Overexpression Of Rad51 Promotes Brain Metastases From Breast Cancer

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The DNA double-strand break (DSB) repair gene RAD51 was found to be overexpressed (1.5-fold, p=0.001) at the mRNA level in resected brain metastases compared to primary breast tumors from the same patients. RAD51 was also overexpressed (1.4-fold, p=0.008) specifically in brain metastases from breast cancer compared to systemic (bone/lung) metastases. We therefore hypothesize that RAD51 promotes brain metastasis development in breast cancer patients and investigated RAD51 as functional driver of brain metastases, independent of its recognized role in resistance to DNA-damaging chemotherapy. RAD51 was overexpressed at physiologically relevant levels in brain-metastatic variants of human MDA-MB-231 breast carcinoma cells (231-BR) and 4T1 murine mammary carcinoma cells (4T1-BR). In vitro, RAD51 overexpression results in a 35-40% (p<0.0001) reduction Gamma-H2AX foci during S-phase, a DNA DSB marker. Furthermore, using a Homologous Recombination (HR) assay we found that the frequency of HR was significantly increased (1.7-fold, p=0.002) in RAD51 overexpressers compared to vector controls. No effects on proliferation, motility or invasion were observed with RAD51 overexpression. In intracardiac-injected mice, overexpression of RAD51 increased formation of large brain metastases, the equivalent of clinically detectable lesions, (3.3-fold, p=0.005; 3.3-fold, p=0.02) and micro-metastases (3.1-fold, p=0.01; 2.0-fold, p=0.04), compared to vector controls in 231-BR and 4T1-BR models, respectively. ShRNA-mediated RAD51 knockdown in 4T1-BR cells had the opposite effect, reducing large brain metastases (2.5-fold, p=0.001) and micro-metastases (2.5-fold, p=0.01). RAD51 modulation had no effect on lung metastasis development in tail-vein injected mouse models of 231-BR or 4T1-BR cells. To distinguish between roles for RAD51 in initiation vs. outgrowth of brain metastasis, we quantified iron-labeled 231-BR cells in the brain microenvironment at 3, 7 and 12 days post-injection. On day 12, we observed significantly more RAD51-overexpressing single cells compared to vector controls, with a clear trend discernable by day 7 (fold-changes over vector: 1.1-fold, p=0.8; 2.4-fold, p=0.07 and 2.8-fold, p=0.001 for days 3, 7 and 12, respectively). Together these data suggest that RAD51 drives initiation of brain metastases from breast cancer. Citation Format: Stephan Woditschka, Diane Palmieri, Renata Duchnowska, Jacek Jassem, Sunil Badve, George W. Sledge, Patricia S. Steeg. Overexpression of RAD51 promotes brain metastases from breast cancer. [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 4589. doi:10.1158/1538-7445.AM2013-4589