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1.  Impact of RING and BRCT Domain Mutations on BRCA1 Protein Stability, Localization, and Recruitment to DNA Damage 
Radiation research  2010;174(1):1-13.
Mutations within the tumor suppressor BRCA1 cause the majority of hereditary breast and ovarian cancer cases. The BRCA1 protein is an important regulator of DNA double strand break repair and BRCA1 deficient cells are highly sensitive to ionizing radiation. Furthermore, BRCA1 function may contribute to G2 cell cycle checkpoint enforcement. E3-ubiquitin ligase activity is the only known enzymatic activity of BRCA1, which is mediated by the N-terminal RING finger domain. The C-terminal BRCT repeat domain, which mediates protein-protein interactions, is the only other identified structural domain. By investigating cancer-linked mutations within each domain, we demonstrate that truncation of the BRCT domain greatly impairs the stability and nuclear localization of BRCA1 protein. A missense mutation within the RING domain does not affect these biochemical properties. However, both mutant forms of BRCA1 fail to co-localize in nuclear foci with the known BRCA1-interacting proteins BARD1 and BACH1, which are important for DNA repair. This failure occurs despite the continued ability of the RING mutant protein to interact with BACH1 and the ability of the BRCT mutant to interact with BARD1. Furthermore, neither mutant form of BRCA1 is recruited into DNA-damage-associated foci marked by γH2AX. Therefore, our data suggests that both the RING and BRCT domains of BRCA1 are required for an early step in the function of BRCA1 during DNA repair: recruitment to the sites of DNA damage.
doi:10.1667/RR1290.1
PMCID: PMC4550207  PMID: 20681793
BRCA1; mutations; breast cancer
2.  Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line 
Breast Cancer Research  2000;2(2):139-148.
We tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo.
Introduction:
Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1, which is located on chromosome 17q21, are associated with a predisposition to the development of cancer in these organs [1,2]. No mutations in the BRCA1 gene have been detected in sporadic breast cancer cases, but mutations have been detected in sporadic cases of ovarian cancer [3,4]. Although there is debate regarding the level of cancer risk associated with mutations in BRCA1 and the significance of the lack of mutations in sporadic tumors, it is possible that alterations in the function of BRCA1 may occur by mechanisms other than mutation, leading to an underestimation of risk when it is calculated solely on the basis of mutational analysis. Such alterations cannot be identified until the function and regulation of BRCA1 are better understood.
The BRCA1 gene encodes a 220-kDa nuclear phosphoprotein that is regulated in response to DNA damaging agents [5,6,7] and in response to estrogen-induced growth [8,9,10,11]. Germline mutations that cause breast and ovarian cancer predisposition frequently result in truncated and presumably inactive BRCA1 protein [12].
BG-1 cells were derived from a patient with stage III, poorly differentiated ovarian adenocarcinoma [13]. This cell line, which expresses wild-type BRCA1, is estrogen responsive and withdrawal of estrogen results in eventual cell death. Previous studies suggest that BRCA1 is stimulated as a result of estrogen treatment [8,9,10,11], and also that BRCA1 may be involved in the cell death process [14]. Therefore, we examined the effect of reduction of BRCA1 levels in BG-1 cells on the cellular response to hormone depletion as well as estrogen stimulation. The results suggest that reduced levels of BRCA1 correlates with a survival advantage when BG-1 cells are placed under growth-restrictive and hormone-depleted conditions. In optimum growth conditions, significantly reduced levels of BRCA1 correlates with enhanced growth both in vitro and in vivo.
Aims:
To test the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as in the inhibition of ovarian cell proliferation.
Materials and methods:
The estrogen receptor-positive, BG-1 cell line [13], which contains an abundant amount of estrogen receptors (600 fmoles/100 μg DNA), was infected using a pLXSN retroviral vector (provided by AD Miller) containing an inverted partial human cDNA 900-base-pair sequence of BRCA1 (from nucleotide 121 in exon 1 to nucleotide 1025 in exon 11, accession #U14680). After 2 weeks of selection in 800 μg/ml of geneticin-G418 (Gibco/Life Technologies, Gaithersburg, MD, USA), BG-1 G418-resistant colonies were pooled, or individually isolated, and assayed for growth in the presence or absence of supplemented estrogen. Virally infected pooled populations of BG-1 cells were examined for BRCA1 message levels by ribonuclease protection assay (Fig. 1a). BRCA1 ribonuclease protection probe was made using an in vitro transcription kit (Ambion, Inc, Austin, TX, USA) as previously described [10] and derived clones were tested for protein levels by Western blot analysis using an anti-BRCA1 (Oncogene Research, Ab-1, Cambridge, MA, USA) antibody. Growth curve analysis of Infected populations and were pretreated for 5 days in phenol red-free, Dulbecco's modified eagle medium (DMEM)/F-12 medium (Gibco/Life Technologies) supplemented with 10% charcoal/dextran treated serum (Hyclone, Logan, UT, USA), then plated at 2.5 × 106 cells per 100mm dish in triplicate in the absence or presence of estrogen (10-8 mol/l; 17β-Estradiol; 1,3,5 (10) - Estratriene 3,17β-diol; Sigma, St Louis, MO, USA). For soft agar assay, clones were plated into 10 60-mm dishes at 1 × 105 cells/dish containing 0.3% bactopeptone agar with or without added estrogen (10-8 mol/l) in phenol red-free medium with 10% stripped serum in order to test for anchorage independent growth. BG-1 infected clones were tested for tumorigenicity by injection of cells (106 cells in 0.1cm2 50% matrigel; Collaborative Biomedical Products, Bedford, MA, USA) into subcutaneous sites in 6-week-old athymic Ncr-nude mice (NCI Animal Program, Bethesda, MD, USA) that were ovariectomized at approximately 4 weeks of age. Half of the ovariectomized mice received an implanted 0.18mg estrogen 60-day pellet (Innovative Research of America, Sarasota, FL, USA).
