Human BRCA1 Research Articles

BRCA2 Regulates Transcription Elongation by RNA Polymerase II to Prevent R-Loop Accumulation

SummaryThe controlled release of RNA polymerase II (RNAPII) from promoter-proximal pausing (PPP) sites is critical for transcription elongation in metazoans. We show that the human tumor suppressor BRCA2 interacts with RNAPII to regulate PPP release, thereby preventing unscheduled RNA-DNA hybrids (R-loops) implicated in genomic instability and carcinogenesis. BRCA2 inactivation by depletion or cancer-causing mutations instigates RNAPII accumulation and R-loop accrual at PPP sites in actively transcribed genes, accompanied by γH2AX formation marking DNA breakage, which is reduced by ERCC4 endonuclease depletion. BRCA2 inactivation decreases RNAPII-associated factor 1 (PAF1) recruitment (which normally promotes RNAPII release) and diminishes H2B Lys120 ubiquitination, impeding nascent RNA synthesis. PAF1 depletion phenocopies, while its overexpression ameliorates, R-loop accumulation after BRCA2 inactivation. Thus, an unrecognized role for BRCA2 in the transition from promoter-proximal pausing to productive elongation via augmented PAF1 recruitment to RNAPII is subverted by disease-causing mutations, provoking R-loop-mediated DNA breakage in BRCA2-deficient cells.

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

‘All-action hero’ or ‘Slap in the face for womankind’? Discursive representations of Angelina Jolie following her prophylactic bilateral mastectomy

When Hollywood actress Angelina Jolie underwent a prophylactic mastectomy, public demands for human tumour suppressor gene BRCA1 testing almost doubled (Evans & Howell, 2015). This paper explores the discursive positioning of Jolie following her procedure in 20 UK newspaper articles using Foucauldian discourse analysis. Jolie was positioned within the discourse in two different ways: the ‘she-ro’ (an agentic decision maker who saved countless lives), and a ‘selfish attention seeker’ (a needy and melodramatic woman who unnecessarily invokes public paranoia and panic). A third (implied) subject position that was identified in the data is the ‘silent and dignified’ post-mastectomy woman who doesn’t need to ‘shout it from the rooftops’. I argue that these subject positions may serve to silence the lived experience of many prophylactic mastectomy patients and render invisible the suffering that some women can experience post-surgery. This implies that counselling and educational services for women who are considering (or who have undergone) prophylactic mastectomy are essential. To conclude, avenues for future research are suggested.

Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters

Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10−10 to 2.4 × 10−6 M with a detection limit (LOD) of 6.4 × 10−11 M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

A novel model to characterize structure and function of BRCA1.

BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1Δ/Δ181-1415 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1Δ/Δ126-136 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1Δ/Δ525-881 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1Δ/Δ872-878 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.

Exosomes isolated from cancer patients\u2019 sera transfer malignant traits and confer the same phenotype of primary tumors to oncosuppressor-mutated cells

BackgroundHorizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients’ sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients’ sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena.MethodsWe used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients’ sera or cancer patients’ sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts’ membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes.ResultsCells treated with exosomes isolated from cancer patients’ sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression, on the plasma membrane of target cells, of receptors, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits.ConclusionThese findings strengthen the hypothesis that oncogenic factors transferred via circulating cancer exosomes, induce malignant transformation of target cells even at distance. Oncosuppressor genes might protect the integrity of the cell genome by inhibiting the uptake of cancer-derived exosomes.

High-grade serous carcinomas arise in the mouse oviduct via defects linked to the human disease.

