Abstract 3448: Association of reactive oxygen species (ROS) levels and radiosensitivity by knockdown of ZEB1 in NSCLC

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The transcriptional repressor zinc-finger E-box binding homeobox 1 protein (ZEB1) governs epithelial-to-mesenchymal transition (EMT) in various human tumors by suppressing the expression of epithelial markers such as E-cadherin and upregulating the expression of Vimentin, thereby promoting invasion and metastasis of tumor cells such as colon and breast cancer. ZEB1 is associated with resistance to conventional chemotherapy in pancreatic cancer and potentially has a predominant role in inducing EMT in NSCLC. However, very little is known about its functional role in the radio-response of NSCLC. We have selected three NSCLC cell lines with high ZEB1 expression (H460, H1299, and Calu-6) and established three matched pair of stable cell lines with and without Zeb1 knockdown (H460/shZeb1 and H460shCV, H1299/shZeb1 and H1299/shCV, Calu6/shZeb1 and Calu6/shCV) using shRNA. The expression of Zeb1, E-cadherin, and vimentin were assessed by western blot analysis, morphological changes associated with EMT phenotype were assessed, and the Zeb1 knocked down cells were tested for radiosensitivity by clonogenic cell survival. Intracellular level of reactive oxygen species (ROS) and subG1-G0 were measured by FACS. The results showed that clear suppression of ZEB1 protein was obtained and reexpression of E-cadherin protein was evident in three stable cell lines with Zeb1 knockdown. Vimentin protein expression were reduced in three stable cell lines with Zeb1 knockdown compared with their non-targeting vector control cell lines respectively. By phase contrast microscopy, Zeb1 knockdown stable cell lines illustrated loss of mesenchymal morphology and restitution of epithelial phenotype. The Zeb1 knocked down cells were substantially radiosensitized compared to the non-targeting vector control cells: the surviving fraction at 2 Gy (SF2) values was reduced from 0.65+/−0.03 in H460/shCV to 0.42+/−0.06 in H460shZeb1 (p=0.02), the SF2 was reduced from 0.70+/−0.03 in Calu6/shCV to 0.52+/−0.02 in Calu6/shZeb1 (p=0.05), and the SF4 was reduced from 0.50+/−0.02 in H1299shCV to 0.32 +/−0.04 in H1299/shZeb1 (p=0.04). H460/shZeb1 and H1299/shZeb1 displayed higher ROS level than H460/shCV and H1299shCV respectively (P<0.05). More importantly, the ROS level of H460/shZeb1 and H1299/shZeb1 at 24 hour after radiation 6Gy were significantly higher than of H460/shCV and H1299shCV after radiation respectively (2.8 fold in H460/shZeb1 to H460/shCV and 2 fold in H1299/shZeb1 to H1299shCV). Moreover, the percentage of subG1-G0 in H460/shZeb1 after radiation (30.9%) was significantly elevated compared to H460/shCV after radiation (11.8%). In conclusion, we have shown that knockdown of ZEB1 is correlated with restoration of the epithelial phenotype and radiation sensitivity in NSCLC through enhanced level of ROS and apoptosis. These results suggest that ZEB1 is a promising therapeutic target for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3448. doi:1538-7445.AM2012-3448