab
Cancer Res
Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab
Cancer Biol Ther
Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain
PLoS Med
EGFR mutation and resistance of non-small-cell lung cancer to gefitinib
N Engl J Med
Lung cancer: intragenic ERBB2 kinase mutations in tumours
Nature
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