Recent studies have suggested that the most common and lethal type of 'ovarian' cancer, i.e. high-grade serous carcinoma (HGSC), usually arises from epithelium on the fallopian tube fimbriae, and not from the ovarian surface epithelium. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen-regulated Cre recombinase in oviductal epithelium-the murine equivalent of human fallopian tube epithelium (FTE). We employed Ovgp1-iCreERT2 mice to show that FTE-specific inactivation of several different combinations of tumour suppressor genes that are recurrently mutated in human HGSCs-namely Brca1, Trp53, Rb1, and Nf1-results in serous tubal intraepithelial carcinomas (STICs) that progress to HGSC or carcinosarcoma, and to widespread metastatic disease in a subset of mice. The cancer phenotype is highly penetrant and more rapid in mice carrying engineered alleles of all four tumour suppressor genes. Brca1, Trp53 and Pten inactivation in the oviduct also results in STICs and HGSCs, and is associated with diffuse epithelial hyperplasia and mucinous metaplasia, which are not observed in mice with intact Pten. Oviductal tumours arise earlier in these mice than in those with Brca1, Trp53, Rb1 and Nf1 inactivation. Tumour initiation and/or progression in mice lacking conditional Pten alleles probably require the acquisition of additional defects, a notion supported by our identification of loss of the wild-type Rb1 allele in the tumours of mice carrying only one floxed Rb1 allele. Collectively, the models closely recapitulate the heterogeneity and histological, genetic and biological features of human HGSC. These models should prove useful for studying the pathobiology and genetics of HGSC in vivo, and for testing new approaches for prevention, early detection, and treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A strategy to apply quantitative epistasis analysis on developmental traits

BackgroundGenetic interactions are keys to understand complex traits and evolution. Epistasis analysis is an effective method to map genetic interactions. Large-scale quantitative epistasis analysis has been well established for single cells. However, there is a substantial lack of such studies in multicellular organisms and their complex phenotypes such as development. Here we present a method to extend quantitative epistasis analysis to developmental traits.MethodsIn the nematode Caenorhabditis elegans, we applied RNA interference on mutants to inactivate two genes, used an imaging system to quantitatively measure phenotypes, and developed a set of statistical methods to extract genetic interactions from phenotypic measurement.ResultsUsing two different C. elegans developmental phenotypes, body length and sex ratio, as examples, we showed that this method could accommodate various metazoan phenotypes with performances comparable to those methods in single cell growth studies. Comparing with qualitative observations, this method of quantitative epistasis enabled detection of new interactions involving subtle phenotypes. For example, several sex-ratio genes were found to interact with brc-1 and brd-1, the orthologs of the human breast cancer genes BRCA1 and BARD1, respectively. We confirmed the brc-1 interactions with the following genes in DNA damage response: C34F6.1, him-3 (ortholog of HORMAD1, HORMAD2), sdc-1, and set-2 (ortholog of SETD1A, SETD1B, KMT2C, KMT2D), validating the effectiveness of our method in detecting genetic interactions.ConclusionsWe developed a reliable, high-throughput method for quantitative epistasis analysis of developmental phenotypes.

Let-7e sensitizes epithelial ovarian cancer to cisplatin through repressing DNA double strand break repair

BackgroundResistance to platinum-based chemotherapy remains a great challenge for ovarian cancer treatment. The human let-7 family contains 13 members located on nine different chromosomes, and most members have been implicated in the modulation of drug sensitivity in cancers. Our previous study showed that deregulation of let-7e in epithelial ovarian cancer (EOC) promoted the development of resistance to cisplatin. In the present study, we aimed to investigate the underlying mechanism and further evaluate the clinical value of let-7e in predicting chemo-response and prognosis in EOC.ResultsIn situ hybridization assays revealed a significantly decreased expression of let-7e in chemo-resistant EOC tissues compared with chemo-sensitive cases. Transfection with let-7e agomir sensitized EOC cells to cisplatin, down-regulated BRCA1 and Rad51 expression, and repressed the repair of cisplatin-induced DNA double strand break, while let-7e inhibitor exerted the opposite effects. In human EOC tissues, BRCA1 and Rad51 levels were increased in the chemo-resistant group compared with the sensitive group and were negatively correlated with let-7e. Low let-7e and high Rad51 were significantly associated with poor progression-free survival and overall survival and multivariate regression analyses showed that let-7e was an independent predictor for overall survival and chemotherapy response in EOC. Receiver operating characteristic analysis indicated that let-7e level was highly predictive of resistance to platinum-taxane chemotherapy with an area under the curve of 0.826.ConclusionsIn EOC, low let-7e leads to activation of BRCA1 and Rad51 expression and subsequent enhancement of DSB repair, which in turn results in cisplatin-resistance. Let-7e is a potential predictor for survival and chemo-response in EOC and re-expression of let-7e might be an effective strategy for overcoming chemo-resistance.

