Abstract 2295: Knockdown of ZEB1, a master EMT gene, suppresses anchorage-independent cell growth of lung cancer cells

Abstract

Abstract Background: Epithelial-to-mesenchymal transition (EMT), an essential process during embryonic development and in wound healing, has been shown to be a key event in tumor migration, invasion and metastasis. EMT program is controlled by several master regulators including Twist, ZEB1, SIP1, Snail, Slug, and Goosecoid which are called master EMT genes1),2). ZEB1 protein binds E-boxes within the promoter region of E-cadherin gene, leading to its transcriptional repression. Also, it directly represses many other genes encoding components of the epithelial junctional complex and cell polarity factors. ZEB1 was shown to promote metastasis in colorectal cancer and breast cancer3), and the association between ZEB1 and tumor progression has been studied in several human cancers. Purpose: To evaluate the association between ZEB1 expression and mesenchymal phenotype in lung cancer, and to test the effect of ZEB1 knockdown with RNA interference on the growth of lung cancer cells. Methods: We analyzed the expression of E-cadherin (epithelial marker), Vimentin (mesenchymal marker), and four master EMT genes (Snail, Slug, ZEB1, SIP1) in 19 NSCLC cell lines and 32 NSCLC tumor tissues. Transient knockdown of ZEB1 with RNA interference was done in three NSCLC cell lines with high expression of ZEB1: H157, H1299, and H460. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin and Vimentin were done. Cellular proliferation was measured by WST-1 assay and clonogenic growth was measured by liquid (anchorage-dependent) and soft agar (anchorage-independent) colony formation assays. Apoptosis was evaluated by FACS and western blot of cleaved caspase-3. Results: In cell lines, of four master EMT genes, only ZEB1 expression significantly correlated with both E-cadherin and Vimentin expression. Most EGFR mutant lines showed epithelial phenotype (ratio of Vimentin to E-cadherin (RVE) < 1.0). ZEB1 expression was also inversely correlated with miR-200c and miR-205. In tumor tissues, ZEB1 and Snail expression correlated with the ratio of Vimentin to E-cadherin, and ZEB1 expression highly significantly correlated with Vimentin expression. After ZEB1 knockdown E-cadherin protein was reexpressed in H460 but not in H1299 and H157 cells. ZEB1 knockdown suppressed cell proliferation and liquid colony formation significantly in H1299 and modestly in H157 and H460. Notably, ZEB1 knockdown dramatically suppressed growth of soft agar in the all three cell lines studied. FACS and western blot of cleaved caspase-3 showed apoptosis induction by ZEB1 knockdown in H460, suggesting that growth inhibitory effect of ZEB1 knockdown in lung cancer was caused in part by apoptosis. Conclusions: ZEB1 has the dominant role in maintaining mesencymal phenotype in NSCLC and removal of ZEB1 in lung cancer cell lines induces inhibition of soft agar growth. These results suggest that ZEB1 could be a therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2295.