Association of BRCA1 and BRCA2 genes in arsenic-induced urinary bladder carcinoma

Objective: Study was performed to determine content of arsenic in urinary bladder tumour tissue and association of BRCA1 and BRCA2 protein expression with urinary bladder carcinoma development. Materials and methods: This study was performed in a tertiary care hospital of Eastern India. Post-operative tumour tissue was analysed for arsenic content as well as BRCA1 and BRCA2 expression. Statistical analysis was done and association between stage, grade and BRCA1 and BRCA2 expression with arsenic level in tumour tissue was done. Results: Total 50 patients were included in study. Out of which 26 were arsenic positive as well as 24 were arsenic negative. Maximum patients in arsenic positive group were from arsenic endemic zones of West Bengal, India. There was significant correlation between higher stage and grade of tumour and arsenic positivity. BRCA1 correlation was significant with arsenic positive group whereas BRCA2 correlation was not significant with arsenic positive group. Conclusion: Bladder carcinomas are more common in arsenic endemic zones of our country. This association can help in future to develop drugs which act on selected mutation of genes such as BRCA1, especially in arsenic-associated bladder cancers.

Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

BRCA2 is a tumor suppressor gene that maintains genome stability by mediating the high fidelity repair of DNA double-strand breaks (DSBs) through homology-directed repair (HDR). Pathogenic mutations in BRCA2 predispose to breast, ovarian, pancreatic, prostate, and other cancers. Mutations in BRCA2 leading to severe protein truncation predict pathogenicity, however, missense mutations with unknown functional consequences, designated Variants of Uncertain Significance (VUS), comprise 60% of BRCA2 sequence changes deposited in clinical databases. Classifying BRCA2 VUS correctly is critical for relaying clinically actionable information to patients concerning future cancer risk or current treatment options. In this study, we identified and biochemically characterized three BRCA2 VUS located in BRC repeats to determine the impact on canonical HDR functions. Two of the germline variants, S1221P and T1980I, map to conserved residues in BRC2 and BRC7, disrupt RAD51 binding, and are diminished in their ability to stabilize RAD51-ssDNA complexes. We provide supporting cellular evidence that S1221P and T1980I are significantly compromised in their response to chemotherapeutics and ionizing radiation. The third variant, T1346I, lies within the spacer region between BRC2 and BRC3 but remains fully functional. We conclude that T1346I has a neutral impact on BRCA2 function, while S1221P and T1980I are hypomorphic alleles that disrupt the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments.

BRCA2 C-terminal RAD51-binding Domain Confers Resistance to DNA-damaging Agents

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand cross-linking agents or X-ray irradiation. These treatments induce double-stranded breaks in genomic DNA. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51-oligomer containing nucleofilaments. RAD51 nucleofilaments are essential for the homologous recombination repair of double-stranded DNA damage. In this study, we investigated the effects of expressing the CTRBD in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, expressing the BRCA2 CTRBD in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. To determine the ability of the BRCA2 CTRBD to mediate DNA damage repair, the endogenous BRCA2 was depleted by shRNA. No significant differences were observed between the sensitivities of the BRCA2-depleted cells with or without expressing BRCA2 CTRBD. Thus, the resistance to X-ray irradiation conferred by the exogenous CTRBD required endogenous BRCA2 expression. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment. This peptide may be useful in the protection of cells against X-ray irradiation or chemotherapeutic agents.

Whole-exome sequencing in eccrine porocarcinoma indicates promising therapeutic strategies

Malignant sweat gland tumours are rare, with the most common form being Eccrine porocarcinoma (EP). To investigate the mutational landscape of EP, we performed whole-exome sequencing (WES) on 14 formalin-fixed paraffin-embedded samples of matched primary EP and healthy surrounding tissue. Mutational profiling revealed a high overall median mutation rate. This was attributed to signatures of mutational processes related to ultraviolet (UV) exposure, APOBEC enzyme dysregulation, and defective homologous double-strand break repair. All of these processes cause genomic instability and are implicated in carcinogenesis. Recurrent driving somatic alterations were detected in the EP candidate drivers TP53, FAT2, CACNA1S, and KMT2D. The analyses also identified copy number alterations and recurrent gains and losses in several chromosomal regions including that containing BRCA2, as well as deleterious alterations in multiple HRR components. In accordance with this reduced or even a complete loss of BRCA2 protein expression was detected in 50% of the investigated EP tumours. Our results implicate crucial oncogenic driver pathways and suggest that defective homologous double-strand break repair and the p53 pathway are involved in EP aetiology. Targeting of the p53 axis and PARP inhibition, and/or immunotherapy may represent promising treatment strategies.

Evaluation of BRCA1 and BRCA2 Protein Dysfunction in Breast Cancer Cells among Women in Osun State, Southwest Nigeria