Results:
Antisense technology was effective in decreasing both RNA and protein levels of BRCA1 in the BG-1 human ovarian adenocarcinoma cells. BRCA1 antisense-infected populations contained significantly less BRCA1 message than control LXSN-infected pools and selected clones contained varying reduced levels of BRCA1 protein compared with control clones (Figs 1a and 1b).
Three independent BRCA1 antisense-infected cultures demonstrated a resistance to cell death induced by withdrawal from estrogen over a 6- to 20-day period (Fig. 2a). The BRCA1 antisense population also exhibited a threefold to sixfold increase in cell growth compared with control cells in the presence of estrogen treatment. BG-1 BRCA1 antisense clones demonstrated a similar response to pooled population studies, enhanced growth with estrogen, and failure to die upon estrogen depletion (Fig. 2b).
The BRCA1 antisense clones were further examined for other associated tumorigenic properties. All of the antisense clones were able to form colonies in soft agar (2-23 colonies per 104 cells plated; data not shown), whereas control clones were deficient in their ability to form colonies (0-0.8 colonies per 104 cells plated). Table 1 shows, in the presence of estrogen, the clone with the lowest levels of BRCA1 (AS-4) produced significantly more colonies (133 ± 17.9 colonies per 104 cells plated) than the control clone (NEO; 6 ± 3.1 colonies per 104 cells plated). Clones AS-4 and NEO were also injected with matrigel subcutaneously into ovariectomized athymic mice. Almost twice as many sites were positive for the AS-4 clone (14 out of 14) as for the NEO clone (eight out of 14) 42 days after injection. In addition, BRCA1 antisense tumors averaged twice the size of control tumors. The BRCA1 reduced cells also formed tumors with half the latency of control cells in the presence of implanted estrogen (11 days versus 21 days until tumor formation).
Discussion:
The present studies show that reduction in BRCA1 levels, using an antisense retroviral vector in the estrogen dependent BG-1 ovarian carcinoma cell line, contributes to confirmation of the hypothesis that BRCA1 plays a pivotal role in the balance between cell death and cell proliferation. BRCA1 RNA and protein levels were successfully reduced in populations and isolated clones of antisense infected BG-1 cells. Decreased BRCA1 levels rescued the BG-1 cells from growth arrest or cell death in adverse growth conditions in monolayer or soft agar conditions. Furthermore, a BRCA1 antisense clone that had significantly low levels of BRCA1 protein was able to form twice as many tumors in ovariectomized nude mice with a decreased latency compared with a control clone.
In multicellular mammalian organisms, a balance between cell proliferation and cell death is extremely important for the maintenance of normal healthy tissues. In support of this hypothesis, it has been shown that p53 and BRCA1 can form stable complexes, and can coactivate p21 and bax genes, which may lead to the activation of the apoptosis pathway [15]. The present data, which show that cells with a reduction of BRCA1 have a survival advantage in conditions where control cells fail to thrive, also supports this hypothesis. BRCA1 levels appear to affect the ability of cells to arrest growth or die in the absence of estrogenic growth-inducing conditions. Although mutations in this gene are uncommon in sporadic breast and ovarian tumors, BRCA1 expression levels and protein levels have been found to be reduced in sporadic human breast carcinomas [16,17,18,19]. In addition it has been demonstrated [20] that hormone-dependent tumors such as breast and ovarian cancers have a decreased ability to undergo apoptosis. Other mechanisms involving gene regulation may allow for decreased expression of BRCA1 in sporadic tumors. The response of BRCA1 mRNA and protein levels to mitogens and hormones in vitro suggests that BRCA1 may play a role in regulation of cell growth or maintenance [21]. The BRCA1 gene product may be involved in the regulation of hormone response pathways, and the present results demonstrate that loss of BRCA1 may result in loss of inhibitory control of these mitogenic pathways. These studies show that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally be acting as a growth inhibitor. Low BRCA1 levels found in sporadic cancers may be an important factor in tumorigenesis. The present data suggest that diminished levels of BRCA1 not only accelerate proliferation in the BG-1 ovarian carcinoma cell line, but also appear to promote tumorigenesis. We propose that the loss or reduction of BRCA1 may predispose a cell population to neoplastic transformation by altering the balance between cell death and proliferation/survival, rendering it more sensitive to secondary genetic changes.
PMCID: PMC13916  PMID: 11056686
antisense; BRCA1; cell death; estrogen; ovarian cancer; proliferation
3.  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 
BMC Cancer  2014;14:73.
Background
Triple-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.
