Defective Stalled Replication Fork Repair And Predisposition To Hereditary Breast Cancer

BRCA1 is a tumor suppressor gene, and germ line BRCA1 mutations increase the risk of breast cancer. While all cells with BRCA1 mutations exhibit a heterozygous BRCA1mut/+ genotype, cancer develops primarily in females, often at young ages and affects almost exclusively the breast and ovaries. Why BRCA1 shows such tissue specificity, and how a normal cell in a BRCA1 mutation carrier (BRCA1mut/+) gives rise to invasive tumor cells are largely unknown. To determine whether BRCA1 heterozygosity in cells confers defect in any of the multiple, known, BRCA1 functions is a potentially valuable step in achieving a better understanding of BRCA1 mutation-driven cancer predisposition. Thus, we have analyzed a collection of primary mammary BRCA1mut/+ epithelial cells and skin fibroblasts obtained from BRCA1 mutation carriers for such functions. We, and others have recently shown that BRCA1 exhibits a new DNA damage repair function – i.e. repair of stalled replication forks (SFR). Stalled forks, when not resolved, lead to mutations, or collapse into double strand breaks (DSBs). Both outcomes result in what is commonly referred to as replication stress (RS), which, when chronic, is a driving force behind cancer development. To determine if SFR is defective in normal/healthy breast cells in BRCA1 mutation carriers, and whether this haploinsufficiency results in the kind of genomic changes that lead to cancer, we have now generated 18 primary fibroblast strains from skin punch biopsies and 10 primary mammary epithelial cell (MECs) strains from prophylactic mastectomies performed on BRCA1 mutation carrying women. This collection includes N=23 different BRCA1 mutations, which, together, span almost the entire BRCA1 gene. BRCA1+/+ control MECs were derived from tissue collected during reduction mammoplasties and control fibroblasts were derived from skin punch biopsies from women with no BRCA1 mutation. Our current data shows that BRCA1mut/+ strains exhibited multiple, normal BRCA1 functions, including the support of homologous recombination- type double strand break repair (HR-DSBR), cell cycle- associated checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. By contrast, nearly all strains were defective in the repair of stalled replication forks and in the suppression of fork collapse, i.e. replication stress. These defects were rescued by reconstituting BRCA1 heterozygous cells with wild-type BRCA1 cDNA, indicating that they are a product of BRCA1 haploinsufficiency. In addition, the development of sufficient replication stalling rendered BRCA1mut/+ cells defective in an otherwise intact BRCA1 function, HR-DSBR. No such ‘conditional’ haploinsufficiency was detected in any of the other non-haploinsufficient functions, noted above. Given the importance of replication stress in cancer development and of an HR defect in breast cancer pathogenesis, these defects, when they develop serially, could contribute to the BRCA1 breast cancer development pathway. Finally, given the important role of BRCA2, another hereditary breast cancer gene, in stalled fork stability, a similar analysis for BRCA2mut/+ cells from BRCA2 mutation carriers is currently underway and will also be reported at the meeting. Citation Format: Shailja Pathania, Sangeeta Bade, Morwenna Le Guillou, Karly Burke, Ying Su, David T Ting, Kornelia Polyak, Andrea L Richardson, Jean Feunteun, Judy E Garber, David M Livingston. Defective stalled replication fork repair and predisposition to hereditary breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S5-05.