Breast Cancer Susceptibility Protein Research Articles

Abstract A16: Requirement of functionally active BRCA2 protein for the expression of IRF9-regulated genes in human breast cells

Abstract Interferons are cytokines that induces antiviral, immunomodulatory and antiproliferative effects to cells by inducing the expressions of many effector proteins. Type I interferons are often used to treat advanced stages of breast cancer. Unfortunately, in many cases interferon treatment does not produce any significant effect. We found that transient ablation of the breast cancer susceptibility gene BRCA2 in the human breast cells impairs the expressions of many type I interferon regulated genes. Thus, it appears that type I interferons need functional BRCA2 for their actions. We tested the promoter activity of several of the interferon target genes, such as GBP1, GBP2, ISG15, MX1, MX2, PKR etc. in the BRCA2 knocked-down human breast cells with or without added INF-alpha2a. The interferon-stimulated activity of the promoter decreased significantly in the BRCA2 knocked-down cells, further suggesting a possible role of BRCA2 in the interferon signal transduction pathway. Over expression of BRCA2 in the BRCA2-deficient pancreatic cancer cell Capan1 significantly increased the interferon-stimulated activity of the IFN-alpha-regulated gene promoters in these cells. We tested whether BRCA2 helps mediating the interferon pathway by interacting with the components of this pathway. The classical signal transduction pathway for the type I interferons involve the formation of the ternary complex ISGF3 among phosphorylated STAT1 and STAT2 with IRF9. Immuno-pull-down experiments revealed the binding of STAT1, STAT2 and IRF9 with BRCA2 in the human breast cells. Our study thus proposes a not-known-before role of the breast cancer susceptibility protein BRCA2 in the type I interferon pathway. Supported by the generous funding from US Department of Defense IDEA grant #W81XWH-08-1-0446 to GC. Citation Information: Cancer Res 2009;69(23 Suppl):A16.

PALB2 Functionally Connects the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2

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 (Delta1-70) disrupt its interaction with BRCA1. We have reconstituted PALB2-deficient cells with PALB2Delta1-70, 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.

Contemplating chemosensitivity of basal-like breast cancer based on BRCA1 dysfunction

Gene-expression profiling classified breast cancer to intrinsic subtypes, including luminal A and B, HER2 positive, normal-breast-like, and basal-like tumors. Of these, basal-like tumors that express basal cytokeratins and that are negative for estrogen receptor alpha, progesterone receptor, and HER2 show the most aggressive phenotype with a poor prognosis. Analyses of clinical samples and basic research indicate that basal-like breast cancer is caused by deficiencies in the breast cancer susceptibility protein, BRCA1. Indeed, conditionally deleting BRCA1 from the mammary gland causes mice to develop basal-like cancers at high rates. One of the major functions of BRCA1 is DNA double-strand break (DSB) repair, and its failure to perform causes increased sensitivity of cells to DNA damage-inducing agents, such as PARP inhibitors, DNA cross-linkers, or topoisomerase inhibitors. Therefore, BRCA1 dysfunction could be a principal target for therapeutic application of basal-like breast cancer. Recently, significant progress has been made in understanding the BRCA1 cascade in response to DSBs, where ubiquitin polymer formation plays critical roles. Ubiquitination was indeed found to be an apparent early response of breast cancer to neoadjuvant treatment with epirubicin and cyclophosphamide. Deducing the role of BRCA1 ubiquitin E3 ligase activity in this pathway is a critical challenge to further clarify its functional mechanism. In individualized treatment of breast cancer, evaluation of the DNA repair capacity by the BRCA1 pathway may be an important issue when determining proper treatment of basal-like breast cancer.

The BRC Repeats of BRCA2 Modulate the DNA-Binding Selectivity of RAD51

The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifested by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA-binding selectivity.

A major switch for the Fanconi anemia DNA damage–response pathway

The Fanconi anemia pathway is part of the DNA-damage network including breast cancer–susceptibility proteins BRCA1 and BRCA2. This pathway is activated by the ataxia telangiectasia and Rad3–related (ATR) kinase, but the underlying mechanism remains unclear. A new study demonstrates that a major switch activating the pathway is the ATR-dependent phosphorylation of FANCI.

A high-throughput RNA interference screen for DNA repair determinants of PARP inhibitor sensitivity

Synthetic lethality is an attractive strategy for the design of novel therapies for cancer. Using this approach we have previously demonstrated that inhibition of the DNA repair protein, PARP1, is synthetically lethal with deficiency of either of the breast cancer susceptibility proteins, BRCA1 and BRCA2. This observation is most likely explained by the inability of BRCA deficient cells to repair DNA damage by homologous recombination (HR) and has led to the clinical trials of potent PARP inhibitors for the treatment of BRCA mutation-associated cancer. To identify further determinants of PARP inhibitor response, we took a high-throughput genetic approach. We tested each of the genes recognised as having a role in DNA repair using short-interfering RNA (siRNA) and assessed the sensitivity of siRNA transfected cells to a potent PARP inhibitor, KU0058948. The validity of this approach was confirmed by the identification of known genetic determinants of PARP inhibitor sensitivity, including genes involved in HR. Novel determinants of PARP inhibitor response were also identified, including the transcription coupled DNA repair (TCR) proteins DDB1 and XAB2. These results suggest that DNA repair pathways other than HR may determine sensitivity to PARP inhibitors and highlight the likelihood that ostensibly distinct DNA repair pathways cooperate to maintain genomic stability and cellular viability. Furthermore, the identification of these novel determinants may eventually guide the optimal use of PARP inhibitors in the clinic.

Crystal Structure of the BARD1 Ankyrin Repeat Domain and Its Functional Consequences

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.

Classical molecular dynamics simulations of the complex between the RAD51 protein and the BRC hairpin loops of the BRCA2 protein

In the repair of double-strand breaks of DNA by homologous recombination the recombinase protein RAD51 has its functions controlled by the breast cancer susceptibility protein BRCA2. BRCA2 can bind to RAD51 via the BRC repeats BRC1–BRC8, which are eight conserved sequence motifs in BRCA2 of about 35 amino acids. We have carried out a series of extensive unrestrained atomistic molecular dynamics (MD) simulations in explicit water for a total time period of 248 ns, in order to study the dynamical behaviour and conformations of the complexes between the hairpin loop region of the BRC repeats and RAD51. Our simulations have allowed us to investigate the conformations adopted by the BRC repeats both while bound to RAD51 and while isolated. These conformations are rationalised through an analysis of the inter- and intra-molecular backbone and side chain bonding interactions in all the eight human BRC repeats as well as in a single-point mutation of BRC4. The differences in sequence result in differences in the interactions between the BRC repeats and the RAD51 protein but these do not appear to disrupt the binding in any of the BRC–RAD51 complexes as there are always a number of key residues remaining which allow a sufficient number of interactions to stabilise the complexes.

Analysis of the DNA Binding Activity of BRCA1 and Its Modulation by the Tumour Suppressor p53

BackgroundThe breast cancer susceptibility protein, BRCA1 functions to maintain the integrity of the genome. The exact mechanisms by which it does so, however, remain unclear. The ability of BRCA1 to bind directly to DNA suggests a more direct role. However, little research has been conducted to understand the functional relevance of this characteristic of BRCA1. In this study we examine the DNA substrate specificity of BRCA1 and how this may be controlled by one of its interacting partners, p53.Methodology/Principal FindingsUsing competition gel retardation assays we have examined the ability of residues 230-534 of BRCA1 to discriminate between different synthetic DNA substrates that mimic those recognised by the DNA damage response i.e. four-way junction DNA, mismatch containing DNA, bulge containing DNA and linear DNA. Of those tested the highest affinity observed was for four-way junction DNA, with a 20 fold excess of each of the other synthetic DNA's unable to compete for any of the bound BRCA1 230-534. We also observed a higher affinity for C∶C and bulge containing DNA compared to linear duplex and G∶T containing DNA. BRCA1 230-534 also has interaction sites for the tumour suppressor p53 and we show that titration of this complex into the DNA binding assays significantly reduces the affinity of BRCA1 for DNA.Conclusions/SignificanceIn this paper we show that BRCA1 can discriminate between different types of DNA damage and we discuss the implications of this with respect to its function in DNA repair. We also show that the DNA binding activity can be inhibited by the tumour suppressor p53 and suggest that this may prevent genome destabilizing events such as HR between non-homologous sequences.

BRC‐1 acts in the inter‐sister pathway of meiotic double‐strand break repair

The breast and ovarian cancer susceptibility protein BRCA1 is evolutionarily conserved and functions in DNA double-strand break (DSB) repair through homologous recombination, but its role in meiosis is poorly understood. By using genetic analysis, we investigated the role of the Caenorhabditis elegans BRCA1 orthologue (brc-1) during meiotic prophase. The null mutant in the brc-1 gene is viable, fertile and shows the wild-type complement of six bivalents in most diakinetic nuclei, which is indicative of successful crossover recombination. However, brc-1 mutants show an abnormal increase in apoptosis and RAD-51 foci at pachytene that are abolished by loss of spo-11 function, suggesting a defect in meiosis rather than during premeiotic DNA replication. In genetic backgrounds in which chiasma formation is abrogated, such as him-14/MSH4 and syp-2, loss of brc-1 leads to chromosome fragmentation suggesting that brc-1 is dispensable for crossing over but essential for DSB repair through inter-sister recombination.

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.

Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.

Activation of BRCA1/BRCA2-Associated Helicase BACH1 Is Required for Timely Progression through S Phase

BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G(1) phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability.

The proteasome is involved in determining differential utilization of double-strand break repair pathways

The DSS1 protein interacts with the breast cancer susceptibility protein BRCA2 that plays an integral role in the repair of DNA double-strand breaks (DSBs). DSS1 has also been shown to interact with components of the 26S proteasome in Saccharomyces cerevisiae and in human tumour cells. This raises the possibility of functional interplay between the DNA repair machinery and the proteasome. We show here that human DSS1 interacts with the RPN3 and RPN7 proteasome subunits and define regions of DSS1 important for the interactions with RPN3, RPN7 and BRCA2. We also show that BRCA2 interacts with RPN3 and RPN7 and that the BRCA2/RPN7 interaction is independent of DSS1. Finally, and most significantly, we demonstrate that the proteolytic activity of the proteasome is a determinant of the choice of DSB repair pathway; inhibition of proteasome proteolytic activity results in an increase in the utilization of potentially mutagenic single-strand annealing at the expense of a reduction in the level of error-free gene conversion. This confirms a functional link between DSB repair and proteasomal activity.

RAD51, BRCA2 and DNA repair: a partial resolution

Cooperation between the DNA recombinase RAD51 and the breast cancer susceptibility protein BRCA2 is crucial for the repair of double-strand DNA breaks. Two papers provide new insight into the BRCA2-RAD51 interaction, revealing two different sets of motifs within BRCA2 that bind structurally distinct forms of RAD51. The balance between these interactions seems to be crucial for the function of BRCA2 in DNA repair.

A requirement of FancL and FancD2 monoubiquitination in DNA repair

The rare hereditary disorder Fanconi anemia (FA) can be caused by mutations in components of the FA core complex (FancA/B/C/E/F/G/L/M), a key regulator FancD2, the breast cancer susceptibility protein BRCA2/FancD1, or the newly identified FancJ/BRIP1 helicase. By performing yeast two-hybrid (Y2H) screens using N-terminal chicken (ch) FancD2 as a bait, we have identified chFancL, the likely ubiquitin E3 ligase subunit of the FA core complex. We also found that ectopically expressed FancD2 and FancL co-immunoprecipitated in 293T cells, and this interaction was dependent on the PHD domain of FancL. FANCL-disrupted chicken DT40 cells displayed defects in both FancD2 monoubiquitination and focus formation. Importantly, cell lines lacking the FANCL or FANCD2 genes, or carrying a "knock-in" mutation of the FancD2 monoubiquitination site (where the Lys 563 residue is changed to Arg), displayed quantitatively identical defects in the repair of I-SceI-induced chromosomal breaks by homologous recombination (HR). These data establish the role of FANCL and FancD2 monoubiquitination in HR repair.

