Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents.

Citation Format: Yoshitaka Yagi, Hiroaki Ozasa, Takahiro Tsuji, Yuichi Sakamori, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5045.