Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

BRCA1 Antibodies Matter.

Breast cancer susceptibility gene 1 (BRCA1) encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes including DNA damage repair, cell cycle checkpoint activation, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation and apoptosis. A large number of BRCA1 antibodies have been generated and become commercially available over the past three decades, however, many commercial antibodies are poorly characterized and, when widely used, led to unreliable data. In search of reliable and specific BRCA1 antibodies (Abs), particularly antibodies recognizing mouse BRCA1, we performed a rigorous validation of a number of commercially available anti-BRCA1 antibodies, using proper controls in a panel of validation applications, including Western blot (WB), immunoprecipitation (IP), immunoprecipitation-mass spectrometry (IP-MS), chromatin immunoprecipitation (ChIP) and immunofluorescence (IF). Furthermore, we assessed the specificity of these antibodies to detect mouse BRCA1 protein through the use of testis tissue and mouse embryonic fibroblasts (MEFs) from Brca1+/+ and Brca1Δ11/Δ11 mice. We find that Ab1, D-9, 07-434 (for recognizing human BRCA1) and 287.17, 440621, BR-64 (for recognizing mouse BRCA1) are specific with high quality performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels.

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

Immunohistochemistry for the detection of BRCA1 and BRCA2 proteins in patients with ovarian cancer: a systematic review.

BACKGROUND: Loss of function in either breast cancer type 1 susceptibility protein (BRCA1) or breast cancer type 2 susceptibility protein (BRCA2) is a major risk factor for epithelial ovarian cancer (EOC) development. BRCA1 or BRCA2 deficiencies are associated with short-term prognosis and might have importance for the treatment of women with the disease. However, the screening of all possible mechanisms of dysfunction is expensive, time-consuming and difficult to apply in clinical practice. On the other hand, immunohistochemistry (IHC) is a simple and reliable method to access the expression of several proteins in tumour tissues. MATERIALS AND METHODS: This systematic review aims to evaluate the current usage of IHC to detect BRCA1 and BRCA2 deficiencies in EOC. We searched and evaluated all primary literature on the use of IHC for evaluating BRCA1 and BRCA2 proteins expression in EOC. The main concepts for the search were: ovarian neoplasms, IHC, BRCA1 and BRCA2. RESULTS: Forty-four studies from 925 unique titles were included. A total of 4206 tumour samples were evaluated for BRCA1 and 1041 for BRCA2 expression. Twelve BRCA1 primary antibodies were used in 41 studies, and the most common was the MS110 clone (75.6%). Seven BRCA2 primary antibodies were used in ten studies. Using the cut-off of 10%, 47.0% of EOCs are associated with loss of BRCA1 and 34.5% with the loss of BRCA2 expression. CONCLUSION: IHC was effective to detect loss of BRCA1 protein expression in EOC; however, data on BRCA2 expression were heterogeneous and difficult to interpret.

Multi-omics profiling of younger Asian breast cancers reveals distinctive molecular signatures.

Breast cancer (BC) in the Asia Pacific regions is enriched in younger patients and rapidly rising in incidence yet its molecular bases remain poorly characterized. Here we analyze the whole exomes and transcriptomes of 187 primary tumors from a Korean BC cohort (SMC) enriched in pre-menopausal patients and perform systematic comparison with a primarily Caucasian and post-menopausal BC cohort (TCGA). SMC harbors higher proportions of HER2+ and Luminal B subtypes, lower proportion of Luminal A with decreased ESR1 expression compared to TCGA. We also observe increased mutation prevalence affecting BRCA1, BRCA2, and TP53 in SMC with an enrichment of a mutation signature linked to homologous recombination repair deficiency in TNBC. Finally, virtual microdissection and multivariate analyses reveal that Korean BC status is independently associated with increased TIL and decreased TGF-ß signaling expression signatures, suggesting that younger Asian BCs harbor more immune-active microenvironment than western BCs.

Expression of breast cancer type 1 and its relation with expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2/neu in breast carcinoma on trucut biopsy specimens.

OBJECTIVE: (1) The objective is to study the immunohistochemical expression of Breast cancer type 1 (BRCA1) in breast carcinoma on trucut biopsy specimens and (2) To relate its expression with that of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER-2)/neu and the clinicopathological parameters. SETTINGS AND DESIGN: A cross-sectional hospital-based study was performed in Lady Hardinge Medical College and Shrimati Sucheta Kriplani Hospital, New Delhi, with collaboration of the Departments of Pathology and Surgery from the period of November 2008 to March 2010. MATERIALS AND METHODS: The study group included 54 cytologically proven cases of breast carcinoma. The immunohistochemical expression of BRCA1 was studied and related with expression of ER, PR, and HER-2/neu on their trucut biopsies. RESULTS: The altered expression of BRCA1 (i.e., reduced or absent expression) was seen in 44.4% cases of breast carcinoma while 55.6% had positive expression. About 83% of breast carcinomas with altered BRCA1 expression were larger than 3 cm in size. The breast carcinomas showing altered expression were found to be mostly high grade (63.6%). This was statistically significant. The ER and PR negativity were seen in 62.5% and 79.2% breast carcinomas with altered BRCA1 expression, respectively. The score 3 positivity of HER-2/neu was more common among carcinomas with altered BRCA1 expression (21% vs. 16.7%). The triple negativity was found in 41.7% breast carcinomas having altered BRCA1 expression. This was statistically significant. CONCLUSION: The combination of immunohistochemical expression of BRCA1, ER, PR, and HER-2/neu and clinicopathological details may be helpful in predicting the individuals more likely to carry BRCA1 mutations and thus selecting the candidate and family members for genetic screening for BRCA1 mutations.

BRCA1, Ki67, and ß-Catenin Immunoexpression Is Not Related to Differentiation, Platinum Response, or Prognosis in Women With Low- and High-Grade Serous Ovarian Carcinoma.

OBJECTIVE: The purpose of this study was to compare the immunohistochemical expression of BRCA1, Ki67, and ß-catenin in women with low-grade (LGSOC) and high-grade serous ovarian carcinomas (HGSOC) and their relationship with clinicopathological features, response to platinum-based chemotherapy, and survival. METHODS: For this study, 21 LGSOC and 85 HGSOC stage I to IV cases, diagnosed and treated from 1996 to 2013 and followed-up until December 2016, were included. BRCA1, Ki67, and ß-catenin expression was assessed using tissue microarray-based immunohistochemistry. RESULTS: Women with HGSOC were significantly more likely to have advanced-stage disease (P < 0.001), higher CA125 levels (P < 0.001), postsurgery residual disease (P < 0.01), and higher rates of disease progression and recurrence (P = 0.001). The percentage of women with HGSOC whose tumors expressed Ki67 was significantly higher compared with women with LGSOC (P < 0.001). The expression of BRCA1 and ß-catenin did not differ between LGSOC and HGSOC (P = 0.12 and P = 1.00, respectively). The clinicopathological features and the response to platinum-based chemotherapy did not differ according to the BRCA1, Ki67, and ß-catenin expression in either group. In HGSOC, only International Federation of Gynecology and Obstetrics stage was independently associated with poor survival (PFS and OS). CONCLUSIONS: Ki67 expression was significantly higher in HGSOC. BRCA1 and ß-catenin expression did not differ between LGSOC and HGSOC samples. BRCA1, Ki67, and ß-catenin expression was neither related to clinicopathological features, response to platinum-based chemotherapy, nor survival. Only International Federation of Gynecology and Obstetrics stage remained associated with poor survival in women with HGSOC.

An Autoimmune Response Signature Associated with the Development of Triple-Negative Breast Cancer Reflects Disease Pathogenesis.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor(+) breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

Autoimmune response to PARP and BRCA1/BRCA2 in cancer.

PURPOSE: To determine the role of autoantibodies to PARP1 and BRCA1/BRCA2 which were involved in the synthetic lethal interaction in cancer. METHODS: Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect autoantibodies to PARP1 and BRCA1/BRCA2 in 618 serum samples including 131 from breast cancer, 94 from lung cancer, 34 from ovarian cancer, 107 from prostate cancer, 76 from liver cancer, 41 from pancreatic cancer and 135 from normal individuals. The positive sera with ELISA were confirmed by Western blot. Immunohistochemistry was used to examine the expression of PARP1 and BRCA1/BRCA2 in breast cancer. RESULTS: Autoantibody frequency to PARP1, BRCA1, and BRCA2 in cancer varied from 0% to 50%. When the sera from cancer patients were tested for the presence of autoantibodies to PARP1 and BRCA1/BRCA2, the autoantibody responses slightly decreased and the positive autoantibody reactions varied from 0% to 50.0%. This was significantly higher autoantibody responses to PARP1 and BRCA1/BRCA2 (especially to PARP1 and BRCA1) in ovarian cancer and breast cancer compared to normal control sera (P < 0.001 and P < 0.01). Immunohistochemistry indicated that Pathology Grade at diagnosis to PARP1 expression in breast cancer was different (P < 0.05). CONCLUSIONS: Different cancers have different profiles of autoantibodies. The autoantibodies to proteins involving the synthetic lethal interactions would be novel serological biomarker in some selective cancers.

AFFIRM--a multiplexed immunoaffinity platform that combines recombinant antibody fragments and LC-SRM analysis.

Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5-1000 ng/mL. Linear responses were demonstrated down to low ng/mL concentrations with high reproducibility. The platform allows for high throughput measurements in 96-well format, and all steps are amendable to automation and scale-up. We believe the use of recombinant antibody technology in combination with SRM MS analysis provides a powerful way to reach sensitivity, specificity, and reproducibility as well as the opportunity to build resources for fast on-demand implementation of novel assays.

No association between BRCA1 immunohistochemical expression and tumor grade, stage or overall survival in platinum-treated epithelial ovarian cancer patients.

BACKGROUND: The aim of this work is to assess the frequency of BRCA1 protein immunohistochemical (IHC) expression in epithelial ovarian cancer (EOC) and to evaluate the association of BRCA1 expression with clinical and pathological characteristics and the overall survival (OS) of patients treated with postoperative platinum- based chemotherapeutic agents. MATERIALS AND METHODS: This retrospective study was conducted on 35 cases of epithelial ovarian cancer selected from the files of the Pathology Department, Faculty of Medicine, Mansoura University, Egypt. Immunohistochemistry (IHC) was performed for BRCA1 gene protein. BRCA1 expression was compared to patient's age, tumor histology, grade, stage and OS time. Statistical analysis was carried out with the SPSS version 16.0 to assess significant associations. RESULTS: BRCA1 nuclear expression was detected in 40% of EOC, in which a mild increase in the percentage of positive cases was observed with serous histology, stage IV, and grade 3 carcinomas. There was a significant statistical difference in BRCA1 expression with regard to histological subtypes of EOC (p=0.048), but not grade or stage. Mean OS and survival rate were slightly better for BRCA1 expressing group, but there was no statistically significant difference (p=0.528). CONCLUSIONS: No association between BRCA1 immunohistochemical expression and tumor grade, stage or overall survival was noted in platinum-treated epithelial ovarian cancer patients.

RAP80 protein is important for genomic stability and is required for stabilizing BRCA1-A complex at DNA damage sites in vivo.

RAP80 (receptor-associated protein 80) is a ubiquitin-binding protein that can specifically recognize and bind to Lys-63-linked polyubiquitin chains, thus targeting the BRCA1-A complex to DNA damage sites. To study the role of RAP80 in vivo, we generated RAP80-deficient mice. The deficient mice are prone to B-cell lymphomagenesis. B-cell lymphomas in RAP80-deficient mice are nearly diploid but harbor clonal chromosome translocations. Moreover, the deficient mice are hypersensitive to ionizing radiation. Repair of ionizing radiation-induced DNA double-strand breaks is impaired in RAP80-deficient mouse embryonic fibroblasts. Mechanistically, loss of RAP80 suppresses recruitment of the BRCA1-A complex to DNA damage sites and abrogates the DNA damage repair process at DNA damage sites. Taken together, these results reveal that RAP80 plays a crucial role in the DNA damage response and in maintaining genomic integrity.

BRCA1 expression and molecular alterations in familial breast cancer.

The aim of the study was to evaluate the performance of immunohistochemical MS110 expression in a series of familial and sporadic breast cancer patients. An immunohistochemical study was performed on TMA samples from 93 sporadic and 94 familial breast cancer patients with (7/94) and without BRCA1 germline mutations. BRCA1 protein expression level was evaluated using the monoclonal MS110 antibody. Immunohistochemistry, performed on TMA samples, showed positive nuclear staining for BRCA1 in 34 sporadic and 37 familial breast tumours, respectively. All the tumours from patients carrying BRCA1 mutations showed complete loss of both BRCA1 and ERalpha expression, regardless of the type of mutation. The percentage of MS110 positive cases was significantly lower in mutated versus wild type BRCA1 familial cases (p=0.02) while the percentage of patients with higher ERalpha expression was significantly lower in BRCA1-mutated versus BRCA1-wild type familial patients (p=0.05). Interestingly, the presence of the E1038G polymorphism in BRCA1 exon 11 was significantly associated with protein expression (p=0.029). The frequency of MS110 negative cases also detected in BRCA1-wild type tumours, points to the inability of the BRCA1 IHC expression in discriminating between familial and sporadic breast cancer.

