BRCA1 haploinsufficiency for replication stress suppression in primary cells

Abstract

BRCA1—a breast and ovarian cancer suppressor gene—promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1+/+ and BRCA1mut/+ mammary epithelial cells and fibroblasts. Here we report that all BRCA1mut/+ cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1mut/+ cells with wt BRCA1. In addition, we observed ‘conditional’ haploinsufficiency for HR-DSBR in BRCA1mut/+ cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue.

Deciphering the BRCA1 Tumor Suppressor Network

“…Conversely, other BRCA1 functions in DSB repair and checkpoint activation are proficient in BRCA1 mut/ϩ cells (60). This observation supports the hypothesis that BRCA haploinsufficiency in resolving replication stress might contribute to high risk of cancer in mutation carriers.…”

“…Consistent with replication stress being the limiting aspect of BRCA1 genome integrity control, primary mammary epithelial cells from patients with heterozygous BRCA1 or PALB2 status experience higher replication stress compared with cells with two wild-type copies of either gene (59,60). Conversely, other BRCA1 functions in DSB repair and checkpoint activation are proficient in BRCA1 mut/ϩ cells (60).…”

Defects in homologous recombination repair behind the human diseases: FA and HBOC

“…In the long run, accumulated knowledge about segregation of the genotype and an individual's cancer susceptibility within families may eventually clarify the significance of VUS. At the moment, careful evaluation of DNA repair capacity in lymphocytes from cases with VUS might be useful (Pathania et al 2014, Vaclová et al 2015. It would be particularly useful to construct a collection of isogenic knock-in cells with candidate variants using the CRISPR-CAS9 system (Paquet et al 2016).…”

“…Thus, in addition to the loss of all BRCA function, BRCA haploinsufficiency may also promote carcinogenesis, and some of the cancers arising in the HBOC patients may not be HR deficient. Indeed, BRCA1 +/− mutated cells are defective in response to replication stress (Pathania et al 2014). As studies on mice showed (Ludwig et al 1997, Jonkers et al 2001, in carcinogenic steps, the cells tend to lose Tp53 (or an equivalent checkpoint gene) before loss of BRCA/HR function to avoid cell death and/or senescence.…”

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…8,9 Together with the central Fanconi anemia protein FANCD2, BRCA1, BRCA2 and Rad51 protect hydroxyurea-stalled replication forks from processing by the MRE11 nuclease. [23][24][25] BRCA1 also promotes the processing of replication forks stalled by UV irradiation. 26 We found that BRCA1, BRCA2 and Rad51 are required for efficient STGC at Tus/Ter stalled replication forks, whereas loss of BRCA1 or BRCA2 led to a paradoxical increase in aberrant "long tract" gene conversions (LTGCs) at Tus/Ter stalled forks.…”