Tumor Suppressor Function Of BRCA1 Research Articles

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7237.

Abstract C102: mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers

Abstract BRCA1 and BRCA2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers, up to 10% BC, are associated with a lack of expression and/or function of BRCA-1 protein, which induces genomic instability. Considering the major role of BRCA1 gene in DNA repair process, therapeutic approaches using cytotoxic agents such as the platinum and PARP inhibitors are used as treatments. However, hypersensitivity to PARPi is limited in the clinic by acquired resistance after restoration of HR. We have developed a new potent strategy combining mRNAs with a tumor selective nanocarrier, to selectively rescue BRCA-1 functions and restore PARPi sensitivity, as potential therapeutic approach in cancers. ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-901, -902 , -903 nanoparticles containing mRNA derived from BRCA-1 were evaluated on breast cancer cells, harboring different BRCA-1 deletion and mutations. Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry and Tunel assay. BRCA-1 and RAD51 protein levels were evaluated by western blot. Sensitivity to Veliparib (PARPi) following ADGN-mRNA treatment was evaluated on PARPI resistance and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of IV-administered ADGN-BRCA NPs (2.0 mg/kg) was evaluated on breast (HCC1937-BRCA-1 deficient, PARPi resistant) cancer mouse xenografts. We have selected specific mRNA sequences encoding for different domains of BRCA-1 protein. We showed than ADGN-901 mediated rescue of BRCA-1 wild type expression inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to-60% and ADGN-903 markedly delay (>60%) the growth of all cells harboring defect in BRCA-1. In contrast, no significant cytotoxicity related to vehicle was observed. ADGN-901 and ADGN-903 resensitize the resistant cells to PARPi Veliparib inhibition. We demonstrated that the expression of domain of BRCA-1 encoded by ADGN-903 mRNA, appears to prevent RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. Intravenous-administration ADGN-901 and ADGN-903 (2.0mg/kg) prevented HCC-1937 tumor growth. ADGN-901 reduced tumor growth by 95% and ADGN-903 resulted in a tumor regression of 50%. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. ADGN-BRCA nanoparticles are effective in rescuing BRCA-1 functions in Breast cancer cells. Our study provides a proof-of-concept that restoration of the expression of the wild type form of BRCA-1 could be combined together with PARP inhibition for potent combinatorial cancer treatment. Citation Format: Gilles DIVITA, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Khadidja Boularache, Veronique Josserand, Neil Desai. mRNA-mediated restoration of BRCA-1 tumor suppressor function prevents tumor growth and reverses resistance to PARP inhibitors in triple-negative breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C102.

Ablation of the Brca1-Palb2 Interaction Phenocopies Fanconi Anemia in Mice.

Heterozygous mutations in the BRCA1 gene predispose women to breast and ovarian cancer, while biallelic BRCA1 mutations are a cause of Fanconi anemia (FA), a rare genetic disorder characterized by developmental abnormalities, early-onset bone marrow failure, increased risk of cancers, and hypersensitivity to DNA-crosslinking agents. BRCA1 is critical for homologous recombination of DNA double-strand breaks (DSB). Through its coiled-coil domain, BRCA1 interacts with an essential partner, PALB2, recruiting BRCA2 and RAD51 to sites of DNA damage. Missense mutations within the coiled-coil domain of BRCA1 (e.g., L1407P) that affect the interaction with PALB2 have been reported in familial breast cancer. We hypothesized that if PALB2 regulates or mediates BRCA1 tumor suppressor function, ablation of the BRCA1-PALB2 interaction may also elicit genomic instability and tumor susceptibility. We generated mice defective for the Brca1-Palb2 interaction (Brca1 L1363P in mice) and established MEF cells from these mice. Brca1 L1363P/L1363P MEF exhibited hypersensitivity to DNA-damaging agents and failed to recruit Rad51 to DSB. Brca1 L1363P/L1363P mice were viable but exhibited various FA symptoms including growth retardation, hyperpigmentation, skeletal abnormalities, and male/female infertility. Furthermore, all Brca1 L1363P/L1363P mice exhibited macrocytosis and died due to bone marrow failure or lymphoblastic lymphoma/leukemia with activating Notch1 mutations. These phenotypes closely recapitulate clinical features observed in patients with FA. Collectively, this model effectively demonstrates the significance of the BRCA1-PALB2 interaction in genome integrity and provides an FA model to investigate hematopoietic stem cells for mechanisms underlying progressive failure of hematopoiesis and associated development of leukemia/lymphoma, and other FA phenotypes. SIGNIFICANCE: A new Brca1 mouse model for Fanconi anemia (FA) complementation group S provides a system in which to study phenotypes observed in human FA patients including bone marrow failure.See related commentary by Her and Bunting, p. 4044.

Abstract P6-08-31: BRCA1 interacting protein C-terminal helicase 1 (BRIP1) as a cancer susceptibility gene

Abstract Background: Breast cancer is the most common cancer as well as the most common cause of cancer associated deaths in women worldwide. National Comprehensive Cancer Network has detailed criteria for identifying patients at risk for hereditary breast and ovarian cancer syndromes. Many multigene genetic testing panels are in use for screening and prognostication purposes. While pathogenic mutation in BRCA 1 and 2 mutations have been established as risk factors for developing breast cancer, the association of other germ line mutations with breast cancer is less defined. BRCA1 interacting protein C-terminal helicase 1 (BRIP1) influences the DNA repair ability as well as tumor suppressor function of BRCA1 and is associated with increased risk of ovarian cancer. Studies looking at association of BRIP1 mutation with risk of breast cancer have provided conflicting data and currently, BRIP1 is not implicated in causing breast cancer. Methods: We conducted chart review of patients who were referred for genetic testing at the Lipson Cancer Institute/Rochester General Hospital, Rochester, NY and tested positive for BRIP1 mutation alone. Samples were sent to Myriad Genetics for Integrated BRACAnalysis® testing with myRisk® panel looking at 35 genes for mutation. BRIP1 mutations with abnormal protein production and/or function thought to increase risk for ovarian cancer (pathogenic or likely pathogenic) are reported as clinically significant. BRIP1 mutations with currently insufficient data to determine an increased risk are reported as variations of uncertain significance. The panel does not report benign and likely benign mutations. Results: We found nine patients who tested positive for BRIP1 mutation and were negative for other genomic alteration including BRCA1 and BRCA2 mutation (Table 1 and 2). Four patients carried the known high risk BRIP1 mutation and five carried BRIP1 mutations of undetermined significance. One of four patients with known high risk BRIP1 mutations had a personal history but no family history of breast cancer while the remaining three had a family history but no personal history of breast cancer. All five patients with BRIP1 mutations of uncertain significance had both personal and family history of breast cancer. Conclusion: The known high risk BRIP1 mutations are probably not associated with increased risk of breast cancer. However, at least some of the BRIP1 mutations of uncertain significance are likely very significant and associated with increased risk of breast cancer. This association needs to be confirmed in a larger patient population. Table 1: Carriers of BRIP1 variations of uncertain significanceSerial NumberAge at time of screeningPositive BRIP 1 mutation Personal history of breast cancerFamily history of breast cancerAncestry164BRIP1 mutation, c.817A>GYesYesGerman/ Irish278BRIP1 mutation, c. 2220G>TYesYesHolland344BRIP1 mutation, c3237T>GYesYesAfrican430BRIP1 mutation, c.380-17T>AYesYesIrish/ English/ German555BRIP1 mutation, c.139C>GYesYesScottish Table 2: Carriers of high risk BRIP1 mutationsSerial NumberAge at time of screeningPositive BRIP 1 mutation Personal history of breast cancerFamily history of breast cancerAncestry169BRIP1 mutation, c.2255_2256delYesNoItalian/ Hungarian263BRIP1 mutation, c548delNoYesFrench Canadian337BRIP1 mutation, c.1045G>CNoYesDutch460BRIP1 mutation, c.2099T>ANoYesUnknown Citation Format: Mukul Singal, Manasi M Godbole, Kim Povenzano, Beth Elmore, Mehul P Patel. BRCA1 interacting protein C-terminal helicase 1 (BRIP1) as a cancer susceptibility gene [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-31.

BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance.

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

Unsolved mystery: the role of BRCA1 in DNA end-joining.

Heritable mutations in the tumor suppressor gene BRCA1 increase a woman's lifetime risk of developing breast and ovarian cancer. BRCA1's tumor suppressor function is directly linked to its myriad of functions in the cellular response to DNA double-strand breaks (DSBs). BRCA1 interacts with an extensive array of DNA damage responsive proteins and plays important roles in DSB repair, mediated by the homologous recombination pathway, and in the activation of cell cycle checkpoints. However, the role of BRCA1 in the other two DSB repair pathways, classical non-homologous end-joining (C-NHEJ) and alternative NHEJ (A-NHEJ), remains unclear. In this review, we will discuss the current literature on BRCA1's potential role(s) in modulating both C-NHEJ and A-NHEJ. We also present a model showing that BRCA1 contributes to genomic maintenance by promoting precise DNA repair across all cell cycle phases via the direct modulation of DNA end-joining.

The BRCA1-Interacting Protein Abraxas Is Required for Genomic Stability and Tumor Suppression

Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C terminus (BRCT) domains interact with multiple proteins and are required for BRCA1's tumor-suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients.

Tissue-specific tumor suppression by BRCA1

Germ-line mutations in the tumor suppressor gene BRCA1 increase the lifetime risk for breast cancer and ovarian cancer by up to ∼80% and ∼50%, respectively. Population-based studies also support a sex- and tissue-specific tumor suppressor function of BRCA1, but the mechanisms of this specificity are not fully understood. Somatic loss of the normal functioning allele in BRCA1 carriers is common in cancer development and additional somatic events, including mutations of PTEN and TP53, occur at high frequencies (detailed below). Research over the last two decades has described the role of the ubiquitously expressed BRCA1 in maintaining genomic stability through its function in DNA repair and cell cycle checkpoint control, in addition to its activity as a transcriptional regulator. In PNAS, Gorrini et al. shed light on the specificity of BRCA1’s tumor suppressor function in breast tissue. The authors identify an estrogen-induced pathway that promotes the survival of BRCA1-deficient mammary epithelial cells (MECs), as well as Brca1-deficient mammary tumor cells from oxidative stress-induced cell death (1).

Further evidence for the contribution of the BRCA1-interacting protein-terminal helicase 1 (BRIP1) gene in breast cancer susceptibility

BRCA1-interacting protein C-terminal helicase 1 (BRIP1) is a DNA helicase that influences the DNA repair ability and tumor suppressor function of BRCA1. Truncating BRIP1 mutations have been described as cancer susceptibility alleles. To evaluate BRIP1 polymorphisms as risk factors for breast cancer, we performed a detailed analysis of possible single nucleotide polymorphisms (rs2048718, rs4988344, rs8077088, rs6504074, rs4986764, rs4986763, rs11079454, rs7213430, rs34289250, rs4988345, and rs12937080) using the MassARRAY system. A total of 319 patients with breast cancer and 306 healthy control females from the Chinese Han population enrolled in the study. A weak association was found between the rs4986764 allele (exon 18) and breast cancer. The frequency of the rs4986764 C allele was significantly higher in breast cancer patients than in healthy controls [χ(2) = 4.089, P = 0.043, odds ratio (OR) = 0.781, 95% confidence interval (CI) = 0.614-0.992]. Additionally, our study is the first to identify a significant association between rs7213430 and breast cancer. Compared to healthy controls, patients with breast cancer had a higher frequency of the rs7213430 A allele (χ(2) = 8.865, P = 0.003, OR = 0.700, 95%CI = 0.553-0.886). Furthermore, linkage disequilibrium was observed in two blocks (D' > 0.9). While significantly more T-A-C haplotypes (P = 0.001, block 1) were found in breast cancer patients, the frequency of T-T haplotypes (P = 0.008, block 2) was significantly higher in healthy controls. The possible association among rs4986764, rs7213430, and breast cancer risk merits further validation in an independent case-control study.

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor GeneBRCA1through GABP Beta

Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the β-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPβ interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

Bcl-2 Inhibits Nuclear Homologous Recombination by Localizing BRCA1 to the Endomembranes

Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability.

