BRCA1 RING Domain Research Articles

Exploring the impact of BRCA1 and BRCA2 mutation type and location on Olaparib maintenance therapy in platinum-sensitive relapsed ovarian Cancer patients: A single center report

ObjectiveIn patients with platinum-sensitive relapsed ovarian cancer (PSROC) harboring pathogenic/likely pathogenic variants (PV) in BRCA1 and BRCA2 genes, olaparib maintenance monotherapy (OMT) is a viable option. Our study aimed to evaluate the impact of different BRCA1/2 PV in survival outcomes and safety of OMT in BRCA1/2-mutated PSROC patients, focusing on the type and location of PV. MethodsWe assessed the outcomes of 100 BRCA1/2-mutated PSROC patients treated at our institute, analyzing progression-free survival (PFS) and overall survival (OS). Germline and tumor BRCA1/2 genotyping was conducted using Illumina's next-generation sequencing (NGS). ResultsPFS and OS were significantly shorter in PSROC patients with PV in BRCA1 compared to those with PV in BRCA2 (PFS:14.0 vs. 38.8 months, p = 0.007, OS: 21.8 vs. 62.0 months, p = 0.011). Notably, there was a significant difference in PFS based on the intragenic location of BRCA1 PV, with shorter PFS in patients with 1st/2nd relapse, harboring PV in BRCA1 RING domain compared to those with PV in the DNA binding domain (DBD) and BRCT domains (12.4 vs. 23.0 months, p = 0.046). No differences in PFS and OS were observed between patients with germline versus somatic BRCA1/2 PV (PFS:14.9 vs.19.3, p = 0.316, OS: not reached vs. 25.8 months; p = 0.224). However, there were significant differences in the reasons for OMT discontinuation between patients with germline and somatic BRCA1/2 PV, primarily due to adverse side effects. ConclusionsIn summary, the type and location of BRCA1 and BRCA2 PV provide additional insight into the expected survival outcomes of olaparib MT in PSROC patients.Trial registration number: ISRCTN42408038, Name of registry: ISRCTN registry, Date of registration: 24/11/2015.

Structural determinants of pathogenesis in the ring domain of BRCA1

Structural determinants of pathogenesis in the ring domain of BRCA1

Simulation and Computational Study of RING Domain Mutants of BRCA1 and Ube2k in AD/PD Pathophysiology.

Lysine-based post-translational modification (PTM) such as acylation, acetylation, deamination, methylation, SUMOylation, and ubiquitination has proven to be a major regulator of gene expression, chromatin structure, protein stability, protein-protein interaction, protein degradation, and cellular localization. However, besides all the PTMs, ubiquitination stands as the second most common PTM after phosphorylation that is involved in the etiology of neurodegenerative diseases (NDDs) namely, Alzheimer's disease (AD) and Parkinson's disease (PD). NDDs are characterized by the accumulation of misfolded protein aggregates in the brain that lead to disease-related gene mutation and irregular protein homeostasis. The ubiquitin-proteasome system (UPS) is in charge of degrading these misfolded proteins, which involve an interplay of E1, E2, E3, and deubiquitinase enzymes. Impaired UPS has been commonly observed in NDDs and E3 ligases are the key members of the UPS, thus, dysfunction of the same can accelerate the neurodegeneration process. Therefore, the aim of this study is firstly, to find E3 ligases that are common in both AD and PD through data mining. Secondly, to study the impact of mutation on its structure and function. The study deciphered 74 E3 ligases that were common in both AD and PD. Later, 10 hub genes were calculated of which protein-protein interaction, pathway enrichment, lysine site prediction, domain, and motif analysis were performed. The results predicted BRCA1, PML, and TRIM33 as the top three putative lysine-modified E3 ligases involved in AD and PD pathogenesis. However, based on structural characterization, BRCA1 was taken further to study RING domain mutation that inferred K32Y, K32L, K32C, K45V, K45Y, and K45G as potential mutants that alter the structural and functional ability of BRCA1 to interact with Ube2k, E2-conjugating enzyme. The most probable mutant observed after molecular dynamics simulation of 50 ns is K32L. Therefore, our study concludes BRCA1, a potential E3 ligase common in AD and PD, and RING domain mutation at sites K32 and K45 possibly disturbs its interaction with its E2, Ube2k.

