Abstract 3336: Comparison of epithelial mesenchymal transition mediated TKI resistanc NSCLC cells containing wild type EGFR and mutant EGFR
Authors: Iderzorig Tsatsral , Singh Sanjana , Chhabra Gagan , Puri Neelu 
Published: July 2017

Abstract
Epithelial to mesenchymal transition (EMT) is a vital process in development and of metastasis and occurs when epithelial cell lose their polarized structure, by reducing adherent junction proteins E-cadherin, Claudin and ZO-1 on the membrane. Cells with EMT are elongated spindle like structures due to upregulation of mesenchymal markers Vimentin and N-cadherin. EMT may be responsible for resistance to molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) against EGFR which is used in patients with activated EGFR mutations. However, these patients acquire resistance to TKIs after prolonged use. This acquired resistance to TKIs may also be due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. EMT is regulated by p120-catenin which interacts with the Kaiso factor in the cell nucleus and inhibits the transcriptional repressor activity of Kaiso factor. Kaiso factor represses Wnt target genes such as ZEB-1. The binding of p120-catenin to Kaiso factor also increases Wnt signaling which results in loss of E-cadherin. Cells undergoing EMT can acquire cancer stem-cell like characteristics by expressing stem-cell marker ABCB1. Thus we investigated EMT characteristics in TKI-resistant NSCLC cells, H2170 ER (Erlotinib resistant), H358 ER and H1975. To determine modulation of EMT biomarkers in TKI-resistant cells, H1975 with L858R and T790M mutations was compared to TKI-sensitive cell line H3255 with L858R mutation, using immunoblotting, and qPCR. Expression of stem-cell markers ABCB1 and EMT biomarker E-cadherin are measured using flow cytometry. Key EMT-related proteins such as PRMT-1, Slug, Snail, Twist, p120-catenin, and Vimentin were upregulated by 3.2, 3.18, 6.2, 1.68, 4 and 6.5 fold, respectively, and E-cadherin, Claudin and ZO-1 were downregulated by 89%, 90% and 99% fold as compared to the H3255 TKI-sensitive cell line. We also observed upregulation of N-cadherin, ZEB-1 and Vimentin by 2.4, 2.6, and 12 fold and downregulation of E-cadherin by 50% in H1975 by qPCR. Immunofluorescence studies for Vimentin showed that H1975 cells were more elongated and stratified as compared to the H3255 that had polarized structures. 90% colocalization of p120-catenin and Kaiso factor was seen H1975 cells whereas 10% colocalization was seen in H3255 cells. Flow cytometry results indicated that there was significant increase in expression of stem cell marker ABCB1 in TKI resistant cells H2170 ER, H358 ER and H1975 in comparison to H2170 P(Parental), H358 P and H3255 TKI sensitive cells. In conclusion, our results indicate that EMT is mediated through PRMT-1, which methylates Twist, transcriptional repressor of E-cadherin along with Slug and Snail in H1975 cells with T790M mutation. Cancer stem-cell marker ABCB1 is specific for TKI resistant NSCLC cells which exhibit EMT.
Citation Format: Tsatsral Iderzorig, Sanjana Singh, Gagan Chhabra, Neelu Puri. Comparison of epithelial mesenchymal transition mediated TKI resistanc NSCLC cells containing wild type EGFR and mutant EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2017-3336