MINIREVIEW BRCA1 16 years later: risk-associated BRCA1 mutations and their functional implications Rebecca J. Linger 1 and Patricia A. Kruk 1,2 1 Department of Pathology and Cell Biology, University of South Florida, Tampa, FL, USA 2 H. Lee Moffitt Cancer Center, Tampa, FL, USA Introduction Family history is the strongest risk factor for the development of ovarian cancer and a major risk factor for the development of breast cancer [1]. Understand- ing how risk-associated mutations contribute to cancer initiation and progression will provide insight into molecular mechanisms and aid in better risk assess- ment, prophylaxis and treatment for carriers. The majority of hereditary ovarian cancers and a significant proportion of hereditary breast cancers are associated with mutation of the breast cancer susceptibility gene 1 (BRCA1) [1,2]. The objective of this review is to pro- vide a brief consideration of the normal functions associated with BRCA1, followed by a discussion of the types of risk-associated BRCA1 mutation and their molecular and cellular impact. Lastly, we will consider the clinical implications of these mutations for breast and ovarian cancer patients. BRCA1 The predominantly nuclear BRCA1 protein, which shuttles between the nuclear and cytoplasmic compart- ments, has multiple functions in the cell [3,4]. BRCA1 plays an important role in the DNA damage response, as evidenced by the fact that BRCA1 null mice die early in embryonic development and exhibit chromo- somal aberrations that are exacerbated by a p53 muta- tion [5] (see also [6–8]). BRCA1’s expression and phosphorylation are cyclic, and BRCA1 plays a role in the cell cycle as well, by regulating key cell cycle Keywords BRCA1; breast cancer; mutation; ovarian cancer; risk Correspondence P. A. Kruk, Department of Pathology and Cell Biology, MDC 11, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA Fax: +813 974 5536 Tel: +813 974 0548 E-mail: pkruk@health.usf.edu (Received 26 January 2010, revised 27 April 2010, accepted 4 June 2010) doi:10.1111/j.1742-4658.2010.07735.x Mutations in the tumor suppressor breast cancer susceptibility gene 1 (BRCA1), an important player in the DNA damage response, apoptosis, cell cycle regulation and transcription, confer a significantly elevated life- time risk for breast and ovarian cancer. Although the loss of wild-type BRCA1 function is an important mechanism by which mutations confer increased cancer risk, multiple studies suggest mutant BRCA1 proteins may confer functions independent of the loss of wild-type BRCA1 through dominant negative inhibition of remaining wild-type BRCA1, or through novel interactions and pathways. These functions impact various cellular processes and have the potential to significantly influence cancer initiation and progression. In this review, we discuss the functional classifications of risk-associated BRCA1 mutations and their molecular, cellular and clinical impact for mutation carriers. Abbreviations BARD1, BRCA1-associated RING domain protein 1; BRAT, BRCA1 185delAG truncation; BRCA1, breast cancer susceptibility gene 1; BRCT, BRCA1 C-terminus. 3086 FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS controllers, including p21, and by physically interact- ing with cell cycle regulators (reviewed in [9]). BRCA1 can also recruit chromatin modifying proteins, such as histone acetyltransferases and histone deacetylases, and directly interact with other transcription factors to alter their function (reviewed in [9]). For example, BRCA1 binds and modulates phosphorylation of p53 to enhance its transactivation function [10,11]. Lastly, BRCA1 is capable of ubiquitin ligase activity when heterodimerized with BRCA1-associated RING domain protein 1 (BARD1) [12]. The loss of these cellular functions of BRCA1 may contribute to cancer by promoting genomic instability and accumulation of cancer-causing mutations [6], a process further accelerated by p53 mutation, a common characteristic of BRCA1 mutant ovarian cancers [13]. BRCA1 mutation carriers have a 30% risk of developing ovar- ian cancer during their lifetime [14] and a 50–80% risk of developing breast cancer before the age of 70 years [6]. Types of BRCA1 mutation All types of BRCA1 mutation have been reported, including frameshift, nonsense, missense, in-frame insertions and deletions, splice altering mutations, mutations in the untranslated regions, as well as silent mutations. The majority of risk-associated mutations are frameshift or nonsense mutations that result in a premature stop codon and truncated protein product BRCA1 DNA damage response Chemosensitivity Apoptosis Proliferation Tu mo r i g e ne s i s Transcription/gene regulation Transactivation BRCT BRCT NLS NLS NES NES domain 185delAG 5382InsC N-terminal 602aa* N-term 302 aa* N-term 771 aa* 185delAG 5382InsC 5677InsA ΔN aa303-1863* 185delAG 185delAG M1775K P1749R Y1853STOP Q1756InsC Δ500-1863* Δ1314-1863* ΔNLS* ΔNLS/C+NLS* Δ515-1091* Δ BamH1 N-terminal 1313aa* Δ Kpn1 N-terminal 771aa* Δ EcoR1 N-terminal 302aa* Δ500-1863* 5083del19 Δ1808-5556* Ser1841Asn 5382InsC M1775K P1749R C64G T826K M1775R ΔN aa303-1863* * Denotes synthetic mutation 1835STOP 340STOP Δ343-1081* Δ 515-1092* 5677InsA ΔEcoR1 N-term 302aa* CT-BRCA1 aa1293-1863* N-terminal 602aa* Δ11 splice variant ΔN aa303-1863* 1835STOP 340STOP Δ343-1081* Δ 515-1092* Δ 542* BRCA1 tr/tr aa1-900* N-terminal 602aa* ΔRING splice variant* trBRCA1 (N-term 300aa)* Δ11 splice variant W1777Stop* ΔRING splice variant* Development Q1756InsC Y1853STOP M1775K/R P1749R C64G T826K 1835STOP 340STOP Ser1841Asn 5083del19 B A RING Fig. 1. BRCA1 mutations and their cellular and physiological impact. (A) Domain structure of BRCA1 protein and the location of risk-associ- ated mutations discussed. NES, nuclear export signal; NLS, nuclear localization signal. (B) BRCA1 mutations categorized by cellular pro- cesses in which each has been found to lack function or exhibit function different from the wild-type. The nomenclature used for each mutation was that used in the original research article, or a structural description if designation was not descriptive of the mutation or mutant structure. R. J. Linger and P. A. Kruk Risk-associated BRCA1 mutations and their functional implications FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS 3087 (NIH Breast Cancer Information Core Database, http://research.nhgri.nih.gov/bic/). Risk-associated trun- cation mutations are found throughout the entire BRCA1 coding sequence (Fig. 1) and result in mutant proteins that vary in length and structural impairment. For example, the nonsense mutation Y1853X, which lacks the last 11 amino acids, is only missing a small portion of the second BRCT (BRCA1 C-terminus) repeat, whereas the 39 amino acid 185delAG mutant lacks all of BRCA1’s known functional domains. A smaller percentage of risk-associated BRCA1 mutations are point mutations classified as missense mutations. Like truncation mutations, missense muta- tions occur throughout the entire BRCA1 coding sequence (Fig. 1) [15], although it is difficult to deter- mine the clinical importance of these mutations because of their rarity and because they do not often result in gross structural or functional loss. Therefore, many missense mutations remain ‘variants of unknown significance’ [16]. The functional significance of the RING and BRCT domains, as well as the substantial conservation of their sequences, fuel speculation that many missense mutations in these areas are probably linked to cancer predisposition. Nonetheless, several missense mutations have already been linked to breast and ⁄ or ovarian cancer predisposition, including C61G, M1775K and P1749R. BRCA1 is thought to act as a classical tumor sup- pressor and the loss of BRCA1’s cellular functions is thought to occur through bi-allelic inactivation. Carri- ers of mutations have one germline hit (the inherited mutated copy of BRCA1) and, in the tumor, a second somatic hit usually through the loss of heterozygosity [6]. The observed phenotype of enhanced breast and ovarian cancer risk is generally thought to result from the loss of some or all wild-type functions of the BRCA1 gene product. However, countless studies have revealed the com- plexities of signaling molecule and transcription factor interactions, as well as cellular adaptations in response to the unique selective pressures of tumor initiation and progression. Therefore, it is important to investi- gate all possible molecular mechanisms by which a mutation may contribute to the disease phenotype. Mutant proteins may antagonize wild-type proteins in a dominant negative manner, resulting in the loss of remaining wild-type function [17], or they may engage in unique molecular interactions and manifest novel functions independent of the loss of wild-type protein function [18]. Likewise, BRCA1 mutations may con- tribute to cancer risk through the loss of wild-type BRCA1 function or through the gain of function asso- ciated with mutant BRCA1 proteins. Loss of function mutations As mentioned previously, several lines of evidence sug- gest the loss of wild-type BRCA1 function as a com- mon mechanism for enhanced breast and ovarian cancer risk (Table 1). Similar to BRCA1 