Oncogene (2000) 19, 6152 6158 2000 Macmillan Publishers Ltd All rights reserved 0950 9232/00 $15.

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BRCA1 and cell signaling

Qiang Wang*,1, Hongtao Zhang1, Richard Fishel2 and Mark I Greene1
1

Department of Pathology and Laboratory Medicine, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA; 2Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jeerson University, Philadelphia, Pennsylvania, PA 19107, USA

The breast cancer and ovarian cancer susceptibility gene BRCA1 encodes a nucleoprotein whose mutations or aberrant expression is associated with both inherited and sporadic cancers. Studies over the last 6 years have suggested that BRCA1 may function as a scaold in the assembly of a multi-protein complex, which plays a role in gene transcription, DNA damage repair, and transcription-coupled DNA damage repair. In this review, we discuss the implications drawn from the studies of BRCA1-interacting proteins and the cellular signaling pathways that may be involved in controlling the functions of BRCA1. Oncogene (2000) 19, 6152 6158. Keywords: BRCA1; gene transcription; DNA damage repair BRCA1 BRCA1 is a tumor suppressor gene that is mutated in a high percentage of hereditary breast and ovarian cancers (Futreal et al., 1994). BRCA1 has also been implicated in some cases of sporadic breast cancers (Merajver et al., 1995; Russell et al., 2000; Thompson et al., 1995; Zheng et al., 2000). The human brca1 gene has been mapped to chromosome 17q21 and encodes a protein of 1863 amino acid residues (Miki et al., 1994). Analysis of the BRCA1 protein has revealed that it contains an N-terminal Ringnger domain and two C-terminal BRCT (BRCA1-CTerminal) domains, both involved in protein protein interactions (Jensen et al., 1998; Li et al., 1999; Wong et al., 1998; Wu et al., 1996; Yarden and Brody, 1999; Yu and Baer, 2000; Yu et al., 1998). The BRCA1 protein is localized to the nucleus of the cell via two nucleus translocation signals (Chen et al., 1995, 1996). The brca1 gene is conserved in mammals, but not in lower animals (Bennett et al., 1995). BRCA1 is essential for embryonic development and brca1 knockout mice die between embryonic day 7.5 and day 13 (Gowen et al., 1996; Hakem et al., 1996; Liu et al., 1996). In order to elucidate the molecular and biochemical functions of BRCA1, a number of researchers have tried to isolate and characterize BRCA1-associated proteins. Several proteins have been shown to interact with BRCA1, which sheds some light on the functions of BRCA1. BRCA1 as a modulator of gene transcription An accumulating body of evidence has implicated that BRCA1 may play a role in controlling gene transcrip*Correspondence: Q Wang

