Abstract 1312: ATM transcriptional regulation mediated by BRCA1/E2F1 axis controls DNA damage response in prostate cancer

Abstract

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death affecting men worldwide. Chromosomal instability is a key feature in PCa progression. Using genome-wide screen for factor binding in combination with expression profiles, we previously reported BRCA1 binding to several promoters involved in cell cycle regulation and DNA damage response. We also generated a BRCA1 depleted xenograft model in order to study the role of BRCA1 role in DNA damage response in vivo. We demonstrated that BRCA1 expression status plays a central role in doxorubicin resistance in PCa. One of the BRCA1 targets that emerged from these analyses was ATM (ataxia telangiectasia mutated).Although it is well accepted that the kinase protein ATM is a pivotal mediator in genotoxic stress, it is unknown if its transcriptional regulation plays a role in the DNA damage response. Our goal was to investigate ATM transcription regulation in PCa under different genotoxic insults. Here we exposed PC3 cells to different genotoxic agents and the ATM promoter activity was determined by a luciferase reporter assay. We found that the topoisomerase II inhibitors doxorubicin and mitoxantrone repressed ATM transcription; however etoposide and methotrexate did not show significant changes. Using BRCA1-overexpressing PC3 cell lines, we found that BRCA1 increases ATM mRNA and promoter activity. Accordingly, BRCA1 depletion by shRNA abolished ATM transcription induction. Furthermore, BRCT domain loss (BRCA1ΔBRCT) impaired the ability of BRCA1 to regulate ATM promoter activity, strongly suggesting that BRCT domain is essential for ATM regulation. Xenograft tumors generated by BRCA1 depleted PC3 cells injected in nu/nu mice demonstrated that BRCA1 knock-down abolished ATM transcriptional induction. Considering ATM phosphorylates BRCA1, we investigated BRCA's ability to activate ATM promoter after inhibition of the ATM kinase activity by KU55933. BRCA1 overexpressing PC3 cells exposed to KU55933 showed significant decreased ATM promoter activity compared to control cells suggesting ATM regulation by BRCA1 is not mediated by ATM kinase activity. In addition, we performed BRCA1-ChIP-qPCR using primers spanning every 500bp along ATM promoter; we found that BRCA1 binds at 500bp upstream of the ATM transcription start site which was disrupted by doxorubicin. We identified one E2F1 putative DNA binding site at this region suggesting that BRCA1 association to ATM promoter could be E2F1 mediated. Finally, E2F1 transfection in PC3 cells significant decreased ATM transcription which was impaired by E2F1 dominant negative (E1-363). In summary, BRCA1/E2F1 complex binds and induces ATM transcription. After genotoxic stress BRCA1 protein is displaced from the ATM promoter and E2F1 downregulates ATM transcription. Thus, BRCA1/E2F1 axis controls DNA damage response in PCa through ATM transcriptional regulation. 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 1312. doi:1538-7445.AM2012-1312