Abstract 2108: Mechanism underlying acquisition of dual resistance to EGFR and MET inhibitors by EGFR-mutant lung adenocarcinoma cells
Authors: Yamaoka Toshimitsu , Ohmori Tohru , Ohba Motoi , Murata Yasunori , Kishino Yasunari , Kusumoto Sojiro , Ishida Hiroo , Ohnishi Tsukasa , Sasakii Yasutsuna 
Published: July 2016

Abstract
BACKGROUND. We established a cell line with acquired resistance to gefitinib by continuously exposing lung adenocarcinoma PC-9 cells to gefitinib. MET amplification was confirmed in this clone, designated as PC-9MET. In PC-9MET, no T790M mutation was detected in EGFR, and combined treatment with EGFR/MET inhibitors suppressed cell proliferation. To investigate further bypass pathways, we established a clone with dual-resistance to EGFR/MET inhibitors by exposing PC-9MET cells to increasing concentrations of MET-TKI (PHA665752) in the presence of 1 μM gefitinib. This clone was designated as PC-9DR2.
EXPERIMENTAL PROCEDURES. To investigate new bypass signals, we used a human phospho-receptor tyrosine kinase (phospho-RTK) array. Cell proliferation and signal transduction were determined by MTT assay and Western-blot analysis, respectively. mRNA expression was quantified by real-time PCR. Mouse embryonic fibroblast cell lines derived from IGF-1R-deficient mice and engineered to overexpress human IGF-1R were used to measure IGF bioactivity in cell culture media.
RESULTS. In PC-9DR2 cells, up-regulation of IGF-1R activity was confirmed on phospho-RTK array, whereas MET expression was partially attenuated. Exposure of PC-9DR2 cells to the IGF-1R-TKI OSI906 plus the MET-TKI PHA665752 suppressed cell proliferation and induced apoptosis. Interestingly, IGF-1R activation leads to PI-3K/AKT via IRS-1, and MET activation separately leads to ERK1/2 via Gab2. Furthermore, combined treatment with the MET-TKI PHA665752 and the IGF-1R inhibitor BI 836845, a humanized monoclonal antibody against IGF-1 and IGF-2, also suppressed cell proliferation, suggesting that increased ligand activation might lead to the phosphorylation of IGF-1R as a bypass signal. In a cell-based human IGF-1R phosphorylation assay, the culture medium of PC-9DR2 more potently phosphorylated IGF-1R than did the medium of PC-9MET cells. However, PC-9DR2 cells were not associated with increased expression of either IGF-1 or IGF-2, the ligands of IGF-1R. The insulin-like growth factor binding proteins (IGFBP) are a family of six proteins that function as transport proteins for IGF-1 and IGF-2 in the circulation and regulate their access to the potentially oncogenic IGF-1R. The expression levels of IGFBP2 and IGFBP4 were significantly attenuated in PC-9DR2 cells. Moreover, exposure of PC-9DR2 cells to human recombinant IGFBP2 or IGFBP4 inhibited IGF-1R phosphorylation, and concurrent treatment with PHA665752 suppressed cell proliferation.
CONCLUSION. Our findings suggest that combined treatment with IGF-1R/MET inhibitors might be of value clinically.
Citation Format: Toshimitsu Yamaoka, Tohru Ohmori, Motoi Ohba, Yasunori Murata, Yasunari Kishino, Sojiro Kusumoto, Hiroo Ishida, Tsukasa Ohnishi, Yasutsuna Sasakii. Mechanism underlying acquisition of dual resistance to EGFR and MET inhibitors by EGFR-mutant lung adenocarcinoma cells. [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 2108.