Architectural plasticity of human BRCA2-RAD51 complexes in DNA break repair.

The tumor suppressor BRCA2 is a large multifunctional protein mutated in 50–60% of familial breast cancers. BRCA2 interacts with many partners and includes multiple regions with potentially disordered structure. In homology directed DNA repair BRCA2 delivers RAD51 to DNA resulting in removal of RPA and assembly of a RAD51 nucleoprotein filament. Dynamic rearrangements of BRCA2 likely drive this molecular hand-off initiating DNA strand exchange. We show human BRCA2 forms oligomers which can have an extended shape. Scanning force microscopy and quantitative single molecule fluorescence define the variety of BRCA2 complexes, reveal dramatic rearrangements upon RAD51 binding and the loading of RAD51 patches on single strand DNA. At sites of repair in cell nuclei, super-resolution microscopy shows BRCA2 and RAD51 arranged in largely separate locations. We identified dynamic structural transitions in BRCA2 complexes suggested to facilitate loading of RAD51 onto RPA coated single strand DNA and subsequent release of BRCA2.

The Impact of M1775K Variant in BRCA1 Regulation

The missense mutations of the BRCT domain of human BRCA1 have been argued to be associated with predisposition to hereditary breast/ovarian cancer. Mutations at the C-terminus of the protein are critical for function and structural excellence. This mutation has been associated with changes in the structure of the protein and loss of interaction with other peptides involved in cell cycle control. In previous studies we have shown that the modification of various mutants in BRCT domain, such as Met1775K is strongly affecting the functional and structural integrity of the domain and the binding affinity with phospopeptides. In the present study we have attempted to determine the effect of the M1775K mutation on the intracellular localization of the protein. For this purpose, GFPBRCA1-BRCT M1775K mutant was created and expressed in MCF-7 cell lines. Due to fluorescence microscopy determined that the mutation affects the protein’s mislocalization in relation to wtBRCA1-BRCT protein. Mutant EGFP-BRCA1-BRCT M1775K form is mainly determined in the cytoplasm in contrast to the EGFPwtBRCA1-BRCT protein that localizes into nucleus and cytoplasm. Cytoplasmic mislocalization of M1775K mutation, presented here, may be related to disruption of BRCA1-BRCT folding. The M1775K mutation influences the nuclear transport of BRCA1-BRCT domain to the nucleus, suggesting that the impact of the integrity of the BRCA1-BRCT domain is crucial in functional and structural level.

Protein-Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

Prediction of Disordered RNA, DNA, and Protein Binding Regions Using DisoRDPbind.

Intrinsically disordered proteins and regions (IDPs and IDRs) are involved in a wide range of cellular functions and they often facilitate interactions with RNAs, DNAs, and proteins. Although many computational methods can predict IDPs and IDRs in protein sequences, only a few methods predict their functions and these functions primarily concern protein binding. We describe how to use the first computational method DisoRDPbind for high-throughput prediction of multiple functions of disordered regions. Our method predicts the RNA-, DNA-, and protein-binding residues located in IDRs in the input protein sequences. DisoRDPbind provides accurate predictions and is sufficiently fast to make predictions for full genomes. Our method is implemented as a user-friendly webserver that is freely available at http://biomine.ece.ualberta.ca/DisoRDPbind/ . We overview our predictor, discuss how to run the webserver, and show how to interpret the corresponding results. We also demonstrate the utility of our method based on two case studies, human BRCA1 protein that binds various proteins and DNA, and yeast 60S ribosomal protein L4 that interacts with proteins and RNA.

A second DNA binding site in human BRCA2 promotes homologous recombination.

BRCA2 tumour-suppressor protein is well known for its role in DNA repair by homologous recombination (HR); assisting the loading of RAD51 recombinase at DNA double-strand breaks. This function is executed by the C-terminal DNA binding domain (CTD) which binds single-stranded (ss)DNA, and the BRC repeats, which bind RAD51 and modulate its assembly onto ssDNA. Paradoxically, analysis of cells resistant to DNA damaging agents missing the CTD restore HR proficiency, suggesting another domain may take over its function. Here, we identify a region in the N terminus of BRCA2 that exhibits DNA binding activity (NTD) and provide evidence for NTD promoting RAD51-mediated HR. A missense variant detected in breast cancer patients located in the NTD impairs HR stimulation on dsDNA/ssDNA junction containing substrates. These findings shed light on the function of the N terminus of BRCA2 and have implications for the evaluation of breast cancer variants.

