The role of the BRCA1 tumor suppressor in DNA double-strand break repair

The tumor suppressor gene BRCA1 was cloned in 1994 based on its linkage to early-onset breast and ovarian cancer. Although the BRCA1 protein has been implicated in multiple cellular functions, the precise mechanism that determines its tumor suppressor activity is not defined. Currently, the emerging picture is that BRCA1 plays an important role in maintaining genomic integrity by protecting cells from double-strand breaks (DSB) that arise during DNA replication or after DNA damage. The DSB repair pathways available in mammalian cells are homologous recombination and nonhomologous end-joining. BRCA1 function seems to be regulated by specific phosphorylations in response to DNA damage and we will focus this review on the roles played by BRCA1 in DNA repair and cell cycle checkpoints. Finally, we will explore the idea that tumor suppression by BRCA1 depends on its control of DNA DSB repair, resulting in the promotion of error-free and the inhibition of error-prone recombinational repair.     

BRCA1 modulates ionizing radiation-induced nuclear focus formation by the replication protein A p34 subunit

Mutations in BRCA1 account for a significant proportion of familial breast and ovarian cancers. BRCA1 has been implicated in DNA damage responses including double-strand break (DSB) repair. However, its exact role in DSB repair and its functional relationship with other known repair proteins remain to be elucidated. In this study, we carried out a cytological analysis of the effect of BRCA1 on damage-induced nuclear focus formation mediated by the replication protein A (RPA). RPA is a multi-functional protein that participates in both DNA replication and various types of DNA repair including DSB repair. Following ionizing radiation (IR), RPA and BRCA1 formed punctate nuclear staining patterns that co-localized with each other, consistent with the implicated roles of both proteins in the same repair process. The number of damage-induced RPA foci in BRCA1-deficient cells, however, was significantly greater than that in BRCA1-positive cells. Moreover, the effect of BRCA1 on the RPA staining pattern appeared to be specific for IR but not ultraviolet (UV) irradiation. These data suggest that BRCA1 plays an important role in processing the RPA-associated intermediates during DSB repair.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide.     

Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer

Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Repair kinetics of genomic interstrand DNA cross-links: evidence for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway

The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.     

Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with potential diagnostic and prognostic value in human breast cancer

Breast cancer 1, early onset (BRCA1) hereditary breast cancer, a type of cancer with defects in the homology-directed DNA repair pathway, would benefit from the identification of proteins for diagnosis, which might also be of potential use as screening, prognostic, or predictive markers. Sporadic breast cancers with defects in the BRCA1 pathway might also be diagnosed. We employed proteomics based on one-dimensional gel electrophoresis in combination with nano-LC-MS/MS and spectral counting to compare the protein profiles of mammary tumor tissues of genetic mouse models either deficient or proficient in BRCA1. We identified a total of 3,545 proteins, of which 801 were significantly differentially regulated between the BRCA1-deficient and -proficient breast tumors. Pathway and protein complex analysis identified DNA repair and related functions as the major processes associated with the up-regulated proteins in the BRCA1-deficient tumors. In addition, by selecting highly connected nodes, we identified a BRCA1 deficiency signature of 45 proteins that enriches for homology-directed DNA repair deficiency in human gene expression breast cancer data sets. This signature also exhibits prognostic power across multiple data sets, with optimal performance in a data set enriched in tumors deficient in homology-directed DNA repair. In conclusion, by comparing mouse proteomes from BRCA1-proficient and -deficient mammary tumors, we were able to identify several markers associated with BRCA1 deficiency and a prognostic signature for human breast cancer deficient in homology-directed DNA repair.     

Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage

The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.     

Identification of sequences that target BRCA1 to nuclear foci following alkylative DNA damage

BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein gamma-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1-304) and the C-terminal sequence aa1078-1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage.     

Recognition of DNA double strand breaks by the BRCA1 tumor suppressor network

DNA double-strand breaks (DSBs) occur in response to both endogenous and exogenous genotoxic stress. Inappropriate repair of DSBs can lead to either loss of viability or to chromosomal alterations that increase the likelihood of cancer development. In strong support of this assertion, many cancer predisposition syndromes stem from germline mutations in genes involved in DNA DSB repair. Among the most prominent of such tumor suppressor genes are the Breast Cancer 1 and Breast Cancer 2 genes (BRCA1 and BRCA2), which are mutated in familial forms of breast and ovarian cancer. Recent findings implicate BRCA1 as a central component of several distinct macromolecular protein complexes, each dedicated to distinct elements of DNA DSB repair and tumor suppression. Emerging evidence has shed light on some of the molecular recognition processes that are responsible for targeting BRCA1 and its associated partners to DNA and chromatin directly flanking DSBs. These events are required for BRCA1-dependent DNA repair and tumor suppression. Thus, a detailed temporal and spatial knowledge of how breaks are recognized and repaired has profound implications for understanding processes related to the genesis of malignancy and to its treatment.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.     

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H₂O₂-induced DNA damage. UVC and H₂O₂ treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G₀, G₁ and S-phase. Rad18 was important for repressing H₂O₂-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G₁, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H₂O₂-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H₂O₂-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G₁. In contrast with G₁-synchronized cultures, S-phase cells were H₂O₂-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G₁ (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.     

BRCA1 participates in DNA decatenation

The tumor suppressor BRCA1 has an important function in the maintenance of genomic stability. Increasing evidence suggests that BRCA1 regulates cell cycle checkpoints and DNA repair after DNA damage. However, little is known about its normal function in the absence of DNA damage. Here we show that BRCA1 interacts and colocalizes with topoisomerase IIalpha in S phase cells. Similar to cells treated with the topoisomerase IIalpha inhibitor ICRF-193, BRCA1-deficient cells show lagging chromosomes, indicating a defect in DNA decatenation and chromosome segregation. More directly, BRCA1 deficiency results in defective DNA decatenation in vitro. Finally, topoisomerase IIalpha is ubiquitinated in a BRCA1-dependent manner, and topoisomerase IIalpha ubiquitination correlates with higher DNA decatenation activity. Together these results suggest an important role of BRCA1 in DNA decatenation and reveal a previously unknown function of BRCA1 in the maintenance of genomic stability.     

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.