Methods
Triple-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.
Results
HCC1937 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.
Conclusions
This 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.
doi:10.1186/1471-2407-14-73
PMCID: PMC3933379  PMID: 24507701
Ruthenium; BRCA1; Triple-negative; Cell cycle; Apoptosis; BRCA1; Ubiquitination
4.  BRCA1 Is a Histone-H2A-Specific Ubiquitin Ligase 
Cell Reports  2014;8(4):999-1005.
Summary
The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.
Graphical Abstract
Highlights
•BRCA1/BARD1 is a histone-H2A-specific ubiquitin ligase•BRCA1/BARD1 ubiquitylates histone H2A on lysines 127 and 129•Specificity for H2A is provided within in a nucleosomal context•Heterodimeric RING domains of BRCA1/BARD1 are sufficient for E3 function in vivo
The ubiquitin ligase (E3) activity of BRCA1 is its only known biochemical activity in vitro. Kalb et al. report that the heterodimeric complex formed by BRCA1/BARD1 ubiquitylates histone H2A in nucleosomes specifically at its C-terminal tail. Moreover, in vitro and in vivo assays identify H2A lysines127 and 129 as the target lysines. These results help to explain the localization and activity of BRCA1/BARD1 on chromatin in cells.
doi:10.1016/j.celrep.2014.07.025
PMCID: PMC4382519  PMID: 25131202
5.  The ubiquitin E3 ligase activity of BRCA1 and its biological functions 
Cell Division  2008;3:1.
The basal-like breast cancer, a new category of breast cancer associated with poor prognosis and possibly unique chemosensitivity, is a current topic in the breast cancer field. Evidence from multiple sources strongly indicate that impairment of BRCA1 pathways is responsible for this phenotype, implying the importance of BRCA1 not only in familial breast cancers but also in sporadic cancers. BRCA1 acts as a hub protein that coordinates a diverse range of cellular pathways to maintain genomic stability. BRCA1 participates in multiple cellular supercomplexes to execute its tasks and, in most of the complexes, BRCA1 exists as a RING heterodimer with BARD1 to provide ubiquitin E3 ligase activity that is required for its tumor suppressor function. It was revealed recently that the BRCA1 RING finger is capable of catalyzing multiple types of ubiquitination depending upon the interacting E2, the ubiquitin carrier protein. BRCA1 may catalyze distinct ubiquitination on different substrates as the situation demands. On the other hand, in response to DNA double-strand breaks where BRCA1 plays its major role for homologous recombination repair, recent evidence showed that ubiquitination is a critical step to recruit BRCA1 to the damaged site through UIM (ubiquitin interacting motif) containing protein RAP80. Thus, ubiquitin and BRCA1 likely affect each other in many ways to perform cellular functions. Elucidation of this mechanism in relation to cell survival is now much anticipated because it could be a key to predict chemosensitivity of basal-like breast cancer.
doi:10.1186/1747-1028-3-1
PMCID: PMC2254412  PMID: 18179693
6.  Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer 
Scientific Reports  2016;6:26273.
BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1β may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer.
doi:10.1038/srep26273
PMCID: PMC4873788  PMID: 27197561
7.  RAP80 Targets BRCA1 to Specific Ubiquitin Structures at DNA Damage Sites 
Science (New York, N.Y.)  2007;316(5828):1198-1202.
Mutations affecting the BRCT domains of the breast cancer–associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-γH2AX–dependent lysine6- and lysine63-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.
doi:10.1126/science.1139516
PMCID: PMC2706583  PMID: 17525341
8.  Drosophila brca2 Is Required for Mitotic and Meiotic DNA Repair and Efficient Activation of the Meiotic Recombination Checkpoint 
PLoS Genetics  2008;4(2):e31.
Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.
Author Summary
Breast cancer can arise due to inherited mutations in a few well-defined breast cancer susceptibility genes. BRCA2 is one of two known human genes in which common mutations are associated with high breast cancer risk. A known function of BRCA2 is the repair of damaged DNA using the homologous recombination repair pathway. In the absence of BRCA2, damaged DNA is repaired by error-prone repair. However, as many other genes required for homologous recombination are not known breast cancer susceptibility genes, BRCA2 likely has additional roles in tumor suppression. In this study, we created fruit flies mutant for the homolog of BRCA2. We show that, like in humans, fruit fly Brca2 is required for DNA repair by homologous recombination. Additionally, we use this new animal model to discover a previously unknown role for Brca2 in the production of a signal that tells cells that damaged DNA is present during meiosis. This “checkpoint” signal may allow the cells to coordinate a protective response after DNA damage.
doi:10.1371/journal.pgen.0040031
PMCID: PMC2233675  PMID: 18266476
9.  BRCA1 Targets G2/M Cell Cycle Proteins for Ubiquitination and Proteasomal Degradation 
Oncogene  2012;32(42):5005-5016.
The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism which is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provides mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and thus, contributes to maintenance of genomic stability.
doi:10.1038/onc.2012.522
PMCID: PMC3796024  PMID: 23246971
BRCA1; cyclin B; Cdc25; ubiquitination; proteasome; G2/M cell cycle checkpoint
10.  The UBXN1 Protein Associates with Autoubiquitinated Forms of the BRCA1 Tumor Suppressor and Inhibits Its Enzymatic Function▿  
Molecular and Cellular Biology  2010;30(11):2787-2798.