Fanconi anemia and breast cancer susceptibility

Two new studies show that the Fanconi anemia complementation group N results from biallelic mutations in PALB2, which encodes a recently identified interaction partner of the breast cancer susceptibility protein BRCA2. A third study shows that monoallelic PALB2 mutations are associated with breast cancer susceptibility, providing yet more links between Fanconi anemia, homologous recombination repair and cancer predisposition.

A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway.

Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds.We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.

Human Immunodeficiency Virus Type 1 Vpr Induces G2 Checkpoint Activation by Interacting with the Splicing Factor SAP145

Vpr, the viral protein R of human immunodeficiency virus type 1, induces G(2) cell cycle arrest and apoptosis in mammalian cells via ATR (for "ataxia-telangiectasia-mediated and Rad3-related") checkpoint activation. The expression of Vpr induces the formation of the gamma-histone 2A variant X (H2AX) and breast cancer susceptibility protein 1 (BRCA1) nuclear foci, and a C-terminal domain is required for Vpr-induced ATR activation and its nuclear localization. However, the cellular target of Vpr, as well as the mechanism of G(2) checkpoint activation, was unknown. Here we report that Vpr induces checkpoint activation and G(2) arrest by binding to the CUS1 domain of SAP145 and interfering with the functions of the SAP145 and SAP49 proteins, two subunits of the multimeric splicing factor 3b (SF3b). Vpr interacts with and colocalizes with SAP145 through its C-terminal domain in a speckled distribution. The depletion of either SAP145 or SAP49 leads to checkpoint-mediated G(2) cell cycle arrest through the induction of nuclear foci containing gamma-H2AX and BRCA1. In addition, the expression of Vpr excludes SAP49 from the nuclear speckles and inhibits the formation of the SAP145-SAP49 complex. To conclude, these results point out the unexpected roles of the SAP145-SAP49 splicing factors in cell cycle progression and suggest that cellular expression of Vpr induces checkpoint activation and G(2) arrest by interfering with the function of SAP145-SAP49 complex in host cells.

Regulation of recombinational repair by the familial breast cancer susceptibility protein BRCA2

Regulation of recombinational repair by the familial breast cancer susceptibility protein BRCA2

Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination

The breast cancer susceptibility protein, Brca2 and the RecQ helicase, Blm (Bloom syndrome mutated) are tumor suppressors that maintain genome integrity, at least in part, through homologous recombination (HR). Brca2 facilitates HR by interacting with Rad51 in multiple regions, the BRC motifs encoded by exon 11 and a single domain encoded by exon 27; however, the exact importance of these regions is not fully understood. Blm also interacts with Rad51 and appears to suppress HR in most circumstances; however, its yeast homologue Sgs1 facilitates HR in response to some genotoxins. To better understand the biological importance of these two proteins, we performed a genotoxic screen on mouse embryonic stem (ES) cells impaired for either Brca2 or Blm to establish their genotoxic profiles (a cellular dose-response to a wide range of agents). This is the first side-by-side comparison of these two proteins in an identical genetic background. We compared cells deleted for Brca2 exon 27 to cells reduced for Blm expression and find that the Brca2- and Blm-impaired cells exhibit genotoxic profiles that reflect opposing activities during HR. Cells deleted for Brca2 exon 27 are hypersensitive to γ-radiation, streptonigrin, mitomycin C and camptothecin and mildly resistant to ICRF-193 which is similar to HR defective cells null for Rad54. By contrast, Blm-impaired cells are hypersensitive to ICRF-193, mildly resistant to camptothecin and mitomycin C and more strongly resistant to hydroxyurea. These divergent profiles support the notion that Brca2 and Blm perform opposing functions during HR in mouse ES cells.