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

MOIOLA C; DE LUCA P; ZALAZAR F; COTIGNOLA J; GARDNER K; VAZQUEZ E; DE SIERVI A

Orlando, Florida. USA

Congreso; AACR - Advances in Prostate Cancer Research; 2012

AACR

Prostate cancer (PCa) is the second leading cause of cancer-related death affecting men in the world. Chromosomal instability is one cause of PCa progression. Previously, using genome-wide assays for factor binding in combination with expression profiles, we reported BRCA1 binding to several promoters involved in cell cycle and DNA damage response. We also generated a BRCA1 depleted xenograft model allowing us to study BRCA1 role in DNA damage response in vivo. Our studies established that BRCA1 expression status plays a central role in doxorubicin resistance in PCa. One BRCA1 target that emerged from these analyses was ATM (ataxia telangiectasia mutated). ATM is a kinase protein that is a pivotal mediator in genotoxic stress; however, it is unknown if the regulation of ATM transcription plays a role in the DNA damage response. Our goal was to investigate ATM transcription regulation in PCa under different genotoxic insults. In this study, we exposed tumor prostate PC3 cells to different genotoxic agents and ATM promoter activity was determined by luciferase reporter assay. We found that topoisomerase II inhibitors doxorubicin and mithoxantrone repressed ATM transcription; however etoposide and methotrexate did not show significant changes in promoter activity. 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 (BRCA1ÄBRCT) impaired the ability of BRCA1 to regulate ATM promoter activity which strongly suggests that BRCT domain is essential for the ATM transcriptional regulation. Xenograft tumors generated by BRCA1 depleted PC3 cells injection into nu/nu mice demonstrated that BRCA1 depletion abolished ATM transcription induction. Due to it is well known that BRCA1 is phosphorylated by ATM, we investigated BRCA’s ability to activate ATM promoter after inhibition of ATM kinase activity by KU55933. BRCA1 overexpressing PC3 cells exposed to KU55933 showed significantly decreased ATM promoter activity compared to control cells suggesting ATM regulation of transcription 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 exposure. We identified one E2F1 putative DNA binding site at this region suggesting that BRCA1 binding to ATM promoter might be mediated by E2F1. Finally, E2F1 transfection in PC3 cells significantly 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 exposure, 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 transcription regulation.