Although the BRCA1 tumor suppressor has been implicated in many cellular processes, the biochemical mechanisms by which it influences these diverse pathways are poorly understood. The only known enzymatic function of BRCA1 is the E3 ubiquitin ligase activity mediated by its highly conserved RING domain. In vivo, BRCA1 associates with the BARD1 polypeptide to form a heterodimeric BRCA1/BARD1 complex that catalyzes autoubiquitination of BRCA1 and trans ubiquitination of other protein substrates. In most cases, BRCA1-dependent ubiquitination generates polyubiquitin chains bearing an unconventional K6 linkage that does not appear to target proteins for proteasomal degradation. Since ubiquitin-dependent processes are usually mediated by cellular receptors with ubiquitin-binding motifs, we screened for proteins that specifically bind autoubiquitinated BRCA1. Here we report that the UBXN1 polypeptide, which contains a ubiquitin-associated (UBA) motif, recognizes autoubiquitinated BRCA1. This occurs through a bipartite interaction in which the UBA domain of UBXN1 binds K6-linked polyubiquitin chains conjugated to BRCA1 while the C-terminal sequences of UBXN1 bind the BRCA1/BARD1 heterodimer in a ubiquitin-independent fashion. Significantly, the E3 ligase activity of BRCA1/BARD1 is dramatically reduced in the presence of UBXN1, suggesting that UBXN1 regulates the enzymatic function of BRCA1 in a manner that is dependent on its ubiquitination status.
doi:10.1128/MCB.01056-09
PMCID: PMC2876507  PMID: 20351172
11.  PALB2 Functionally Connects the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2 
Molecular cancer research : MCR  2009;7(7):1110-1118.
BRCA1 and BRCA2 are prominently associated with inherited breast and ovarian cancer. The encoded proteins function in DNA damage responses, but no functional link between BRCA1 and BRCA2 has been established. We show here that PALB2 physically and functionally connects BRCA1 and BRCA2 into a DNA damage response network that also includes the RAD51 recombinase. PALB2 directly binds BRCA1, as determined with bacterially expressed fragments of each protein. Furthermore, PALB2 independently interacts with BRCA1 and BRCA2 through its NH2 and COOH termini, respectively. Critically, two point mutants (L21P and L24P) of the PALB2 coiled-coil domain or an NH2-terminal deletion (Δ1–70) disrupt its interaction with BRCA1. We have reconstituted PALB2-deficient cells with PALB2Δ1–70, PALB2-L21P, or PALB2-L24P, or with COOH-terminally truncated PALB2 that is deficient for interaction with BRCA2. Using extracts from these cells, we find that PALB2 mediates the physical interaction of BRCA2 with a COOH-terminal fragment of BRCA1. Analysis of the assembly of foci in these cells by BRCA1, PALB2, BRCA2, and RAD51 suggests that BRCA1 recruits PALB2, which in turn organizes BRCA2 and RAD51. Resistance to mitomycin C and the repair of DNA double-strand breaks by homologous recombination require the interaction of PALB2 with both BRCA1 and BRCA2. These results suggest that BRCA1 and BRCA2 cooperate in DNA damage responses in a PALB2-dependent manner, and have important implications for the genesis of breast/ovarian cancer and for chemotherapy with DNA interstrand cross-linking agents.
doi:10.1158/1541-7786.MCR-09-0123
PMCID: PMC4928587  PMID: 19584259
12.  Quantitative proteomic identification of the BRCA1 ubiquitination substrates 
Journal of proteome research  2011;10(11):5191-5198.
Mutation of the BRCA1 tumor suppressor gene predisposes women to hereditary breast and ovarian cancers. BRCA1 forms a heterodimer with BARD1. The BRCA1/BARD1 heterodimer has ubiquitin ligase activity, considered to play crucial roles in tumor suppression and DNA damage response. Nevertheless, relevant BRCA1 substrates are poorly defined. We have developed a new approach to systematically identify the substrates of ubiquitin ligases by identifying proteins that display enhanced incorporation of His-tagged ubiquitin upon ligase co-expression; using this method, we identified several candidate substrates for BRCA1. These include scaffold attachment factor B2 (SAFB2), Tel2, as well as BARD1. BRCA1 was found to enhance SAFB protein expression and induce Tel2 nuclear translocation. Identification of the ubiquitination substrates has been a major obstacle to understanding the functions of ubiquitin ligases. The quantitative proteomics approach we devised for the identification of BRCA1 substrates will facilitate the identification of ubiquitin ligase-substrate pairs.
doi:10.1021/pr200662b
PMCID: PMC3208807  PMID: 21950761
BRCA1; BARD1; ubiquitination; substrate; quantitative proteomics
13.  Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction 
PLoS ONE  2008;3(1):e1448.
BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.
doi:10.1371/journal.pone.0001448
PMCID: PMC2174531  PMID: 18197258
14.  HP1 regulates the localization of FANCJ at sites of DNA double‐strand breaks 
Cancer Science  2016;107(10):1406-1415.
