Abstract 1599: Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines
Authors: Ichwaku Rastogi, Tsatsral Iderzorig, Gagan Chhabra, Gregory M. Botting, Andrew Webb, Brad Foster, Brian Webb, Marie Nlend, Neelu Puri
Journal: Cancer Research (2016)

Abstract NSCLC cells acquire resistance to EGFR and c-Met TKIs after prolonged use. Our studies indicate that resistance maybe due to upregulation of alternative signaling pathways such as Wnt and mTOR. We have found that activation of Wnt/β-Catenin is also associated with EMT which results in loss of cell adhesion properties and gain of motility and invasiveness. To understand the mechanism of TKI resistance in NSCLC cells with wild type EGFR, we have developed and used H2170-P (parental) cells and the TKI resistant H2170-ER (erlotinib resistant) and H2170-SR (SU11274 resistant) cells. We aim to study EMT and determine if inhibition of β-Catenin, a key regulator of transcription by siRNA or inhibition of ZEB-1 a transcriptional repressor of cell adhesion proteins by inducing mir-200a will help overcome the TKI resistance in NSCLC cells. Using immunoblotting, we observed modulations in key EMT-related proteins in H2170-ER cells which showed upregulation of ZEB-1, N-cadherin, active beta-catenin, Vimentin, PRMT1 and ZO1 by 1.8, 2.4, 2, 1.8, 2.6 and 2 fold, respectively, and downregulation of E-cadherin (1.8 fold) as compared to H2170-P cells. Similar results were observed for H2170-SR cells. These results were verified using qPCR where we show that β-catenin (3.4-3.2 fold) and N-cadherin (2-1.9 fold) have increased gene expression while E-cadherin (1.7-2.4 fold) has a decreased gene expression in TKI resistant H2170-ER and SR cells when compared to H2170-P cells. miR-200a induction in H2170-ER cells showed significant downregulation of ZEB-1 (3 fold) at 72 hr and an upregulation of E-cadherin (2 fold) when compared to the mock transfected cells. Morphological changes indicative of EMT were detected using immunofluorescence with Vimentin and E-Cadherin antibodies, which displayed upregulation of Vimentin filaments (2 fold) and downregulation of E-Cadherin (3 fold) in H2170-ER and H2170-SR cells when compared to H2170-P cells. We then conducted experiments where we suppressed ZEB-1 by inducing miR-200a in TKI resistant cells. The immunoblotting results suggested recovery of E-Cadherin, and downregulation of ZEB-1 and N-Cadherin in TKI resistant cells. We also observed increased sensitivity towards erlotinib and SU11274 by 20-25%. Additionally, we observed decrease in levels of β-Catenin and upon siRNA knockdown of β-Catenin, suppression of levels of ZEB-1. This indicates a direct correlation between nuclear accumulation of β-Catenin and occurrence of EMT in TKI resistant cells by increase in expression of ZEB-1. Our results indicate that increased activation of Wnt/β-Catenin pathway in the TKI resistant NSCLC cells is due to the EMT. In NSCLC patients, L858R and T790M mutations are associated with TKI resistance which could be responsible for inducing EMT. We are further studying cell lines with EGFR mutations to determine their role in induction of EMT this may provide clinicians with novel targets to overcome TKI resistance in NSCLC patients. Citation Format: Ichwaku Rastogi, Tsatsral Iderzorig, Gagan Chhabra, Gregory M. Botting, Andrew Webb, Brad Foster, Brian Webb, Marie Nlend, Neelu Puri. Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines. [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 1599.