knockout mice and cell lines, elevated levels of aneuploidy and loss of heterozygosity indicative of an impaired DNA damage response have been noted in breast cancer tis- sue from mutation carriers compared with control breast cancers, as well as in the human BRCA1 trun- cated breast cancer cell line, HCC1937 (reviewed in [6]). In structural protein studies, Tischkowitz et al. [19] suggested that structural alterations in the BRCT phosphopeptide-binding pocket caused by the BRCA1 M1775K missense mutation contributed to enhanced breast and ovarian cancer risk through diminished transactivation and binding to other DNA damage response proteins. Likewise, Williams et al. [20] found that decreased stability of BRCA1 missense and trun- cation mutants resulting from aberrant protein folding contributed to the loss of BRCA1 function and enhanced cancer risk. Expression of mutant BRCA1 constructs in the absence of wild-type BRCA1 frequently fails to restore wild-type BRCA1 function. Scully et al. [21] utilized the c radiation-sensitive HCC1937 breast cancer cell line, which lacks wild-type BRCA1 and carries two 5382InsC BRCA1 alleles that code for a frameshift and premature stop signal at codon 1829, and were able to decrease c radiation sensitivity with restoration of wild-type BRCA1. However, transfection of several BRCA1 mutants into these cells failed to alter radia- tion sensitivity. In agreement, the addition of wild-type BRCA1 expression into breast cancer cell lines that exhibit low wild-type BRCA1 expression due to the presence of a single wild-type BRCA1 allele inhibited growth. However, expression of the risk-associated truncation mutants 1835STOP and 340STOP, as well as the synthetic internal deletion mutants D343-1081 and D 515-1092, failed to alter cell growth, tumor for- mation and tumor progression in nude mice [22]. Lastly, introduction of wild-type BRCA1 into HCC1937 breast cancer cells and IGROV 1 ovarian cancer cells inhibited tumor initiation and growth, whereas a synthetic BRCA1 mutant lacking the last 542 amino acids did not [23]. Interestingly, Cousineau & Belmaaza [24] hypothesized that reduced gene dos- age of wild-type BRCA1 in mutation carriers is solely responsible for altered DNA damage repair, subse- quent mutation accumulation and increased cancer risk. Using MCF7 breast cancer cells that harbor a single copy of wild-type BRCA1 and exhibit enhanced Risk-associated BRCA1 mutations and their functional implications R. J. Linger and P. A. Kruk 3088 FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 1. Studies supporting loss or gain of function mutation as mechanisms of enhanced breast cancer and ovarian cancer risk. Mutation Result of mutation In vitro In vivo Model system Endpoint Summary Reference Loss of function Various X NA Number of genetic changes Mutant breast cancers more chromosomal gain ⁄ loss events versus control breast cancers 59 P1749R C64G T826K M1775R Missense P>R Missense C>G Missense T>K Missense M>R X Breast cancer DNA damage Wild-type BRCA1 rescued c radiation sensitivity of HCC1937 cells; mutants did not 21 5382InsC Truncated: 1828 amino acids X Breast cancer DNA damage, chemosensitivity Wild-type BRCA1 rescued hyper-recombination, chemosensitivity of MCF7 cells; mutants did not 24 P1749R Q1756InsC Y1853STOP Missense P>R Truncated: 1828 amino acids Truncated: 1852 amino acids X COS-7, colon cancer Gene regulation Wild-type BRCA1 increased p21 expression in COS-7, cancer cells; mutants did not 26 1835STOP 340STOP Truncated: 1834 amino acids Truncated: 339 amino acids X X Breast cancer Cell growth, tumor growth Wild-type BRCA1 inhibited growth, tumor growth in nude mice; mutants did not 22 Gain of function 5677InsA Truncated: 1852 amino acids X Prostate cancer Proliferation Mutant inhibited proliferation more efficiently than wild-type BRCA1 38 N-terminal 602 amino acids Synthetic mutant: 602 amino acids X X Mouse ovarian epithelium Proliferation, chemosensitivity, tumorigenesis Mutant BRCA1 enhanced proliferation, chemosensitivity, tumorigenesis; wild-type BRCA1 suppressed 41 5677InsA N-terminal 302 amino acids N-terminal 771 amino acids Truncated: 1852 amino acids Synthetic mutant: 302 amino acids Synthetic mutant: 771 amino acids X Prostate cancer Proliferation, chemosensitivity 5677InsA and wild-type BRCA1 impaired proliferation, enhanced chemosensitivity; synthetic truncations decreased sensitivity 39 185delAG Truncated: 39 amino acids X Ovarian epithelium Apoptosis 185delAG decreased cIAP1, XIAP, P-Akt, and enhanced cleaved caspase 3, apoptosis after drug treatment 46 5382InsC 5677InsA Truncated: 1828 amino acids Truncated: 1852 amino acids X Breast, ovarian cancer Apoptosis Co-expression of mutants with wild-type BRCA1 inhibited wild-type BRCA1’s ability to enhance apoptosis 50 5083del19 Truncated: 1669 amino acids X X HeLa Gene regulation Mutant increased periostin mRNA, protein and mutation carrier serum, breast cancer tissue 52 R. J. Linger and P. A. Kruk Risk-associated BRCA1 mutations and their functional implications FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS 3089 spontaneous recombination or ‘hyper-recombination’, they showed that transfection of MCF7 cells with wild-type BRCA1 diminished hyper-recombination and chemosensitivity, whereas addition of the 5382InsC BRCA1 mutation affected neither endpoint. These studies further support a role for the loss of wild-type BRCA1 function as a contributing factor to enhanced breast and ovarian cancer risk. It is important to note that many of the aforemen- tioned studies attempted to delineate BRCA1 mutant function in model systems lacking normal levels of wild-type BRCA1, which makes it difficult to discrimi- nate between the contribution of BRCA1 mutants and the loss of wild-type BRCA1 to disease risk. However, several studies utilizing a wild-type BRCA1 back- ground clearly support the loss of BRCA1 wild-type function for cancer risk. For example, although the overexpression of wild-type BRCA1 in several wild- type BRCA1 cancer cell lines and COS cells upregulat- ed p21 expression, several synthetic deletion and trun- cation mutants and risk-associated BRCA1 mutants, including P1749R, Q1756InsC (aka 5382InsC) and Y1853STOP (aka 5677InsA), a frameshift mutation resulting in a premature stop codon that lacks the last 11 amino acids [25], failed to alter p21 expression [26]. Gain of function mutations Although mutations resulting in a premature stop codon are typically susceptible to nonsense-mediated mRNA decay, mounting evidence suggests that mutant mRNA and proteins are not uniformly degraded. Per- rin-Vidoz et al. [27] found that several BRCA1 muta- tions were unaffected by mRNA decay, including 185delAG and 5382InsC, two of the most common risk-associated BRCA1 mutations [28]. Truncation mutant mRNAs may avoid decay by translation re-ini- tiation at a methionine codon downstream of the pre- mature stop codon [29], and consequently, may contribute aberrant gene products coding for trunca- tion proteins exhibiting varying degrees of protein sta- bility that may impart novel cellular functions [30]. It is important to consider that detection of some mutant BRCA1 proteins in clinical samples has proven unsuc- cessful due to technical challenges such as cross-reac- tivity of antibodies with wild-type BRCA1. However, validation studies of mutant proteins in tissue samples are ongoing and will provide a framework within which to view experimental studies of mutant function. BRCA1 mutant proteins may participate in novel protein–protein interactions as a result of aberrant cel- lular localization. Rodriguez et al. [31] found that exogenous missense and truncation mutants lacking a small portion of the BRCA1 C-terminal, including 5382InsC, exhibited aberrant cytoplasmic localization in breast cancer cells, whereas larger truncations resulted in enhanced nuclear localization of mutants. Aberrant localization may result from mutation or loss of the nuclear localization or export signals, impaired recognition of these signals as a result of improper protein folding, or altered interaction with binding partners that impact BRCA1 localization, such as BARD1 [31]. Mutant BRCA1 proteins may convey unique pheno- types by inhibiting the normal function of wild-type BRCA1 in a dominant negative manner by binding BRCA1 and inhibiting its interaction with other pro- teins, or by sequestering BRCA1 binding partners. Likewise, mutant proteins may also convey unique functions by interacting with novel proteins and ⁄ or regulating alternative genes. Indeed, a significant pro- portion of BRCA1-associated breast cancer tissue sam- ples [32], as well as primary cells from mutation carrier-derived ovarian cancer cell xenograft tumors [33], exhibit loss of the wild-type BRCA1 allele con- comitant with increased mutant allele copy number. Consequently, mutant BRCA1 proteins have been shown to impact a range of cellular functions, includ- ing development, proliferation, chemosensitivity, apop- tosis and gene regulation (Fig. 1, Table 1). Role of gain of function mutations for development, cellular proliferation, chemosensitivity, apoptosis and gene regulation Essentially all BRCA1 knockouts are embryonic lethal