tion. For example, the C-terminus of BRCA1 can activate transcription when it is fused to a protein domain that binds to a promoter (Chapman and Verma, 1996; Monteiro et al., 1996, 1997). This segment of the BRCA1 polypeptide is rich in acidic amino acid residues, a character found in the activation domain of many transcription factors. BRCA1 was found to be associated and co-puried with the RNA polymerase complex (Schlegel et al., 2000; Scully et al., 1997a). In addition, BRCA1 also interacts with RNA Helicase A, which is involved in gene transcription (Anderson et al., 1998). These observations suggest that the BRCA1 polypeptide is a component of the transcription machinery. Moreover, the BRCA1-binding protein BARD1 can associate with polyadenylation factor CstF50 and inhibit polyadenylation in vitro (Kleiman and Manley, 1999). BARD1 was found co-localized with BRCA1 during S phase but not in G1 (Jin et al., 1997). Therefore, the BARD1-BRCA1 complex may also play a role in gene transcription at the level of polyadenylation of mRNA. BRCA1 has also been found to interact with a number of transcription factors, such as p53 (Ouchi et al., 1998; Somasundaram et al., 1997; Zhang et al., 1998) and c-Myc (Wang et al., 1998). In these studies, however, it appears that BRCA1 can activate transcription in some cases and repress transcription in some others. For example, BRCA1 can bind to p53 and enhance its transcription activity at the promoter of p21WAF/CIP or BAX (Ouchi et al., 1998; Somasundaram et al., 1997; Zhang et al., 1998). Furthermore, BRCA1-induced cell cycle arrest is dependent on functional p21WAF/CIP (Somasundaram et al., 1997). In contrast, BRCA1 inhibits the transcriptional activity of c-Myc at the CDC25A promoter and reverse the cellular transformation activity of c-Myc (Wang et al., 1998). Interestingly, CtIp, a protein implicated in repression of gene transcription, can bind to the C-terminal portion of BRCA1 (Li et al., 1999; Wong et al., 1998; Yu and Baer, 2000; Yu et al., 1998). Furthermore, CtIP is phosphorylated in response to radiation, which is dependent on ATM (Yu and Baer, 2000). Hence, it is likely that CtIp is a negative regulator on the activity of BRCA1 in transcription, in response to DNA damage. Although BRCA1 may inhibit c-Myc transcription activity by aecting Myc binding to DNA (Wang et al., 1998), it remains possible that CtIP is also involved in repression of Myc activity. Ectopically expressed BRCA1 can induce expression of GADD45 in a manner independent of p53 (Harkin et al., 1999). The BRCA1-responsive element was mapped to a small region in the GADD45 promoter, which contains a potential binding site for C/EBP (Jin et al., 2000).

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Notably, previous works have demonstrated that cMyc can repress GADD45 expression by inhibiting C/ EBP activity at the promoter (Amundson et al., 1998; Constance et al., 1996; Li et al., 1994; Marhin et al., 1997). In this regard, suppression of c-Myc by BRCA1 may be important for activation of GADD45, since BRCA1 may thus de-repress the GADD45 promoter. In addition to c-Myc, BRCA1 can inhibit transcription events mediated by the estrogen receptor (ER-alpha) through an unknown mechanism (Fan et al., 1999). Finally, BRCA1 can associate with the hyperphosphorylated form of pRb, which may be essential for BRCA1 induced suppression of proliferation (Aprelikova et al., 1999). BRCA1 may also modulate gene transcription by aecting chromatin structures through acetylation or deacetylation of histones. Chromatin remodeling is involved in biological processes such as activation and repression of gene transcription, DNA damage repair, and DNA replication. First, BRCA1 has been found to associate with components of the histone deacetylase complex, including HDAC1, HDAC2, RbAp46 and RbAp48 (Yarden and Brody, 1999). Two other BRCA1-interacting proteins, pRb and CtIP, have been shown to repress gene transcription by recruiting HDAC complexes to the promoter (Aprelikova et al., 1999; Li et al., 1999; Yarden and Brody, 1999). Hence, BRCA1 may be present in a large protein complex that deacetylates histone and thereby limits gene transcription. These ndings may provide another explanation on how BRCA1 suppresses gene transcription mediated by c-Myc or estrogen receptors. In addition, the BRCA1-CtIP complex may also suppress gene transcription by a mechanism that involves histone deacetylation activity. Second, BRCA1 also appears to associate with a protein complex that activates gene transcription by histone acetylation. For example, CBP/p300 associates with BRCA1 in a cell cycleindependent manner, and activates the Rous sarcoma virus repeat promoter (Pao et al., 2000). Thirdly, the C-terminal portion of BRCA1 changes the chromatin structure and stimulates local DNA replication in a yeast model (Hu et al., 1999). Finally, the presence of histone acetylase or deacetylase activity in the BRCA1containing complex may be important for chromatin remodeling during DNA damage repair. Although BRCA1 can associate with the RNA polymerase complex and some transcription factors, it does not appear that BRCA1 is a universal component of the transcription machinery. For instance, BRCA1 has little eect on transcription controlled by USF, Jun, Fos, or Gal4 (Fan et al., 1999; Jin et al., 2000; Wang et al., 1998). It is likely that BRCA1 only modulate a subset of transcription factors that are involved in cell proliferation or apoptosis, such as p53, c-Myc, or pRb. It is also possible that BRCA1's role in transcription is coupled with its role in DNA damage repair. BRCA1 in double-strand break DNA damage repair Over the last 3 years, it was found that BRCA1 can form complexes with a number of proteins directly involved in DNA damage repair (Figure 1). Scully et al. (1997b,c) rst observed that BRCA1 can associate

with RAD51, a gene involved in homologous DNA recombination and repair. In a study of cellular localization of BRCA1, both BRCA1 and RAD51 were found localized to a nuclear speckle structure. These nuclear dots, whose function is unknown, are relocated at the S phase of the cell cycle or in response to DNA damaging reagents (Scully et al., 1997c). Secondly, BRCA1 has also been shown to interact and co-localize with RAD50/MRE11/NBS1 (Zhong et al., 1999), a protein complex implicated in homologous recombination, non-homologous end joining (NHEJ), and meiotic recombination DNA damage response (Haber, 1998). Notably, formation of irradiationinduced foci was only seen in BRCA1 positive cells. Thirdly, BRCA1 can interact with BRCA2, which is also implicated in DNA damage repair (Chen et al., 1998). All these observations suggest that BRCA1 may play a role in repair of double strand DNA breaks (DSBs). This hypothesis is further supported by genetic evidence. For example, BRCA1 is essential for

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Figure 1 Regulation of BRCA1 functions. (a) BRCA1 becomes serine phosphorylated by ATM in response to g irradiation. Tyrosine phosphorylated of BRCA1 may be inhibited by the oncogenic form of p185neu tyrosine kinase receptor. BRCA1 is involved in DNA damage repair through its interaction with RAD51, BRCA2, the Rad50/Mre11/NBS protein complex, or the MSH proteins. BRCA1 can also bind to p53 and pRb, which may lead to cell cycle arrest or apoptosis. (b) BRCA1 can bind to cMyc and inhibit its transcription and transformation activity. CtIP and HDAC1/2 bind to BRCA1, respectively, and may be involved in transcription repression. BRCA1 may activate GADD45 by reversing the inhibitory eect of c-Myc on GADD45 promoter. BRCA1 also represses transcription mediated by the estrogen receptors
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homology-directed DNA repair and stem cells from BRCA1 knockout mice showed defects in repair of DNA double-stand breaks by homologous recombination (Moynahan et al., 1999). In contrast, repair of DSBs based on non-homologous end joining (NHEJ) was not aected, which may cause aberrant repair events and lead to translocation (Moynahan et al., 1999). Furthermore, ectopic expression of BRCA1 by retroviral vectors can decrease cellular sensitivity to radiation and increase the eciency for DNA double break repair (Scully et al., 1999). Taken together, the current evidence supports the hypothesis that BRCA1 is phosphorylated in response to DNA damage, which in turn activates or assembles a set of proteins involved in DNA damage repair, cell proliferation, or apoptosis (Figure 1). BRCA1 in DNA mismatch repair and transcription-coupled DNA damage repair More recently, we and others have shown that BRCA1 can interact with MSH2, MSH3 and MSH6 (Wang et al., submitted, 2000). These MSH proteins are involved in repair of mismatched DNA lesions as a result of spontaneous errors during DNA replication or during repair of double-strand DNA breaks. (Evans et al., 2000; Fishel and Wilson, 1997; Paques and Haber, 1999). Using the yeast two-hybrid system, we isolated MSH2 as a BRCA1-interacting protein. We further demonstrated that both BRCA1 and BARD1 can associate with MSH2 and MSH6 (Wang et al., submitted.) Interestingly, the BRCA1-MSH2 interaction is dependent on the adenine-nucleotide-binding motif of MSH2. More importantly, BRCA1 appears to have high anity to the ATP-bound form of the MSH2-MSH6 complex, but not to the ADP-bound form (Figure 2). In an independent study, Wang et al. (2000) have reported co-purication of BRCA1 with a number of proteins involved in DNA damage repair, including MSH2, MSH6, MLH1, ATM, BLM, and the RAD50-MRE11-NBS1 protein complex. It has been postulated that the ADP-bound form of the MSH2MSH6 heterocomplex can recognize and bind mismatched DNA lesions, which is followed by exchange of ADP with ATP (Fishel, 1998; Gradia et al., 1997, 1999, 2000). The ATP-bound form of the MSH2MSH6 heterocomplex acts as a sliding clamp on DNA until hydrolysis of ATP occurs in the presence of yet unidentied co-factors (Fishel, 1998; Gradia et al., 1997, 1999, 2000). Hence, binding of BRCA1 to the ATP-bound form of MSH2/MSH6 may reect a regulatory role of BRCA1 in DNA mismatch repair (Figure 2). More importantly, it is plausible that the sliding clamp of MSH2-MSH6 may transmit a signal via BRCA1 to downstream events such as cell cycle arrest or apoptosis, as a consequence of accumulation of mismatched DNA lesion, (Figure 2). Therefore, as a sensor for mismatched DNA lesions, the MSH2-MSH6 multi-protein complex may not only interact with the mismatch repair machinery, but also modulate checkpoints for cell cycle progression and apoptosis. Indeed, it was found that overexpression of MSH2 causes apoptosis, just like that of BRCA1 does (Toft et al., 1999; Zhang et al., 1999). MSH2-induced apoptosis is partially dependent on p53 (Toft et al., 1999; Zhang et

