Genes are the instruction manuals of life and contain the information needed to build the proteins that keep cells alive. Over time, genes can accumulate errors or mutations and eventually become faulty, which can lead to diseases like cancer. Sometimes mutations can be passed on through generations and increase the chances of getting cancer. The BRCA1 gene, for example, provides instructions for making a protein that helps to repair or remove damaged DNA and stops cells from growing uncontrollably. When the BRCA1 gene becomes faulty, cells could continue to grow with damaged DNA. This makes it more likely for cancer to develop, especially breast cancer and ovarian cancer.

However, not all changes in BRCA1 gene cause the protein to become faulty or lead to cancer. In fact, about 30% of BRCA1 gene changes identified by genetic tests are referred to as ‘variants of uncertain clinical significance’, meaning that it is not clear if these variants are indeed mutations that could affect the clinical outcome of the people that carry them. Software predictions based largely on patient data have categorized many of these variants as not cancer-causing, but the majority still need to be experimentally tested and confirmed. Many studies have tried to determine the effect of selected variants on the BRCA1 protein, but a complete picture remains lacking.

Now, Anantha et al. have tested the top 22 common variants in the BRCA1 gene, some of which had known effects and some did not. The study tested how these variants affect the ability of the protein to repair damaged DNA and the efficacy of chemotherapies targeting cancer cells with a DNA repair defect. The experiments revealed that three specific parts of the protein must remain intact in order for the protein to carry out this activity, i.e. mutations that affect these three areas are likely to cause cancer and also make cancer cells vulnerable to these chemotherapies. Anantha et al. also generated a series of 10 artificially shortened BRCA1 proteins, each missing a specific part, to determine the possible effects of other variants in those missing parts.

Together the findings reveal previously unknown effects of certain variants that are commonly seen in cancer patients as well new insights into how the BRCA1 protein repairs DNA. The next step will be to assess rarer variants where little data is available. A better understanding of how these variants affect DNA repair and drug response will help to improve the genetic counseling and treatment of patients with breast cancer and ovarian cancer.

Germline, heterozygous mutations in BRCA1 confer high risk of breast and ovarian cancer development in an autosomal dominant fashion (Couch et al., 2014; Fackenthal and Olopade, 2007). BRCA1 has been implicated in numerous cellular processes including DNA repair, cell cycle checkpoints, centrosome duplication, and transcriptional regulation, etc. (Deng, 2006; Mullan et al., 2006; Roy et al., 2012). Ever since BRCA1 was found to localize to discrete nuclear foci and colocalize with the recombination enzyme RAD51 (Scully et al., 1997), its function in homologous recombination (HR)-based repair of DNA double strand breaks (DSBs) has been a subject of intense study (Moynahan and Jasin, 2010). Tumors arising from BRCA1 mutation carriers usually show loss of the wild-type (wt) allele, which renders tumor cells biallelically null for the gene. It is generally believed that genome instability resulting from the DNA repair defect following the loss of BRCA1 is a driver of tumor development (Li and Greenberg, 2012; Venkitaraman, 2014). Importantly, the very DNA repair defect that leads to tumor development is also an ‘Achilles’ Heel’ of the resulting tumor cells, which can be selectively killed by suitable DNA-damaging agents that target HR defect, such as platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors (Lord and Ashworth, 2016).

The human BRCA1 gene consists of 24 exons encoding a large polypeptide of 1863 amino acid residues. BRCA1 contains a RING domain at the N terminus and a tandem BRCT domain at the C terminus (Figure 1A). Two nuclear localization signals (NLSs) facilitate the localization of BRCA1 primarily to the nucleus (Chen et al., 1996), whereas a nuclear export signal (NES) can mediate its cytoplasmic export (Rodríguez and Henderson, 2000). The RING domain of BRCA1 binds to a similar domain in its close partner BARD1 (Wu et al., 1996), leading to the formation of a stoichiometric complex that possesses substantial ubiquitin E3 ligase activity (Hashizume et al., 2001; Ruffner et al., 2001). At the same time, binding of BARD1 to BRCA1 shields the NES and helps retain BRCA1 in the nucleus (Fabbro et al., 2002). The BRCT domain directly binds, in a phosphorylation-dependent manner, to at least three other proteins, namely BRIP1, CtIP and Abraxas, all of which have function in DNA repair (Huen et al., 2010; Jiang and Greenberg, 2015). Additionally, BRCA1 contains a highly conserved coiled-coil (CC) motif that directly binds PALB2, the partner and localizer of BRCA2 (Xia et al., 2006), which links BRCA1 and BRCA2 in the HR pathway (Sy et al., 2009; Zhang et al., 2009a, 2009b).

Figure 1 with 2 supplements see all

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Figure 1—source data 1

HR activities of the BRCA1 variants and mutants analzyed in this study.

See texts and materials and methods for details.

https://doi.org/10.7554/eLife.21350.004

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The Breast Cancer Information Core (BIC) database was the first major public database into which patient-derived mutations and variants were deposited. It contains a total of 15,311 entries of BRCA1 sequence alterations, among which 6133 (40%) are frameshift or protein-truncating mutations, which can generally be classified as pathogenic. Importantly, 4577 (30%) are missense variants, most of which are considered to be of ‘unknown clinical importance’, or commonly known as variants of unknown (or uncertain) clinical significance (VUSs), the interpretation of which remains a challenge (Couch et al., 2014). Recently, more data have been deposited into the ClinVar database, which now includes all BIC cases along with ones from multiple other sources. Based largely on genetic and epidemiological evidence, a large number of VUSs has been designated by ClinVar as ‘benign’. However, the majority of the variants have not been functionally characterized.

A number of studies have been conducted to assess the functions of select BRCA1 VUS in transcription, DNA repair, and in supporting the viability of mouse embryonic stem (ES) cells (Bouwman et al., 2013; Chang et al., 2009; Millot et al., 2012; Ransburgh et al., 2010; Towler et al., 2013). However, data on the impact of VUS on drug sensitivity have only begun to emerge. In particular, a recent study used a recombinase-mediated cassette exchange (RMCE) approach to profile 74 patient-derived missense variants for their activities in conferring resistance to cisplatin and a PARP inhibitor in mouse ES cells, and it found that all functionally deleterious variants were confined to the RING and BRCT domains (Bouwman et al., 2013).

In this study, we systematically assessed HR and SSA activities of 32 natural variants, including 11-patient-derived missense mutations that affect known protein-protein interactions and the top 22-patient-associated missense variants, as well as 10 overlapping deletion mutations that span the entire length of the protein. Our data clearly demonstrate that the HR function of BRCA1 requires the RING, CC and BRCT domains. Interestingly, deletion of residues 1056–1357 (BD7) caused ~50% reduction in both HR and SSA activity without affecting the binding of any of the above known partners. This segment of BRCA1 does not contain any recognizable domains and has not been implicated in HR before, suggesting the existence of a novel binding partner or regulatory mechanism. Together, these results establish a functional landscape of BRCA1 for its HDR function.