The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1‐associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double‐strand breaks. Here, we investigated the importance of HP1 and BARD1–HP1 interaction in the localization of FANCJ together with the other BRCA1–BRCT binding proteins to clarify the separate role of the HP1‐mediated pathway from the RNF8/RNF168‐induced ubiquitin‐mediated pathway for BRCA1 function. FANCJ interacts with HP1γ in a BARD1‐dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double‐strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1–HP1 interaction. Taken together, the results suggest that the BRCA1–FANCJ and BRCA1–CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.
doi:10.1111/cas.13008
PMCID: PMC5084677  PMID: 27399284
BRCA1; CtIP; DNA damage response; FANCJ; HP1
15.  BRCA1 and Its Network of Interacting Partners  
Biology  2012;2(1):40-63.
BRCA1 is a large multi-domain protein with a pivotal role in maintaining genome stability and cell cycle progression. Germline mutations in the BRCA1 gene confer an estimated lifetime risk of 60%–80% for breast cancer and 15%–60% for ovarian cancer. Many of the germline mutations associated with cancer development are concentrated in the amino terminal RING domain and the carboxyl terminal BRCT motifs of BRCA1, which are the most well-characterized regions of the protein. The function of BRCA1 in DNA repair, transcription and cell cycle control through the DNA damage response is orchestrated through its association with an impressive repertoire of protein complexes. The association of BRCA1 with ATM/ATR, CHK2 and Aurora A protein kinases regulates cell cycle progression, whilst its association with RAD51 has a direct impact on the repair of double strand DNA breaks (DSBs) by homologous recombination (HR). BRCA1 interactions with the MRN complex of proteins, with the BRCC complex of proteins that exhibit E3 ligase activity and with the phosphor proteins CtIP, BACH1 (BRIP1) and Abraxas (CCDC98) are also implicated in DNA repair mechanisms and cell cycle checkpoint control. BRCA1 through its association with specific proteins and multi-protein complexes is a sentinel of the normal cell cycle control and DNA repair.
doi:10.3390/biology2010040
PMCID: PMC4009870  PMID: 24832651
BRCA1; DNA repair damage response; HR; cell cycle checkpoint regulation; phosphorylation; E3 ligase; ubiquitination
16.  RAP80 and RNF8, key players in the recruitment of repair proteins to DNA damage sites 
Cancer letters  2008;271(2):179-190.
Chromosomal double-strand breaks (DSBs) in eukaryotes provoke a rapid, extensive modification in chromatin flanking the breaks. The DNA damage response (DDR) coordinates activation of cell cycle checkpoints, apoptosis, and DNA repair networks, to ensure accurate repair and genomic integrity. The checkpoint kinase ATM plays a critical role in the initiation of DDR in response to DSBs. The early ATM-mediated phosphorylation of the histone variant H2AX proteins near DSBs leads to the subsequent binding of MDC1, which functions as a scaffold for the recruitment and assembly of many DDR mediators and effectors, including BRCA1. Recent studies have provided new insights into the mechanism by which BRCA1 and associated proteins are recruited to DNA damage foci and revealed key roles for the receptor-associated protein 80 (RAP80) and the E3 ligase RNF8 in this process. RAP80 is an ubiquitin-interaction motif (UIM) containing protein that is associated with a BRCA1/BARD1 complex through its interaction with CCDC98 (Abraxas). The UIMs of RAP80 are critical for targeting this protein complex to DSB sites. Additional studies revealed that after binding γ-H2AX, ATM-phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. This complex catalyzes K63-linked polyubiquitination of histones H2A and γH2AX, which are then recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites. Depletion of RAP80 or RNF8 impairs the translocation of BRCA1 to DNA damage sites and results in defective cell cycle checkpoint control and DSB repair. In this review, we discuss this cascade of protein phosphorylation and ubiquitination and the role it plays in the control of cellular responses to genotoxic stress by regulating the interactions, localization, and function of DDR proteins.
doi:10.1016/j.canlet.2008.04.046
PMCID: PMC2749678  PMID: 18550271
double strand breaks; RAP80; RNF8; ubiquitination; CCDC98/Abraxas; BRCA1; UIM; MDC1
17.  DNA damage-induced cytotoxicity is dissociated from BRCA1’s DNA repair function but dependent on its cytosolic accumulation 
Cancer research  2010;70(15):6258-6267.
The tumor suppressor BRCA1 is a nuclear shuttling protein. However, the role of BRCA1 localization in the control of its functions remains to be elucidated. Given the central role of BRCA1 in DNA damage repair, we hypothesized that depletion of nuclear BRCA1 will compromise its nuclear function in DNA repair and thereby result in enhanced cytotoxic response to DNA damage. In this study, we showed that repair of DNA double strand breaks (DSBs) required BRCA1 in the nucleus. In addition, sequestering BRCA1 in the cytosol enhanced the cytotoxic response to ionizing radiation (IR) or cisplatin in human breast and colon cancer cells. However, further genetic dissection of the mechanism of this enhanced cytotoxicity using BRCA1 mutants deficient in DSB repair unexpectedly revealed a dissociation of BRCA1’s function in DNA repair from its effects on cellular sensitivity to DNA damage. Interestingly, we observed a dependence of the DNA damage-induced cell killing on the translocation and accumulation of BRCA1 in the cytosol. Together, these data suggest a novel role of cytoplasmic translocation of BRCA1, not only in controlling its DNA repair functions, but also in the regulation of cell death processes following DNA damage. Further dissection of the mechanism of cytotoxicity induced by BRCA1 cytoplasmic translocation revealed involvement of the apoptotic pathway. We propose that the status of BRCA1 nuclear/cytoplasmic shuttling may provide a molecular marker to predict tumor response and a potential novel target to sensitize cancer cells to DNA damage-based therapy.