Introduction/Objective Breast cancer among Nigeria women had been found to occur at a much younger age compared with their Caucasian age groups. BRCA1 and BRCA2 were suspected to responsible for breast cancers at a young age, therefore this work examined the BRCA1 and BRCA2 dysfunction among women suffering from breast cancer in Osun State, Nigeria. Methods This cross-sectional study was carried out at the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife and Ladoke Akintola University Teaching Hospital, Osogbo, Nigeria. The request cards and tissue blocks were sorted from the year 2014 to 2017. The breast tissue blocks were sectioned, stained with H&E. A representative tissue block was selected for each patient. Sections obtained from the blocks were stained with BRCA 1 and BRCA 2 antibody using a diaminobenzidine horseradish peroxidase technique. The cells were semi-quantitatively scored as percentage of tumour cells stained brown. Score 0-5% were taken as negative as proposed by Karuna et al., 2013. The value obtained was tabulated against the age of the patient. Results Out of 240 breast cancer patients sampled 16(6.7%), 32(13.3%), 85(35.4%), 43(17.9%), 64(26.7%) patients were between ages 21-30, 31-40, 41-50, 51-60 and 60+ years respectively. 54(22.5%) showed loss of BRCA 1 staining with only 1(0.4%) patient between age 21-40 years while 18(7.5%), 16(6.67), 19(7.9) patients between age 41-50, 51-60 and 60+ years respectively. 82(34.2%) showed loss of BRCA 2 staining; 7(2.9%), 12(5%), 31(12.9%), 15(6.25%),17(5.8%) patients for age between 21-30, 31-40, 41-50, 51-60 and 60+ years respectively. Among women aged 50 years and below, BRCA 1 and BRCA 2 dysfunctions were responsible for 14.3% and 37.6% of breast cancers respectively. Conclusion Increase in age increases the rate of BRCA 1 dysfunction while BRCA 2 dysfunction is not more associated to with any age. BRCA1 and BRCA2 are responsible for more than thirty percent of breast cancers among women less than 50 years of age in Osun state.

Checkpoint kinase inhibition in prostate cancer cells resistant to poly ADP-ribose polymerase inhibitors.

150 Background: Poly ADP-ribose polymerase inhibitors (PARPi) have shown promise in the treatment of metastatic castration-resistant prostate cancer patients with DNA damage response defects . The phase 3 PROfound trial showed olaparib delayed the time to radiographic progression or death as compared with abiraterone or enzalutamide. In addition to olaparib, three other PARPi are in Phase 3 trials in prostate cancer (PC): rucaparib, talazoparib, and niraparib. Despite responses, resistance is common and treatment options for PARPi-resistant patients are limited. In this study, we observed de novo activation of checkpoint kinases (CHEK) in talazoparib-resistant (TR) PC cells. Therefore, we hypothesized that targeting CHEK may mitigate resistance to PARPi in PC. Methods: We developed TR human prostate cancer PC3 (low BRCA2 protein due to heterozygous deletion of BRCA2) cells. We performed phosphoproteomic analysis to identify possible mechanisms of talazoparib resistance in PC3 cells and validated the results with Western blot. Results: TR-PC3 cells proliferated slower and had a significant increase in the phosphorylation of CHEK2 compared to parental (p) PC3. Treatment with a CHEK2-selective inhibitor, CCT241533, did not affect cell growth in TR-PC3 cells. Conversely, treatment with a CHEK 1/2 inhibitor, prexasertib, led to significant cell growth inhibition in TR-PC3 at a much lower IG 50% concentration compared to pPC3. RNAi-mediated knockdown validated the superior efficacy of combined CHEK1 and CHEK2 inhibition since this combination produced the greatest cell growth inhibition seen in both TR-PC3 and de novo PARPi-resistant p22RV1. Treatment of pPC-3 and p22RV1 with combinations of talazoparib and prexasertib showed greater cell growth inhibition compared to either drug alone. Conclusions: Resistance to PARPi in PC cells with deletion of BRCA2 may potentially be overcome with CHEK inhibition. Moreover, our preliminary data suggested that the effect of PARPi and CHEK inhibitors on PARPi/CHEK inhibitor-naïve PC cells was greatest when used together, indicating that patients with PC may experience greatest anti-tumor activity of the two drugs when they are used in combination.

The BRCA2 p.N372 H i.a.1342A>C Could Regulate the Sensitivity of Ovarian Cancer Cells to Platinum-Based Drugs

Background and Objective: We have previously reported that BRCA2 N372 H i.a.1342A>C heterozygous variation presented in platinum-resistant patients. This study aimed to further investigate the mechanism of BRCA2 N372 H mutation in the development of platinum resistance in ovarian cancer. Methods: The BRCA2 N372 H i.a.1342A>C was synthesized and used to exchange 1 wildtype allele followed by sequencing to confirm the mutant allele sequence. Plasmids were constructed and transfected into the OVCAR-3 cells after lentiviral packaging. BRCA2 N372 H mRNA was detected by qPCR. BRCA2 protein was assessed by immunoblotting. Binding of the BRCA2 to Rad51 was detected by immunofluorescence staining. Sensitivity of the cells to cisplatin treatment was assessed with CCK-8 assay. Results: It was found that expression of BRCA2 protein in ovarian cancer cells transfected with BRCA2 N372 H i.a.1342A>C gene (2.177 ± 0.003) was significantly increased compared to that of the cells transfected with lenti-EGFP only (1.227 ± 0.003, P < 0.001). Binding of the BRCA2 and Rad51 proteins was significantly increased in the cells with BRCA2 N372 H i.a.1342A>C mutation (3.542 ± 0.24) than that in the cells transfected with lenti-EGFP (1.29 ± 0.32) or empty cells (1.363 ± 0.32, P < 0.001). Cell viability significantly increased in the cells transfected with BRCA2 N372 H mutant gene. The IC50 value was significantly higher in the cells transfected with BRCA2 N372 H mutant gene (1.963 ± 0.04) than that of the cells transfected with lenti-EGFP (0.955 ± 0.03, P < 0.01) or empty cells (1.043 ± 0.007, P < 0.01). Conclusion: Over expression of mRNA and protein of BRCA2 was detected in the cells with BRCA2 N372 H i.a.1342A>C mutation but not in the lentivirus negative control (lenti-EGFP) or the cells without transfection (empty cells), which may lead to resistance to platinum-based drugs in ovarian cancer cells through homologous recombination repair pathway.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

The Drosophila melanogaster PIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair

PIF1 is a 5’ to 3’ DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair.