Is Hexo1 a Cancer Predisposing Gene?

Subject Review Is hEXO1 a Cancer Predisposing Gene?

Sascha Emilie Liberti and Lene Juel Rasmussen

Department of Life Sciences and Chemistry, Roskilde University, Roskilde, Denmark

Introduction like enzymes reported in yeast, fly, and mammals and have Changes in the efficiency of DNA repair and recombination been ascribed roles in DNA repair, replication, and recombi- activities can cause predisposition to cancer. The finding that nation (7-12, 14-16). The RAD2 domain of hEXO1 exhibits a hereditary nonpolyposis colorectal cancer (HNPCC) families 5V-3V nuclease activity on both ssDNA and dsDNA substrates. frequently harbor mutations in DNA mismatch repair (MMR) The specific 5V-3V exonuclease activity is higher on blunt-end genes has generated widespread interest in this research area and 5V recessed dsDNA substrates compared with duplex DNA (1-5). At the time of writing, germ line mutations in at least containing a 5V overhang or ssDNA (12). The exonuclease four genes, hMSH2, hMSH6, hMLH1, and hPMS2, all homo- activity of hEXO1 at nicked and gapped duplex DNA logues of bacterial components involved in MMR, have been substrates is similar or slightly higher than that observed for found in HNPCC patients (6). However, because some HNPCC blunt-end duplexes. hEXO1 does not discriminate between families fail to display mutations in the known MMR genes, RNA and DNA substrates in vitro (17). Whereas hEXO1 changes in other components of the MMR pathway may be clearly possesses a 5V-3V activity, its involvement in 3V nick- responsible. One such new candidate gene is the human exo- directed repair suggests that the enzyme has a cryptic 3V-5V nuclease 1 (hEXO1). The exact functional and biological roles activity or that it is necessary for activation of a distinct activity of hEXO1 remain unclear, but this exonuclease has been that is responsible for excision in the 3V-5V direction (13). In implicated in DNA replication, recombination, and repair. The addition to an exonucleolytic function, hEXO1 has a flap recent discovery that hEXO1 plays a role in MMR and the structure specific endonuclease activity analogous to FEN1 identification of germ line mutations in hEXO1 in patients (12). hEXO1 does not incise endonucleolytically at bubble-like with atypical HNPCC suggest a possible nexus between structures, G/T mismatches, or uracil residues but degrades hEXO1 mutations and HNPCC. hEXO1 may also be linked these duplex substrates exonucleolytically (12). Both 5V-3V to carcinogenesis through its role in recombinational events exonuclease and flap endonuclease activities require a divalent such as repair of DNA double-strand breaks (DSB) and telo- metal cofactor, with Mg2+ being the preferred metal ion (12). mere stabilization. hEXO1 has been shown to display a RNase H activity (17). This observation indicates that the enzyme may also be Exonuclease 1 involved in DNA replication, in agreement with studies in The hEXO1 gene is located at 1q42-43 and consists of 14 yeast suggesting a backup function of EXO1 to FEN1 in RNA exons that yield a f3 kb transcript (7-9). Two different forms primer removal. Overexpression of yeast EXO1 or hEXO1 of hEXO1 exist (HEX1/hEXO1a and hEXO1b) that arise from protein complements the defects of RAD27 (FEN1) mutants, alternatively spliced mRNA transcripts, involving only the suggesting that these two RAD2 family members have COOH-terminal content outside the nuclease domain of the analogous functions (8, 11, 17). Moreover, a study of yeast protein. Thus far, functional differences between the two splice mutants shows that EXO1 RAD27 double mutants are nonviable, variants have not been observed. EXO1 belongs to class III of unlike the corresponding single mutants. The lethality could be the RAD2 family of endonucleases and exonucleases and is caused by an inability to remove primer RNA during lagging generally described as a 5V-3V exonuclease (9-12). Recent strand DNA synthesis (11). Localization of hEXO1 is restricted work suggests that it also plays a role in 3V-5V exonucleolytic to the nucleus and it colocalizes with proliferating cell nuclear degradation of DNA (13). The strong sequence homologies antigen (PCNA; refs. 18, 19). The colocalization of hEXO1 and within the RAD2 family are restricted to two discrete regions, PCNA during S phase suggests that this enzyme could be involved in events associated with DNA replication, perhaps as designated the N (NH2-terminal) and I (internal) regions, which comprise the catalytic domain responsible for nuclease a backup to FEN1 in the removal of RNA primers, similar to activities. The RAD2 class III subfamily consists of EXO1- that proposed for the yeast orthologues. However, the colocalization of hEXO1 and PCNA could also reflect the participation of these proteins in the MMR process, because results have shown that PCNA is required in the resynthesis Received 4/27/04; revised 7/2/04; accepted 7/8/04. step of MMR (20) and also is involved in MMR steps prior to The costs of publication of this article were defrayed in part by the payment of DNA resynthesis (20, 21). page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Lene Juel Rasmussen, Department of Life Sciences DNA MMR and Chemistry, Roskilde University, DK-4000 Roskilde, Denmark. Phone: 45- 46742728; Fax: 45-46743011. E-mail: [email protected] The MMR system corrects base-base mispairs and small Copyright D 2004 American Association for Cancer Research. insertion/deletion loops, which have escaped the DNA

polymerase proofreading activity (22). The MMR system is Genetic recombination is involved in several different cel- responsible for maintaining the genomic integrity, and the lular processes, such as meiotic recombination, repair of DSBs, activity of the repair system lowers the spontaneous mutation antibody generation, and telomere maintenance (45). EXO1 is frequency up to a 1,000-fold (23). The mutator phenotype of involved in recombinational events, although the exact function EXO1-deficient yeast is not as significant as that observed in of EXO1 during recombination remains uncertain. Proposed MSH2 strains, but the mutation spectrum suggests a MMR activities include generation of 5V resected ends needed for defect (11, 24-26). In bacteria, redundant exonucleases have strand exchange (10, 15, 46), resolvation of DNA intermediates been shown to be active in MMR (27-29), and the fact that formed during recombination (12), and repair of mismatches in EXO1-deficient yeast strains are weak mutators indicate that heteroduplex DNA that lead to gene conversion events (25, 47). alternate activities may support the excision step in this lower Furthermore, hEXO1 interacts with the Werner syndrome eukaryote as well. To date, hEXO1 is the only exonuclease protein, and this interaction stimulates the nucleolytic activities identified as part of the human MMR system and it is believed of hEXO1 (48). EXO1 has also been proposed a role in meiotic to play an essential role by functioning both as a 5V-3V and as a recombination (24, 44, 46, 47, 49), and transcription of the gene 3V-5V nuclease as well as contributing to the overall stability of encoding EXO1 is highest in cell types and tissues associated the MMR complex (13, 30-34). with meiosis (8, 9, 14, 16, 36). Along these lines, it was shown that both male and female Exo1 mutant mice are sterile pre- Regulation of Exonuclease Activity in DNA MMR sumably because of a meiotic defect (49). The yeast and the hEXO1 have been shown to interact with MMR proteins (7, 11, 18, 34-36). Both hMSH2 and hMLH1 bind to the COOH-terminal domain of hEXO1, whereas Gene Expression of EXO1 in Mammalian Tissues hMSH3 interacts with a hEXO1 domain in the NH2-terminal part of the protein. Furthermore, experimental evidence hEXO1 is highly expressed in fetal tissues of liver and suggests the formation of a ternary complex between hEXO1- thymus, whereas expression in adult liver, spleen, and kidney hMLH1-hPMS2 (hEXO1-hMutLa; ref. 19) as well as between has not been detected, indicating a role for hEXO1 in devel- hEXO1-hMSH2-hMSH6 (hEXO1-hMutSa; ref. 37). opment of these tissues (9, 36). The high expression of hEXO1 The influence of MMR proteins on the activity and function in fetal liver and adult bone marrow, areas of active hemo- of hEXO1 has been studied. It was shown that hMutSa ac- poiesis, suggests that hEXO1 could act in DNA metabolic tivates the 5V-3V activity of hEXO1 in a mismatch-dependent processes that occur during differentiation of various stem cell manner, an effect that is best explained by protein-protein in- lineages (9, 36). This hypothesis is supported by results teractions on a heteroduplex (13). Furthermore, the hEXO1 demonstrating that transcription factors Sp1/Sp3 and cAMP- activity on heteroduplex DNA is significantly enhanced in the responsive element binding protein-1 are required for hEXO1- presence of hMutLa (13). Whereas hMutSa is sufficient for promoter activity (50). The high expression of hEXO1 in hEXO1 activation on substrates nicked 5V to a mismatch, both thymus and bone marrow also indicates that this exonuclease hMutSa and hMutLa are required for excision on substrates might participate in V(D)J rearrangement and the generation containing a nick 3V to the mismatch (3V-5V nuclease activity; of antibody diversity. Experimental evidence to support this ref. 13). hMutSa and hMutLa not only activate excision on hypothesis is provided by studies of Exo1 mutant mice (51), a G/T heteroduplex but also suppress hEXO1 action on which display decreased class-switch recombination and homoduplex DNA (19, 30). The observation that hEXO1 is changes in the characteristics of somatic hypermutation similar also required for excision directed by a strand break located 3V to those observed in Msh2 mutant mice (51, 52). to the mismatch (13) indicates that the 5V-3V hydrolytic activity of hEXO1 must be regulated in a manner that depends on the relative orientation of strand signal and mismatch. This implies Telomeres the existence of additional factors that regulate hEXO1 activity Telomeres have important roles in cellular processes when mismatch-provoked excision is directed by a 3V strand including chromatin organization and control of cell prolifera- break. Such factors have not yet been identified but could tion (53-55). The common pathway for telomere maintenance in include PCNA, replication factor A, and ATP, which have been human cells involves the ribonucleoprotein telomerase (53, 56), shown to have significant impact on the efficiency and but alternative mechanisms for lengthening of telomeres, which regulation of excision in eukaryotic MMR (30). is dependent on homologous recombination, are functional in human cells under selective pressure. Such alternative mecha- Genetic Recombination nisms are detectable in a small percentage of tumors, including DSBs are a very disruptive form of DNA damage and, if left bone and soft tissue sarcomas, glioblastomas, and carcinomas unrepaired, can lead to broken chromosomes and cell death. of the lung, kidney, breast, and ovary (57-60). A role for EXO1 On the other hand, if repaired improperly, DSBs can lead to has been proposed in end resection at telomeres (61-63), and chromosome translocation and cancer (38). Two independent deletion of EXO1 reduced the mutation rate in telomerase pathways carry out repair of DSBs: homologous recombination deficient cells, suggesting that EXO1 contributes to the end and nonhomologous DNA end joining. EXO1 does not seem resection that generates terminal deletions in cells with to be involved in repair of DSBs through nonhomologous dysfunctional telomeres (64). The observation that EXO1 pro- DNA end joining (39-42) but functions via the homologous motes genetic instability in telomerase-deficient cells might recombination pathway (42-44). have interesting implications for carcinogenesis and emphasizes

the importance of cellular mechanisms, which regulate exo- hEXO1 variants could be explained by reduced MMR activity. nuclease activity such as inhibition of hEXO1 activity on However, another study reported that five of nine hEXO1 homoduplex DNA by the hMutSa and hMutLa MMR variants identified by Wu et al. (79) were detected in both complexes. CRC and control individuals with equal frequency, indicating that these are naturally occurring polymorphisms unrelated to cancer predispositions (81). Interestingly, four hEXO1 variants, Cancer V27A, E109K, L410R, and P770L, were only detected in Inactivation of the human MMR pathway has been samples from CRC patients and not in control individuals. implicated in onset of both hereditary and sporadic cancers Based on these results, the authors concluded that hEXO1 (23, 65, 66). HNPCC is caused by mutations in MMR genes, variants either do not predispose to HNPCC or account for a and HNPCC individuals are heterozygous for a MMR gene very small proportion of such cases (81). mutation. An acquired somatic mutation in or chromosomal loss of the wild-type allele (loss of heterozygosity) is believed to promote tumorigenesis as a consequence of MMR defi- EXO1-Deficient Mice ciency, increasing the likelihood of inactivation of key Knockout mice provide a useful model system to study the regulatory genes involved in growth suppression, apoptosis, function of individual mammalian genes and to model a range or signal transduction (67). Estimated incidences of HNPCC of human inherited disorders. The Exo1 mutant mice are char- vary between 0.5% and 13% of all colorectal cancers (CRC; acterized by reduced survival, increased susceptibility to the refs. 68-70) and are characterized by an early onset of ma- development of lymphomas, and higher mutation rates, lignancy (age <50 years) and high penetrance (71). Further- although the mutation frequency is lower than in Msh2-deficient more, HNPCC individuals have an increased frequency of mice cells (49, 82). Furthermore, Exo1 mutant cells show ele- certain extracolorectal malignancies in the endometrium, uter- vated MSI at mononucleotide repeats and incomplete in vitro us, ovary, breast, and small intestine (72). The International MMR of base-base mismatches and single-base insertion/ Collaborative Group on HNPCC has defined criteria to provide deletion loops but normal repair of dinucleotide insertion/ a basis for uniformity of the diagnosis for HNPCC, known as deletion loops (49). Comparable observations come from Msh6 the Amsterdam criteria (73, 74). Families that fulfill several mutant mice, which develop lymphomas or epithelial tumors but not all of these criteria are defined as having atypical of the skin and uterus (82). Extracts from Msh6-deficient cells HNPCC. Mutations in hMSH2 and hMLH1 account for the are defective in repair of base-base mismatches but are capable majority of clinically defined HNPCC mutations, whereas of repairing insertion/deletion loops (83). Germ line mutations mutations in hMSH6 and hPMS2 are rare. Germ line mutations in hMSH6 have been identified at a high incidence in patients in these genes have been identified in only a proportion of with atypical HNPCC, and these cases are characterized by a families with atypical HNPCC (75, 76). In a significant num- late onset of cancer as well as low rates of MSI in the tumors ber of the cases, mutations in known MMR genes have not (84, 85). In addition, hMSH6 mutations have been linked to a been identified, which suggests that essential components in high incidence of endometrial cancers and incomplete pene- the MMR pathway remain to be identified (70, 77). trance compared with classic HNPCC (86). It is therefore possible that a similar pathology may explain the relation between hEXO1 Mutations in HNPCC Patients hEXO1 mutations and HNPCC resulting in late onset of disease, Microsatellite instability (MSI) often indicates that a tumor deviating tumor spectrum, and/or incomplete penetrance. If so, a has developed as a result of MMR deficiency, and the majority significant number of individuals carrying hEXO1 mutations of sporadic MSI cancers are caused by inactivation of hMLH1 would not be recognized as HNPCC but instead diagnosed as by promoter hypermethylation rather than somatic mutations or sporadic cancer. Furthermore, it is possible that the weak muta- loss of heterozygosity (70, 78). Because hEXO1 is involved tor phenotype of hEXO1 mutations is only pathogenic in combi- in MMR, it is expected that hEXO1 mutations contribute to nation with other mutator alleles. This multimutation hypothesis HNPCC and/or sporadic CRC. Germ line variants of hEXO1 is substantiated by studies in yeast, demonstrating that the weak were detected in a proportion of patients with atypical HNPCC mutator phenotype of EXO1 mutant cells becomes robust when but were absent from >200 healthy control individuals, combined with other weak mutator alleles in genes encoding suggesting a role for hEXO1 in CRC. All hEXO1 variants MLH1, PMS1, MSH2, PCNA (POL30), or DNA polymerase were identified in families in which no germ line hMHS2, y (POL3; refs. 26, 33). Interestingly, tumor samples from two hMSH6,orhMLH1 mutations had been detected and all tumor HNPCC families with the same hEXO1 mutation (L410R) samples displayed various degrees of MSI, suggesting a MMR showed different MSI status (79). This difference in phenotypes compromised background (79). The identification of both high may well reflect the presence of unidentified secondary and low MSI tumors with hEXO1 mutations indicates a mutations that in combination with the hEXO1-L410R mutation difference in the pathogenic background between the various cause cancer progression in these patients. Along these lines, the individuals carrying potential hEXO1 mutations. Eight of the hEXO1-L410R mutation has been identified in an individual germ line variants identified by Wu et al. (79) were examined from a suspected HNPCC family carrying a missense mutation using functional assays. It was shown that the variants E109K in hMSH2 of unknown pathology (81). and L410R were defective in exonuclease activity, whereas The contribution of mutations in hEXO1 to cancer in a set of P640S, G759E, and P770L showed reduced ability to interact 21 families that fulfill the diagnostic criteria for HNPCC was with hMSH2 (80). Therefore, the pathogenic effect of certain recently examined (87). These families carried no mutations in

hMSH2 or hMLH1, but hEXO1 had four changes that were Is hEXO1 an Oncogene? identified previously and one that is a novel mutation (79, 81, The surprising observation that 12 of 13 tumors examined 87). The new missense mutation identified was not considered from atypical HNPCC patients with hEXO1 mutations had lost causative because it was observed in three unrelated patients and the mutant allele and retained the normal one (79) suggests that did not involve a highly conserved amino acid. Based on these a functional active hEXO1 provides an advantage for cancer observations, it was concluded that hEXO1 is not linked to progression. This speculation agrees with expression analysis HNPCC (87). However, the transgenic mouse model system demonstrating that hEXO1 is expressed in some human cancer suggests that mutations in hEXO1 are likely linked to late onset tissues but not in the corresponding nonneoplastic tissues (36). of CRC or atypical HNPCC. All families included in the Overexpression of hEXO1 might increase genetic instability study by Thompson et al. (87) fulfill the Amsterdam criteria for either by changing the MMR activity or by affecting re- HNPCC. The selection of families based on these criteria would combinational events at telomeres or at DSBs. The MMR pro- exclude patients carrying pathogenic hEXO1 mutations, because teins have been shown to regulate the nuclease activity of these would not be expected to fulfill the Amsterdam criteria if hEXO1, and any change in the interaction between hEXO1 and indeed hEXO1 is linked to late onset CRC or atypical HNPCC. other MMR proteins may result in genetic instability. This Polymorphisms in the hEXO1 gene, which are not rec- may occur if the nuclease is not inhibited from acting on homo- ognized as actual mutations, may have relevance for the duplex DNA during MMR. The hEXO1 could also cause development of sporadic cancers. It can be speculated that genetic instability through its involvement in recombinational certain gene variations in combination with individual genetic events, promoting carcinogenesis by increasing chromosome backgrounds may promote carcinogenesis at an advanced state rearrangements, amplifications, or deletions. Recently, nine of life. This phenomenon is known to apply for some weak individuals from two British families with skin and uterine mutations in hMLH1, which result in a late onset of cancers leiomyomata were analyzed for tumor MSI status and family that are diagnosed as sporadic cancers and characterized by a history of HNPCC spectrum cancers (88). Leiomyomata is a high MSI status (78). One way to explain the multimutation benign condition resulting from heterozygous germ line hypothesis is to invoke that hEXO1 physically interacts with deletions of 1q42.3-q43 encompassing several genes including other MMR proteins to stabilize the repair complex. Destabi- hEXO1. None of these tumors displayed MSI, and none of lization of this complex and the resulting onset of cancer will the nine individuals from the two families had any personal or depend on the combination and the nature of hEXO1 and other family history of HNPCC spectrum cancers (88). These results MMR gene variants. indicate that hEXO1 deficiency does not confer a genetic

FIGURE 1. Proposed roles for hEXO1 in human cancer.

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Sascha Emilie Liberti and Lene Juel Rasmussen

Mol Cancer Res 2004;2:427-432.

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