Cell cycle regulation of the BRCA1/acetyl-CoA-carboxylase complex.

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. The BRCA1 protein has been implicated in multiple cellular functions. We have recently demonstrated that BRCA1 reduces acetyl-CoA-carboxylase alpha (ACCA) activity through its phospho-dependent binding to ACCA, and further established that the phosphorylation of the Ser1263 of ACCA is required for this interaction. Here, to gain more insight into the cellular conditions that trigger the BRCA1/ACCA interaction, we designed an anti-pSer1263 antibody and demonstrated that the Ser1263 of ACCA is phosphorylated in vivo, in a cell cycle-dependent manner. We further showed that the interaction between BRCA1 and ACCA is regulated during cell cycle progression. Taken together, our findings reveal a novel mechanism of regulation of ACCA distinct from the previously described phosphorylation of Ser79, and provide new insights into the control of lipogenesis through the cell cycle.

Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics.

INTRODUCTION: Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known. METHODS: We generated and characterized 16 cell lines from five distinct Brca1deficient mouse mammary tumors with respect to their cancer stem cell characteristics. RESULTS: All cell lines derived from one tumor included increased numbers of CD44+/CD24- cells, which were previously identified as human breast cancer stem cells. All cell lines derived from another mammary tumor exhibited low levels of CD44+/CD24- cells, but they harbored 2% to 5.9% CD133+ cells, which were previously associated with cancer stem cells in other human and murine tumors. When plated in the absence of attachment without presorting, only those cell lines that were enriched in either stem cell marker formed spheroids, which were further enriched in cells expressing the respective cancer stem cell marker. In contrast, cells sorted for CD44+/CD24- or CD133+ markers lost their stem cell phenotype when cultured in monolayers. As few as 50 to 100 CD44+/CD24- or CD133+ sorted cells rapidly formed tumors in nonobese diabetic/severe combined immunodeficient mice, whereas 50-fold to 100-fold higher numbers of parental or stem cell depleted cells were required to form few, slow-growing tumors. Expression of stem cell associated genes, including Oct4, Notch1, Aldh1, Fgfr1, and Sox1, was increased in CD44+/CD24- and CD133+ cells. In addition, cells sorted for cancer stem cell markers and spheroid-forming cells were significantly more resistant to DNA-damaging drugs than were parental or stem cell depleted populations, and they were sensitized to the drugs by the heat shock protein-90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride). CONCLUSION: Brca1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44+/CD24- cells represent a population that correlates with human breast cancer stem cells.

Dynamics of DNA repair suggested by the subcellular localization of Brca1 and Brca2 proteins.

The localization of proteins to specific subcellular compartments often reveals clues regarding their biological functions. Although significant progress has been made towards understanding how damaged DNA is repaired, experiments to date have primarily focused on signal transduction pathways that activate DNA repair protein complexes and on how these complexes are assembled. Current evidence suggests that certain DNA repair processes are spatially organized such that aberrant DNA structures can be brought into proximity with DNA repair proteins at fixed sites. Since biochemical evidence suggests that the tumor suppressor proteins, Brca1 and Brca2, may mediate the assembly of protein complexes involved in the repair of damaged DNA, we have performed subcellular fractionation experiments to determine the subnuclear localization of these proteins. The majority of Brca1 and Brca2 proteins were found to interact tightly with the nuclear matrix. Furthermore, within the limits of detection, localization of Brca1 and Brca2 to the nuclear matrix was not altered following treatment of cells with DNA damaging agents that activate homology-mediated double-stranded DNA break and transcription-coupled repair pathways. Our findings suggest that Brca1 and Brca2 may perform their DNA repair-related functions from positions that are anchored to the nuclear matrix. These data are consistent with proposed models that suggest that components of specific repair complexes residing on the nuclear matrix function to recruit damaged DNA.