Figure 2 BRCA1 in mismatched DNA repair and transcriptioncoupled DNA damage repair. BRCA1 has low anity to the ADP-bound-form of the MSH2-MSH6 complex, which can recognize mismatched DNA. The exchange of ADP for ATP occurs after binding of the complex to mismatched DNA lesion, and the ATP-bound form of the MSH2-MSH6 complex forms a sliding clamp on the DNA. BRCA1 bind to the ATP-bound form of the MSH2-MSH6 complex with a high anity. The C-terminal portion of BRCA1 can interact with the RNA polymerase complex. Other MSH2-interacting proteins involved in mismatch repair are not shown

al., 1999). It remains to be determined if BRCA1 is required for MSH2-induced apoptosis. Since MSH2 has been implicated in transcriptioncoupled DNA damage repair (Leadon and Avrutskaya, 1997), the nding of BRCA1-MSH2 interaction suggests that BRCA1 may also play a role in these biological events (Figure 2). There is genetic evidence showing that BRCA1 is essential for transcriptioncoupled repair of oxidative DNA damage (Gowen et al., 1998). BRCA1 expression also restored radiation resistance in Brca1-defective cells by enhancing transcription-coupled DNA damage repair (Abbott et al., 1999). We have also found that BRCA1 associates with the valosin-containing protein (VCP) in the nucleus, which may function as an ATP transporter in these events (Zhang et al., 2000). Conceivably, BRCA1 is a component of both the DNA damage repair machinery and the transcription-coupled repair machinery. It is also possible that the BRCA1 associated ensemble of polypeptide is a much more complicated superstructure involved in both DNA damage repair and gene transcription. Is BRCA1 involved in chromosome segregation? Some recent studies have demonstrated that BRCA1 may be required for preventing abnormal chromosome segregation. For example, human tumor cells which lack functional BRCA1 demonstrate high frequency of chromosome aneuploidy (Tirkkonen et al., 1997; Tomlinson et al., 1998). In mouse embryonic cells that have defective exon 11 of BRCA1, the checkpoint at G2-M phase of the cell cycle is impaired, leading to a phenotype of unequal chromosomal segregation, abnormal nuclear division, and aneuploidy (Xu et al., 1999). The mechanism by which BRCA1 controls chromosome segregation is not clear. Since BRCA1 is probably involved in gene transcription and/or DNA