in mice (reviewed in [34]). However, mice homozygous for a specific synthetic mutation truncating the BRCA1 protein by half are viable, although highly susceptible to multiple tumor types, including lymphomas, sarco- mas, and carcinomas ⁄ adenocarcinomas of the colon, endometrium, lung, liver and mammary gland [35]. Interestingly, introduction of a synthetic BRCA1 trun- cation mutant encoding the first 300 BRCA1 amino acids inhibits mammary gland differentiation and structural formation during murine development, despite the presence of wild-type BRCA1 [36]. Like- wise, when injected into the cleared murine mammary fat pad, primary human breast epithelial cells trans- fected with the BRCA1 D11 splice variant or murine BRCA1-W1777Stop (which mimics the human 1835STOP mutation), undergo limited differentiation and branching and develop extensive hyperplasia [37]. The 5677InsA insertion mutation, resulting in a frameshift and premature stop signal at codon 1853, Risk-associated BRCA1 mutations and their functional implications R. J. Linger and P. A. Kruk 3090 FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS inhibits proliferation of DU145 human prostate cancer cells expressing a low level of wild-type BRCA1 more efficiently than exogenous wild-type BRCA1 [38], whereas a synthetic N-terminal mutant was found to inhibit physical interaction of wild-type BRCA1 and cyclin D1 [39]. In contrast, an exogenous C-terminal fragment of BRCA1 can enhance normal breast epithe- lial cell growth, possibly by acting in a dominant nega- tive manner to inhibit wild-type BRCA1’s growth suppressive function [40]. Similarly, whereas over- expression of wild-type BRCA1 in the ID8 mouse ovarian epithelial cell line diminished proliferation, chemosensitivity and tumorigenicity of intraperitone- ally injected cells, expression of a synthetic truncation mutant encoding the first 602 amino acids of BRCA1 yielded enhanced proliferation and chemosensitivity. Furthermore, when injected intraperitoneally, cells expressing the mutant were significantly more tumori- genic [41]. It should be noted, however, that BRCA1 mutants have also been shown to exhibit some residual wild-type growth function as a result of remaining intact domains. For example, mouse embryonic fibro- blasts homozygous for D11 BRCA1 exhibited a failed G2-M checkpoint [42], whereas breast cancer cells expressing only the 5382InsC mutant maintained an intact G2-M checkpoint [21]. Fan et al. [39] reported that in DU145 prostate cancer cells expressing low levels of wild-type BRCA1, overex- pression of wild-type BRCA1 or 5677InsA increased to- poisomerase inhibitor cytotoxicity, which could be reversed by transfection of synthetic mutants DEcoRI (amino acids 1-302) and DKpnI (amino acids 1-771), yielding chemoresistant cells. Likewise, in the HCC1937 breast cancer cell model system lacking endogenous wild-type BRCA1 , the addition of exogenous wild-type BRCA1 enhanced chemoresistance, which was reversed by cotransfection of DEcoRI and DKpnI [39]. This suggests that mutants can, at least in part, overturn wild-type BRCA1 function, thereby supporting a role for gain of function BRCA1 mutations. The 185delAG (BRAT) mutation, which imparts upon carriers a 66% lifetime risk of developing ovar- ian cancer [43], arises from the deletion of two nucleo- tides (AG) in the second exon of the BRCA1 gene. This deletion results in a reading frame shift that pro- duces a premature stop signal at codon 39 and a trun- cated protein product. Using SV-40 transfected ovarian surface epithelial cells from women with the BRAT mutation, we found that mutant cells exhibited enhanced apoptosis and caspase 3 activation in response to staurosporine [44], possibly related to diminished levels of phospho-Akt, XIAP and cIAP1 [45]. To rule out the possible contribution of wild-type BRCA1 haploinsufficiency to altered apoptosis in 185delAG cells, BRAT was expressed in wild-type BRCA1 ovarian surface epithelial cells. In agreement with our earlier studies, BRAT enhanced caspase 3-mediated apoptosis and diminished levels of phospho-Akt, cIAP1 and XIAP [46]. In more recent studies, we found that BRAT upregulated the expres- sion of maspin [47], a tumor suppressor important in apoptosis, invasion and metastasis that is uniquely overexpressed in several tumor types, including ovarian cancer [48]. Maspin expression has been correlated with cisplatin sensitivity in ovarian cancer cell lines and longer progression-free and overall survival times in ovarian cancer patients [49], and may be involved in BRAT-mediated enhanced chemosensitivity [47]. Lastly, Thangaraju and colleagues [50] found that co-expression of 5382InsC and 5677InsA with wild- type BRCA1 inhibited the wild-type protein’s ability to enhance apoptosis in breast and ovarian cancer cells. Several studies support a role for BRCA1 mutants in gene regulation. For example, wild-type BRCA1 and 5677InsA inhibited exogenous estrogen receptor alpha transactivation, but co-transfection of DBamHI, DKpnI and DEcoRI reversed this phenomenon [39]. Similarly, the synthetic BRCA1 mutant (D500-1863), which encodes a protein less than a third the length of the wild-type, inhibited wild-type BRCA1-mediated activation of a p53 reporter [10]. Likewise, using the mouse mammary gland-specific expression of wild-type BRCA1, a risk-associated mutation that truncates the protein at amino acid 340, or a BRCA1 splice variant that omits the N-terminal 72 amino acids, Hoshino et al. [51] showed that the splice variant mediated hyperproliferation and enhanced lobule formation in the mammary gland. In addition, tumorigenesis and death were accelerated in mice expressing the splice variant. In separate studies, Quaresima and colleagues [52] performed microarray analysis on HeLa cells stably expressing vector, wild-type BRCA1 or the founder mutation 5083del19, which encodes a BRCA1 protein missing the last 193 amino acids, and, conse- quently both BRCT domains, and found differential regulation of multiple genes, including upregulation of periostin. Furthermore, periostin levels were also increased in serum and breast cancer tissue from a small number of patients carrying this mutation. In other studies, expression of a synthetic truncation mutant maintaining the first third of the BRCA1 pro- tein enhanced p53 expression in 1D8 mouse epithelial ovarian cancer cells and downregulated constituents of the SAPK ⁄ JNK and MAPK ⁄ ERK1⁄ 2 pathways [53]. Finally, the missense mutation Ser1841Asn, which is associated with enhanced breast cancer risk, upregu- R. J. Linger and P. A. Kruk Risk-associated BRCA1 mutations and their functional implications FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS 3091 lates D52 (TD52) and the folate receptor alpha (FOL1) in HeLa cells [54]. This regulation is clinically relevant, as expression of these genes correlates with tumor progression in breast [55,56] and ovarian cancers [57,58]. Taken together, these studies support a gain of func- tion role for some mutations. The presence or absence of a mutant function, as well as its impact on the cell, is probably very specific to each mutation and factors impacting mutant function, including mutant protein size, loss ⁄ maintenance of various domains, or struc- tural changes resulting in novel domains. These studies must also be viewed in a cautionary manner. Gain and loss of function experiments provide valuable insight into the mechanism of BRCA1 mutant functions. However, until the presence of stable mutant proteins is validated clinically, it is necessary to remain mindful of the limitations, as well as the promise, of this type of experimental study. Clinical impact of gain of function mutations Studies investigating the effect of BRCA1 mutant pro- teins in the context of wild-type BRCA1 are clinically important. They represent the genotypic and pheno- typic state of disease-free mutation carriers before the loss of both wild-type BRCA1 alleles. Novel functions mediated by mutant proteins have been shown in vari- ous model systems to significantly impact proliferation and apoptosis and, therefore, have the potential to influence cancer initiation, progression and, ultimately, prognosis for patients carrying mutations. Although some mutants may retain specific wild-type BRCA1 functions, others may enhance the risk of cancer devel- opment by antagonizing BRCA1’s tumor suppressive functions. Further investigation of mutant protein function is warranted, as a better understanding of the function of specific mutations could greatly improve risk assessment and prognostic value for mutation carriers. A better understanding of BRCA1 mutant functions may also help to identify novel drug targets for treat- ment and prophylaxis of mutation carriers. Novel interacting proteins and signaling pathways, as well as downstream target genes, may reveal as yet unidenti- fied players in BRCA1 mutation-associated breast and ovarian cancer. Data from our laboratory suggest that genes important for cancer initiation and progression, such as maspin, are differentially regulated in normal human ovarian epithelial cells expressing the BRAT mutation [47]. Furthermore, compared with sporadic breast cancer