Efficacy of maintenance olaparib plus bevacizumab in patients with newly diagnosed advanced ovarian cancer according to BRCA mutation genotype in the phase III PAOLA-1/ENGOT-ov25 trial.

5571 Background: In the Phase III PAOLA-1/ENGOT-ov25 trial, the addition of maintenance olaparib (ola) to bevacizumab (bev) in patients (pts) with newly diagnosed advanced high-grade ovarian cancer (HGOC) resulted in prolonged progression-free survival (PFS), particularly for pts with HRD-positive tumors including a BRCA1/ BRCA2 mutation (BRCAm; Ray-Coquard et al. NEJM 2019). Preclinical data suggest that pts with mutations (mut) in the RING domain of BRCA1 are less sensitive to ola. The magnitude of benefit from ola + bev, according to the location of mut in the functional domains (FD) of BRCA , remains to be explored. Methods: Pts with newly diagnosed advanced HGOC in response after platinum-based chemotherapy + bev received maintenance bev (15 mg/kg q3w for 15 months [mo]) + either ola (300 mg bid for 24 mo) or placebo (pbo). In this post hoc exploratory analysis, PFS was analyzed in pts with BRCAm according to mut location in the FDs of BRCA1 (RING, DNA-binding domain [DNA-BD], or BRCA1 C terminus [BRCT]) and BRCA2 (RAD51-BD; DNA-BD). Results: Among the 806 randomized pts, 235 (29.2%) harbored a BRCAm: 160 (19.9%) with a BRCA1 mut and 76 (9.4%) with a BRCA2 mut. BRCA1 mut in FDs of RING, DNA-BD and BRCT were detected in 19 (11.8%), 41 (25.6%) and 34 (21.2%) pts, respectively. BRCA2 mut were detected in FDs of RAD51-BD and DNA-BD in 37 (48.7%) and 14 (18.4%) pts, respectively. With a median follow-up of 25.5 mo, 24-mo PFS rates and hazard ratios (HRs) according to mut locations are reported in the Table. In pts with a BRCA1 DNA-BD mut, 24-mo PFS was 89% and 14% (ola + bev vs pbo + bev; HR 0.08, 95% confidence interval [CI] 0.02–0.26) compared with 64% and 24% for pts with mut in RING + BRCT + other domains (HR 0.33, 95% CI 0.19–0.57). In pts with BRCA2 mut, 24-mo PFS for pts with mut in the DNA-BD was 91% vs 100% (ola + bev vs pbo + bev) compared with 82% and 44% for pts with mut in RAD51-BD + other domains (HR 0.21, 95% CI 0.08–0.54). Conclusions: In this exploratory analysis, pts with newly diagnosed advanced HGOC and a BRCAm had a PFS benefit from maintenance ola + bev regardless of mut locations in BRCA1/BRCA2. Sensitivity to ola + bev maintenance was particularly high for pts with mut in the DNA-BD of BRCA1. Pts with a mut in the DNA-BD of BRCA2 commonly had excellent outcomes. Clinical trial information: NCT02477644. [Table: see text]

Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Functional analysis of BRCA1 RING domain variants: computationally derived structural data can improve upon experimental features for training predictive models.

Advancements in the interpretation of variants of unknown significance are critical for improving clinical outcomes. In a recent study, massive parallel assays were used to experimentally quantify the effects of missense substitutions in the RING domain of BRCA1 on E3 ubiquitin ligase activity as well as BARD1 RING domain binding. These attributes were subsequently used for training a predictive model of homology-directed DNA repair levels for these BRCA1 variants relative to wild type, which is critical for tumor suppression. Here, relative structural changes characterizing BRCA1 variants were quantified by using an efficient and cost-free computational mutagenesis technique, and we show that these features lead to improvements in model performance. This work underscores the potential for bench researchers to gain valuable insights from computational tools, prior to implementing costly and time-consuming experiments.

Abstract 5457: Structural and functional assessment of mutations identified in different breast cancer genes

Abstract Atomic protein structure helps in understanding the functions associated with protein molecule. Therefore, it is very important to correlate the structural information from the biological functions of proteins for translational research. So far, BRCA1 and BRCA2 (Breast Cancer Susceptibility genes 1 and 2) genes have been extensively studied genes for hereditary breast and ovarian cancers. BRCA1 comprises 1863 amino acids and is involved in the tumour suppressor function. It also plays an important role in cell cycle checkpoint control and DNA damage repair process. Germ-line mutations discovered in BRCA1 and BRCA2 genes are distributed all over gene coding regions. BRCA1 encompasses different functional domains like RING-domain at N- terminus, DNA binding domain at the centre and tandem repeats of BRCT at C-terminus. RING-domain of BRCA1 interacts with BARD1, BAP1 whereas C-terminal region of BRCA1 interacts with BACH1, CtIP, Abraxas and controls the cell -cycle checkpoint through the protein-protein interactions. Germ-line mutations identified from the larger cohort of patients is available in different databases. However, unreported mutations are also regularly detected in clinical settings. We have evaluated the folding pattern of mutations discovered in different domains of BRCA1 and BRCA2 using in-silico, in-vitro and biophysical approaches. Furthermore, studies of protein-protein interactions (PPIs) with the binding partners of BRCA1 such as RAP80, MERIT40, and ABRAXAS have been explored using isothermal titration calorimetry. The slight differences between wild-type and mutant proteins of BRCA1 and BRCA2 have been observed. The data obtained from Circular- Dichroism, protein-protein interactions and structural analysis confirm that an alterations in protein folding pattern although not able to serve as a sole tool for mutation pathogenicity assessment, could provide valuable information to aid the classification of uncertain, potentially disease-causing, variants in the human cancer-relevant genes. Grant number: DST (Grant DST/INT/RUS/RSF/11), Russian foundation for Basic research (grant number 19-515-25001) Citation Format: Neha Mishra, Mudassar Ali Khan, Ekaterina S. Kuligina, Syed Hasan, Ashok K. Varma. Structural and functional assessment of mutations identified in different breast cancer genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5457.

Structure-based functional analysis of BRCA1 RING domain variants: Concordance of computational mutagenesis, experimental assay, and clinical data

A significant impediment to the improvement of clinical outcomes in treating breast and ovarian cancers rests with the lack of available interpretations for BRCA1 variants of unknown significance. Two research groups recently implemented large-scale functional assays for quantifying effects of single missense mutations on homology-directed DNA repair activity of BRCA1 variants, which is critical for tumor suppression and strongly correlates with cancer risk, and their results are significantly concordant with each other as well as with known pathogenic and benign variant clinical data. In this work, we implemented an established computational mutagenesis procedure to characterize structural impacts of single residue replacements to the BRCA1 RING domain. The computational data showed similarly strong concordance with known clinical data as well as with experimental data from both functional assays. Predictions made by models trained on our computational data offer a complementary and orthogonal approach for classifying all remaining unexplored BRCA1 RING domain variants.

BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes

Missense mutations that disrupt the RING domain of the tumor suppressor gene BRCA1 lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes CYP1A1 and CYP3A4; breast epithelial cells edited to create heterozygous loss of BARD1 showed significantly higher expression of CYP1A1 and CYP3A4 Reintroduction of wild-type BARD1 into these cells restored CYP1A1 and CYP3A4 transcription to normal levels, but introduction of the cancer-predisposing BARD1 RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.

Abstract AP29: BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE

Abstract Although cancers harboring BRCA1 mutations initially respond well to platinum and poly(ADP-ribose)-polymerase inhibitor (PARPi) therapy, resistance invariably arises and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. In this study, we utilized the SUM1315MO2 cancer cell line that harbors a hemizygous BRCA1185delAG mutation to study PARPi and platinum resistance. SUM1315MO2 cells were initially PARPi and cisplatin sensitive but readily acquired resistance. PARPi and cisplatin resistant clones did not harbor secondary reversion mutations. Rather, increased expression of a RING domain-deficient BRCA1 protein (Rdd-BRCA1) was required for resistance. Translation initiation occurred downstream of the frameshift mutation, likely at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BARD1 interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci-formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance in vitro and in vivo. Furthermore, Rdd-BRCA1 protein expression was detectable in recurrent carcinomas from germline BRCA1185delAG mutation carriers. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic, and when expressed at high enough levels are capable of contributing to PARPi and platinum resistance. Citation Format: Yifan Wang, John J. Krais, Andrea J. Bernhardy, Emmanuelle Nicolas, Kathy Q. Cai, Maria I. Harrell, Hyoung H. Kim, Erin George, Elizabeth M. Swisher, Fiona Simpkins and Neil Johnson. BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP29.

Biochemical and biophysical characterization of ruthenation of BRCA1 RING protein by RAPTA complexes and its E3 ubiquitin ligase activity

RAPTA compounds, ([Ru(η6-arene)(PTA)Cl2], PTA = 1,3,5-triaza-7-phosphaadamantane), have been reported to overcome drug resistance in cisplatin resistant cells. However, the exact mechanism of these complexes is still largely unexplored. In this study, the interaction of some RAPTA compounds with the N-terminal fragment of the BRCA1 RING domain protein was investigated. The binding of the RAPTA compounds to the BRCA1 protein resulted in a release of Zn2+ ions in a dose and time dependent manner, as well as thermal alteration of ruthenated-BRCA1 proteins. Electron Transfer Dissociation (ETD) fragmentation mass spectrometry revealed the preferential binding sites of the RAPTA complexes on the BRCA1 zinc finger RING domain at a similar short peptide stretch, Cys24Lys25Phe26Cys27Met28Leu29 and Lys35 (residues 44–49 and 55 on full length BRCA1). Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were established, resulting in inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase function. These findings could provide mechanistic insight into the mode of action of RAPTA complexes for on tested BRCA1 model protein.

Abstract B33: BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC

Abstract Breast cancer is the second leading cause of cancer-related deaths amongst women with 231,840 new cases projected for 2015. Majority of young African American women with BRCA1 mutations have a so-called Triple negative breast cancer with an aggressive phenotype. Currently there is no targeted therapy for TNBC. BRCA1 encodes a large protein and the two highly conserved regions are located at both ends, an N-terminal RING domain and two BRCT repeats at the C-terminus. The RING domain through interaction with BARD1 contains E3 ubiquitin ligase activity. Various BRCA1 mutations have been found throughout BRCA1 coding region, but the majority of cancer-associated mutations are nonsense or frame shift mutations which lead to chain termination. The C61G mutation has been linked to both breast and ovarian cancer development. In people from Ashkenazi Jewish descent, the most common mutations are 185delAG and 5382 ins C in BRCA1 and is 1:40. Our lab has cloned two naturally occurring splice variants of BRCA1 (BRCA1a and BRCA1b). These isoforms are the most evolutionary conserved of all the splice variants and code for multifunctional proteins. Our group has previously reported that BRCA1 RING domain, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins interact with the sole SUMO E2-conjugating enzyme Ubc9 and this facilitates both the entry of BRCA1 proteins to the nucleus and mediates ubiquitination of ER-alpha. The disease associated mutants do not bind Ubc9, remain stalled in the cytoplasm and have lost their growth suppressor function. We have identified a new nuclear trafficking pathway and malfunction of this by BRCA1 dysfunction can result in TNBC. The BARD1-dependent -E3 ubiquitin ligase activity of BRCA1 has been predicted to be required for its tumor suppressor function, as certain cancer –associated mutations in BRCA1 RING domain ablate this activity. Several mutations in BRCA1 RING domain have been identified however; their role in TNBC has yet to be elucidated. This work is based on the hypothesis that BRCA1 is a tumor suppressor gene and its RING domain can harbor several mutations some of which can result in loss of BRCA1 function resulting in TNBC and others can be gain of function mutants similar to WT BRCA1. We tested this hypothesis by introducing C61G, K109R and I26A mutations into TNBC cells and studied their growth inhibitory activity using colony suppression assays. Our results demonstrate for the first time that BRCA1 I26A to be a gain of function mutant similar to WT BRCA1. I26A mutant associates with Ubc9, lacks E3 ubiquitin ligase activity and suppresses growth of BRCA1 mutant TNBC and sporadic TNBC cells unlike K109R and C61G mutants. Clinically, the ability to predict which of these mutations can result in TNBC offers unprecedented prospects for early detection and cancer prevention. This is the first study demonstrating the physiological link between Ubc9 binding, loss of BARD1- dependent E3 ubiquitin ligase activity and growth suppression of I26A mutant BRCA1 protein in TNBC cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of TNBC. This study will accelerate precision medicine and reduce cancer health disparities in health outcomes. Work supported in part by Georgia Cancer Coalition Distinguished Cancer Scholar award, NIH-NCRR-RCMI grant G-12-RR003034, U54 RR02613, 5P20RR11104 and NIHMD research endowment grant 2S21MD000101, MSM/TU/UAB CCC Partnership/U54 CA118638 and ING foundation to V.N.R. Patent issued No:8372,580; 2013. Citation Format: Jingyao Xu, Shanekkia Black, Kartik Aysola, Yunlong Qin, Vaishali Reddy, Karan Singh, Joel Okoli, Derrick Beech, Uma Krishnamurthy, Gabriela Oprea, Valerie M. Rice, E Shyam Reddy, Douglas Moellering, Yuchang Fu, Veena N. Rao. BRCA1 RING domain mutations and function-based tools to predict risk for the development of TNBC. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B33.

A84a: Role of BRCA1 dysfunction in sporadic triple-negative breast cancer

Background There is increasing evidence that BRCA1-related DNA-repair defects determine sensitivity to certain agents, such as platinum-based chemotherapy. There is a lot of evidence about a link between TNBC and BRCA1 deficiency. Many clinical characteristics and molecular features are shared by sporadic triple-negative breast cancer and BRCA1-associated breast cancer. The majority of BRCA1-related breast cancers are of basal-like/triple-negative phenotype. Identification of specific markers of BRCA1-dysfunction will be essential to translate an understanding of defective DNA repair into targeted treatments for this poor prognosis subtype. Materials and methods Twenty-two patients with triple-negative breast cancer (TNBC) were treated with neoadjuvant platinum-based chemotherapy followed by surgery. Pathologic treatment response was assessed in correlation with biomarkers of BRCA1-dysfunction. Pathological response was evaluated according to the Chevallier classification (Ch). All patients were screened for germ-line mutations in BRCA1 (185delAG, 5382insC, 4153delA, 4158A/G, C61G) and BRCA2 (6174delT). All samples negative for germ-line mutations in BRCA1 and BRCA2 then submitted to BRCA1-dysfunction screening. Biomarkers of BRCA1-dysfunction included: BRCA1 somatic mutations (C61G, 185delAG, 5382insC), promoter methylation of BRCA1, low BRCA1 mRNA expression, high ID4 mRNA expression, low RAD51 mRNA expression, PTEN (T4721C) mutation, p53 (Arg72Pro, Pro72Pro) mutations. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), methylation-specific PCR were used for BRCA1-dysfunction analysis. Results Twelve patients (54.5%) achieved a pathological complete response (pCR) (Ch1+ Ch2). Ten patients (45.5%) had a residual disease (Ch3+ Ch4). BRCA1 mRNA expression was absent in 16/22 (72.7%) patients, low in 5/22 (22.7%). 11/21 (52.4%) patients with other than normal BRCA1 mRNA expression achieved a pCR. BRCA1 promoter methylation was detected in 9/22 (40.9%). 6/9 patients with BRCA1 promoter methylation achieved a pCR. RAD51 mRNA levels were low in 14/22 (63.6%), high in 1/22 (4.5%). 8/14 (57.1%) patients with other than normal RAD51 mRNA expression achieved a pCR. High ID4 mRNA levels were determined in 5/22 (22.7%). 3/5 (60%) patients with high ID4 mRNA expression achieved a pCR. p53 (Arg72Pro, Pro72Pro) mutations were identified in 12/22 (54.5%). No patient had PTEN (T4721C) mutation. No statistically significant correlation was found between the BRCA1 mRNA expression, BRCA1 promoter methylation, RAD51 mRNA expression, high ID4 mRNA levels, p53 (Arg72Pro, Pro72Pro) mutations and pCR to neoadjuvant platinum-based chemotherapy ( p > 0.05). Eleven patients had BRCA1 somatic mutations: 6/22 (27.3%) – BRCA1 5382insC, 4/22 (18.2%) – BRCA1 185delAG, 3/22 (13.6%) – BRCA1 C61G. 3/4 patients with BRCA1 185delAG mutation had BRCA1 C61G mutation. BRCA1 LOH was detected in 1/22 (4.5%) patient. All patients with BRCA1 5382insC mutation achieved a pCR ( p = 0.01). All patients with BRCA1 185delAG mutation and BRCA1 C61G mutation had residual disease (0.04 and 0.1, accordingly). Conclusion BRCA1 5382insC somatic mutation is associated with good response to neoadjuvant platinum based chemotherapy ( p = 0.01). BRCA1 185delAG and BRCA1 C61G mutations in the BRCA1 RING domain may predict resistance to neoadjuvant platinum-based chemotherapy (0.04 and 0.1, accordingly).

