Small Molecule Therapeutics Inhibition of ABCB1 Overcomes Cancer Stem Cell–like Properties and Acquired Resistance to MET Inhibitors in Non–Small Cell Lung Cancer Teppei Sugano, Masahiro Seike, Rintaro Noro, Chie Soeno, Mika Chiba, Fenfei Zou, Shinji Nakamichi, Nobuhiko Nishijima, Masaru Matsumoto, Akihiko Miyanaga, Kaoru Kubota, and Akihiko Gemma Abstract Patients with non–small cell lung cancer (NSCLC) EGFR muta- tions have shown a dramatic response to EGFR inhibitors (EGFR- TKI). EGFR T790M mutation and MET amplification have been recognized as major mechanisms of acquired resistance to EGFR- TKI. Therefore, MET inhibitors have recently been used in NSCLC patients in clinical trials. In this study, we tried to identify the mechanism of acquired resistance to MET inhibi- tors. We analyzed the antitumor effects of two MET inhibitors, PHA-665752 and crizotinib, in 10 NSCLC cell lines. EBC-1 cells with MET amplification were the only cells that were sensitive to both MET inhibitors. We established PHA- 665752–resistant EBC-1 cells, namely EBC-1R cells. Activation of KRAS, EGFR, and FGFR2 signaling was observed in EBC-1R cells by FISH and receptor tyrosine kinase phosphorylation antibody arrays. EBC-1R cells also showed overexpression of ATP-binding cassette subfamily B member 1 (ABCB1) as well as phosphorylation of MET. EBC-1R cells grew as cell spheres that exhibited cancer stem cell–like (CSC) properties and epithelial–mesenchymal transition (EMT). The level of miR- 138 that targeted ABCB1 was decreased in EBC-1R cells. ABCB1 siRNA and the ABCB1 inhibitor elacridar could reduce sphere numbers and suppress EMT. Elacridar could also reverse resis- tance to PHA-665752 in EBC-1R cells. Our study demonstrated that ABCB1 overexpression, which was associated with CSC properties and EMT, was involved in the acquired resistance to MET inhibitors. Inhibition of ABCB1 might be a novel ther- apeutic strategy for NSCLC patients with acquired resistance to MET inhibitors. Mol Cancer Ther; 14(11); 2433–40. Ó2015 AACR. Introduction Lung cancer is the most frequent cause of cancer-related death in Japan and worldwide (1). Recently, oncogenic driver mutations in non–small cell lung cancer (NSCLC) patients, such as EGFR mutation and anaplastic lymphoma kinase gene (ALK) fusion gene, have been identified (2–4). Several tyrosine kinase inhibi- tors (TKI) are currently approved or are under clinical develop- ment for the treatment of NSCLC. Our group and others have recently reported that first-line gefitinib treatment in advanced NSCLC patients with EGFR mutations improved progression-free survival (PFS) in randomized phase III studies (5, 6). Unfortu- nately, despite this initial and marked response, most NSCLC patients become resistant to EGFR-TKIs. Two major mechanisms of acquired resistance to EGFR-TKI were identified in patients with NSCLC (7, 8). About half of resistant tumors develop a secondary EGFR mutation in exon20 T790M, which prevents effective inhi- bition by EGFR TKIs due to steric hindrance or an increased binding affinity for ATP (7). An additional 5% to 10% of tumors from refractory patients undergo MET gene amplification, which causes HER3-dependent activation of the signaling cascade down- stream of EGFR despite its inhibition by TKIs (8). MET is a proto-oncogene that encodes a receptor tyrosine kinase, c-MET. c-MET is the receptor for hepatocyte growth factor (HGF). The binding of HGF to c-MET leads to cellular responses, including cell proliferation, motility, migration, and invasion (9, 10). In lung cancer, MET can be activated by HGF stimulation (11). Our recent study demonstrated that MET amplification and gene copy number gains showed a short response to gefitinib treatments in lung adenocarcinoma with EGFR mutation (12). Recently, MET inhibitors have been administered to NSCLC patients who are na € ve or resistant to EGFR TKIs in a clinical trial (13). This phase II study showed that PFS was longer in the group treated with erlotinib plus the MET inhibitor tivantinib than in the group treated with erlotinib alone, especially among patients with KRAS mutations (13). Recent studies showed mechanisms of resistance to MET inhibitors, including mutation in the MET activation loop (Y1230), bypassed EGFR activation, and MET and KRAS gene amplification (14, 15). However, the molecular mechanisms of the acquired resistance to MET inhibitors in NSCLC are not completely understood. In this study, we aimed to identify a novel molecular mechanism for acquired resistance to MET inhibitors and demonstrate potential therapeutic strategies. We established MET inhibitor–resistant NSCLC cells (EBC-1R). EBC-1R cells Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, Japan. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Masahiro Seike, Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Phone: 813-3822-2131; Fax: 813-5685-3075; E-mail: mseike@nms.ac.jp doi: 10.1158/1535-7163.MCT-15-0050 Ó2015 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org 2433 on June 6, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst September 8, 2015; DOI: 10.1158/1535-7163.MCT-15-0050