doi:10.1158/0008-5472.CAN-09-4713
PMCID: PMC2912952  PMID: 20631074
BRCA1; nuclear export; apoptosis; DNA repair; homologous recombination; radiation sensitization; checkpoint
18.  AKT Regulates BRCA1 Stability in Response to Hormone Signaling 
BRCA1, with its binding partner BARD1, regulates the cellular response to DNA damage in multiple tissues, yet inherited mutations within BRCA1 result specifically in breast and ovarian cancers. This observation, along with several other lines of evidence, suggests a functional relationship may exist between hormone signaling and BRCA1 function. Our data demonstrates that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling. Further, we have identified a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. This rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis and treatment with the clinically utilized proteasome inhibitor bortezomib similarly leads to a rapid increase in BRCA1 protein levels. Together, these data suggest that AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. We conclude that AKT regulates BRCA1 protein stability and function through direct phosphorylation of BRCA1. Further, the responsiveness of the AKT-BRCA1 regulatory pathway to hormone signaling may, in part, underlie the tissue specificity of BRCA1 mutant cancers. Pharmacological targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for the treatment of breast and ovarian cancers.
doi:10.1016/j.mce.2010.01.019
PMCID: PMC4548798  PMID: 20085797
BRCA1; Estrogen; Breast Cancer; AKT; Proteasomal degradation; Phosphorylation
19.  Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains 
PLoS Genetics  2011;7(12):e1002409.
The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.
Author Summary
The breast tumor suppressor BRCA2 has a major role in DNA repair by homologous recombination (HR). BRCA2 is a large protein with multiple domains that interact with several proteins as well as with DNA, complicating our understanding of how the protein functions in cells. To investigate the mechanism by which BRCA2 functions in HR in cells, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that DNA binding by BRCA2 is critical when a BRCA2 peptide is deficient in binding another breast cancer suppressor, PALB2, but not when the peptide can bind PALB2, suggesting alternative mechanisms of activity. Binding the small peptide DSS1 is also essential for HR only in some contexts, as are activities in the BRCA2 C terminus. Our results reveal redundancy of BRCA2 domains and emphasize plasticity within this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.
doi:10.1371/journal.pgen.1002409
PMCID: PMC3240595  PMID: 22194698
20.  BRCA1185delAG tumors may acquire therapy resistance through expression of RING-less BRCA1 
The Journal of Clinical Investigation  null;126(8):2903-2918.
Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain–less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.
doi:10.1172/JCI70196
PMCID: PMC4966325  PMID: 27454287
21.  RING domain–deficient BRCA1 promotes PARP inhibitor and platinum resistance 
The Journal of Clinical Investigation  null;126(8):3145-3157.
Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain–deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (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. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.
doi:10.1172/JCI87033
PMCID: PMC4966309  PMID: 27454289
22.  Blood cellular mutant LXR-α protein stability governs initiation of coronary heart disease 
World Journal of Cardiology  2013;5(8):305-312.
AIM: To investigate the role of [breast and ovarian cancer susceptibility 1 (BRCA1)-associated RING domain 1 (BARD1)]/BRCA1 E3-ubiquitin ligase complex in governing the stability of mutant liver X receptor-α (LXR-α) protein in coronary heart disease (CHD) subjects.
METHODS: The expression analysis of various genes was carried out by quantitative real time polymerase chain reaction and western blotting within blood mononuclear cells of human CHD subjects at various stages of coronary occlusion and their corresponding normal healthy counterparts. Immunoprecipitation experiments were performed to establish protein interactions between LXR-α and BARD1. Peripheral blood mononuclear cells were cultured and exposed to Vitamin D3 and Cisplatin to validate the degradation of mutant LXR-α protein in CHD subjects by BARD1/BRCA1 complex.
RESULTS: The expression of mutant LXR-α protein in CHD subjects was found to decrease gradually with the severity of coronary occlusion exhibiting a strong negative correlation, r = -0.975 at P < 0.001. Further, the expression of BARD1 and BRCA1 also increased with the disease severity, r = 0.895 and 0.873 respectively (P < 0.001). Immunoprecipitation studies established that BARD1/BRCA1 complex degrades mutant LXR-α via ubiquitination. The absence of functional LXR-α protein resulted in increased expression of inflammatory cytokines such as interleukin (IL)-6, IL-8 and interferon-γ and decreased expression of ABCA1 (ATP-binding cassette A1) (r = 0.932, 0.949, 0.918 and -0.902 with respect to Gensini score; P < 0.001). Additionally, cell culture experiments proved that Vitamin D3 could prevent the degradation of mutant LXR-α and restore its functional activity to some extent.
