Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer

Subject Review Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer

Ceshi Chen,1 Arun K.Seth, 2 and Andrew E.Aplin 1

1The Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York and 2Laboratory of Molecular Pathology, Department of Anatomic Pathology, Division of Molecular and Cellular Biology, Sunnybrookand Women’s College Health Science Center, Toronto, Ontario, Canada

Abstract Genetics Alterations of Breast Cancer Recent studies revealed that E3 ubiquitin ligases play Many genetic studies of breast cancer show that tumor important roles in breast carcinogenesis.Clinical development and progression involves the accumulation of research studies have found that (epi)-genetic (deletion, various genetic defects, including amplification and concomi- amplification, mutation, and promoter methylation) and tant overexpression of certain oncogenes and mutation or loss expression aberration of E3s are frequent in human of various tumor suppressor genes. Accumulated evidence breast cancer.Furthermore, many studies have suggests that amplification and overexpression of Her-2/ErbB-2 suggested that many E3s are either oncogenes or tumor (17q12), cyclin D1 (11q13), and Myc (8q24) contributes to a suppressor genes in breast cancer.In this review, we subset of breast cancer. Simultaneously, germ line mutations of provide a comprehensive summary of E3s, which have BRCA1 and BRCA2 play important roles in a subset of familial genetic and/or expression aberration in breast cancer. and sporadic breast cancer. Most cancer-related E3s regulate the cell cycle, p53, However, breast cancer is heterogeneous, and many other transcription, DNA repair, cell signaling, or apoptosis. genetic alterations have also been detected. For example, gene An understanding of the oncogenic potential of the E3s amplification at 17q23(2) and 20q11-13(3)and chromosome may facilitate identifying and developing individual E3s loss at 8p22 (4), 11q23(5), and 16q22 (6) are frequently as diagnosis markers and drug targets in breast cancer. detected in breast cancer by fluorescent in situ hybridization, (Mol Cancer Res 2006;4(10):695–707) comparative genomic hybridization, and loss of heterozygosity studies. The implicated target genes of these genetic alterations have not been firmly revealed, although a number of interesting Introduction candidate genes may contribute to progression of breast cancer. Breast Cancer In addition to chromosomal instability and gene mutation, Breast cancer is the second leading cause of cancer death in epigenetic alteration by abnormal promoter methylation seems American women. The American Cancer Society estimates that to be another approach to inactivate some tumor suppressor there will be about 212,920 new cases of breast cancer, and that genes in breast cancer (7). about 41,430 women will die of this disease in the United States There are an overwhelming number of abnormalities that in 2006 (1). Hormone therapy and chemotherapy have been have been identified in breast cancer in molecules involved in shown to improve survival of patients with breast cancer; protein degradation through the ubiquitin proteasome system however, the serious side effects of these treatments inspire the (UPS). The field of protein ubiquitination and degradation has development of targeted therapy. It is important to understand experienced explosive growth in the past decade. The UPS the mechanism/s involved in breast cell transformation and to regulates an enormous range of biological processes, including identify novel molecular targets for use in prevention, diagnosis, and therapy. the cell cycle, apoptosis, transcription, protein trafficking, signaling, DNA replication and repair, and angiogenesis (see refs. 8, 9 for reviews). Consistently, defects in the UPS have already been directly implicated in many diseases, including Received 6/27/06; revised 8/3/06; accepted 8/28/06. breast cancer (10-13). Well-known examples are the deregula- Grant support: Susan G. Komen Breast Cancer Foundation grant BCRT#0503705 (C. Chen), CBCRA grant (A.K. Seth), and NIH grant R01- tion of the ubiquitin ligases BRCA1, Mdm2, and Skp2 (see GM067893(A.E. Aplin). below in detail). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. UPS Note: We apologize to the many authors who we could not cite because of the Protein homeostasis in mammalian cells is tightly controlled space limit. Requests for reprints: Ceshi Chen, The Center for Cell Biology and Cancer by protein synthesis and degradation. Rapid protein turnover is Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY a typical feature for many important proteins that regulate the 12208. Phone: 518-262-2936; Fax: 518-262-3065. E-mail: [email protected] kip1 Copyright D 2006 American Association for Cancer Research. cell cycle, apoptosis, and transcription, such as p53, p27 , doi:10.1158/1541-7786.MCR-06-0182 and Myc. Selective degradation of these proteins through the

UPS plays an essential role in normal cell growth and As several extensive reviews of ubiquitination in breast differentiation, whereas abnormal accumulation or hyperactive cancer are available (12, 13), here, we provide a comprehensive degradation of these regulatory proteins are associated with review on the role of E3s in breast cancer. We focus on recent carcinogenesis. advances concerning aberrant expression and genetic alterations Proteins degraded through the proteasome are often first of E3s in breast cancer and also common mechanisms of E3 tagged by a K48 polyubiquitin chain (below in detail), through action in breast cancer. Finally, we discuss the challenge of the action of three classes of enzymes: the ubiquitin-activating identifying and developing E3s as drug targets and diagnosis enzyme (E1), ubiquitin-conjugating enzymes (E2), and ubiq- markers in breast cancer. uitin ligases (E3). Although the human genome encodes a unique E1 and limited numbers of E2s (f50), there are hundreds of different E3s in human cells. Ubiquitin is a small E3 Ligase Subfamilies conserved protein with 76 amino acids. E1 binds to and E3s carry out the key rate-limiting step in ubiquitin-mediated activates ubiquitin and then passes the activated ubiquitin to an proteolysis. In mammalian cells, abundant ubiquitin ligases E2. The E2s have only limited substrate specificity. Typically, guarantee specific substrate recognition. Researchers have now an E2 transfers the ubiquitin to a substrate and conjugates the identified >500 E3s (Table 1). Many of these E3s contain the ubiquitin moieties to the NH2 groups of the substrate’s Lys homologous to E6-associated protein (E6-AP) COOH terminus through interaction with an E3. The E3 is a scaffold protein that (HECT) domain or the really interesting new gene (RING) recognizes specific substrates. finger domain. Although all HECT domain and some RING Attachment of a single ubiquitin to a single lysine of a finger E3s can function independently, some RING finger substrate protein is called monoubiquitination. Most membrane proteins form E3complexes with Cullins and several other receptors, such as epidermal growth factor receptor and platelet- proteins, such as F-box; bric a brac, tramtrack, and broad derived growth factor receptor, with monoubiquitin modifica- complex (BTB)-box; or suppressor of cell signaling (SOCS)- tion undergo endocytosis and are degraded in lysosome box proteins. Recently, two RING-like domains, the U-box and (reviewed in refs. 14, 15). Additionally, monoubiquitin the plant homeo domain (PHD), have also been shown to have modification for transcription factors, such as Smad4 (16) and E3activity. Here, we outline the basic features of each E3ligase p53(17), can also serve as a signal for transcription activation subfamily. or trafficking, respectively. Moreover, monoubiquitination of FANCD2 participates in DNA repair (reviewed in ref. 18). HECT Domain E3s Following monoubiquitination, the second ubiquitin mol- The number of proteins containing HECT domain in the ecule can be conjugated to previous one through an iso- human genome is estimated to be about 82 according to the 76 peptide bond between Gly of a new ubiquitin molecule and Pfam database. The actual number that has been characterized q the -NH2 groups of one of the seven lysines (K6, K11, K27, from humans is around 30 (23). The HECT domain K29, K33, K48, and K63) of the previous conjugated containing proteins can form thiol intermediates with ubiquitin. After several rounds of catalysis by the E2/E3 ubiquitin and function independently as E3s (Fig. 1). Several complex, a polyubiquitin chain will be conjugated to a HECT domain E3s, such as human papilloma virus gene substrate protein. The predominant polyubiquitin chains in product E6-AP, Smurf2, WWP1, and ARF-BP1, have been cells are through the K48 and K63branching points. As implicated in human breast cancer. For example, E6-AP is an mentioned earlier, proteins tagged with a K48 polyubiquitin E3enzyme that targets the p53protein (24). The catalytic chain are able to be rapidly degraded by a large cytosolic cysteine near the COOH terminus of E6-AP can transfer protease complex named the 26S proteasome. On the other ubiquitin from E2 to p53, leading to the ubiquitin-dependent hand, proteins with a K63polyubiquitin chain will not be degradation of the p53. targeted for degradation but rather have been shown to play important role in nuclear factor-nB signaling transduction (reviewed in ref. 19). Table 1.Classification and Estimated Numbers of Human Protein ubiquitination and degradation are usually regulated E3s in a temporal and spatial manner. In most cases, the substrate must be modified first, through mechanisms such as phos- E3s Subfamily No. proteins References phorylation, prolylhydeoxylation, acetylation, glycosylation, or HECT f30-82 (23) cleavage before the destruction domain is recognized by E3s RING finger f350-510 (23) (20). Alternatively, the E3itself could be activated by U box 6-16 (27) posttranslational modification. For example, the anaphase- PHD domain 211 Cullin-based Cullins f5-24 (202) promoting complex/cyclosome (APC/C) is phosphorylated E3s and APC/C and activated when cells enter mitosis. Activity of AIP4/ITCH F-box f70 (33, 203) is dramatically activated by c-Jun NH -terminal kinase– SOCS f40-72 (204) 2 BTB f183-273 (38) mediated phosphorylation and negatively regulated by Src Skp1, ElonginC, >15 kinase Fyn-mediated tyrosine phosphorylation (21, 22). By DDB1/2, ubiquitin-mediated proteolysis, cells quickly regulate the cell APC/C, etc cycle, transcription, migration, and apoptosis in response to NOTE: The estimated numbers from the cited literature and the Pfam data base internal and external signaling. are shown.

ubiquitinating extracellular signal-regulated kinase 1/2 after activating them by phosphorylation. This activity is PHD domain dependent (30). Although there are hundreds of PHD domain–containing proteins in the human genome, whether all these proteins have E3activity remains to be explored.

Cullin-BasedandAPC/C E3 Complexes Cullin-based E3s contain multiple subunits (for a review, see ref. 33). Cullins serve a scaffold function within E3 ligase complexes. Typically, the NH2-terminal domain of cullins recruits the substrate through one or two adaptor proteins (F-box/SOCS/BTB proteins), whereas the COOH-terminal domain of the cullins binds to an E2 through a zinc-binding RING domain protein (Roc1 or Roc2). There are at least seven cullins in human cells. Two well-studied cullin-based E3s are the Cul1-based E3SCF complex and the Cul2-based E3ECS (ElonginC, Cul2/5, SOCS) complex. Available results support that Cul3and Cul4A also form E3complexes. Additionally, the APC/C E3complex also contains a protein (APC-2) with cullin domain.

FIGURE 1. Classification of E3 ubiquitin ligases. Three major types of E3 are illustrated. The PHD domain and U-box E3 are included in RING F-Box Proteins finger E3s because of structure similarity. The adaptor for Cullin-based E3 The F-box is an f40-amino-acid motif, originally identified could be Skp1-F-box, Elongin C-SOCS, or BTB proteins. Right, examples in cyclin F (34). F-box–containing proteins mediate substrate for each type of E3. S, substrate of an E3. recognition for SCF complexes. F-box proteins bind to Skp1, which, in turn, binds Cul1. In addition to interaction with Skp1 RING Finger E3s through the F-box motif, F-box proteins recruit substrates There are >350 potential RING finger proteins; hence, this through distinct protein-protein interaction domains, such as class represents the largest subfamily of ubiquitin ligases. WD40 repeats and leucine-rich repeats. There are >70 F-box Consistently, many RING finger E3s, including Mdm2, proteins in the human genome; however, few have been BRCA1, BARD1, and estrogen-inducible RING finger protein characterized in detail (33). The most well studied h (EFP), have been suggested to play important roles in F-box proteins in breast cancer are Skp2, -TrcP, and Fbw7. mammary carcinogenesis. The RING finger motif contains an octet of cysteine and histidine residues that constitute a zinc SOCS Proteins binding domain. It has become clear that many RING finger Proteins with the SOCS-box motif, similar to F-box proteins, containing proteins function as E3enzymes. Some RING can bridge the specific substrates in ECS-type E3s. Besides finger proteins can function alone (Fig. 1), such as Mdm2 and interacting with Cul2/5, SOCS proteins interact specific Parkin, whereas others form complexes with other proteins to substrates through either WD40 repeats, ankyrin repeats, SPRY function as E3s (Fig. 1). For example, Roc1 is associated domains, or tubby domains. More than 40 members from nine with E2 and Cullin1 (Cul1) in the Skp1, Cul1, F-box (SCF) families have been identified as SOCS proteins, the most well complexes. studied being von Hippel-Lindau. In the case of von Hippel- Additionally, two types of RING finger–like domains, such Lindau, a major substrate is HIF1-a. It has been well documented as U-box and PHD, also have E3activity. The predicted three- that von Hippel-Lindau is frequently mutated in renal carcinoma. dimensional structure of the U-box is similar to that of the It has been reported that deficiency of SOCS1 leads to RING finger except there are no metal-chelating residues in the accelerated mammary gland development (35), and methylation U-box (25, 26). Several U-box containing proteins, including of SOCS1 contributes to breast tumorigenesis (36). Recently, carboxy terminus of Hsc70-interacting protein (CHIP), UFD2, SOCS1 was shown to inhibit tumor necrosis factor-a signaling PRP19, UIP5, and CYC4, have been shown to function as through targeting apoptosis signal-regulating kinase 1 for E3s using their U-box (27). U-box type E3s are likely ubiquitin-mediated degradation (37). participants in protein quality control through interacting with chaperones. Interestingly, CHIP may play an important role in BTB-Box Proteins breast cancer by promoting ErbB2 (28) and estrogen receptor a Similar to Skp1 in the SCF complex and ElonginC in the (29) ubiquitination and degradation together with the chaper- ECS complex, BTB proteins function as subunits of Cul3-based ones (Fig. 2). The PHD motif is also similar to the RING finger E3s. Although Cul1 and Cul2 ligases each use a two-protein domain. Several PHD domain–containing proteins, such as module in substrate recognition and cullin binding, Cul-3 mitogen-activated protein kinase kinase kinase 1 (30), c-MIR ligases use single BTB proteins. BTB proteins possess a (31), and AIRE (32), have been reported to have E3 activity. protein-protein interaction domain so that they can function as Mitogen-activated protein kinase kinase kinase 1 is capable of substrate adaptors like F-box proteins and SOCS proteins.

FIGURE 2. The well-studied E3s and their common mechanisms of regulating breast cancer. !, promoting; ?, blocking. HECT-type E3s are in ellipse. RING finger E3s are in hexagon. F-box proteins are in octagon. Transcription factors are in O-vertex.

Human BTB proteins typically recruit substrates through APC/C E3 MATH, Kelch, or Zn-Finger domains. Although there are The APC/C comprises f12 subunits. Similar to SCF E3s, it >183BTB proteins in the human genome (38),at present, contains a cullin domain protein APC-2 and a RING protein only a few of them have been shown to connect to the APC-11. The substrate recognition is mediated by two different UPS. Furthermore, their in vivo substrates remain to be activators: Cdc20 and Cdh1. APC/C is temporally regulated identified. during the cell cycle (39). Cdc20 is up-regulated in late G2 and

Table 2.Oncogenic E3 Ligases in Human Breast Cancer

E3Type Gene locus Alteration in breast cancer Major targets Function

ARF-BP1 HECT Xp11.22 Overexpression P53Mcl1 Promoting cell growth and antiapoptosis WWP1 HECT 8q21 Amplification and overexpression Smad2, 4, ThR1, KLF2, KLF5 Transcription and signal transduction Mdm2 RING 12q14.3-q15 Amplification, mutation, overexpression, P53Antiapoptosis and aberrant RNA splicing RNF11 RING 1p31-32 Overexpression AMSH Signal transduction EFP RING 17q23.1 Overexpression 14-3-3j Cell cycle XIAP RING Xq25 Smac, Caspases Antiapoptosis BCA2 RING 1q21.1 Amplification and overexpression Skp2 F-box 5p13 Amplification and overexpression P27, p21, FoxO1, Cell cycle, transcription h-TrcP F-box 10q24.32 Amplification and overexpression InB, h-catenin, CDC25A, Cell cycle, transcription Cul4A Cullin 13q34 Amplification and overexpression DDB2, Jun DNA repair and antiapoptosis

NOTE: For references, please see E3s as Oncogenes in Breast Cancer.

mediates the degradation of securins and cyclins to induce entry breast (55-58). It has been shown that expression of into and progression through mitosis, whereas Cdh1 levels aberrantly spliced Mdm2 mRNA in breast carcinoma was increase during anaphase and control mitotic exit through associated with a shortened overall patient survival (59). the degradation of cyclin B and other substrates. Thus, inhibi- Although Mdm2 has been characterized as a RING finger E3 tion of the APC/C complex is believed to be a good strategy for the tumor suppressor p53(60), Mdm2 also has trans- for an anticancer therapy because it will block cell cycle forming potential independent on the p53(61, 62). Inhibiting progression. the interaction of Mdm2 with p53has therefore been a focus in drug discovery (63, 64). Recently, several potent and E3s as Oncogenes in Breast Cancer selective small-molecule antagonists of Mdm2 have been identified (65-67). Many E3s could be oncogenes or tumor suppressor genes because frequent deregulation of E3s has been shown in human carcinogenesis. Some E3s have established roles in cell cycle EFP and apoptosis, such as the Mdm2 and the SCFSkp2 complex. The EFP stimulates proliferation of breast cancer through More recently discovered E3s, such as ARF-BP1, WWP1, and facilitating ubiquitin-mediated destruction of a tumor suppres- breast cancer–associated gene 2 (BCA2), may be similarly sor 14-3-3j (68). Recently, EFP was shown to function as an important in breast tumorigenesis. These E3s are overexpressed E3for ISGylation of 14-3-3 j in response to IFNs (69). EFP in human breast cancer, and their inhibition leads to growth may regulate a switch from estrogen-dependent to estrogen- arrest or apoptosis. The well-studied oncogenic E3s in breast independent growth of breast tumors because EFP overex- cancer are summarized in Table 2 and discussed below in detail. pressed estrogen-dependent MCF-7 breast cancer cells gained the ability to form tumors in nude mice in the absence of ARF-BP1/HUWE1 estrogen (68). Inhibition of EFP expression by antisense The protein ARF-BP1 is a HECT domain E3that targets oligonucleotides reduced tumor growth (68). The protein the p53protein for ubiquitin-mediated proteolysis (40-42). expression of EFP is significantly correlated with poor ARF-BP1 was shown to be a critical mediator of both the prognosis of breast cancer patients (70). Therefore, EFP may p53-independent and p53-dependent tumor suppressor func- be a new valuable biomarker and molecular target for breast tions of ARF (42). The gene encoding ARF-BP1 is located at cancer (71). Xp11, and gains on chromosomes Xp11-13have been detected in breast cancer (43). In agreement with this, ARF-BP1 has X-LinkedInhibitor of Apoptosis Protein/BIRC4 been shown to be overexpressed in colorectal and breast cancer. Several inhibitor of apoptosis proteins (IAP) possess RING finger domains that are able to bind E2s, ultimately promoting WWP1 ubiquitination of target proteins. In addition to directly binding WWP1 is located at 8q21, a region frequently amplified in and inhibiting caspases, IAPs may cause ubiquitination and human breast cancer. Indeed, copy number gain and over- subsequent proteolysis of caspases and other apoptotic expression is frequently detected in human prostate and breast regulators. For example, X-linked IAP (XIAP) targets cas- cancer (44, 45). WWP1 is also a HECT domain–containing E3. pase-3, caspase-7, and caspase-9 for degradation and may also Growing evidence suggests that WWP1 negatively regulates enhance its own activity by mediating ubiquitination of its the transforming growth factor-h (TGF-h) tumor suppressor antagonists, including second mitochondria-derived activator of pathway by mediating the ubiquitination and degradation of caspases (Smac; refs. 72-75). Overexpression of XIAP may Smad2 (46), Smad4 (47), and TGF-h receptor 1 (48). Besides cause the resistance to apoptosis induced by cytotoxic drugs in inhibiting the above components of the TGF-h pathway, breast cancer. It has been shown that inhibition of XIAP by WWP1 has recently been shown to function as an E3for two RNA interference suppresses MCF-7 xenograft tumor growth transcription factors: KLF2 (49) and KLF5 (44). KLF2 and and sensitizes cells to etoposide and doxorubicin (76, 77). Thus, KLF5 have been shown to be frequently down regulated in the role of IAP proteins in ubiquitination is an emerging field ovarian and breast tumors, respectively (50, 51). Both with important implications for resistance to apoptosis in tumor transcription factors were shown to induce apoptosis through cell chemotherapy. regulating transcription of their target genes (52, 53). WWP1 may also play a role in other biological processes, including RING Finger Protein 11 regulation of epithelial sodium channels, viral budding, and Both mRNA and protein of RING finger protein 11 (RNF11) receptor trafficking (54). Many studies have found that WWP1 are highly expressed in breast cancer cells (78), although its directly binds to specific targets through its WW domains, role in breast tumorigenesis remains to be elucidated. RNF11 which can interact with a proline-rich motif in the target was found to cooperate with Smurf2 to degrade an enzyme proteins (44, 46). Alternatively, WWP1 targets Smad4 and AMSH, a positive regulator of both TGF-h and epidermal TGF-h receptor 1 through adaptor proteins Smad2 (47) and growth factor receptor signal pathways (79). RNF11 interacts Smad7, respectively (48). with multiple HECT-domain E3s and Cul1 (80). Like the role of Skp1/2 in SCF E3, RNF11 may function as an adaptor to Mdm2 bridge some substrates to HECT domain E3s for ubiquitination Gene amplification, mutation, overexpression, and aberrant because a large number of interacting partners (f80) have been splicing have been identified in 19 tumor types, including revealed (80).

BCA2/ZNF364 Cul-4A BCA2 is another E3with a RING domain. The BCA2 gene The Cul-4A gene is located at 13q34 and is frequently amplified is located at 1q21.1, a region frequently amplified in breast and overexpressed in breast cancer (100). Recently, Cul4A protein cancer. Using immunohistochemistry and tissue microarray, was shown to participate in the Mdm2-mediated proteolysis of Burger et al. showed that BCA2 protein was overexpressed in p53(101) and DET1-regulated c-Jun degradation (102). Addition- invasive breast cancer but correlated with positive estrogen ally, Cul-4A expression is critical for early embryonic development receptor, negative lymph node metastasis, and increased because the homozygous deletion or heterozygous deletion of survival (81). Inhibition of BCA2 by small interfering RNA Cul-4A is lethal in a knock-out mouse model (103). suppressed T-47D and MCF7 cell proliferation (81). The targets In addition to the E3s described above, many other E3s have of BCA2 E3have not been identified. also been shown to be oncogenic proteins in breast cancer, such as E6-AP (24), COP1 (104), and PirH2 (105). All of these E3s Skp2 target p53for ubiquitin-mediated degradation. The Skp2 gene is located at 5p13, a region found to be amplified in 11% of breast cancer cell lines in a comparative E3s as Tumor Suppressor Genes in Breast genomic hybridization study (82). Skp2 has been reported to Cancer be overexpressed in a subset of breast carcinomas (estrogen In contrast to oncogenic E3s, many other E3s, including receptor and Her-2 negative), and Skp2 expression inversely BRCA1 and Fbw7, have been shown to be tumor suppressors in KIP1 correlates with p27 levels in a variety of human tumors, breast cancer. Frequently inactivating mutations or down- including breast carcinomas (83, 84). Thus, Skp2 may be a regulated expression of these E3s has been detected in breast potential specific biomarker and therapeutic target in a subset cancer. Several recently discovered E3s, such as CHFR, of aggressive breast carcinomas. Skp2 is an extensively SIAH1, and CHIP, may play a significant role in regulating studied F-box protein required for the ubiquitin-mediated breast tumorigenesis. Besides mutation and gene copy loss, KIP1 CIP1 degradation of the p27 (85), p21 (86), p130 (87), and epigenetic alteration (i.e., promoter methylation) also contrib- FoxO1 (88) by SCF E3. Interestingly, the ubiquitination of utes to inactivation of these tumor suppressors. The E3s with KIP1 skp2 p27 by SCF requires an accessory protein Cks1 (89). tumor suppressor function in breast cancer were summarized in Recent structural studies show that Cks1 binds to the Table 3and discussed below in detail. phosphorylated Thr187 side chain of p27KIP1 (90). Expression of Cks1 is associated with decreased tumor differentiation and BRCA1-BARD1 poor disease-free and overall survival outcome in breast BRCA1 is a well-known tumor suppressor gene and is cancer (91). mutationally inactivated in familial forms of breast and ovarian cancer. BRCA1 protein contains a RING finger domain in its b -TrcP1/BTRC1 NH2 terminus. This RING finger domain has been shown to h-TrcP1 is another F-box protein but differs from Skp2 in autoubiquitinate and monoubiquitinate histone H2A in vitro that it harbors a h-transducin repeat domain. Overexpression (106). BRCA1 interacts with another RING finger protein, of h-TrcP1 in mouse mammary gland epithelium promotes tumor suppressor BARD1, which is also mutated albeit with low nuclear factor-nB activity and epithelial cell proliferation (92). frequency in breast cancer (107, 108). BARD1 has BRCA1- Thirty-eight percent of transgenic mice develop tumors, independent and p53-dependent proapoptotic activity (109). including mammary, ovarian, and uterine carcinomas (92). BARD1 mRNA is lower in invasive breast cancers compared Consistently, targeting of h-TrcP1 either by RNA interference with normal breast tissues (110). Interestingly, BARD1 or by forced expression of a dominant-negative h-TrcP1 expression is highly up-regulated in the cytoplasm of most mutant suppresses cell growth and sensitizes human breast breast cancer cells and correlates with poor clinical outcome cancer cells to the antiproliferative effects of anticancer drugs (111). The RING heterodimer BRCA1-BARD1 is a ubiquitin (93). h-TrcP1 mediates ubiquitination and degradation of ligase, and the mutations in the RING finger domain of h-catenin (94), InB (95), CDC25A (96), Smad4 (97, 98), and BRCA1 found in familial breast cancer abolish the E3activity Emil1 (99). (112, 113). The activity of BRCA1-BARD1 was shown to be

Table 3.E3 Ligases as Tumor Suppressors in Human Breast Cancer

E3Type Locus Alteration in breast cancer Major targets Function

BRCA1 RING 17q21 Mutation H2A, Rpb1, g-tubulin DNA repair, genomic stability BARD1 RING 2q34-q35 Mutation, expression aberration H2A, Rpb1, g-tubulin DNA repair, apoptosis SIAH1 RING 16q12-13 h-Catenin, OBF-1, CtIP, Kid, Numb Cell cycle, transcription, and apoptosis Parkin RING 6q25 Deletion and loss of expression aSP22, p38/JTV1, Pael-R CHFR RING 12q24.33 Loss of expression by promoter methylation Plk1, Aurora A Cell cycle Cul5 Cullin 11q22-23 Loss of expression Fbw7 F-box 4q31 Mutation and loss of expression Cyclin E, Myc, Notch Cell cycle CHIP U-box 16q13.3 ErbB2, estrogen receptor a, AR, Smads Signaling

NOTE: For references, please see E3s as Tumor Suppressor Genes in Breast Cancer.

down-regulated by CDK2 (114) and enhanced by BRCC36 Fbw7/hCDC4 whose mRNA expression is elevated in breast tumors (115). Human F-box protein Fbw7/hCdc4 has been reported to BRCA1-BARD1 E3was shown to ubiquitinate g-tubulin, which promote ubiquitin-mediated degradation of several onco- regulates centrosome number and genome stability (116). proteins involved in the control of cell division and growth, Following that, the largest subunit of RNA polymerase II such as cyclin E (137), c-Myc (138), c-Jun (139), and Notch (Rpb1) was shown to be ubiquitinated by this E3complex (117). (140). Mice lacking Fbw7 die at about day 10 with multiple abnormalities (140, 141). Although cyclin E expression is SIAH1 frequently dysregulated in breast cancer (142), a truncated The SIAH1 gene was first identified as a candidate tumor mutant form of Fbw7, which can not bind to phosphorylated suppressor gene at 16q12.1 in human hepatocellular carcinomas cyclin E target, has been found in one breast cancer cell line because of frequent loss of heterozygosity and expression (143). However, the frequency of Fbw7 mutation in breast down-regulation (118). Overexpression of SIAH1 in MCF7 cancer has not been studied. Proteasomal degradation of cyclin cells suppresses cell growth by altering the mitotic process E via the UPS is distinct from elastase, and calpain-mediated (119, 120). SIAH1 is a RING finger E3targeting h-catenin for NH2-terminal proteolysis of cyclin E that generates low ubiquitin-mediated degradation in response to activation of p53 molecular weight forms of cyclin E, which are frequently (121). The ubiquitin ligase complex contains SIAH1, SIAH1- detected in breast tumor tissue (144-146). interacting protein, Skp1, and the F-box protein Ebi (122). Importantly, this degradation pathway is different from CUL-5 degradation of h-catenin by SCFh-TrcP. The latter requires the CUL-5 is located at 11q22-23, which is frequently deleted h-catenin phosphorylation by glycogen synthase kinase 3h.Itis in breast cancer (147). A statistically significant decrease in well documented that h-catenin promotes cell cycle progression CUL-5 expression versus the matched normal tissue was and inhibits cell cycle arrest. Because SIAH1 is a p53-induced detected in 82% (41 of 50) of the breast cancers (148). Over- gene (123), SIAH1-mediated h-catenin degradation may expressing Cul-5 significantly attenuated cellular proliferation contribute to p53-dependent cell cycle arrest and apoptosis. In of the T-47D breast cancer cell line (149). addition to h-catenin, SIAH1 has been reported to promote degradation of several substrate proteins, such as transcriptional CHIP/STUB1 coactivator OBF-1 (124), transcriptional corepressor CtIP The chaperone-dependent U-box E3CHIP mediates a (125), kinesin-like DNA-binding protein Kid (120), and cell degradative pathway for ErbB2/Neu (28). ErbB2 overexpres- fate regulator Numb (126). sion is frequent in breast cancer and associated with poor prognosis. In addition to ErbB2, CHIP also promotes estrogen CHFR receptor a and glucocorticoid receptor degradation and Epigenetic inactivation of CHFR by aberrant promoter attenuates receptor-mediated gene transcription (29, 150). h methylation was found in several types of carcinomas, However, CHIP negatively controls the sensitivity of TGF- including breast (127, 128). In a knock-out mouse model, signaling by targeting Smads for ubiquitin-mediated degrada- CHFR was suggested to be a tumor suppressor because mice tion (151, 152). Interestingly, CHIP was found to be associated deficient in its expression spontaneously develop a variety with Parkin and enhances Parkin E3activity (153).The CHIP of epithelial tumors in major organs, including lung, liver, gene is located at 16p13.3, which is frequently deleted in gastrointestinal tract, and reproduction system (129). The papillary carcinomas of the breast (154). CHFR gene also encodes a RING finger domain containing E3, which was shown to play an important role in mitosis Functional Mechanisms of E3s in Breast Cancer through targeting key mitotic proteins Plk and Aurora A for Every E3specifically targets one or several substrate ubiquitin-mediated proteolysis (129, 130). proteins for ubiquitination. However, if an E3plays an important role in breast tumorigenesis, the targets are usually Parkin involved in the cell cycle, p53actions, transcription, DNA Although mutations of the Parkin gene are the most repair, signaling transduction, or apoptosis (Fig. 2). All these common cause of hereditary parkinsonism, Parkin has also cellular processes are known to be critical in breast cancer been suggested as a candidate tumor suppressor gene on initiation and progression. chromosome 6q25-q27 in human breast cancer, ovarian cancer, and lung cancer (131, 132). The expression of the Parkin gene Cell Cycle has been shown to be frequently down-regulated or absent in The cell cycle plays a central role in regulating cell breast tumor cells (131). Although no mutations or in vitro proliferation and tumorigenesis. It is well documented that changes in cell proliferation or cell cycle were observed, forced alterations of cell cycle regulators occur in breast cancer, such expression of Parkin was shown to suppress tumorigenicity in as overexpression of cyclin E (155) and down-regulation of nude mice (132). Parkin is a RING-type E3 involved in the the p27KIP1 (156). Because rapid protein turnover by ubiquitin- ubiquitination pathway for misfolded proteins (133). Although mediated proteolysis is the most important mechanism of aSp22, Pael receptor (Pael-R), and p38/JTV1 were identified as controlling protein abundance for cell cycle regulators, the the Parkin substrates in brain cells (134-136), the targeted frequent genetic and expression alterations of their E3s substrate in breast cancer cells remains unclear. contribute to breast tumorigenesis. Typically, cullin-based

E3s, including SCF complexes, function through regulating the to CHIP, which promotes ligand-independent degradation of cell cycle. For example, F-box proteins Fbw7 and Skp2 target growth factor receptors, c-Cbl is another E3that can negatively cyclin E and p27 KIP1 for degradation, respectively. In addition, regulate the epidermal growth factor receptor by monoubiquiti- APC/C facilitates proteolysis of cyclin B, securin, and several nating them for endocytosis upon ligand stimulation (173, 174). other proteins to regulate mitosis exit. It has been reported that degradation of ErbB-2 by Herceptin (a humanized ErbB-2 antibody) involves the recruitment of p53 c-Cbl to ErbB-2 (174). The Cbl gene is located at 11q23.3, p53is a key tumor suppressor targeted by multiple E3s, a region frequently lost in breast cancer (175). Mutation of Cbl including Mdm2 (60), ARF-BP1 (40-42), E6-AP (24), Pirh2 is oncogenic in many types of cancer (176). Additionally, (105), p300 (157), Topo1 (158), and COP1 (159). Interestingly, controlling TGF-h receptors by WWP1/Smurfs is implicated in p53itself induces expression of Mdm2 and COP1 to negatively the loss of sensitivity of tumorigenic cells to TGF-h–induced control p53activity. Besides Mdm2 and ARF-BP1, COP1, a growth inhibition (48, 177, 178). RING finger-containing E3, is also significantly overexpressed in breast cancer (104). Additionally, Pirh2 protein expression is Apoptosis up-regulated in major human lung neoplasms (160). It would be Inhibition and resistance to apoptosis is one of the major interesting to know if Pirh2 is also overexpressed in human obstacles in cancer therapy. Apoptotic proteins have been breast cancer. identified as substrates of E3s and are frequently altered in breast cancer. In addition to p53and the IAP family described above, Transcription InB and Bcl2 family proteins are frequently deregulated by E3s Protein instability is a typical feature of many transcription in human cancer (9). It is well known that degradation of InB h factors, such as HIF1-a (161), Myc (162), h-catenin (163), by the SCF -TrcP complex is a critical step for the activation of c-Jun (164), and KLF5 (165). Each of these transcription nuclear factor-nB and up-regulated expression of antiapoptotic factors regulates expression of a large number of target genes, genes. Additionally, the proapoptotic proteins Bax, Bid, and and alterations of these transcription factors are frequently Bim are degraded through the UPS, which is believed to be a involved in tumorigenesis. Besides proteolysis, accumulating survival mechanism in human cancer cells, although their E3has evidence suggests that ubiquitination of transcription factors not been revealed thus far (179, 180). Up-regulated expression can enhance their transcriptional activation function (8). of Bim following detachment of normal breast epithelial cells Therefore, it is not surprising that E3s of these transcription from the extracellular matrix has been shown to be required for factors, such as von Hippel-Lindau, Fbw7, SIAH1, and WWP1, detachment-induced apoptosis (181). Recently, an antiapoptotic play important roles in tumorigenesis through controlling the Bcl-2 family member, Mcl-1, was shown to be an ARF-BP1 E3 abundance and activity of transcription factors. substrate (182). Therefore, alteration of apoptosis by E3s is an important mechanism for breast carcinogenesis. DNA Repair DNA damage is an important cause of gene mutation and Future Directions carcinogenesis. Many proteins participating in DNA repair are In conclusion, ubiquitin-mediated protein degradation plays regulated by the ubiquitin-proteasome pathway. For example, an important role in many cancer-related cellular processes. E3s UV-induced p21 degradation is essential for DNA repair (166). play critical roles because they control the substrate specificity. Growing results indicate that BRCA1-BARD1 E3is required Accumulating evidence suggests that genetic and expression for double-strand break repair after exposure to ionizing alteration of E3s contributes to breast carcinogenesis. In >500 radiation (167). Additionally, a BRCA1-dependent zinc finger human E3s, a number of E3s have been characterized as either transcriptional repressor ZBRK1 is degraded through the UPS oncogenes or tumor suppressor genes in breast cancer. Cell upon DNA damage (168). Interestingly, altered expression of cycle, p53, transcription, signaling transduction, DNA repair, the ZBRK1 gene has been frequently found in human breast and apoptosis are major targets of E3s in breast cancer carcinomas (169). No E3has been identified for ZBRK1 development. Several directions listed below should be ubiquitination upon DNA damage, although RNF11 has been important to develop E3s as diagnostic and therapeutic targets shown to be a ZBRK1-associated protein (80). Recently, DNA in the future. damage–induced degradation of Cdt1 (170, 171) and DDB2 (172), two key proteins in DNA repair mechanisms, has been Identification of Novel Breast Cancer Related E3s as shown to link to Cul4A and DDB1. Therefore, compromise of Biomarkers andDrug Targets DNA damage could be an important deregulating mechanism Although there are >500 E3s in the human genome, to date, for BRCA1 and Cul4A in breast cancer. only a limited number of E3s have been examined for their specific targets, genetic changes, and expression pattern in a Growth Factor Signaling large number of human breast tumors. High-throughput Many growth factor signal receptors, such as epidermal screening using E3cDNA and small interfering RNA libraries growth factor receptor, estrogen receptor, and TGF-h receptors, have been developed to identify the cell cycle–related E3s. are frequently altered in breast cancer. For example, ErbB-2 is However, systematic screening of the E3s with genetic and frequently amplified and overexpressed in breast tumors, and expression alteration in human tumors has not been reported. this alteration is associated with poor prognosis. In addition With development of microarray technology, a cDNA chip with

all E3s should be a useful tool to identify more cancer-related Acknowledgments E3s. Tissue microarray is another powerful tool to detect We thank Karen Adamo for critical reading of the article. expression of a candidate E3or its substrates simultaneously in large numbers of tumors by immunohistochemical staining of References a single microscope slide (183). An E3 with genetic and 1. American Cancer Society. Cancer facts and figures 2006. Oakland (CA): expression alteration in breast cancer could be developed as a American Cancer Society; 2006. biomarker for breast cancer diagnosis. Emerging technologies, 2. Sinclair CS, Rowley M, Naderi A, Couch FJ. The 17q23amplicon and breast such as array-based comparative genomic hybridization, cDNA cancer. Breast Cancer Res Treat 2003;78:313 – 22. microarray, tissue microarray, and RNA interference, will 3. Guan XY, Xu J, Anzick SL, Zhang H, Trent JM, Meltzer PS. 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Mol Cancer Res 2006;4(10). October 2006 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer

Ceshi Chen, Arun K. Seth and Andrew E. Aplin

Mol Cancer Res 2006;4:695-707.

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