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damage repair, loss of BRCA1 function may lead to de-regulation or mutation of genes that mediate checkpoints for appropriate chromosome segregation, such as p53. We also noted that a number of protooncogenes, which may be inhibited by BRCA1, can cause chromosome aberration. For example, transient overexpression of c-Myc leads to genomic instability, including aneuploidy and gene amplication (Felsher and Bishop, 1999; Felsher, 2000). Loss of p53 in these cells does not aect myc-induced aneuploidy (Felsher, 2000). Similarly, the mammary tumors developed in MMTV-myc transgenic mice were characterized with chromosome aneuploidy and translocation, a phenotype independent of p53 status (McCormack et al., 1998; Weaver et al., 1999). Recently, a family of Aurora-like serine/threonine kinases has been found to inuence microtubule dynamics during mitosis (Bischo et al., 1998; Terada et al., 1998; Zhou et al., 1998). For example, STK15/ BTAK, which is overexpressed in breast tumors, can induce centrosome amplication, followed by aneuploidy and cellular transformation (Zhou et al., 1998). However, it is not known how BRCA1 or c-Myc is related to the functions of these kinases. BRCA1 may also directly regulate the appropriate distribution of chromosomes into the daughter cells. A recent report showed that the BRCA1 protein appears to be co-localized and co-puried with the centrosome and that the hyperphosphorylated form of BRCA1 can be co-immunoprecipitated with g-tubulin, a component of the centrosome essential for microtubule nucleation (Hsu and White, 1998). However, it is yet to be demonstrated whether dislocation of BRCA1 from the centrosome would cause any defects in chromosome segregation. BRCA1 in cell cycle arrest and apoptosis The linkage of its loss of heterozygosity (LOH) with cancers has suggested that brca1 is a tumor suppressor gene (Futreal et al., 1994). Some reports have demonstrated that BRCA1 has an inhibitory eect on cell proliferation and growth. For example, overexpression of BRCA1 causes growth retardation in breast and ovarian cell lines (Holt et al., 1996). There has been conicting evidence on the potential role of BRCA1 in apoptosis. In a study using a tetracyclineinducible expression system, ectopic expression of BRCA1 was found to induce apoptosis (Harkin et al., 1999). Moreover, cell death was also induced in a stable cell line transfected with a deletion mutant of BRCA1 when coupled with serum withdrawal (Shao et al., 1996). In contrast, when ectopic BRCA1 expression was introduced by adenoviral vectors, little apoptosis was observed and some cell lines were arrested in the G2/M phase of the cell cycle (MacLachlan et al., 2000). The varied participatory roles of BRCA1 in apoptosis may arise from one or a combination of the following factors. First, the genetic background of the cell lines used in these studies was dierent. Second, the expression levels of BRCA1 in the inducible system and the adenoviral system may not be comparable. It is likely that the protein levels of BRCA1, or in particular, the phosphorylation levels or localization of BRCA1, may determine the fate of cells

in the decision between cell cycle arrest and apoptosis. It has been postulated that BRCA1 may transmit a signal from sensors of DNA damage to a `switch' that determines cell cycle arrest versus apoptosis (Wang et al., submitted; MacLachlan et al., 2000). The DNA damage may be `sensed' by protein complexes involved in damage repair, such as RAD51, RAD50/MRE11/ NBS1, or MSH2/MSH6, which eventually may lead to apoptosis in a BRCA1-dependent fashion, when DNA damages are beyond repair (Figures 1 and 2). BRCA1-induced apoptosis is dependent on functional Jun N-terminal Kinase (JNK/SAPK), accompanied with induction GADD45 expression (Harkin et al., 1999). A recent study have shown that GADD45 and its homologues physically associate with MTK1, a MAPKKK that activates JNK-SAPK and is partially required for GADD45-induced apoptosis (Takekawa and Saito, 1998). Hence, BRCA1 may mediate apoptosis through the GADD45-JNK/SAPK pathway. Although it is required for directly activating GADD45 transcription upon some environmental stress, p53 is not essential for apoptosis or GADD45 expression induced by BRCA1 (Harkin et al., 1999). Regulation of BRCA1 functions How is BRCA1 regulated? This is an important issue, since any deciency in appropriate modulation of BRCA1 activity may predispose to tumorigenesis. One mechanism that may regulate the function of BRCA1 is through phosphorylation. Some reports have shown that BRCA1 is phosphorylated in a cell cycle-dependent manner. For instance, BRCA1 is hyperphosphorylated during the late G1 and S phase of the cell cycle, and is transiently de-phosphorylated at early M phase (Runer et al., 1999; Runer and Verma, 1997). BRCA1 can also be phosphorylated on certain serine residues in response to ionizing radiation, which required functional ATM (Ataxia Teleangiectasia Mutated) proteins (Cortez et al., 1999; Gatei et al., 2000). Indeed, it was found that ATM can bind and phosphorylate BRCA1 directly. Furthermore, phosphorylation of BRCA1 is required for rescuing radiation hypersensitivity of brca1-defective cells, since mutations of corresponding phosphorylation sites of BRCA1 abolish its ability to protect cells from radiation (Cortez et al., 1999; Gatei et al., 2000). Finally, BRCA1 also becomes tyrosine phosphorylated at the G2/M phase (Zhang et al., 1997). In contrast, the tyrosine phosphorylation levels of BRCA1 are substantially low in cells transformed by the oncogenic form of p185neu receptor (Zhang et al., 1997). It is not clear how the functions of BRCA1 are modulated by phosphorylation. Perhaps, the phosphorylation states of BRCA1 determine its ability to bind to other proteins and aect its biochemical activities in DNA damage repair or gene transcription. Phosphorylation of BRCA1 may aect its cellular localization or stability. Another way to modulate the function of BRCA1 is through phosphorylation of proteins that are associated with BRCA1. For example, CtIP is phosphorylated in response to radiation, which is also dependent on ATM (Yu and Baer, 2000). Since CtIP is implicated in transcription repression, it is possible that the

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transcription repression activity of BRCA1 is modulated by phosphorylation of CtIP. The BRCA1 polypeptide may also be susceptible to modications such as ubiquitination or SUMOlyation. Jensen et al. (1998); Jensen and Rauscher (1999) reported that BRCA1 can interact with BAP1, a protein that is possibly involved in removing ubiquitin from BRCA1 and increase protein stability by preventing ubiquitin-mediated protein degradation. We have also found that BRCA1 can associate with Ubc9, a protein that plays a role in an analogous process termed SUMOlyation (Wang et al., unpublished). Similar to ubiquitination, proteins may be modied by covalently binding of a ubiquitin-like protein SUMO1, mediated by the conjugating factor Ubc9. Hence, it is likely that BRCA1 is also susceptible to SUMOlyation, which may either protect the protein from degradation or aect its cellular localization. Hence, ubiquitination or SUMOlyation may be another level of regulation on BRCA1 functions. Although the molecular functions of BRCA1 in tumorigenesis are not fully understood, there is an emerging picture that BRCA1 may be regulated by certain signaling pathways. First, ATM can phosphorylate BRCA1 and may modulate its function in response to g-irradiation (Cortez et al., 1999). ATM becomes activated by DNA damage and is required for repair of double-strand DNA breaks. Phosphorylation of BRCA1 by ATM does not appear to aect its nuclear translocation, but mutations of the ATMphosphorylation sites abrogate BRCA1's ability to rescue cell after radiation (Cortez et al., 1999). Therefore, the ATM pathway is likely to be required for BRCA1-mediated repair of double-strand DNA breaks, a process which probably also involves RAD51 and/or the RAD50-MRE11-NBS1 protein complex. It should also be noted that ATM is not required for phosphorylation of BRCA1 by other factors, such as UV irradiation or treatment with the DNA replication-blocking reagent hydroxyurea (Cortez et al., 1999; Scully et al., 1997b). Hence, BRCA1 may be activated by other ATM-like proteins or through other unidentied pathways. Secondly, the tyrosine receptor p185her2/neu may represent a signaling
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Acknowledgments This work is supported by the Abramson Family Institute for Cancer Research.

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