Novel Regulatory Role of BRCA2 in Endothelial Cell Function and Survival Following Genotoxic Stress
Krishna SINGH, St. Michael's Hospital, University of Toronto, Canada,
AL-OMRAN M. 2,4
, VERMA S. 2,3,4
, LEONG-POI H. 1,4
, SINGH K. 2,3,4
, MATKAR P. 1,4
1 Department of Cardiology, St. Michael's Hospital
2 Department of Vascular Surgery, St. Michael's Hospital
3 Department of Cardiac Surgery, St. Michael's Hospital
4 Keenan Research Centre for Biomedical Science at St. Michaelís Hospital, Toronto, Ontario
Background: Germ-line mutations in the tumor suppressor gene BRCA2 (breast cancer 2, early onset) predispose carriers not only to breast cancer, but also to other cancers. BRCA2 plays crucial role in the genome integrity maintenance and is central to DNA-damage repair. BRCA2-associated DNA-damage responses are not only specific to cancer syndromes but also represent a pathophysiological basis for diverse cardiovascular diseases (CVDs). We hypothesize that BRCA2 is an essential regulator of endothelial function and apoptosis following genotoxic stress.
Methods: BRCA2 was silenced using siRNA and DharmaFECT-4 in human umbilical vein endothelial cells (ECs). Key indices of EC function; tube formation and proliferation, DNA-damage/repair and apoptosis were measured by MatrigelTM assay, MTT assay, qPCR, immunoblotting and immunofluorescence following doxorubicin (DOX) treatment.
Results: Basal BRCA2 expression and its successful silencing in ECs were confirmed at transcript and protein levels by qPCR and immunoblotting, respectively. Genotoxic stress in the form of DOX exacerbated DNA-damage in BRCA2-silenced ECs, evident by increased expression and activation of DNA double-stranded breaks (DSBs) marker H2A.X and reduced RAD51-foci formation, an essential regulator of DSB repair. Increased DSBs were associated with significantly increased expression and activation of p53, and p53-upregulated modulator of apoptosis PUMA. Elevated levels of DNA-damage and p53 were further associated with significantly increased DOX-induced apoptosis in BRCA2-silenced ECs as measured by immunoblotting for cleaved-caspase-3 and TUNEL-staining. Key indices of endothelial function, including tube formation and proliferation, were significantly reduced following DOX-treatment in BRCA2-deficient ECs, which was accompanied with significantly increased expression of cell cycle inhibitor, p21 at transcript and protein levels and decreased nitric oxide levels.
Conclusion: Our data for the first time, demonstrate an entirely novel role of BRCA2 as a regulator of endothelium function, and provide important clues regarding a potential susceptibility of BRCA2 mutation carriers to anthracycline-induced CVDs, a cornerstone of cancer chemotherapy.