Interplay among BRCA1, SIRT1, and Survivin during BRCA1-associated tumorigenesis

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. However, the downstream mediators of BRCA1 function in tumor suppression remain elusive. We found that human BRCA1-associated breast cancers have lower levels of SIRT1 than their normal controls. We further demonstrated that mammary tumors from Brca1 mutant mice have low levels of Sirt1 and high levels of Survivin, which is reversed by induced expression of Brca1. BRCA1 binds to the SIRT1 promoter and increases SIRT1 expression, which in turn inhibits Survivin by changing the epigenetic modification of histone H3. Absence of SIRT1 blocks the regulation of Survivin by BRCA1. Furthermore, we demonstrated that activation of Sirt1 and inhibition of Survivin expression by resveratrol elicit a more profound inhibitory effect on Brca1 mutant cancer cells than on Brca1-wild-type cancer cells both in vitro and in vivo. These findings suggest that resveratrol treatment serves as an excellent strategy for targeted therapy for BRCA1-associated breast cancer.     

Haploinsufficiency of Parp1 accelerates Brca1-associated centrosome amplification, telomere shortening, genetic instability, apoptosis, and embryonic lethality

The breast tumor associated gene-1 (BRCA1) and poly(ADP-ribose) polymerase-1 (PARP1) are both involved in DNA-damage response and DNA-damage repair. Recent investigations have suggested that inhibition of PARP1 represents a promising chemopreventive/therapeutic approach for specifically treating BRCA1- and BRCA2-associated breast cancer. However, studies in mouse models reveal that Parp1-null mutation results in genetic instability and mammary tumor formation, casting significant doubt on the safety of PARP1 inhibition as a therapy for the breast cancer. To study the genetic interactions between Brca1 and Parp1, we interbred mice carrying a heterozygous deletion of full-length Brca1 (Brca1(+/Delta11)) with Parp1-null mice. We show that Brca1(Delta11/Delta11);Parp1(-/-) embryos die before embryonic (E) day 6.5, whereas Brca1(Delta11/Delta11) embryos die after E12.5, indicating that absence of Parp1 dramatically accelerates lethality caused by Brca1 deficiency. Surprisingly, haploinsufficiency of Parp1 in Brca1(Delta11/Delta11) embryos induces a severe chromosome aberrations, centrosome amplification, and telomere dysfunction, leading to apoptosis and accelerated embryonic lethality. Notably, telomere shortening in Brca1(Delta11/Delta11);Parp1(+/-) MEFs was correlated with decreased expression of Ku70, which plays an important role in telomere maintenance. Thus, haploid loss of Parp1 is sufficient to induce lethality of Brca1-deficient cells, suggesting that partial inhibition of PARP1 may represent a practical chemopreventive/therapeutic approach for BRCA1-associated breast cancer.     

Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.     

Breast cancer cell response to genistein is conditioned by BRCA1 mutations

Soy phytoestrogens, among which genistein, seem to protect from breast cancer development. In order to study the role of the breast tumour suppressor BRCA1 in response to genistein, we used a new breast cancer cell model: the SUM1315MO2 cell line carrying the 185delAG BRCA1 mutation, which we stably transfected with a plasmid encoding wild-type BRCA1. We showed that growth of BRCA1 mutant cells was strongly inhibited by genistein whereas it only had a weak effect in cells expressing wild-type BRCA1 protein. BRCA1 mutant cells hypersensitivity could be linked to higher expression of ERβ gene, which suggests that genistein may be an efficient inhibitor of cancer development in BRCA1 mutant breast cancer cells.     

Preclinical evaluation of radiation therapy of BRCA1-associated mammary tumors using a mouse model

Although germline mutations in BRCA1 highly predispose women towards breast and ovarian cancer, few substantial improvements in preventing or treating such cancers have been made. Importantly, BRCA1 function is closely associated with DNA damage repair, which is required for genetic stability. Here, we examined the efficacy of radiotherapy, assessing the accumulation of genetic instabilities, in the treatment of BRCA1-associated breast cancer using a Brca1-mutant mouse model. Treatment of Brca1-mutant tumor-engrafted mice with X-rays reduced tumor progression by 27.9% compared with untreated controls. A correlation analysis of irradiation responses and biomarker profiles in tumors at baseline identified differences between responders and non-responders at the protein level (pERα, pCHK2, p53, and EpCAM) and at the SOX2 target expression level. We further demonstrated that combined treatment of Brca1-mutant mammary tumors with irradiation and AZD2281, which inhibits PARP, significantly reduced tumor progression and extended survival. Our findings enhance the understanding of DNA damage and biomarker responses in BRCA1-associated mammary tumors and provide preclinical evidence that radiotherapy with synthetic DNA damage is a potential strategy for the therapeutic management of BRCA1-associated breast cancer.     