The RING domain of BRCA1 binds to the RING domain of BARD1, and the BRCA1/BARD1 heterodimer formation is critical for their stability, their E3 ubiquitin ligase activity and the nuclear retention of BRCA1 (Fabbro et al., 2002; Hashizume et al., 2001; Ruffner et al., 2001). Conditional knock out of either Brca1 or Bard1 in murine mammary epithelial cells led to the development of mammary carcinomas that are indistinguishable from each other (Shakya et al., 2008), and the BRCA1 C61G mutant that abrogates BARD1 binding failed to suppress mammary tumor development in mice (Drost et al., 2011). Moreover, the I26A mutation that abolishes its E3 ligase activity did not affect HR activity or mitomycin C (MMC) sensitivity (Reid et al., 2008), nor did it affect tumor suppression in mice (Shakya et al., 2011). These findings suggested that the BARD1-binding function of the RING domain, rather than its E3 ligase activity, plays a key role in BRCA1 HR and tumor suppression activity. In this regard, our findings on the I26A mutation are worth noting. First, it caused a significant impairment in BARD1 binding (Figure 5A); second, it moderately reduced HR efficiency (Figure 5B); and third, it caused sensitivity to both cisplatin and olaparib under the condition used (Figure 5C). The reduced BARD1 binding is consistent with the fact that I26A mutant mouse cells had substantially reduced amount of both BRCA1 and BARD1 (Reid et al., 2008). Also, a recent report showed that HeLa cells expressing only BRCA1-I26A were similarly sensitive to olaparib as BRCA1 knockdown cells (Densham et al., 2016). Thus, it is safe to conclude that the I26A mutation affects HR and drug resistance in human cells. The discrepancy in observed HR activities and drug sensitivities in mouse ES cells and human cancer cells may be explained by the different tolerance for partial loss of BARD1 binding in the two systems or the presence of other mutations in cancer cells. At the same time, although I26A mutant mouse cells showed no defect in apparent HR activity or MMC resistance, reduced gene targeting efficiency and increased chromosomal abnormalities after MMC treatment were noted (Reid et al., 2008), indicative of a possible defect in HR in mouse cells as well. Despite the discrepancy, it should be noted that our results do not contradict the argument that the E3 ligase activity of BRCA1 is dispensable for tumor suppression (Shakya et al., 2011).

Mutations of the BRCT domain dramatically abolish HR activity, and the mechanism is also complex. First, this domain directly interacts with at least three proteins, Abraxas, a scaffold protein (Wang et al., 2007), BRIP1, a DNA helicase (Cantor et al., 2001), and CtIP, an endonuclease involved in resection (Sartori et al., 2007). It has recently been shown that CtIP binding to BRCA1 is dispensable for CtIP-mediated DNA resection (Polato et al., 2014; Reczek et al., 2013), yet to what extent BRCA1 HR activity depends on Abraxas and BRIP1 remains unclear. Second, mutation of the BRCT domain causes a major defect in the nuclear accumulation of BRCA1 (Figure 4A–B), supporting a key role of the BRCT domain for BRCA1 nuclear entry. Our finding that BARD1 can significantly rescue nuclear localization of BRCT mutants but not their HR defects (Figure 4F) also supports an important and direct role of the BRCT domain for BRCA1 DNA repair activity. Collectively, our data demonstrate the dual role of the BRCT domain and underscore a complex regulation of BRCA1 localization and repair function by the RING and BRCT domains and their binding partners (Figure 7A).

Figure 7

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Consistent with early observations made in mouse cells (Stark et al., 2004), BRCA1 depletion reduced the efficiency of both HR and SSA, whereas depletion of BRCA2 led to severe loss of HR (data not shown) but increased SSA (Figure 2B). As efficient resection is necessary for both repair mechanisms, the data is consistent with the notion that BRCA1 plays a key role in resection (Bunting et al., 2010). Importantly, we found that depletion of PALB2 showed similar, if not more dramatic, induction of SSA as did BRCA2 depletion (Figure 2B). Moreover, all three point mutations in BRCA1 that affect PALB2 binding led to increased SSA usage (Figure 2A), and two different PALB2 mutants with greatly impaired BRCA1-binding capacity both failed to suppress the upregulation of SSA in PALB2-depleted cells (Figure 2F). These results clearly demonstrate that the direct interaction between BRCA1 and PALB2 is required for suppressing, or at least preventing, SSA, while promoting HR (Figure 7A–B). As such, our results support two critical roles of BRCA1 function in DSB repair, promoting resection and recruiting/stimulating PALB2, which then channels the ssDNA down the HR path (Figure 7B). Based on the available data, PALB2 is the key partner of BRCA1 specifically for its HR function and the primary switch point, acting upstream of BRCA2, for HDR pathways following resection.