CONCLUSION: Mutant LXR-α protein in CHD subjects is degraded by BARD1/BRCA1 complex and Vitamin D3 can rescue and restore its function.
doi:10.4330/wjc.v5.i8.305
PMCID: PMC3761184  PMID: 24009820
Mutant liver X receptor-α; Ubiquitination; Breast and ovarian cancer susceptibility 1-associated RING domain 1/breast and ovarian cancer susceptibility 1; Mononuclear Cells; Coronary heart disease subjects; Vitamin D3
23.  BRCA2 Is Ubiquitinated In Vivo and Interacts with USP11, a Deubiquitinating Enzyme That Exhibits Prosurvival Function in the Cellular Response to DNA Damage 
Molecular and Cellular Biology  2004;24(17):7444-7455.
Individuals carrying a germ line mutation of the breast cancer susceptibility gene BRCA2 are predisposed to breast, ovarian, and other types of cancer. The BRCA2 protein has been proposed to function in the repair of DNA double-strand breaks. Using an immunopurification-mass spectrometry approach to identify novel proteins that associate with the BRCA2 gene product, we found that a deubiquitinating enzyme, USP11, formed specific complexes with BRCA2. Moreover, BRCA2 was constitutively ubiquitinated in vivo in the absence of detectable proteasomal degradation. Mitomycin C (MMC) led to decreased BRCA2 protein levels associated with increased ubiquitination, consistent with proteasome-dependent degradation. While BRCA2 could be deubiquitinated by USP11 in transient overexpression assays, a catalytically inactive USP11 mutant had no effect on BRCA2 ubiquitination or protein levels. Antagonism of USP11 function either through expression of this mutant or through RNA interference increased cellular sensitivity to MMC in a BRCA2-dependent manner. All of these results imply that BRCA2 expression levels are regulated by ubiquitination in the cellular response to MMC-induced DNA damage and that USP11 participates in DNA damage repair functions within the BRCA2 pathway independently of BRCA2 deubiquitination.
doi:10.1128/MCB.24.17.7444-7455.2004
PMCID: PMC506974  PMID: 15314155
24.  The BARD1 Cys557Ser Variant and Breast Cancer Risk in Iceland 
PLoS Medicine  2006;3(7):e217.
Background
Most, if not all, of the cellular functions of the BRCA1 protein are mediated through heterodimeric complexes composed of BRCA1 and a related protein, BARD1. Some breast-cancer-associated BRCA1 missense mutations disrupt the function of the BRCA1/BARD1 complex. It is therefore pertinent to determine whether variants of BARD1 confer susceptibility to breast cancer. Recently, a missense BARD1 variant, Cys557Ser, was reported to be at increased frequencies in breast cancer families. We investigated the role of the BARD1 Cys557Ser variant in a population-based cohort of 1,090 Icelandic patients with invasive breast cancer and 703 controls. We then used a computerized genealogy of the Icelandic population to study the relationships between the Cys557Ser variant and familial clustering of breast cancer.
Methods and Findings
The Cys557Ser allele was present at a frequency of 0.028 in patients with invasive breast cancer and 0.016 in controls (odds ratio [OR] = 1.82, 95% confidence interval [CI] 1.11–3.01, p = 0.014). The alleleic frequency was 0.037 in a high-predisposition group of cases defined by having a family history of breast cancer, early onset of breast cancer, or multiple primary breast cancers (OR = 2.41, 95% CI 1.22–4.75, p = 0.015). Carriers of the common Icelandic BRCA2 999del5 mutation were found to have their risk of breast cancer further increased if they also carried the BARD1 variant: the frequency of the BARD1 variant allele was 0.047 (OR = 3.11, 95% CI 1.16–8.40, p = 0.046) in 999del5 carriers with breast cancer. This suggests that the lifetime probability of a BARD1 Cys557Ser/BRCA2 999del5 double carrier developing breast cancer could approach certainty. Cys557Ser carriers, with or without the BRCA2 mutation, had an increased risk of subsequent primary breast tumors after the first breast cancer diagnosis compared to non-carriers. Lobular and medullary breast carcinomas were overrepresented amongst Cys557Ser carriers. We found that an excess of ancestors of contemporary carriers lived in a single county in the southeast of Iceland and that all carriers shared a SNP haplotype, which is suggestive of a founder event. Cys557Ser was found on the same SNP haplotype background in the HapMap Project CEPH sample of Utah residents.
Conclusions
Our findings suggest that BARD1 Cys557Ser is an ancient variant that confers risk of single and multiple primary breast cancers, and this risk extends to carriers of the BRCA2 999del5 mutation.
Editors' Summary
Background.
About 13% of women (one in eight women) will develop breast cancer during their lifetime, but many factors affect the likelihood of any individual woman developing this disease, for example, whether she has had children and at what age, when she started and stopped her periods, and her exposure to certain chemicals or radiation. She may also have inherited a defective gene that affects her risk of developing breast cancer. Some 5%–10% of all breast cancers are familial, or inherited. In 20% of these cases, the gene that is defective is BRCA1 or BRCA2. Inheriting a defective copy of one of these genes greatly increases a woman's risk of developing breast cancer, while researchers think that the other inherited genes that predispose to breast cancer—most of which have not been identified yet—have a much weaker effect. These are described as low-penetrance genes. Inheriting one such gene only slightly increases breast cancer risk; a woman has to inherit several to increase her lifetime risk of cancer significantly.
