Abstract 1876: Mechanism of tyrosine kinase inhibitors resistance and role of epithelial mesenchymal transition in NSCLC
Authors: Singh Sanjana , Khan Nabiha H. , Puri Neelu 
Published: August 2019

EMT is a vital process in the development of metastasis and occurs when epithelial cells lose their polarized structure, by downregulation of adherens junction proteins, E-cadherin and Claudin1, located on the cell membrane. Cells with EMT are elongated spindle-like structures due to upregulation of mesenchymal markers Vimentin and N-cadherin. EMT may be responsible for tyrosine kinase inhibitor (TKIs) resistance to epidermal growth factor receptor (EGFR) in patients with activated EGFR mutations. Our earlier studies indicate that the presence of T790M mutation may induce EMT in erlotinib resistant (ER) cells (H1975). When p120-catenin, a key EMT regulator, is no longer bound to membranous E-cadherin, a complex is formed with Kaiso factor, suppressing its transcription repressor activity and promoting oncogenesis. PRMT1, another key EMT inducer, is also overexpressed in non-small cell lung cancer (NSCLC). Cells undergoing EMT also acquire cancer stem-cell (CSC) like characteristics by expressing CSC markers ABCB1, BMI-1 and Oct-4. Thus, we investigated EMT characteristics in wild type EGFR ER H358 and H2170 cells. The modulation of EMT biomarkers was determined by immunoblotting (WB) and Quantitative real-time PCR (qPCR). Expression of CSC markers was measured using flow cytometry. PRMT1 and p120-catenin were upregulated 1.5 and 6.1-fold in H358ER cells. Hence, knockout of p120-catenin and PRMT1 were performed using CRISPR-Cas9 or an inhibitor and was examined by WB and cell proliferation assay (MTT). Other EMT regulators such as Snail, PRMT-5, Vimentin and Kaiso factor were also upregulated by 2.6 to 10.7-folds and E-cadherin and Claudin-1 were downregulated by 2.6 to 10-fold in H358ER cells. RhoA, a small GTPase downstream of p120-catenin and a negative EMT regulator, was also downregulated by 2.9-fold promoting EMT in these cells. Immunofluorescence (IF) studies showed that there was 95% colocalization of p120-catenin and Kaiso factor in H358ER cells whereas 70% colocalization was seen in H358 parental cells. In ER NSCLC cells with wild type EGFR, flow cytometry studies showed increased expression of CSC markers ABCB1, BMI-1 and Oct-4. Knockdown of PRMT1 by Furamidine increased erlotinib efficacy by 16.5%% in H358ER and 21% in H2170ER cells. Similarly, knockout of p120-catenin by CRISPR-Cas9 increased erlotinib efficacy by 19% in H358ER cells. Also, there was a 54.7% increase in erlotinib’s efficiency in p120-catenin knockout H358ER cells treated with Furamidine. Moreover, immunohistochemistry (IHC) studies with p120-catenin and PRMT-1 biomarkers showed that a higher expression of these biomarkers could be related to poor prognosis in NSCLC patients. In conclusion, EMT maybe mediated through biomarkers such as PRMT1, which also upregulates other EMT regulators, and p120-catenin, which represses E-cadherin and Kaiso factor, thus activating EMT in TKI resistant cells.

Citation Format: Sanjana Singh, Nabiha H. Khan, Neelu Puri. Mechanism of tyrosine kinase inhibitors resistance and role of epithelial mesenchymal transition in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1876.