VUSs are commonly found during clinical genetics tests; however, their biological and clinical significance is often difficult to ascribe with confidence, and this uncertainty poses significant challenges for both clinicians and patients. Our results demonstrate that relatively common VUSs outside the RING and BRCT domains do not significantly affect the HDR activity of BRCA1. Our findings are mostly consistent with several previous studies (Table 2), including the above noted mouse ES cell-based study that covered 6 of the top 22 variants (Bouwman et al., 2013); on the rare PALB2-binding mutants L1407P and M1411T, however, our results showed that they are severely defective in HR (Figure 1D) and conferring drug resistance (Figure 3C), whereas their effects in mouse cells were less pronounced. Moreover, M1400V, which was found to be neutral in the mouse cell system, shows clear sensitivity to olaparib treatment in the human cell line. This discrepancy could again be due to a difference in the thresholds of human and mouse cells to tolerate partial loss of BRCA1-PALB2 interaction or the presence of other mutations in the human cancer cells.

Platinum-based therapeutics have been commonly used for treatment of ovarian cancer, and olaparib has recently been approved for the treatment of BRCA1/2 mutant ovarian cancer, with multiple trials on breast and other cancers ongoing or recently completed. These agents generate lesions that block or collapse DNA replication forks, leading to DSBs that require HR and therefore BRCA1/2 for repair (Lord and Ashworth, 2016). Under the condition used, olaparib appeared to have a better selectivity in killing MDA-MB-436 cells expressing mutant BRCA1 proteins. Although this could be due to a slightly lower than optimal concentration of cisplatin used, the possibility that cisplatin is intrinsically less selective than olaparib cannot be ruled out. Based on data from this study and other recent studies, patients with relatively common missense mutations in the RING and BRCT domains, such as C61G and A1708E, respectively, or rare mutations in the CC motif, such as L1407P or M1411T, are likely to respond to platinum- or PARPi-based therapies, unless resistance mechanisms have already occurred prior to treatment or are induced by the treatment (Lord and Ashworth, 2013). However, except for C61G and A1708E, all other top 22 missense variants are fully or mostly functional both in HR and in conferring cisplatin and olaparib resistance (Figure 3C), which corroborates with clinical genetics evidence to further cast doubts on their pathogenicity and diminish the likelihood of a positive response of the patients to the above regimens. Based on the results of our deletion analyses and other available data, it is unlikely for any VUS outside the RING, CC and BRCT domains to be strongly pathogenic or to elicit high sensitivity to platinum or PARPi therapies (unless it destabilizes the transcript or protein). However, the region deleted in BD7 (residues 1056–1,307) may harbor VUS that have intermediate effects.

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Author details

1. Rachel W Anantha

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   RWA, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Srilatha Simhadri and Tzeh Keong Foo

   Competing interests

   The authors declare that no competing interests exist.

2. Srilatha Simhadri

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States
   3. Department of Medicine, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   SS, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Rachel W Anantha and Tzeh Keong Foo

   Competing interests

   The authors declare that no competing interests exist.

3. Tzeh Keong Foo

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   TKF, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Rachel W Anantha and Srilatha Simhadri

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-0168-7054

4. Susanna Miao

   Department of Genetics, School of Arts and Sciences, Rutgers, The State University of New Jersey, Piscataway, United States

   Contribution

   SM, Data curation, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Jingmei Liu

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   JL, Resources, Investigation

   Competing interests

   The authors declare that no competing interests exist.

6. Zhiyuan Shen

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   ZS, Resources, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-2834-0309

7. Shridar Ganesan

   1. Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States
   2. Department of Radiation Oncology, Rutgers, The State University of New Jersey, New Brunswick, United States
   3. Department of Medicine, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   SG, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Bing Xia

   Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, United States

   Contribution

   BX, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   xiabi@cinj.rutgers.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-3259-6139

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