Abstract 1782: Regulation of ATM-dependent DNA damage signaling in human breast cancer cells by the RhoGEF Net1A .

Abstract

Abstract The Neuroepithelioma transforming gene 1 (Net1) is a RhoGEF that is frequently overexpressed in human cancer, including breast cancer. We have previously reported that DNA damage activates Net1 to control p38 MAPK signaling and protect cells from ionizing radiation (IR) induced apoptosis. However, others have shown that Net1 activation contributes to cell death by activating a RhoB-Bim signaling pathway. Thus, the role of Net1 in controlling IR responses and cell survival is controversial. Here we show that the Net1A isoform is uniquely important for DNA double-strand break (DSB)-induced signaling and DNA repair. SiRNA-mediated knockdown of Net1A in human breast cancer cells reduced IR-stimulated ATM activation and signaling to its substrates Chk2 and H2AX. In addition, suppression of Net1A expression adversely affected cell survival after IR. We also observed that Net1A overexpression significantly reduced γH2AX foci formation after IR, which required the unique N-terminal region of Net1A. Importantly, this effect did not require Rho GTPase activation by Net1A, and was not recapitulated by overexpression of RhoA or RhoB. Using comet assays, we found that Net1A depletion substantially slowed DNA DSB repair in response to IR. Intriguingly, knockdown of Net1A promoted non-homologous end joining (NHEJ) in the absence of DNA damage, and Net1A was found to co-immunoprecipitate with the DNA-PK complex in an IR-regulated manner. Taken together, these data suggest a model in which Net1A functions as a non-catalytic binding protein to control DNA damage response signaling and DNA repair to affect cell survival after IR. Citation Format: Wonkyung Oh, Jeffrey A. Frost. Regulation of ATM-dependent DNA damage signaling in human breast cancer cells by the RhoGEF Net1A . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2013-1782