Abstract 337: Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC

Abstract

Abstract Current EGFR/c-Met tyrosine kinase inhibitor (TKI) combination therapies are shown to be susceptible to acquired resistance in the majority of NSCLC patients. To understand how cells develop EGFR/c-Met TKI resistance, we used three model NSCLC cell lines: H2170, H3255 and H1975. H2170 cell line express high levels of EGFR and c-Met and are EGFR wild-type. The H3255 cell line is positive for an EGFR TKD mutation, L858R, and is sensitive to initial TKI treatment. The H1975 cell line is positive for two EGFR TKD mutations, T790M and L858R, which confer resistance to erlotinib. Previous studies in our lab have shown that activation of alternative signaling pathways lead to the development of TKI resistance. In the wild type TKI resistant cells, H2170 erlotinib resistant (ER) and H2170 SU11274 resistant(SR), we observed activation of Wnt and mTOR pathway along with increased accumulation of β-catenin in the nucleus. However, in H1975 cells with mutated EGFR (L858R and T790M), we observed activation of only mTOR pathway. β-catenin is associated with Epithelial-Mesenchymal Transition (EMT), during which cells lose tight junction proteins such E-Cadherin and gain expression of transcriptional proteins such as β-catenin and ZEB1, which may lead to aberrant activation of Wnt signaling pathway and increased motility and invasion. Activation of mTOR pathway leads to increased expression of p70S6K and 4E-BP1 which are key translational regulators. We found increased accumulation of active β-catenin in the nucleus by 1.9-2.5 folds and 2.9-3.1 folds in H2170 ER and SR cells, respectively, compared to H2170 parental cells. In H2170 ER cells we observed modulations in key EMT-related proteins, N-Cadherin (2-3.3 fold) and Claudin-1 (2.5-14 fold) were upregulated and Snail was downregulated 1.7-2.5 fold in the presence and absence of erlotinib and EGF, when compared to H2170 parental cells. Similarly in H2170 SR cells we observed upregulation of N-Cadherin (1.3-2.3 fold) and Zeb-1 (1.2-1.4 fold) and downregulation of ZO-1 (1.4-1.9 fold) and E-Cadherin (1.5-2.0 fold) in the presence and absence of HGF and SU11274 when compared to H2170 parental cells. Morphological changes indicative of EMT were detected using immunostaining with Vimentin and E-Cadherin antibodies, which displayed increased Vimentin filaments (2-fold) and loss of E-Cadherin (5-10 fold) in H2170 ER cells when compared to H2170 parental cells. In H1975 cells we observed that proteins related to mTOR signaling pathway such as p-mTOR, p-GSK3-β, p-4EBP1 and p-p70S6K were upregulated 2.5 fold, 4-5 fold, 2.2-2.4 fold, and 4.4-10.4 fold, respectively, when compared to H3255 cells in the presence of erlotinib. Based on these results, we treated H1975 cells with combination of EGFR and mTOR inhibitors. We observed that 1 μM everolimus with 2.5 μM erlotinib synergistically inhibited cell growth by 53%. These studies may provide clinicians with novel targets for improving treatment options for future NSCLC patients. Citation Format: Ichwaku Rastogi, Gregory M. Botting, Andrew Webb, Brian L. Webb, Marie C. Nlend, Neelu Puri. Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 337. doi:10.1158/1538-7445.AM2015-337