Erlotinib Resistance in Lung Cancer Cells Mediated by Integrin b1/Src/Akt-Driven Bypass Signaling

EGF receptor (EGFR) kinase inhibitors, including gefitinib and erlotinib, exert potent therapeutic efficacy in non–small cell lung cancers harboring EGFR-activating mutations. However, most patients ultimately develop resistance to these drugs. Here, we report a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors and the reversal of which could improve clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFRmutations that we established, there was increased expression of Src, integrin b1, a2, and a5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrin b1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Furthermore, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrin b1, a5, and/or a2 was also observed in refractory tumor samples from patients with lung cancer treated with erlotinib and/or gefitinib. Together, our findings identify the integrin b1/Src/Akt signaling pathway as a keymediator of acquired resistance to EGFR-targeted anticancer drugs.Cancer Res; 73(20); 1–11. 2013 AACR. Introduction Patients with non–small cell lung cancer (NSCLC) harboring activating somatic mutations in the EGF receptor (EGFR) gene show dramatic clinical responses. Of the somatic mutations, in-frame deletions in exon 19 (del E746-A750) and the L858Rpoint mutation are most commonly observed in NSCLC (1). These EGFR mutations are closely associated with sensitivity to EGFR tyrosine kinase inhibitors (TKI), erlotinib and gefitinib. Both gefitinib and erlotinib have shown to improve progression-free survival as compared with chemotherapy alone when given as first-line treatment for patients with NSCLCs harboring activating EGFR mutations (2–6). However, one of the serious problems encountered during clinical treatment with EGFR-TKI is the appearance of drugresistant tumors (7). Well-characterized mechanisms for the acquired resistance to EGFR-TKIs include theT790Mmutation in exon 20 of the EGFR-TKI domain (8, 9) and Met amplification (10). Furthermore, the loss of PTEN and increased expression of mitogen-activated protein kinase (MAPK), ABCG2, insulin growth factor 1 receptor (IGF-IR), and TGF-b have also been reported (7, 11, 12). In addition to these wellcharacterized mechanisms, further elucidation of novel mechanisms for acquired drug resistance is essential for the development of personalized therapeutics and strategies to circumvent drug resistance. In our laboratory, we have established various drug-resistant cell lines from human lung cancer cell lines harboring activating EGFR mutations by selecting for resistance to EGFRTKIs (13). Previously, we have reported the loss of PTEN expression with the loss of nuclear translocation of EGR-1, a transcription factor responsible for PTEN gene expression, in gefitinib-resistant clones (14–16). We also reported that either the complete or partial loss of the activated EGFR gene allele could also result in the acquisition of erlotinib resistance (17). In this present study, we further established erlotinib-resistant clones by step-wise selection following exposure to erlotinib and observed enhanced expression and activation of integrin b1 and Src. Herein, we present a novel bypass mechanism through which the integrin b1/Src/Akt signaling may play a pivotal role in the acquisition of erlotinib resistance in lung cancer cells. Materials and Methods Cell culture PC9 cells were kindly provided by Dr. Y Ichinose, National Hospital Organization, Kyushu Cancer Center (Fukuoka, Japan) and 11–18 cells were provided by Dr. K Nakagawa, Authors' Affiliations: Department of Pharmaceutical Oncology and Laboratory of Molecular Cancer Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka; Department of Diagnostic Pathology, Kurume University Hospital, Kurume; Section of Functional Morphology, Faculty of Pharmaceutical Science, Nagasaki International University, Nagasaki; SecondDepartment of Surgery, School ofMedicine, University of Occupational and Environmental Health, Kitakyushu; St. Mary's Institute of Health Science, St. Mary's Hospital, Kurume, Japan; and Pangaea Biotech, Dexeus University Institute, Barcelona, Spain Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Mayumi Ono, Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Phone/Fax: 81-92-642-6296; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-4502 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 19, 2017. © 2013 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst July 19, 2013; DOI: 10.1158/0008-5472.CAN-12-4502