Abstract 718: Acquired epithelial-mesenchymal transition like feature in an EGFR-mutant lung cancer cell line with acquired resistance to erlotinib

Abstract

Abstract Introduction: Almost all patients with lung cancer harboring a mutation in the epidermal growth factor receptor (EGFR) gene become refractory to the EGFR-tyrosine kinase inhibitors (TKIs), after showing an initial dramatic response. Secondary mutation of the EGFR gene (T790M) and MET gene amplification are the two main molecular mechanisms underlying this resistances, however about 30 percents of acquired resistance mechanisms are currently unknown. The purpose of the present study was to investigate novel acquired resistance mechanisms by analyzing in vitro acquired resistance models to EGFR-TKIs. Methods: We developed erlotinib resistant cells (HCC4006ER) from erlotinib-sensitive HCC4006 cells (IC50: 51nM) harboring an EGFR deletion mutation via chronic exposure to increasing concentrations of erlotinib. Erlotinib resistance mechanisms were analyzed using candidate or comprehensive approaches. Results: HCC4006ER cells were >200 times more resistant to erlotinib (IC50: >10uM) compared with the parent HCC4006 cells, although the resistant cells preserved EGFR exon19 deletion mutation. At first, we denied the involvement of known resistance mechanisms (secondary mutation of EGFR, MET activation by either MET gene amplification or HGF, activation of IGF-IR, downregulation of PTEN, or mutation of KRAS) in HCC4006ER cells. Next, we identified that HCC4006ER cells lost their dependency to EGFR; siRNA-mediated EGFR knockdown suppressed the survival of the parental cells effectively, but not of the resistant cells. However, comprehensive analyses (phospho-receptor tyrosine kinase array, phospho-kinase array, and gene expression assay) failed to identify novel candidate molecules that may confer erlotinib resistance in HCC4006ER cells. On the other hand, we found that HCC4006ER cells acquired a mesenchymal-like feature in their appearance and molecular markers and exhibited increased expression of a gene set that is related to the transforming growth factor beta (TGFbeta) signaling pathway. We also found that TGFbeta treatment led to moderate erlotinib resistance in HCC4006 parental cells. Conclusions: Recent reports have shown that the EMT plays a role in acquired resistance to EGFR-TKIs, although these previous reports utilized cell lines (A549 or H1650) that are not sensitive to these drugs originally. This is the first report of a relationship between EMT and EGFR-TKI acquired resistance in a TKI sensitive EGFR-mutant lung cancer cell line. Our results indicate that it would be important to consider the influence of EMT in the development of treatments against acquired resistance to EGFR-TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 718. doi:10.1158/1538-7445.AM2011-718