Abstract 5456: Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR
Authors: Singh Sanjana , Iderzorig Tsatsral , Kumar Pragya , Puri Neelu 
Published: August 2018

Abstract
EMT is a vital process in development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens 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, N-cadherin and transcription repressor of E-cadherin, ZEB1. EMT may be responsible for Tyrosine kinase inhibitor (TKI) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing EMT. An EMT regulator, p120-catenin when it is no longer bound to membranous E-cadherin forms a complex with Kaiso factor suppressing its transcription repressor activity, promoting oncogenesis. PRMT1, another EMT inducer is also overexpressed in NSCLC. Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing a CSC marker ABCB1. Thus, we investigated EMT characteristics in wild type EGFR TKI resistant H358, H2170 cells and TKI resistant EGFR mutant H1975 cells (L858R and T790M mutation) and TKI sensitive EGFR mutant H3255 cells (L858R mutation). The modulation of EMT biomarkers was determined by immunoblotting and qPCR. Expression of ABCB1 and E-cadherin was measured using flow cytometry. Key EMT regulators such as PRMT1 and p120-catenin were upregulated 4.0 and 3.2-fold, respectively. Hence, knockdown of p120-catenin and PRMT1 were performed using siRNA or an inhibitor and examined by immunoblotting and MTT. EMT transcription factors such as Slug, Snail and Twist were also upregulated 3.18, 6.2 and 1.68-folds and E-cadherin, Claudin and ZO-1 were downregulated by 8.6, 11.6 and 15.2-folds in H1975 cells compared to H3255 cells. Also, N-cadherin, ZEB1 and Vimentin were upregulated by 4.9, 3.0 and 10.7-fold. Immunofluorescence studies with Vimentin showed that H1975 cells were elongated and have a mesenchymal phenotype compared to the H3255. 90% colocalization of p120-catenin and Kaiso factor was seen in H1975 cells whereas 10% colocalization was seen in H3255 cells. In resistant NSCLC cells with wild type and mutated EGFR, flow cytometry studies showed significant increase in expression of CSC marker ABCB1 whereas E-cadherin expression was decreased. Knockdown of PRMT1 by Furamidine, increased erlotinib efficacy by 27% in H1975 cells. Similarly, knockdown of p120-catenin by siRNA increased erlotinib efficacy by 40% in H1975 cells. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which methylate's Twist, upregulates transcription factors and p120-catenin which represses E-cadherin and Kaiso factor, activating EMT in H1975 cells.
Citation Format: Sanjana Singh, Tsatsral Iderzorig, Pragya Kumar, Neelu Puri. Mechanism of epithelial-mesenchymal transition in erlotinib resistance in NSCLC cells containing both wild-type and mutant EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5456.