Abstract 1778: C-MYC plays a novel role in driving the error-prone double-strand break repair in triple negative breast cancers.
Authors: Nagaria Pratik K. , Chowdhury Khadiza , Brodie Angela , Rassool Feyruz 
Published: August 2014

Abstract
Approximately 10-20% of breast cancers lack estrogen, progesterone or HER2 receptors, the so-called triple negative breast cancer cells (TNBCs). These women have a poor prognosis and treatment options are limited. Therefore, the development of novel therapies are urgently required. It is well established that repair of DNA double-strand breaks (DSBs), one of the most lethal forms of DNA damage, is abnormal in breast cancers and can lead to genomic instability. We have recently reported that an alternative and highly error-prone form of NHEJ (ALT NHEJ) is found to be upregulated in TNBCs, as evidenced by increased protein levels of ALT NHEJ components, DNA ligase III (LIG3) and poly ADP ribose polymerase 1 (PARP1) and increased ALT NHEJ activity. Notably, these cells are highly sensitive to a combination of DNA ligase and PARP inhibitors in colony survival assays. This suggests that ALT NHEJ may not only contribute to the genomic instability, but it is also important for survival. The aim of this study is to determine the mechanisms underlying ALT-NHEJ upregulation in TNBCs that will likely identify potential novel targets for therapy. Herein, we demonstrate that c-MYC plays a novel role in driving error-prone repair by ALT NHEJ. A significant decrease in PARP1 and LIG3 is observed with siRNA-mediated knockdown (KD) of c-MYC in several TNBC cell lines (N=4). Furthermore, overexpression of c-MYC in wild-type breast epithelial cells (WT) with c-MYC-pcDNA3 construct leads to &gt;2-fold increase in steady-state levels of PARP1 and LIG3. To determine whether c-MYC expression affects the functional outcome of DSB repair via ALT NHEJ, we performed in vivo and in vitro plasmid-based end-joining assays following knockdown or overexpression of c-MYC. Notably, c-MYC KD led to an increase in fidelity of NHEJ repair in TNBCs. In contrast, c-MYC overexpression leads to an increase in DSB repair errors, characterized by larger DNA deletions. Given the pro-proliferative role of c-MYC we determined whether error-prone ALT NHEJ was S-phase specific. In vivo NHEJ assays on cells released from synchronization at S- and G2/M phases of cell cycle reveal that DSB repair errors in the plasmid are introduced mainly in the S-phase. Putative c-MYC binding sites are present on the promoters of both PARP1 and LIG3. To investigate whether c-MYC is involved in transcriptional regulation of ALT NHEJ proteins, we cloned the PARP1 promoter into a luciferase vector and transfected this construct into WT and TNBCs (N=4). PARP1 promoter activity decreases upon c-MYC knockdown in TNBCs and increases upon c-MYC overexpression in WT cells, suggesting that c-MYC is likely involved in direct regulation of PARP1. Our findings demonstrate a novel role of c-MYC in driving error-prone DSB repair by ALT NHEJ pathway, which could have important implications in genomic instability, disease progression and the development of novel therapies in TNBCs.
Citation Format: Pratik K. Nagaria, Khadiza Chowdhury, Angela Brodie, Feyruz Rassool. C-MYC plays a novel role in driving the error-prone double-strand break repair in triple negative breast cancers. [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 1778. doi:10.1158/1538-7445.AM2013-1778