Bovine BRCA1 shows classic responses to genotoxic stress but low in vitro transcriptional activation activity.

Human BRCA1 has a genetically demonstrated role in DNA repair, and has been proposed to act as a transcriptional activator in a limited number of specialized settings. To gain insight into biologically conserved functional motifs, we isolated an ortholog of BRCA1 from cattle (Bos taurus). The predicted protein product shows 72.5% sequence identity with the human protein and conservation of amino acids involved in BRCA1 structure and function. Although the bovine C-terminus is truncated by seven amino acids as compared to human, bovine BRCA1 protein exhibited a similar cell cycle-regulated nuclear expression pattern. Expression was characteristically low and diffuse in the nucleus of G1/G0 cells, followed by increasing BRCA1-positive nuclear speckles in late S phase and G2/M phase cells. Bovine BRCA1 was phosphorylated and nuclear speckling was enhanced in response to DNA-damaging agents. Consistent with evidence from studies of human BRCA1, bovine BRCA1 was shown to interact with RNA polymerase II in vivo, an activity that was mapped to the C-terminal domain (CTD) (bBRCA(1364-1849)). Interestingly, when tested in the GAL4 transcriptional activation assay, full-length bovine and human BRCA1 lacked any ability to act as transcriptional activators and the CTD of bovine BRCA1 had five-fold lower activity when compared to the more acidic human C-terminus. These results provide evidence that phosphorylation and nuclear relocalization are highly conserved features of the BRCA1 response to genotoxic stress. In addition, bovine BRCA1 binds the RNA polymerase II holoenzyme, but this interaction lacks significant ability to correctly orient or recruit RNA polymerase II for transcription in the classic GAL4 transcriptional activation system.

Expression of BRCA1 and BRCA2 in male breast cancers and gynecomastias.

BRCA1 and BRCA2 breast cancer susceptibility genes are responsible for most of the hereditary breast cancers. Mutations in BRCA1 account for up to 40-50% of families with hereditary breast cancer only. Mutations in BRCA2 are linked to the other half of inherited breast cancer families and also to male breast cancer. On the contrary, no sporadic breast tumors have been shown to harbor mutations in BRCA1 and BRCA2 genes. It seems that altered expressions of BRCA1 and BRCA2 genes may contribute to breast cancer development. Moreover, BRCA1 and BRCA2 expressions are regulated in human breast cancer cell lines by estrogen. We addressed the issue of BRCA1 and BRCA2 expression in male breast cancers and gynecomastias. We investigated the presence of BRCA1 and BRCA2 proteins in male breast specimens by immunohistochemical analysis with a panel of antibodies elicited against BRCA1 and BRCA2. The specificity of each antibody has been verified by Western blotting in cell lines from different origins. The characterization of 6 anti-BRCA1 antibodies revealed a BRCA1 200-kDa protein detected in breast cell lines (MDA-MB 231, HBL 100, T-47D and MCF7) or in an acute leukemia (MOLT 4), known to overexpress BRCA1. All 5 anti-BRCA2 antibodies detected a BRCA2 384-kDa protein in the HBL100 and MCF7 breast cell lines. By immunohistochemistry, we found nuclear, perinuclear, endoplasmic reticulum, Golgi vesicle, secretion and apical cytoplasmic stainings in gynecomastias and sporadic and hereditary male breast cancers, for BRCA1 and BRCA2 protein expressions. We report an extensive expression of BRCA1 and BRCA2 proteins in different compartments of the mammary gland cells in male breast carcinomas and gynecomastias. This is consistent with the estrogen-dependent expression of BRCA1 and BRCA2 in human breast cells.

Presence of BRCA1 and BRCA2 proteins in human milk fat globules after delivery.

We evaluated BRCA1 and BRCA2 oncosuppressor protein expression in 26 milk samples in women just after delivery. The quantification of BRCA1 and BRCA2 proteins was performed in isolated milk fat globules using an affinity chromatography strategy. The amounts of BRCA1 and BRCA2 proteins were found to be similar. We explained the presence of BRCA1 and BRCA2 proteins in human milk fat globules by the fact that they are formed by exocytosis of lipids from epithelial cells of the mammary gland and are enveloped by plasma membrane from the apical part of the milk-secreting cells. This raises the possibility that BRCA1 and BRCA2 proteins are a protective response to proliferation and play a possible role in newborn nutrition.