Why Was This Study Done?
It is important to identify these additional predisposing gene variants because they might provide insights into why breast cancer develops, how to prevent it, and how to treat it. To find low-penetrance genes, researchers do case–control association studies. They find a large group of women with breast cancer (cases) and a similar group of women without cancer (controls), and examine how often a specific gene variant occurs in the two groups. If the variant is found more often in the cases than in the controls, it might be a variant that increases a woman's risk of developing breast cancer.
What Did the Researchers Do and Find?
The researchers involved in this study recruited Icelandic women who had had breast cancer and unaffected women, and looked for a specific variant—the Cys557Ser allele—of a gene called BARD1. They chose BARD1 because the protein it encodes interacts with the protein encoded by BRCA1. Because defects in BRCA1 increase the risk of breast cancer, defects in an interacting protein might have a similar effect. In addition, the Cys557Ser allele has been implicated in breast cancer in other studies. The researchers found that the Cys557Ser allele was nearly twice as common in women with breast cancer as in control women. It was also more common (but not by much) in women who had a family history of breast cancer or who had developed breast cancer more than once. And having the Cys557Ser allele seemed to increase the already high risk of breast cancer in women who had a BRCA2 variant (known as BRCA2 999del5) that accounts for 40% of inherited breast cancer risk in Iceland.
What Do These Findings Mean?
These results indicate that inheriting the BARD1 Cys557Ser allele increases a woman's breast cancer risk but that she is unlikely to have a family history of the disease. Because carrying the Cys557Ser allele only slightly increases a woman's risk of breast cancer, for most women there is no clinical reason to test for this variant. Eventually, when all the low-penetrance genes that contribute to breast cancer risk have been identified, it might be helpful to screen women for the full set to determine whether they are at high risk of developing breast cancer. This will not happen for many years, however, since there might be tens or hundreds of these genes. For women who carry BRCA2 999del5, the situation might be different. It might be worth testing these women for the BARD1 Cys557Ser allele, the researchers explain, because the lifetime probability of developing breast cancer in women carrying both variants might approach 100%. This finding has clinical implications in terms of counseling and monitoring, as does the observation that Cys557Ser carriers have an increased risk of a second, independent breast cancer compared to non-carriers. However, all these findings need to be confirmed in other groups of patients before anyone is routinely tested for the BARD1 Cys557Ser allele.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030217.
• MedlinePlus pages about breast cancer
• Information on breast cancer from the United States National Cancer Institute
• Information on inherited breast cancer from the United States National Human Genome Research Institute
• United States National Cancer Institute information on genetic testing for BRCA1 and BRCA2 variants
• GeneTests pages on the involvement of BRCA1 and BRCA2 in hereditary breast and ovarian cancer
• Cancer Research UK's page on breast cancer statistics
In a population-based cohort of 1090 Icelandic patients, a Cys557Ser missense variant of the BARD1 gene, which interacts with BRCA1, increased the risk of single and multiple primary breast cancers.
doi:10.1371/journal.pmed.0030217
PMCID: PMC1479388  PMID: 16768547
25.  Combinatory effect of BRCA1 and HERC2 expression on outcome in advanced non-small-cell lung cancer 
BMC Cancer  2016;16:312.
Background
BRCA1 is a main component of homologous recombination and induces resistance to platinum in preclinical models. It has been studied as a potential predictive marker in lung cancer. Several proteins modulate the function of BRCA1. The E3 ubiquitin ligase HERC2 facilitates the assembly of the RNF8-UBC13 complex to recruit BRCA1 to DNA damage sites. The combined analysis of multiple components of the pathway leading to the recruitment of BRCA1 at DNA damage sites has the potentiality to improve the BRCA1 predictive model.
Methods
We retrospectively analyzed 71 paraffin-embedded tumor samples from advanced non-small-cell lung cancer patients treated with first-line platinum based chemotherapy and measured the mRNA expression levels of BRCA1, RNF8, UBC13 and HERC2 using real-time PCR. The mRNA expression was categorized using median value as cut-off point.
Results
The median progression-free survival of all 71 patients was 7.2 months whereas the median overall survival of the study population was 10.7 months. Among patients with low BRCA1 expression, the median PFS was 7.4 months in the presence of low HERC2 levels and 5.9 months for patients expressing high HERC2 levels (p = 0.01). The median OS was 15.3 months for patients expressing low levels of both genes and 7.4 months for those with low BRCA1 but high HERC2 (p = 0.008). The multivariate analysis showed that among patients with Eastern Cooperative Oncology Group performance status 0–1, the combined low expression of both BRCA1 and HERC2 clearly reduced the risk of progression (p = 0.03) and of death (p = 0.004).
Conclusions
These findings confirm the potentiality of integrated DNA repair components analysis in predicting the sensitivity to platinum in lung cancer. The study indicates a predictive role for HERC2 mRNA expression and paves the way for further refinement of the BRCA1 predictive model.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2339-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-016-2339-5
PMCID: PMC4868003  PMID: 27179511
BRCA1; HERC2; Non-small-cell lung cancer; Platinum; Predictive markers; DNA repair

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