Secretome proteomics reveals candidate non-invasive biomarkers of BRCA1 deficiency in breast cancer.

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.

Analysis of alternative lengthening of telomere markers in BRCA1 defective cells.

Telomeres are specialized structures responsible for the chromosome end protection. Previous studies have revealed that defective BRCA1 may lead to elevated telomere fusions and accelerated telomere shortening. In addition, BRCA1 associates with promyelocytic leukemia (PML) bodies in alternative lengthening of telomeres (ALTs) positive cells. We report here elevated recombination rates at telomeres in cells from human BRCA1 mutation carriers and in mouse embryonic stem cells lacking both copies of functional Brca1. An increased recombination rate at telomeres is one of the signs of ALT. To investigate this possibility further we employed the C‐circle assay that identifies ALT unequivocally. Our results revealed elevated levels of ALT activity in Brca1 defective mouse cells. Similar results were obtained when the same cells were assayed for the presence of another ALT marker, namely the frequency of PML bodies. These results suggest that BRCA1 may act as a repressor of ALT. © 2016 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

Abstract 3669: “DNA binding region” of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint

Abstract Introduction. BRCA1, a tumor suppressor gene in breast or ovarian cancer, is a large multifunctional protein implicated in DNA double-strand break repair, centrosome duplication, transcription regulation, DNA damage response, and cell cycle control, all of which are important for maintaining genomic stability, BRCA1 contains 3 domains; a N-terminal RING domain, C-terminal BRCT domain and a central region (CR). RING and BRCT domains are well addressed, but the function of the CR remains unclear. The CR is reported to have DNA binding region (DBR). Our aim of this study is to identify DBR and to clarify the biological significance of BRCA1 binding to DNA. Materials and Methods. 1) Identification of DBR of BRCA1. First, electrophoretic mobility shift assay (EMSA) was performed to identify an essential region responsible for BRCA1 binding to DNA using purified tagged full length BRCA1 protein or the fragments and DNA oligos, and to investigate features of the DNA binding using various structures or lengths of DNA. Next, atomic force microscopy (AFM) was performed to see how BRCA1 binds to DNA. 2) Analysis of the biological significance of BRCA1 binding to DNA using mouse ES cells which are deficient in the DBR of BRCA1(ΔDBR) by TALEN-mediated Gene Targeting. 1. MTT survival assay under PARP1 inhibitor or Hydroxyurea (HU) to see the ability to homologous recombination (HR) repair or the sensitivity to replication stress, respectively. 2. HR assay in vitro. 3. EdU incorporation assay in the presence of HU to see cell cycle under replication stress. 4. Western blot to evaluate the phosphorylation status of CHK1 which regulates intra-S-phase checkpoint. 5. Karyotyping analysis to check for the genetic instability. Results. 1) We identified an essential DBR (421-701 amino acids) within the CR of human BRCA1 by EMSA or AFM, which preferably binds to splayed-arm DNA (mimic of replication fork) in a sequence-independent manner and brings DNA strands together. These in vitro data suggest that binding of BRCA1 to DNA may affect protection of the DNA replication fork. 2) 1. The ΔDBR cells exhibited decreased survival as compared to wild type (WT) cells treated with a PARP1 inhibitor or HU in MTT assay. 2. There was not statistical difference with a HR assay between WT cells and ΔDBR cells. 3. ΔDBR cells continued to incorporate more EdU in the presence of HU in EdU incorporation assay. 4. Phosphorylation of CHK1 was moderately decreased in ΔDBR cells in Western blot. 5. The ΔDBR cells exhibited an increase in abnormal chromosome structures in Karyotyping analysis. Conclusion. In this study, we demonstrate that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint that is activated by replication stress. Thus, our finding refines a functional domain of BRCA1, DBR, deficiency of which results in moderately impaired DNA replication upon replicative stress, leading to accumulative DNA mutations and cancer development. Citation Format: Takaaki Masuda, Koshi Mimori, Chuxia Deng. “DNA binding region” of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3669.

Persistent Activation of NF-κB in BRCA1-Deficient Mammary Progenitors Drives Aberrant Proliferation and Accumulation of DNA Damage

Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-κB) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-κB, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKKα. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. Invivo, NF-κB inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-κB. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-κB signaling.

Screening for Del 185 AG and 4627C>A BRCA1 Mutations in Breast Cancer Patients from Lahore, Pakistan.

Breast cancer contributes to approximately 23% of the cancer cases identified and 14% of cancer related deaths worldwide. Including a strong association between genetic and environmental factors, breast cancer is a complex and multi factorial disorder. Two high penetration breast cancer susceptibility genes (BRCA1 and BRCA2) have been identified, and germ line mutations in these are thought to account for between 5% and 10% of all breast cancer cases. The human BRCA1 gene, located on 17q, is involved in the regulation of cell proliferation by aiding in DNA repair, transcriptional responses to DNA damage and cell cycle check points. Mutations in this gene enhance cell proliferation and facilitate formation of tumors. Two mutations, the 185 deletion of AG and the 4627 substitution from C to A, are founder mutations in the BRCA1 gene for breast cancer in Asian populations. Allele specific PCR was performed to detect these selected mutations in 120 samples. No mutation of 4627 C to A was detected in the samples and only one of the patients had the 185 del AG mutation in the heterozygous condition. Our collected samples had lower consanguinity and family history indicating the greater involvement of environmental as compared to genetic factors.

Abstract A26: Telomere length in normal and tumor breast tissue from BRCA2 mutation carriers and sporadic breast cancer cases

Abstract Germline mutations in the BRCA genes are associated with highly increased risk of breast and ovarian cancers and to a lesser extent to prostate and pancreatic cancers. Great number of mutations with variable impact on cancer risk and progression are known in these genes worldwide. A single founder mutation has been detected in each of the BRCA genes in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is more frequent and can be found in approximately 6-7% of female breast cancer patients and 40% of male breast cancer patients in Iceland. The mutation leads to a non-functional protein product and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation has been associated with poor prognosis, both in breast and prostate cancers, although mutation carriers seem to differ with respect to age of onset and severity of disease. Dysfunctional telomere maintenance can lead to excessive telomere shortening which causes chromosome instability. It is therefore a driving force behind cancer progression and is considered a hallmark of many human cancers. Telomere shortening has been shown to be an early and a common molecular alteration in epithelial cancers, including breast cancers. Previous results from our laboratory, amongst others, have shown that BRCA2 is associated with telomere protection and maintenance. Telomere dysfunction induced foci (TIFs) formation was detected in cell lines derived from heterozygous BRCA2999del5 mutation carriers1. Telomere length has been proposed as a possible predictive factor for cancer risk for various cancers. In the case of breast cancer and BRCA mutation carriers, the results have been conflicting. The aim of the study was to investigate if telomere length in normal and tumor breast tissue is correlated with breast cancer progression and survival in a well-defined group of BRCA2 mutation carriers and sporadic breast cancer cases. The study group consisted of breast cancer patients carrying the BRCA2999del5 Icelandic founder mutation and sporadic cases, matched with respect to age and year of diagnosis. Tumor and normal adjacent tissue samples from these patients were embedded in paraffin and Quantitative Fluorescence in Situ Hybridization method (Q-FISH) was used to carry out telomere length measurements. Telomeres were visualized using a telomeric sequence-specific fluorescence peptide nucleic acid (PNA) probe. A centromere-specific PNA probe was used as an inner control for hybridization efficiency. The results show a clear difference in telomere length of different cell types in normal breast tissue with inner epithelial cells having shorter telomeres than both myoepithelial cells and fibroblasts. These findings support previously reported data in the literature2. The majority of examined tumors have very short telomeres. In mutation carriers, there are indications that less variable telomere length in normal adjacent tissue is associated with reduced breast cancer specific survival. This seems to be more pronounced in the mutation carrier group compared to the sporadic group. Telomere length in adjacent normal tissue appears to be shorter in cases with luminal type than with basal-like tumors.