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1.
Nucleic Acids Res ; 52(12): 6964-6976, 2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38142462

ABSTRACT

BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.


Subject(s)
BRCA2 Protein , DNA, Single-Stranded , Protein Binding , Humans , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Animals , Mice , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , Chromosomal Instability , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cisplatin/pharmacology , DNA Damage , Mutation, Missense , Female , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Mouse Embryonic Stem Cells/metabolism , Cell Line, Tumor , Mitomycin/pharmacology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Proteasome Endopeptidase Complex
2.
Cell Death Dis ; 14(11): 753, 2023 11 18.
Article in English | MEDLINE | ID: mdl-37980415

ABSTRACT

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.


Subject(s)
Antineoplastic Agents , Breast Neoplasms , Ovarian Neoplasms , Humans , Female , Mice , Animals , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Genetic Testing , Ovarian Neoplasms/genetics , Homozygote , Breast Neoplasms/genetics , BRCA1 Protein/genetics , Genetic Predisposition to Disease
3.
Sci Rep ; 9(1): 3816, 2019 03 07.
Article in English | MEDLINE | ID: mdl-30846724

ABSTRACT

Soft-tissue sarcomas (STS) are an uncommon and heterogeneous group of malignancies that result in high mortality. Metastatic STS have very bad prognosis due to the lack of effective treatments. Dinaciclib is a model drug for the family of CDK inhibitors. Its main targets are cell cycle regulator CDK1 and protein synthesis controller CDK9. We present data supporting Dinaciclib ability to inactivate in vitro different STS models at nanomolar concentrations. Moreover, the different rhythms of cell death induction allow us to further study into the mechanism of action of the drug. Cell death was found to respond to the mitochondrial pathway of apoptosis. Anti-apoptotic Bcl-xL was identified as the key regulator of this process. Already natural low levels of pro-apoptotic proteins BIM and PUMA in tolerant cell lines were insufficient to inhibit Bcl-xL as this anti-apoptotic protein showed a slow decay curve after Dinaciclib-induced protein synthesis disruption. Combination of Dinaciclib with BH3-mimetics led to quick and massive apoptosis induction in vitro, but in vivo assessment was prevented due to liver toxicity. Additionally, Bcl-xL inhibitor A-1331852 also synergized with conventional chemotherapy drugs as Gemcitabine. Thus, Bcl-xL targeted therapy arises as a major opportunity to the treatment of STS.


Subject(s)
Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Death/drug effects , Pyridinium Compounds/pharmacology , Sarcoma/pathology , bcl-X Protein/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cyclic N-Oxides , Humans , Indolizines , Mice
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