Massively Parallel Functional Analysis of BRCA1 RING Domain Variants.

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.

Cellular responses of BRCA1-defective and triple-negative breast cancer cells and in vitro BRCA1 interactions induced by metallo-intercalator ruthenium(II) complexes containing chloro-substituted phenylazopyridine

BackgroundTriple-negative breast cancer (TNBC) is defined by the absence of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Breast cancers with a BRCA1 mutation are also frequently triple-negative. Currently, there is a lack of effective therapies and known specific molecular targets for this aggressive breast cancer subtype. To address this concern, we have explored the cellular responses of BRCA1-defective and triple-negative breast cancer cells, and in vitro BRCA1 interactions induced by the ruthenium(II) complexes containing the bidentate ligand, 5-chloro-2-(phenylazo)pyridine.MethodsTriple-negative MDA-MB-231, BRCA1-defective HCC1937 and BRCA1-competent MCF-7 breast cancer cell lines were treated with ruthenium(II) complexes. The cytoxoxicity of ruthenium-induced breast cancer cells was evaluated by a real time cellular analyzer (RTCA). Cellular uptake of ruthenium complexes was determined by ICP-MS. Cell cycle progression and apoptosis were assessed using propidium iodide and Annexin V flow cytometry. The N-terminal BRCA1 RING protein was used for conformational and functional studies using circular dichroism and in vitro ubiquitination.ResultsHCC1937 cells were significantly more sensitive to the ruthenium complexes than the MDA-MB-231 and MCF-7 cells. Treatment demonstrated a higher degree of cytotoxicity than cisplatin against all three cell lines. Most ruthenium atoms were retained in the nuclear compartment, particularly in HCC1937 cells, after 24 h of incubation, and produced a significant block at the G2/M phase. An increased induction of apoptotic cells as well as an upregulation of p53 mRNA was observed in all tested breast cancer cells. It was of interest that BRCA1 mRNA and replication of BRCA1-defective cells were downregulated. Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were observed, causing inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase activity.ConclusionsThis study has revealed the ability of ruthenium complexes to inhibit cell proliferation, induce cell cycle progression and apoptosis. Ruthenium treatment upregulated the marker genes involved in apoptosis and cell cycle progression while it downregulated BRCA1 mRNA and replication of HCC1937 cells. Our results could provide an alternative approach to finding effective therapeutic ruthenium-based agents with promising anticancer activity, and demonstrated that the BRCA1 RING domain protein was a promising therapeutic target for breast cancers.

DNA Damage: Placing BRCA1 in the Proper Context

How does BRCA1's evolutionarily conserved E3 ligase activity contribute to DNA damage responses? Genetically engineered cells containing a BRCA1 RING domain mutation have been used to identify Claspin as a new target of BRCA1 E3 ligase activity in response to specific forms of DNA damage.

STRUCTURE-FUNCTION OF THE TUMOR SUPPRESSOR BRCA1

BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11–13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11–13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11–13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.

BRCA1 RING Function Is Essential for Tumor Suppression but Dispensable for Therapy Resistance

Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.

Functional differences among BRCA1 missense mutations in the control of centrosome duplication

We analyzed the effects of 14 different missense mutations in the RING domain of BRCA1 on the function of the protein in the control of centrosome number in tissue culture cells. Whereas 2 of the 14 BRCA1 variant proteins were neutral in the centrosome duplication assay, missense mutations of zinc-coordinating residues (C24R, C27A, C39Y, H41F, C44F and C47G) and mutations encoding BRCA1 variants M18T and I42V resulted in BRCA1 proteins that caused centrosome amplification. BRCA1 variant proteins I21V, I31M, L52F and D67Y had an intermediate effect on centrosome duplication. In addition, one of the variants, L52F, caused a peculiar phenotype with amplified centrosomes but the centrioles remained paired. By comparison, other BRCA1 variants that caused centrosome amplification had clustering of supernumerary centrosomes with unpaired centrioles. This surprising phenotype suggests that the BRCA1 protein regulates two functions in the control of centrosome duplication: regulation of centrosome number and regulation of centriole pairing. The L52F is unusual as it is defective in only one of these processes. This study analyzes the function of BRCA1 missense mutations in the control of centrosome duplication, a critical step in the maintenance of genetic stability of mammary epithelial cells, and indicates a new function of BRCA1 in the control of centriole pairing.