Abstract 2490: Silencing of TCF7L2 sensitizes Wnt/β-catenin signaling-dependent colorectal cancer cells to radiation

Abstract

Abstract Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We recently profiled a series of responsive and resistant rectal carcinomas using gene expression microarrays and identified TCF7L2, the main downstream effector of the Wnt/β-catenin pathway, as over-expressed in the resistant tumors. The aim of this study was to explore the relevance of TCF7L2 for resistance to radiation and to study the underlining mechanisms. Methods: We transfected three microsatellite-stable colorectal cancer cell lines (SW837, SW480 and HT-29) with two different shRNA-vectors targeting TCF7L2 and a non-silencing. Next, we established single cell clone (SCC) populations, and after confirming the reduction of TCF7L2 protein levels by Western blotting, we irradiated the SCCs at 0, 1, 2, 4, 6 and 8 Gy and calculated survival fractions. To gain functional insight, we chose SW837 and HT-29 for further experiments. Using γH2AX staining we evaluated DNA double strand repair after irradiation at 2 Gy and analyzed cell cycle changes before and after radiation. Furthermore, we studied Wnt/β-catenin pathway activity using the TOPFLASH/FOPFLASH reporter assay. Results: RNAi-mediated silencing of TCF7L2 led to a pronounced reduction in TCF7L2 protein levels. In radiation experiments we observed a highly significant radiosensitization in SW837 and SW480 SCCs, whereas no effect was observed in HT-29 SCCs. Well-fitting, we observed significantly more γH2AX foci 24h after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to impaired DNA damage repair in the SCCs from SW837. Furthermore, in SW837 SCCs but not in HT-29 SCCs we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation and compromised cell cycle control after radiation. Finally, using the TOPFLASH/FOPFLASH reporter assay we found SW837 wild-type cells to show high Wnt/β-catenin pathway activity, while HT-29 wild-type cells only showed very low activity. Conclusion: TCF7L2 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization, mediated by DNA damage repair deficiencies and impaired cell cycle control. Interestingly, radiosensitization effetcts were only observed in cell lines showing Wnt/β-catenin pathway activity. Thus, we have uncovered a novel role of Wnt/β-catenin signaling for mediating resistance to multimodal cancer therapy. Moreover, these data suggest that TCF7L2 is a potential molecular target for sensitizing Wnt/β-catenin-dependent tumor cells to radiation. 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 2490. doi:10.1158/1538-7445.AM2011-2490