tissue, BRCA1 mutation-associated breast cancer samples reveal more chromosomal aberrations in specific regions, potentially containing additional tumor suppressors important in BRCA1- dependent tumor initiation and progression [59]. An understanding of specific interacting proteins, signaling pathways and target genes involved in the mechanism of enhanced breast and ovarian cancer risk conveyed by each mutation provides the opportunity for muta- tion-specific personalized therapy for mutation carriers. Similar mutations may also share common functions and respond to similar therapeutic strategies. Further- more, targeting functions of BRCA1 mutants that probably contribute to premalignancy, cancer initiation and the early stages of tumor growth holds great promise for effective prophylactic measures that are less invasive than oophorectomy and mastectomy. It is interesting to speculate that cells heterogeneous for risk-associated mutations, although nontumorigenic in their current state, may represent an initial step towards cellular transformation, although additional changes may be necessary for these cells to become malignant. Likewise, early changes that may promote malignant transformation, including enhanced telo- meric instability, have been observed in cell lines gener- ated from normal ovarian surface epithelial cells of women with a strong family history of ovarian cancer [60] (reviewed in [61]). Furthermore, several studies have found more frequent occurrence of deep invagin- ations in the ovary surface, dysplasia, hyperplasia and ⁄ or surface papillae in high-risk prophylactically removed ovaries versus normal ovaries [62–64], suggest- ing that early ‘premalignant’ changes may already exist in those carriers. The possibility of independent mutant BRCA1 functions does not exclude the contribution of other oncogenes, tumor suppressors or invasion ⁄ metastasis-promoting proteins. Conversely, these early changes probably facilitate further cellular changes that manifest in the aggressive phenotype seen clinically in hereditary breast and ovarian cancer. Lastly, there are salient differences between the mechanisms of tumor initiation and progression of breast and ovarian cancer in BRCA1 mutation carriers. The lifetime risk for development of breast cancer is higher than that for ovarian cancer [14], and carriers do not always develop both types of disease. Further- more, the importance of differential expression and stoichiometry of transcription factors and signaling molecules in different tissues is also well established. The impact of specific mutants is, therefore, probably context specific. Holt and colleagues [22] observed a series of BRCA1 mutants to be largely ineffective in inhibiting the growth of breast cancer cells. However, one mutant was shown to inhibit the growth of three Risk-associated BRCA1 mutations and their functional implications R. J. Linger and P. A. Kruk 3092 FEBS Journal 277 (2010) 3086–3096 ª 2010 The Authors Journal compilation ª 2010 FEBS ovarian cancer cell lines. You et al. [65] also found cell type-specific BRCA1 mutant functions. Although expression of the 185delAG mutation in immortalized ovarian surface epithelial cells and ovarian cancer cells revealed multiple downstream effectors and physiologic impacts [46,47], primary and immortalized cells derived from normal breast tissue of a 185delAG mutation carrier did not show a significant difference in growth, stress response, growth in soft agar or tumorigenicity when compared with normal breast epithelial cells homozygous for wild-type BRCA1 [66]. Several epide- miological studies have observed differential ovarian and breast cancer risk based on the location of the truncation mutation within the BRCA1 gene [67,68]. Disparate risk levels may represent tissue-specific degrees of importance for the specific functions lost or gained as a result of each mutation, and the interplay of these factors. In conclusion, it is clear from a wide range of model systems and endpoints that BRCA1 mutations are capable of significant physiological impacts. Further- more, molecular and phenotypic changes are evident in mutation carriers. These changes may result from loss of wild-type BRCA1 function, gain of function muta- tions or both. Consequently, further experimental and clinical studies of mutant BRCA1 proteins are war- ranted, and will provide a better understanding of mutation-associated breast and ovarian cancer and improve the strength of prognosis and efficacy of pro- phylaxis and treatment for mutation carriers. 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