Hyperplasia and spontaneous tumor development in the gynecologic system in mice lacking the BRCA1-Delta11 isoform

Alternative splicing in the BRCA1 locus generates multiple protein products including BRCA1-Delta11, which is identical to the BRCA1 full-length isoform (BRCA1-FL) except for the absence of exon 11. Mutation analysis using gene targeting to create null mutations or disrupt BRCA-FL has provided much of our understanding of BRCA1 functions; however, targeted mutation of specific short forms of BRCA1 has not been reported. To understand the physiologic functions of BRCA1-Delta11, we used a knock-in approach that blocks alternative splicing between exons 10 and 12 to prevent the formation of this form of BRCA1. We showed that homozygous mutant mice (Brca1(FL/FL)) were born at a Mendelian ratio without obvious developmental defects. However, the majority of Brca1(FL/FL) female mice showed mammary gland abnormalities and uterine hyperplasia after one year of age with spontaneous tumor formation. Cultured Brca1(FL/FL) cells exhibited abnormal centrosome amplification and reduction of G(1) population that was accompanied by accumulation of cyclin E and cyclin A. Accumulation of cyclin E was also found in epithelial layers of dilated ducts and hyperproliferative lobular regions in the mammary glands of Brca1(FL/FL) mice. These observations provide evidence that BRCA1 splicing variants are involved in BRCA1 functions in modulating G(1)/S transition, centrosome duplication, and repressing tumor formation.     

Genetic steps of mammalian homologous repair with distinct mutagenic consequences

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.     

Can soy phytoestrogens decrease DNA methylation in BRCA1 and BRCA2 oncosuppressor genes in breast cancer?

Although soy phytoestrogens have been postulated to exert a protective effect against breast cancer, the attendant mechanisms, in particular epigenetics underpinnings, have remained elusive. We investigated the putative effects on DNA methylation by two naturally occurring isoflavones, genistein and daidzein, in a study of the BRCA1 and BRCA2 oncosuppressor genes in breast cancer cell lines (MCF-7, MDA-MB 231, and MCF10a). A demethylant agent, the 5-azacytidine, and a methylant, the budesonide, were used as treatment controls. DNA methylation of BRCA1 and BRCA2 was investigated with methylated DNA immunoprecipitation coupled with PCR. In parallel, protein expression was determined by Western blot, immunohistochemistry, and confocal microscopy. Our results suggest that treatment with 18.5?μM Genistein or 78.5?μM Daidzein might reverse DNA hypermethylation and restore the expression of the oncosuppressor genes BRCA1 and BRCA2. 5-Azacitydine also enhanced the reexpression of these genes while budesonide had an opposite effect. To the best of our knowledge, these observations, while requiring replication, provide new evidence on potential epigenetic mechanisms by which genistein and daidzein might contribute to regulation of the BRCA1 and BRCA2. Future studies are warranted on whether the demethylating effect of genistein and daidzein is global or focused on select candidate genes.     

Mouse models for BRCA1 associated tumorigenesis: From fundamental insights to preclinical utility

Germline mutations in BRCA1 result in a significant predisposition for breast and ovarian cancer, with frequent LOH of the remaining wild type allele. Soon after the identification of BRCA1, several different knockout mice were generated to study its biological function in vivo. BRCA1, which is involved in DNA double-strand break (DSB) repair, appeared to be essential for embryonic proliferation and survival during mid-gestation. In contrast to human mutation carriers however, heterozygous mouse mutants did not show spontaneous cancer development. Therefore, a number of conditional mouse models were developed. While tumors of these mice show varying degrees of similarity with their human counterparts, two mouse models develop mammary tumors that lack expression of estrogen and progesterone receptors and ERBB2. This ‘triple negative’ signature is a characteristic feature of BRCA1-associated breast cancers, which can therefore not be treated with endocrine agents or ERBB2-targeting therapeutics. Promising drugs for treating BRCA1-mutated tumors include platinum compounds and PARP inhibitors, which are specifically toxic to DSB repair deficient cells. Although encouraging results have been reported, recent findings indicate that BRCA1/2 deficient ovarian tumors can escape from such targeted treatment by genetic reversion. This resistance mechanism might be studied in future mouse tumor models based on Brca1 truncating mutations mimicking defined human founder mutations.     

