Abstract 1834: The molecular characters of acquired resistant non-small cell lung cancer cells to afatinib
Authors: Hashida Shinsuke , Toyooka Shinichi , Ohtsuka Tomoaki , Suzawa Ken , Shien Kazuhiko , Yamamoto Hiromasa , Soh Junichi , Asano Hiroaki , Tsukuda Kazunori , Miyoshi Shinichiro 
Published: May 2015

[Background] The non-small cell lung carcinomas (NSCLCs) with activating epidermal growth factor receptor (EGFR) gene mutation show significant response to EGFR-tyrosine kinase inhibitors (TKIs). However, almost all of them acquire resistance to EGFR-TKIs due to various mechanisms, such as T790M mutation or MET amplification. Afatinib is an irreversible TKI for EGFR and HER2 and known to be effective to the EGFR T790M variant. However, a half of the patient responded afatinib acquire resistance by 12 months in clinical use. Thus, to unravel the mechanisms of the resistances and to overcome them are still important issues of EGFR-TKI treatment. In this study, we established various kinds of afatinib resistant cell lines with changing drug exposing condition as we previously reported (Shien K. et al Cancer Res. 2013). We also investigated these cell lines to understand mechanisms of afatinib resistances in NSCLC.

[Materials and Methods] Afatinib resistant cell lines were established using four NSCLC cell lines with activating EGFR mutations by exposing afatinib with different procedures, escalation from 1 nM, escalation from 10 nM, intermittent 2 µM and continuous 2 µM procedures. These cell lines were investigated about previously reported resistant related features, T790M mutation, MET amplification, and epithelial to mesenchymal transition (EMT). ALDH1A1 and ABCB1 expression, which were putative stem cell markers, were also investigated. Proliferative inhibitions with various drugs were evaluated using MTS assay.

[Results] There were no T790M mutations in all afatinib resistant cell lines. HCC827 sublines exposed afatinib with escalation from 1 nM and intermittent 2 µM, and HCC4011 sublines showed MET amplification. HCC827 sublines exposed afatinib with escalation from 10 nM and continuous 2 µM, and HCC4006 sublines showed EMT features. Furthermore, ALDH1A1 and ABCB1 expression were up-regulated in HCC827 afatinib resistant sublines with EMT features. Next, we examined the effect of afatinib or crizotinib for the afatinib resistant cell line with MET amplification. We found that combination therapy with afatinib and crizotinib was effective to the MET amplified cell. The combination exposure was continued to establish a resistant subline (HCC827-ACR) to combination therapy. The HCC827-ACR was also showed EMT features and up-regulation of stem cell markers.

[Conclusions] As in the cases of reversible EGFR-TKI, MET amplification, EMT, and stem cell-like features were emerged in afatinib resistant cells. To develop strategies to resistant cells with stem cell-like property may be critical part to overcome EGFR-TKI resistance.

Citation Format: Shinsuke Hashida, Shinichi Toyooka, Tomoaki Ohtsuka, Ken Suzawa, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. The molecular characters of acquired resistant non-small cell lung cancer cells to afatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2014-1834