Abstract 4381: EGFR-Src signaling regulates self-renewal of cancer stem like cells from NSCLC through Sox2

Abstract

Abstract NSCLC is the most common form of lung cancer with 5-year survival rate of approximately 10%. Cancer stem cell model has been proposed for tumor-hetrogeniety, carcinogenesis and relapse after therapy. According to this model, cancer stem like cells (CSCs) are defined as self-renewing tumor cells, able to initiate and maintain the tumor. Here, we isolated CSCs based on “side populaton” (SP) phenotype, a functional property of adult stem cells to efflux Hoechst-33352 dye through ABCG2 transporter. Irrespective of genotype of the cells, we observed SP-cells in H358, H292, H1650, H1975, A549 and H460 cell lines. SP phenotype was also displayed by clinical-human xenografts, which could be specifically blocked by ABCG2 inhibitor. Relative tumorigenic potential of SP and MP (main population) cells was determined by subcutaneous or orthotopic xenograft-implantation in SCID mice. As low as 1×103 SP-cells isolated from H1650 cells could form subcutaneous tumor. Similarly, 5×104 SP-cells from A549 could establish orthotopic tumors in lung. Flow-cytometry analysis of subcutaneous tumors demonstrated the asymmetric division of SP-cells generating MP-cells within the tumors. In vitro analysis showed higher expression of ABCG2 and anti-apoptotic protein MCL1 in SP-cells as compared to MP cells. Further, SP-cells displayed higher expression of mesenchymal marker vimentin and lower levels of epithelial marker E-cadherin, suggesting the EMT-like features in SP-cells. SP-cells were found to express embryonic self-renewal factors Oct4, Sox2 and Nanog and demonstrated self-renewal capability by growing as suspended spheres in serum free, stem cell-selective medium. To successfully eliminate these CSCs from tumors, it is necessary to understand the mechanisms facilitating its self-renewal. Based on the favorable clinical outcome of EGFR- and Src-targeted therapy against certain NSCLCs, we hypothesized that the activated EGFR and Src signaling might play important roles in CSCs of NSCLC. This hypothesis was tested using biochemical as well as genetic inhibitor tools against EGFR and Src. SiRNA against EGFR significantly blocked the SP phenotype by downregulating ABCG2 protein levels in all the tested cell lines. Importantly, inhibitor of EGFR (Gefitinib, Erlotinib, BIBW2992) and Src (Dasatinib, PP2) completely blocked the self-renewal capability of SP-cells, as assessed by sphere formation assays. Further, blocking of EGFR and Src signaling by SiRNA or inhibitors resulted in downregulation of the protein levels of Sox2 in SP cells. Importantly, SiRNA against Sox2 significantly blocked the SP phenotype as well as self-renewal capacity of SP-cells. Our findings reveal an important role of EGFR-Src signaling axis in self-renewal and expansion of NSCLC-CSCs by regulating the Sox2 activity in the cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2011-4381