MMTV-trBrca1 mice display strain-dependent abnormalities in vaginal development

The breast cancer susceptibility gene Brca1 encodes a large multi-functional protein which is implicated as a caretaker of the genome, through its role in regulation of DNA damage response pathways, including apoptosis. Here we show that in mice expressing a dominant-negative Brca1 transgene on a BALB/c background, vaginal entrance remodeling is inhibited, and that the incidence of this phenotype is increased on a p53 +/- genotype. Given that this developmental process is mediated primarily by apoptosis, we hypothesized that disruption of BRCA1 may confer a resistance to apoptosis in normal epithelial cells. Consistent with this, we show that expression of this transgene in vitro leads to resistance to ionizing radiation induced cell killing in mammary epithelial cells. This is the first time that BRCA1 has been implicated in an apoptosis-mediated normal developmental process.     

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2‐deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N‐terminal RING domain. This “RING‐less” BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING‐less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING‐less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING‐less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1–BARD1 in genomic integrity that is independent of RAD51 loading.     

The DNA double-strand break response pathway: becoming more BRCAish than ever

Breast carcinoma is the leading cause of cancer incidence, and second in cancer mortality to lung cancer, in women of the Western hemisphere. Germ line mutations in the breast cancer susceptibility gene, BRCA1, is responsible for half of all cases of hereditary breast cancer, which constitutes about 5-10% of all cases of breast cancer. Current hypothesis has ascribed a role for Brca1 in maintaining genomic stability, through its involvement in cellular response pathway to the DNA double-strand breaks (DSB). DNA DSB, which are the most deleterious form of DNA damage, are repaired through a series of coordinated steps embedded in a signal transduction pathway that ultimately ensure the elimination of potentially harmful mutations to the genome. This pathway can be crudely divided into a primary and secondary phase. The primary response phase is initiated by sensor proteins that activate transducer protein kinases Atm and Atr, which target downstream effector proteins, such as Chk1 and Chk2, to elicit the secondary response phase. Brca1 has been intimately linked with various aspects of this signaling pathway. However, the precise role of Brca1 in this process remains unclear. In this review, we will provide a simple model in an attempt to clarify the role of Brca1 during cellular response to DNA DSB.     

Brca2 and Trp53 Deficiency Cooperate in the Progression of Mouse Prostate Tumourigenesis

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.     

Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor,results in early embryonic lethality and chromosomal instability

The BRCA1 tumor suppressor has been implicated in many cellular pathways, but the mechanisms by which it suppresses tumor formation are not fully understood. In vivo BRCA1 forms a heterodimeric complex with the related BARD1 protein, and its enzymatic activity as a ubiquitin ligase is largely dependent upon its interaction with BARD1. To explore the genetic relationship between BRCA1 and BARD1, we have examined the phenotype of Bard1-null mice. These mice become developmentally retarded and die between embryonic day 7.5 (E7.5) and E8.5. Embryonic lethality results from a severe impairment of cell proliferation that is not accompanied by increased apoptosis. In the absence of p53, the developmental defects associated with Bard1 deficiency are partly ameliorated, and the lethality of Bard1; p53-nullizygous mice is delayed until E9.5. This result, together with the increased chromosomal aneuploidy of Bard1 mutant cells, indicates a role for Bard1 in maintaining genomic stability. The striking similarities between the phenotypes of Bard1-null, Brca1-null, and double Bard1; Brca1-null mice provide strong genetic evidence that the developmental functions of Brca1 and Bard1 are mediated by the Brca1/Bard1 heterodimer.     

In Vitro Repression of Brca1-associated RING Domain Gene,Bard1, Induces Phenotypic Changes in Mammary Epithelial Cells

BRCA1-associated RING domain (BARD1) was identified as a protein interacting with the breast cancer gene product BRCA1. The identification of tumorigenic missense mutations within BRCA1 that impair the formation of BARD1–BRCA1 complexes, and of BARD1 mutations in breast carcinomas, sustain the view that BARD1 is involved in BRCA1-mediated tumor suppression. We have cloned the murine Bard1 gene and determined that its expression in different tissues correlates with the expression profile of Brca1. To investigate the function of Bard1, we have reduced Bard1 gene expression in TAC-2 cells, a murine mammary epithelial cell line that retains morphogenetic properties characteristic of normal breast epithelium. Partial repression of Bard1, achieved by the transfection of TAC-2 cells with plasmids constitutively expressing ribozymes or antisense RNAs, resulted in marked phenotypic changes, consisting of altered cell shape, increased cell size, high frequency of multinucleated cells, and aberrant cell cycle progression. Furthermore, Bard1-repressed cell clones overcame contact inhibition of cell proliferation when grown in monolayer cultures and lost the capacity to form luminal structures in three-dimensional collagen gels. These results demonstrate that Bard1 repression induces complex changes in mammary epithelial cell properties which are suggestive of a premalignant phenotype.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.