Regulated recruitment of tumor suppressor BRCA1 to the p21 gene by coactivator methylation

Tumor suppression by p53 and BRCA1 involves regulation of cell cycle, apoptosis, and DNA repair and is influenced by transcriptional coactivators and post-translational modifications. Here we show that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Arg 754 in the KIX region of coactivator p300. Methylated p300 and p300 protein fragments are preferentially recognized by BRCT domains of BRCA1, identifying the BRCT domain as a novel methylarginine-binding module. CARM1 and p300 cooperate with BRCA1 and p53 to induce expression of the critical cell cycle and proliferation regulator p21(WAF1/CIP1) in response to DNA damage. This induction was severely attenuated by elimination of CARM1 or its methyltransferase activity, or by mutation of Arg 754 of p300. Absence of CARM1 methyltransferase activity led to failure of cells to arrest in the G1 phase of the cell cycle in response to DNA damage. CARM1 methyltransferase activity was required for induction of some p53 target genes (p21 and Gadd45) but not others (Bax) by DNA damage. Recruitment of BRCA1 to the p53-binding region of the p21 promoter in response to DNA damage required methylation of Arg 754 of p300 by CARM1. Thus, coactivator methylation may be crucial for fine-tuning the tumor suppressor function of BRCA1 and other BRCT domain proteins.

Structural Evidence for Direct Interactions between the BRCT Domains of Human BRCA1 and a Phospho-peptide from Human ACC1

The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 A resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

BRCT Domains: phosphopeptide binding and signaling modules

The BRCA1 C-terminus (BRCT) domains are essential for the tumor suppressor function of BRCA1, and have been found in a variety of proteins from bacteria to men. Recent studies demonstrate that the BRCT domain constitutes a novel phosphopeptide binding region. In this review we seek to discuss the recent biochemical and structural data that have helped elucidate the molecular basis of BRCT domain function and BRCT-mediated interactions, with special emphasis on the role of phospho-specific interactions in key networks that regulate DNA repair. Finally we offer predictions on additional phospho-interacting BRCT domains and potential in vivo binding sites for several BRCT domains.

Missense mutations of BRCA1 gene affect the binding with p53 both in vitro and in vivo

Women with BRCA1 gene mutations have an increased risk for breast and ovarian cancer (BOC). Classification of missense variants as neutral or disease causing is still a challenge and has major implications for genetic counseling. BRCA1 is organized in an N-terminal ring-finger domain and two BRCT (breast cancer C-terminus) domains, involved in protein-protein interaction. The integrity of the C-terminal, BRCT repeat region is also critical for BRCA1 tumor suppressor function. Several molecular partners of BRCA1 have so far been identified; among them, the tumor suppressor protein p53 seems to play a major role. This study was aimed at evaluating the impact of two missense mutations, namely the W1837R and the S1841N, previously identified in BOC patients and located in the BRCT domain of the BRCA1 gene, on the binding capacity of this protein to p53. Co-immunoprecipitation assays of E. coli-expressed wild-type and mutated BRCTs challenged with a HeLa cell extract revealed, for the S1841N variant a significant reduction in the binding activity to p53, while the W1837R mutant showed an inverse effect. Furthermore, a clonogenic soft agar growth assay performed on HeLa cells stably transfected with either wild-type or mutant BRCA1 showed a marked decrease of the growth in wild-type BRCA1-overexpressing cells and in BRCA1S1841N-transfected cells, while no significant changes were detected in the BRCA1W1837R-transfected cells. These results demonstrate that: i) distinct single nucleotide changes in the BRCT domain of BRCA1 affect binding of this protein to the tumor suppressor p53, and ii) the two missense mutations here described are likely to play a role in breast tumorigenesis. We suggest that in vitro/in vivo experiments testing the effects of unclassified BRCA1 gene variants should therefore be taken in to consideration and that increased surveillance should be adopted in individuals bearing these two BRCA1 missense alterations.

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.

Structure of the BRCT Repeats of BRCA1 Bound to a BACH1 Phosphopeptide: Implications for Signaling

The recognition of the phosphorylated BACH1 helicase by the BRCA1 C-terminal (BRCT) repeats is important to the tumor suppressor function of BRCA1. Here we report the crystal structure of the BRCT repeats of human BRCA1 bound to a phosphorylated BACH1 peptide at 2.3 Å resolution. The phosphorylated serine 990 and phenylalanine 993 of BACH1 anchor the binding to BRCA1 through specific interactions with a surface cleft at the junction of the two BRCT repeats. This surface cleft is highly conserved in BRCA1 across species, suggesting an evolutionarily conserved function of phosphopeptide recognition. Importantly, conserved amino acids critical for BACH1 binding are frequently targeted for missense mutations in breast cancer. These mutations greatly diminish the ability of BRCA1 to interact with the phosphorylated BACH1 peptide. Additional structural analysis revealed significant implications for understanding the function of the BRCT family of proteins in DNA damage and repair signaling.

Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and Missense Mutations

Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms.