Polymorphisms in the p53 pathway - Nature

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Oncogene (2006) 25, 1602–1611

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REVIEW

Polymorphisms in the p53 pathway EC Pietsch1, O Humbey1and ME Murphy Division of Medical Sciences, Fox Chase Cancer Center, Philadelphia, PA, USA

The p53 tumor suppressor gene continues to be distinguished as the most frequently mutated gene in human cancer; this gene can be found mutated in up to 50% of human tumors of diverse histological type. It is generally accepted that the ability of p53 to induce either growth arrest or programmed cell death in response to diverse stimuli underlies the powerful selection against this protein in the development of cancer. It is somewhat surprising, then, to ﬁnd p53 and several target genes in this pathway containing polymorphisms that impair their function. The nature of these polymorphic variants, and the mechanism whereby they impair the function of the p53 pathway, are reviewed here-in. The impact of these polymorphisms on cancer risk and the efﬁcacy of therapy are only now becoming unraveled. Of particular relevance in these efforts will be the generation of mouse models of polymorphic variants in p53 and its target genes. Equally important will be better-controlled human studies, wherein haplotypes for p53 (that is, combinations of different polymorphisms in the p53 gene) and for p53-target genes are taken into account, instead of analyses of single gene variants, which have largely predominated to date. Studies in both regards should shed light on an emerging area in cancer biology, the signiﬁcance of inter-individual differences in genotype on cancer risk, prognosis, and the efﬁcacy of cancer therapy. Oncogene (2006) 25, 1602–1611. doi:10.1038/sj.onc.1209367 Keywords: p53; apoptosis

p21/waf1;

MDM2; polymorphism;

The tumor suppressor protein p53 The p53 gene Tp53 represents one of the most studied tumor suppressor genes in biology; its product, the p53 protein, is referred to as ‘the guardian of the genome’, and represents a key regulator of cellular growth control. p53 is a 53 kDa phosphoprotein encoded by 393 amino acids forming ﬁve highly conserved regions and four functional domains (Harris and Hollstein, 1993) (see Figure 1). In response to a variety of stress Correspondence: Dr ME Murphy, Program in Cell and Developmental Biology, Division of Medical Sciences, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. E-mail: [email protected] 1 These authors contributed equally to this work.

signals, including genotoxic stress, hypoxia, and oncogene activation, the p53 protein is post-translationally stabilized, leading to its activation as a sequence-speciﬁc transcription factor. This stabilization can then lead to different programs, depending on the cell of origin or cellular context; these include cell cycle arrest, senescence, or apoptosis (Jin and Levine, 2001). In the case of apoptosis, this process requires both transcription-dependent and -independent activities of p53 (see Schuler and Green (2005) for a review). p53 is well-known as the most frequently mutated gene in human cancer, as it is found inactivated by mutation in over 50% of human tumors. Some of these mutations have already been correlated to speciﬁc clinical phenotypes. It is therefore conceivable that the existence of natural variants of p53 could be linked with the development of speciﬁc diseases, owing to differences in the activity of variant proteins in this pathway, and could then represent an interesting predictive marker for preventive and early intervention strategies. The natural genetic variants of p53 have thus emerged as a resource to be studied in the understanding of inter-individual differences in cancer risk and therapy. By deﬁnition, a polymorphism has a minor allele frequency of greater than 1% in at least one population. Several polymorphisms have been identiﬁed in the Tp53 gene (for reference, see Olivier et al., 2002). Most of these polymorphisms are single-nucleotide polymorphisms (SNPs) affecting a single base. A great number of these natural variants are localized in non-coding regions of the gene (introns). Among the polymorphisms found in the coding regions (exons) of Tp53, only two alter the amino-acid sequence of its product, proline (P) to serine (S) at residue 47 (Figure 2) and arginine (R) to proline (P) at residue 72 (Figure 3). In this review, we will focus on both coding region and non-coding region polymorphisms, but will discuss only those that have been shown to be functionally relevant.

Polymorphisms that alter the coding sequence of p53 protein The serine 47 polymorphism The SNP P47S changes an evolutionarily conserved proline residue of p53 to serine. Initially identiﬁed by Felley-Bosco et al. (1993), the S47 polymorphic variant is very rare, with an allele frequency under 5% in African Americans and undetectable in Caucasians.

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Figure 1 Functional domains of the p53 tumor suppressor protein. Noted are the conserved functional domains of p53, with amino-acid residues for each functional domain listed below. The location of the two coding region polymorphic variants (codon 47 and codon 72) are denoted with an asterisk.

Figure 2 Amino-acid sequences of the p53 polymorphism at residue 47. The two p38 MAPK sites of phosporylation (serines 33 and 46), adjacent to proline residues at amino acids 34 and 47, are denoted.

Figure 3 Amino-acid sequences of the p53 polymorphism at residue 72. This region contains several SH3-binding motifs (PXXP), which are postulated to be important for the ability of p53 to induce apoptosis.

These authors demonstrated that the S47 form of p53 did not affect the growth suppression activity of this protein as compared to wild-type p53 (P47, or wt). However, the authors did not investigate the ability of p53 to induce apoptosis; further, the importance of phosphorylation of the adjacent residue, serine 46, had not yet been demonstrated to be critical for p53dependent apoptosis. Indeed, several years later, several groups demonstrated the importance of serine 46 phosphorylation to p53-dependent apoptosis (Bulavin et al., 1999; Oda et al., 2000; Sanchez-Prieto et al., 2000; Takekawa et al., 2000; Okamura et al., 2001). The phosphorylation of p53 on serine 46 is mediated by the proline-directed kinases p38 MAPK and HIPK2 (homeo-domain-interacting protein kinase 2); these require an adjacent proline to direct phosphorylation. Serine 46 phosphorylation is predicted to alter the transactivation function of p53, and confer increased ability of this protein to transactivate a subset of p53 target genes, including p53AIP1 (Okamura et al., 2001). We recently investigated the ability of the S47 variant to induce apoptosis. Our results showed an S47 allele frequency of 1% in a sample of 200 healthy African Americans, conﬁrming the low frequency of this germline polymorphism (Li et al., 2005). Our data indicated that, compared to wt p53, the S47 variant represents a poorer substrate for phosphorylation on serine 46 by the proline-directed kinase p38 MAPK. Consistent with the importance of serine 46 phophorylation to apoptosis

induction, we found that cell lines engineered to inducibly express the S47 variant demonstrated decreased ability to induce apoptosis, relative to wt p53. Mechanistically, we showed that the S47 and wt proteins did not differ in their ability to bind to DNA, trafﬁc to mitochondria, or regulate the overwhelming majority of p53 target genes. Rather, we found that the decrease in apoptotic activity of the S47 variant was because of an impaired transactivation of a subset of p53-target genes, including PUMA and p53AIP1, both of which are known to be important mediators of p53-dependent apoptosis. In particular, our studies highlighted the impaired transactivation of PUMA as critical for the decreased apoptotic efﬁciency of the S47 variant (Li et al., 2005). Currently, the impact of the S47 polymorphism on cancer risk and therapy is not known. While the ethnic differences in S47 allele frequency may explain some of the well-established differences in cancer risk and prognosis between Caucasian and African-American populations, the low frequency of the S47 allele has precluded population studies on this variant. Rather, the impact of this variant on cancer risk and cancer therapy awaits the generation of a mouse model for this polymorphism. The generation of such a model is facilitated by the recent creation and characterization of humanized p53 knock-in (Hupki) mice, in which coding sequences for the human p53 gene replace exons from the mouse gene (Luo et al., 2001). The codon 72 polymorphism This common SNP results in a non-conservative change of an arginine (R72) to a proline (P72) at amino acid 72 that results in a structural change of the protein giving rise to variants of distinct electrophoretic mobility (Figure 3) (Harris et al., 1986; Matlashewski et al., 1987). This polymorphism occurs in a proline-rich region of p53, which is known to be important for the growth suppression and apoptotic functions of this protein (Walker and Levine, 1996; Sakamuro et al., 1997). Beckman and co-workers were the ﬁrst to demonstrate a signiﬁcant difference in the allelic distribution of the R72 and P72 variants. They ﬁrst noted a signiﬁcant difference in the P72 allele frequency between a Nigerian population (African Black) and a Swedish population (Western Europe), which were 17 and 63%, respectively; in contrast, they did not note any differences between populations living on the same geographical latitude (Beckman et al., 1994). The authors went on to demonstrate that the frequency of the P72 allele differs with latitude, increasing in a linear manner as populations near the equator (Sjalander et al., 1995). These observations led the authors to suggest that the codon 72 variants differed in biological activity, and further that these differences in activity might be subject to selection in areas of high ultraviolet light exposure. Banks and co-workers subsequently demonstrated the existence of biochemical and biological differences between the R72 and P72 isoforms of p53. In this study, the authors demonstrated that the E6 proteins from Oncogene

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both low- and high-risk subtypes of human papillomavirus (HPV) targeted enhanced ubiquitin-dependent degradation of the R72 protein, compared to P72 (Storey et al., 1998). In a subsequent study, the authors went on to demonstrate that the P72 form of p53 had enhanced ability to function as a sequencespeciﬁc transactivator, owing, in part, to its stronger interaction with two TFIID-associated factors, TAFII32 and TAFII70 (Thomas et al., 1999). In contrast, the authors found that the R72 variant of p53 was a markedly better suppressor of cellular transformation, an activity commonly associated with p53’s apoptotic function. Differences in the biological activity of R72 and P72 proteins have also been described for certain tumorderived mutant forms of p53. Speciﬁcally, the p53homolog p73 has been reported to physically interact with certain tumor-derived mutant forms of p53 (but not wild-type p53). More to the point, the authors demonstrated that these mutant forms of p53 interacted with p73 preferentially when they occurred in cis with the R72 p53 polymorphism (Marin et al., 2000). This study went on to show that, in tumors from individuals heterozygous for the codon 72 polymorphism (R72/ P72), the R72 allele was most commonly subject to mutation, while the other allele (P72) was more frequently lost by deletion (Marin et al., 2000). These data suggested that the R72 variant of p53, when in cis with certain tumor-derived mutations, might have enhanced tumor suppressive function owing to increased ability to inactivate p73. Subsequent studies suggest that the ability of R72 to target and inhibit p73 may be cell type dependent (Vikhanskaya et al., 2005). Speciﬁcally, these authors demonstrated that some of the p53 tumorderived mutants that are unable to bind and inhibit p73 are still able to confer resistance to drug treatment, suggesting that R72-containing mutants may possess other mechanisms to disrupt chemotherapy-induced apoptosis. Two groups found that, for non-mutated forms of p53, the R72 variant has a signiﬁcantly increased ability to induce programmed cell death, in cells containing inducible versions of p53, as well as in cells homozygous for R72 and P72 (Bonafe et al., 2002; Dumont et al., 2003). The absence of differences in speciﬁc DNA binding or transcriptional ability of these two polymorphic variants led our group to discover that the enhanced apoptotic potential of the R72 variant was owing to increased trafﬁcking to the mitochondria, resulting from enhanced interaction with, and ubiquitylation by, the MDM2 ubiquitin ligase (Dumont et al., 2003). Such mitochondrial localization of p53, leading to cytochrome c release, was ﬁrst described by Moll and co-workers, and later conﬁrmed by our group (Marchenko et al., 2000; Dumont et al., 2003). Our group in association with the group of George has identiﬁed the pro-apoptotic protein BAK, an important member from the Bcl-2 family involved in cytochrome c release from mitochondria, as a mitochondrial p53-interacting protein (Leu et al., 2004). Interestingly, we found that the two p53 isoforms R72 and P72 demonstrate the same Oncogene

afﬁnity for BAK, suggesting that the enhanced ubiquitylation and nuclear export of the R72 underlies its enhanced mitochondrial function in cell death. In sum, the combined data from several groups has conﬁrmed the altered apoptotic potential of the codon 72 polymorphic variants, with the R72 variant demonstrating enhanced apoptotic ability, and the P72 variant demonstrating enhanced growth arrest (Pim and Banks, 2004). Based on these ﬁndings, a number of studies have tried to establish a correlation between the p53 codon 72 polymorphism and the risk to develop certain types of cancer. In general, these studies have not yielded consistent results; this may be accounted for by the fact that the R72 variant, when found in mutant forms of p53, might be predicted to enhance tumor development (increased inactivation of p73), but when found in the context of wild-type p53, might be predicted to better inhibit tumor development (increased apoptotic ability). The impact of the codon 72 polymorphism on cancer risk One of the ﬁrst studies to demonstrate a correlation between the codon 72 polymorphism of p53 and the risk to develop cancer was by Banks and co-workers, who reported that women with the R72 variant of p53 (better targeted for degradation by HPV E6 protein) had a seven-fold increased risk to develop cervical cancer (Storey et al., 1998). To date, dozens of studies have failed to conﬁrm these results, possibly because of differences in subtypes of HPV, so an association between cervical cancer and the codon 72 polymorphism of p53 is not currently accepted. Several groups have reported an association between the R72 p53 variant (binds and inactivates p73 better) and increased risk for epithelial cancer, including gastric cancer (Shen et al., 2004) and cancer of the breast (Langerod et al., 2002; Bonafe et al., 2003; Buyru et al., 2003; Ohayon et al., 2005), ovary (Pegoraro et al., 2002), esophagus (Kawaguchi et al., 2000), skin (Dokianakis et al., 2000; Bastiaens et al., 2001; Shen et al., 2003; De Oliveira et al., 2004), lung (Wu et al., 2002), bladder (Soulitzis et al., 2002), prostate (Henner et al., 2001), and larynx (Sourvinos et al., 2001). In other studies, however, authors have found the opposite correlation, instead demonstrating an association between the P72 (lesser apoptotic) variant and increased risk for other cancer types, including cancer of the thyroid (Granja et al., 2004), nasopharynx (Tsai et al., 2002a, b; Tiwawech et al., 2003), prostate (Suzuki et al., 2003), skin (Chen et al., 2003), urogenital region (Kuroda et al., 2003), and lung (Wang et al., 1999; Fan et al., 2000; Zhang et al., 2003). Still other groups have failed to demonstrate any association between codon 72 variants of p53 and cancer risk. Again, these discrepancies may be inﬂuenced by a failure to determine the mutational status of p53 in these tumors. Other researchers suggest that these discrepancies may be accounted for by a failure to conduct meta-analyses, or owing to poorly controlled ‘normal’ populations that do not take into account the latitudinal differences in allele

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frequency (Makni et al, 2000; Klug et al., 2001; Koushik et al., 2004). While correlations between cancer risk and the codon 72 polymorphism have been inconsistent, more consistent have been the correlations between these polymorphic variants and cancer progression, survival, and age of onset of cancer. In particular, several groups have found that patients homozygous for P72 (lesser apoptotic allele) were diagnosed at an earlier median age of onset for their cancer. The median age varied from 6 years earlier for squamous cell carcinoma of the head and neck, to 13 years earlier for non-polyposis colorectal cancer, and between 10 and 11 years earlier for oral cancer (Gottschlich et al., 2000; Shen et al., 2002; Jones et al., 2004). These data are consistent with the hypothesis that the R72 allele, which has greater apoptotic ability, consequently possesses enhanced tumor suppression function. Also consistent with this hypothesis are ﬁndings that individuals with the R72 genotype have higher response rates and better survival after receiving chemo- and radiation therapy for advanced head and neck cancer (Sullivan et al., 2004) and for cancers of the breast and lung (Biros et al., 2002; Bonafe et al., 2002; Nelson et al., 2005; Tommiska et al., 2005; Xu et al., 2005). Therefore, while correlations between cancer risk and p53 polymorphic variants have not been clear, more consistent correlations exist for cancer progression, survival, age of onset, and response to therapy.

Polymorphisms in p53 non-coding regions The IARC TP53 Mutation Database lists 15 common polymorphisms in the non-coding region of Tp53. Some of these natural variations have also been associated with increased risk of cancer development, although in the absence of clear indications that such variants alter the function of p53, it remains possible that these ﬁndings are the result of linkage to other, functionally signiﬁcant, polymorphisms of p53. Here, we will present only two polymorphisms that have been shown to impact the function or level of expression of p53, an allele that carries a 16 base-pair (bp) duplication in intron 3 localized at nucleotide 11 951 (denoted þ 16), and an allele that carries a G to C transversion in intron 6 at nucleotide 13 964 (denoted G-C). The allele frequencies of these two non-coding polymorphisms, which show signiﬁcant differences between populations from Russia and Belarus, have recently been correlated to ethnical variations (Khrunin et al., 2005). Polymorphism of intron 3 ( þ 16 bp) Among all of the polymorphisms identiﬁed in the Tp53 gene, the polymorphism in intron 3 (16 bp duplication) has been, along with codon 72 polymorphism, the most frequently studied. However, only a single work has demonstrated an altered activity of this natural variant. Harboring the assumption that the þ 16 variant might inﬂuence alternative splicing of p53, Gemignani et al.

(2004) instead reported a reduced amount of steadystate RNA for this allele in immortalized lymphoblastoid cell lines, relative to wild type. These results were re-capitulated with mRNA extracted directly from patient lymphocytes. Other investigators have reported that the þ 16 allele is associated with decreased apoptotic and DNA repair capacity in lymphoblastoid cell lines (Wu et al., 2002). Consistent with these altered functional activities, several studies have correlated the intron 3 duplication with an increased risk of various cancers, including cancer of the colon (Gemignani et al., 2004), lung (Wu et al., 2002), breast (Weston and Godbold, 1997; Wang-Gohrke et al., 1999; Powell et al., 2002), and ovary (Runnebaum et al., 1995; WangGohrke et al., 1999). However, other groups have failed to conﬁrm these results (Khaliq et al., 2000; Kang et al., 2004; Mitra et al., 2005). As in the case of the codon 72 polymorphism, better study design, better-controlled normal populations, and efforts to include haplotype analyses of other p53 polymorphic variants will be needed to conﬁrm the importance of this variant on cancer risk. Analysis of haplotypes remains a much more powerful approach than single polymorphism investigations, since the integration of an increasing number of common genetic variations in the analysis should allow for increased statistical power in such studies. Polymorphism of intron 6 (G-C) In a study trying to establish the frequency of germline mutations of p53 in familial breast cancer patients, Lehman et al. (2000) ﬁrst provided evidence that an intron 6 G-C variant form of p53 is associated with altered p53 function. In this work, the authors analysed the effect of this polymorphism on apoptosis and cell survival following cisplatin treatment of EBV-immortalized lymphoblastoid cells from patients carrying wildtype p53 that was heterozygous or homozygous for the intron 6 C genotypes. Their in vitro data demonstrated that the p53 intron 6 G-C variant was associated with a prolonged cell survival and decreased apoptosis. Interestingly, the germline substitution of intron 6 G-C was only found in patients presenting with a family history of cancer, but not in cases of sporadic cancer (Lehman et al., 2000). Other intron 6 base substitutions have been reported, suggesting that this locus represents a potential hot spot region for mutation. Several epidemiological studies have conﬁrmed the correlation between the intron 6 polymorphism and increased risk for the development of various cancer types (Hillebrandt et al., 1997; Biro et al., 2000; Biros et al., 2001; Fiszer-Maliszewska et al., 2003; Buyru et al., 2005).

Polymorphisms in p53-induced genes p21/Waf1 Transcriptional activation of p21/Waf1 by the p53 tumor suppressor protein occurs via a p53 response Oncogene

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element located 2.4 kb upstream of the p21/Waf1 transcription start site in response to genotoxic stress resulting in arrest of the cell cycle in G1 (el-Deiry et al., 1993). The function of p21/Waf1 together with p27kip1 and p57kip2 during the cell cycle is to inhibit progression of the cell cycle from G1 to S phase by inhibiting the activity of cyclin-dependent kinases (cdks) (Massague, 2004; Deshpande et al., 2005). p21/Waf1 has been identiﬁed as a universal cdk inhibitor, and binding of p21/Waf1 to cdk/cyclin complexes prevents these protein complexes from phosphorylating and inactivating the retinoblastoma (RB) protein, a central regulator of G1–S progression (Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993; Massague, 2004; Deshpande et al., 2005). Inactivation of RB results in release of E2F proteins, which transcriptionally activate several genes necessary for cell cycle progression (Cobrinik, 2005). In addition to exerting regulatory inﬂuences on the cell cycle, p21/Waf1 affects DNA replication by interaction with the proliferating cell nuclear antigen (PCNA) (Flores-Rozas et al., 1994; Waga et al., 1994). PCNA is an accessory factor to replicative DNA polymerases and ensures polymerase processivity during DNA replication and repair (Maga and Hubscher, 2003). Within the cell, p21/Waf1, PCNA, cdk, and cyclins form a tetrameric complex in which the N-terminus of p21/ Waf1 binds cdk while the C-terminus binds PCNA (Chen et al., 1995). Interestingly, interaction of p21/ Waf1 with PCNA seems to inhibit DNA synthesis during elongation, but not short gap DNA synthesis, such as those required for DNA repair processes (Flores-Rozas et al., 1994; Li et al., 1994). Hence, p21/ Waf1 is an important downstream regulator of p53 and functions as a unique link for p53 to cell cycle arrest and DNA repair. This is further evidenced by the observation that partial or complete deletion of the p21/Waf1 gene completely abrogates the DNA-damage-induced G1 arrest mediated by p53 (Brugarolas et al., 1995; Deng et al., 1995). The p21/Waf1 gene is located on chromosome 6p21.2 and consists of three exons which are 68 (exon 1), 450 (exon 2), and 1600 (exon 3) bp in length (el-Deiry et al., 1993). The translation start codon is located at nucleotide 76 in exon 2 and the translation stop signal is located at nucleotide 567 in exon 3 (el-Deiry et al., 1993). Human and mouse p21/Waf1 share 79% identity at the amino-acid level and contain two regions that are highly conserved and predicted to represent regions that are critical for protein function (el-Deiry et al., 1993; Huppi et al., 1994). These regions lie between codons 21–60 (95% identity) and 130–164 (89% identity); additionally, a putative zinc-ﬁnger-like motif has been identiﬁed between amino acids 13–42. As genes with growth suppressive function are frequently prone to mutation in human cancers, several studies have investigated the possibility that the p21/ Waf1 gene may be mutated in human cancer. Surprisingly, with the exception of a few rare cases, including Burkitt’s lymphoma (Bhatia et al., 1995), primary prostate cancer (Gao et al., 1995), primary cervical cancer (Harima et al., 2001), and breast cancer Oncogene

(McKenzie et al., 1997), mutations in the p21/Waf1 gene are generally rare (Shiohara et al., 1994; Facher et al., 1995; Gong et al., 1995; Koopmann et al., 1995; Li et al., 1995; Mousses et al., 1995; Sun et al., 1995; Terry et al., 1996; Hachiya et al., 1999). However, four polymorphisms in the p21/Waf1 gene have been identiﬁed and have been assessed with respect to their effects on cancer susceptibility. Two of these polymorphisms are non-synonymous SNPs located in the p21/Waf1 coding region, at codons 31 and 149. The other two polymorphisms are located in the 30 untranslated region (UTR), 20 bp from the translation stop site and in intron 2, 16 bp from the 50 splice site. These latter two polymorphisms will be discussed brieﬂy, and only in context with the coding region polymorphisms. Codon 31 of p21/waf1 The polymorphism at codon 31 arises from a C to A transition at nucleotide 168 changing codon 31 from AGC to AGA (Chedid et al., 1994). This results in a non-conservative amino-acid change from serine (Ser) to arginine (Arg) (Figure 4). Interestingly, like the coding region polymorphisms of p53, the frequency of the Arg allele varies dramatically between major ethnic groups (Birgander et al., 1996). In Caucasians, the frequency of the Arg allele ranges from 4 to 19% (Facher et al., 1995; Koopmann et al., 1995; Birgander et al., 1996; Sjalander et al., 1996; Lukas et al., 1997; Keshava et al., 2002) while in African and Asian populations the frequency ranges from 22 to 55% (Koopmann et al., 1995; Birgander et al., 1996; Hachiya et al., 1999). While some studies suggest that the p21/Waf1 codon 31 polymorphism may occur preferentially in tumors that do not contain mutations in p53 (Facher et al., 1995; Mousses et al., 1995), other studies have failed to corroborate these ﬁndings (Koopmann et al., 1995; Milner et al., 1996). Reasoning that codon 31 is part of a highly conserved area within p21/Waf1 that contains the cdk interaction area, several research groups have performed functional studies to investigate if these polymorphic variants demonstrate altered functional activity (Chedid et al., 1994; Sun et al., 1995; Terry et al., 1996). These studies

Figure 4 Genomic organization of the p21/Waf1 gene. Exon– intron boundaries, and start/stop codons for translation, are denoted, along with the location of each of the four polymorphic variants discussed here-in.

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revealed that the serine and arginine polymorphic variants have very similar kinase inhibitory activity and growth suppression abilities, and suggest that the codon 31 polymorphism does not affect the structure or function of the protein. More recently, Su et al. (2003b) reported that the codon 31 polymorphism may not affect the functional activity of the protein, but rather alters mRNA expression of p21/Waf1. These researchers found that individuals who carried at least one p21/ Waf1 Arg allele had an approximate geometric mean decrease of 38% in p21/Waf1 mRNA expression as compared to individuals with the p21/Waf1 Ser/Ser genotype. Interestingly, individuals heterozygous for codon 72 variants (Arg/Pro) in addition to p21/Waf1 Arg/Ser variant demonstrated a mean geometric reduction in p21/Waf1 mRNA levels of 57% as compared to the individuals carrying p53 Arg/Arg-p21/Waf1 Ser/Ser (Su et al., 2003b). A number of studies have been performed to examine the involvement of the p21/Waf1 codon 31 polymorphism Arg allele on cancer susceptibility. Approximately half of these studies support the idea that this polymorphism correlates with cancer risk (Facher et al., 1995; Mousses et al., 1995; Sjalander et al., 1996; Hachiya et al., 1999; Harima et al., 2001; Chen et al., 2002; Keshava et al., 2002; Huang et al., 2004) while others refute this claim (Gong et al., 1995; Li et al., 1995; Sun et al., 1995; Wan et al., 1996; Lukas et al., 1997; McKenzie et al., 1997; Konishi et al., 2000; Shih et al., 2000; Tsai et al., 2002a, b; Su et al., 2003a; Wu et al., 2004). Discrepancies among these studies may be explained by inadequate sample sizes or inclusion of different ethnic groups in single studies (Facher et al., 1995; Wu et al., 2004). Additionally, although most studies have tested whether the codon 31 Arg allele is a risk factor for cancer development, three studies have identiﬁed the Ser allele as a risk factor for development of cervical cancer (Roh et al., 2001) and endometrial cancer (Roh et al., 2004) in a Korean population and esophageal cancer in a Taiwanese population (Wu et al., 2003). Most studies with respect to the codon 31 polymorphism have focused on cancer susceptibility and only a few have examined inﬂuences of the codon 31 polymorphism on cancer prognosis and progression. One study on a Taiwanese population demonstrated that individuals carrying the Ser/Ser genotype tended to have a shorter postoperative survival compared to those with the Ser/Arg or Arg/Arg genotype (Shih et al., 2000). In contrast, this polymorphism was not found to affect survival in a population of Caucasian Australian breast cancer patients (Powell et al., 2002). Other researchers report that the codon 31 polymorphism may inﬂuence tumor type and grade. For example, in a Taiwanese population with bladder cancer, the serine heterozygote was found more often in patients with invasive bladder cancer when compared to the non-invasive group (Chen et al., 2002). Likewise, in a population of 54 Japanese women with endometrial cancer, the codon 31 Arg (heterozygote or homozygous) allele was associated with the development of advanced adenocarcinomas that were histologically undifferen-

tiated, while patients with clinically localized, welldifferentiated adenocarcinomas were more frequently homozygous for the serine allele at codon 31 (Hachiya et al., 1999). The variant codon 31 polymorphism was found present three times more in samples from Caucasian Australian breast cancer patients whose tumors lacked progesterone receptors compared with patients whose tumors expressed progesterone receptors (Powell et al., 2002). In a Chinese population with prostate cancer, the p21/Waf1 Arg/Arg genotype was found to be signiﬁcantly associated with localized/ locally advanced cancer, but not with bone metastasis. Additionally, benign prostate hyperplasia patients with the Arg/Arg genotype were found to have larger prostate volumes compared to patients with the Ser/ Ser genotype (Huang et al., 2004). Other groups suggest there may be a correlation between the codon 31 polymorphism and a susceptibility to cancer development based on environmental inﬂuences. For example, in a Taiwanese population of 47 patients with nasopharyngeal carcinoma, the serine form of codon 31 was more predominant in smokers than in non-smokers (Tsai et al., 2002a, b). The codon 31 polymorphism is linked to a polymorphism in the 30 UTR of the p21/Waf1 gene that is located 20 nucleotides downstream of the translation stop codon (nucleotide 590) (Shiohara et al., 1994; Facher et al., 1995; Mousses et al., 1995; Milner et al., 1996; Konishi et al., 2000). This polymorphism is a C-T base substitution which results in deletion of a PstI restriction enzyme site (Law and Deka, 1995). The allele frequency of this polymorphism ranges from 3% in a Brazilian population to 9% in Caucasians (Law and Deka, 1995; Rodrigues et al., 2003). Interestingly, the codon 31 polymorphism combined with the polymorphism in the 30 UTR may contribute jointly to the development of squamous cell carcinoma of the head and neck, prostate adenocarcinoma, and breast tumors (Facher et al., 1995; Mousses et al., 1995). In addition, Kibel demonstrated that the 30 UTR polymorphism was by itself associated with the development of prostate carcinoma in a Caucasian population (Kibel et al., 2003). The functional signiﬁcance of the 30 UTR polymorphism of p21/Waf1 may be an inﬂuence on mRNA stability. Since the 30 UTR is a region that controls mRNA stability and degradation in many genes, it has been suggested that the polymorphism in the 30 UTR may be located at a site necessary for mRNA degradation and may prevent timely degradation of the p21/ Waf1 mRNA, therefore altering the cellular DNA damage-induced cell cycle arrest response (Facher et al., 1995; Kibel et al., 2003). Another polymorphism that has been identiﬁed in the non-coding region of the p21/Waf1 gene may also be linked to the codon 31 polymorphism; this polymorphism is located in intron 2 of the p21/Waf1 gene, 16 bps downstream from the splice donor site of the second intron. This C to G transition is hypothesized to affect the mRNA splicing of p21/Waf1 (Shi et al., 1996; Powell et al., 2002). Powell et al. (2002) demonstrated that the C to G transition alone increases the risk for breast Oncogene

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cancer in a Caucasian Australian population. Additionally, this polymorphism was more frequently detected in patients with well-differentiated tumors as compared to patients whose tumors demonstrated poorly differentiated histology (Powell et al., 2002). Interestingly, Xi et al. (2004) demonstrated that the codon 31 polymorphism, the polymorphism in the 30 UTR, and the polymorphism in intron 2, when each was analysed alone, were not sufﬁcient to inﬂuence gastric cancer risk. However, patients with the combined haplotype of codon 31 serine, the cytidine allele of the second intron, and the cytidine allele in the 30 UTR demonstrated a higher risk to develop gastric cancer than to develop precancerous lesions, such as intestinal metaplasia and dysplasia (Xi et al., 2004). Since p21/Waf1 is a downstream target of p53, several studies have investigated if there is a correlation between p53 polymorphisms and p21/Waf1 polymorphisms. It seems that for a few tumor types, a combination of the p53 and p21/Waf1 polymorphisms correlates with an increased risk for cancer development. For example, the combination of the Ser genotype of p21/Waf1 codon 31 and the Pro genotype of p53 codon 72 was found associated with increased susceptibility for the development of endometrial cancer in a Korean population (Roh et al., 2004). Likewise, in a Chinese population, patients with the combination of the p21/Waf1 codon 31 Ser/Ser genotype, cytidine at the second intron, and the cytidine in the 30 UTR of p21/Waf1 exon 3 plus p53 codon 72 Pro allele were found to have increased risk for development of gastric cancer (Xi et al., 2004), but not lung cancer (Shih et al., 2000). Codon 149 of p21/waf1 The polymorphism at codon 149 is the result of an A to G transition at nucleotide 521 changing codon 149 from GAT to GGT (Bahl et al., 2000), which changes the predicted amino acid from aspartate to glycine. To date the codon 149 polymorphism has only been detected in an Indian population with an allele frequency of 16% (Bahl et al., 2000). It has been suggested that this polymorphism may affect the ability of p21/Waf1 protein to interact with PCNA, and this may alter the antiproliferative activity of p21/Waf1, since the polymorphism is located in the PCNA interaction area (Bahl et al., 2000; Ralhan et al., 2000). However, functional studies have not been performed on the codon 149 polymorphic variants and it is not clear whether or not this polymorphism affects the p21/Waf1–PCNA interaction. Results of two separate studies have shown that the codon 149 polymorphism is associated with an increased risk for the development of esophageal squamous cell carcinoma and oral squamous cell carcinoma (Bahl et al., 2000; Ralhan et al., 2000). Polymorphism in the MDM2 promoter Levine and co-workers were the ﬁrst to discover a functionally signiﬁcant SNP in the promoter for MDM2; this polymorphism occurs at nucleotide 309 of intron 1 of the MDM2 gene, and changes a T to a G Oncogene

(the ‘G’ allele is denoted SNP309). The G allele frequency was reported to be 0.32, making this a common polymorphism. Notably, this change is predicted to create a higher-afﬁnity binding site for the transcription factor Sp1, which is an important controller of MDM2 RNA levels. This group demonstrated that the G allele bound with 2–4-fold enhanced afﬁnity to puriﬁed Sp1 both in vitro and in vivo. This Sp1 site lies close to a site recognized for binding to the estrogen receptor, and this polymorphism may inﬂuence estrogen receptor alpha-induced expression of the MDM2 gene as well (Phelps et al., 2003). Notably, Levine’s group observed a consistent correlation between cell lines homozygous for the G allele with increased steady-state levels of MDM2 protein (Bond et al., 2004). In line with this ﬁnding, cell lines homozygous for the G allele of SNP309 were shown to have an attenuated p53 transcriptional and apoptotic response, owing to a decreased ability of p53 to stabilize following DNA damage. In addition to impaired stabilization of p53 associated with the G allele of SNP309, Bargonetti and co-workers recently reported that cell lines homozygous for this allele have an impaired transcriptional response of p53, independent of the attenuated stabilization response. Speciﬁcally, chromatin immunoprecipitation analysis of p53 target genes indicated that in cells homozygous for the G allele of SNP309, p53 could be found bound to the promoters of p53-target genes following treatment with chemotherapeutic agents. However, p53 binding to these response elements was not accompanied by transactivation of such genes because MDM2 was found complexed as well (Arva et al., 2005); MDM2 is known to inhibit p53 transactivation by concealing the transactivation domain of this protein. Downregulation of MDM2 in these cells, using siRNA, restored the p53 responsiveness of p53 target genes in these cells (Arva et al., 2005). The take-home message from such studies is that the increased expression of MDM2 caused by SNP309 impairs the p53 pathway. Levine and co-workers analysed SNP309 in individuals from families with Li–Fraumeni syndrome, which have one mutated copy of the p53 gene, and therefore already have an attenuated p53 response. This group hypothesized that the MDM2 SNP might further attenuate the p53 pathway in these individuals, and therefore affect cancer incidence in these families. Analysis of 88 individuals from Li–Fraumeni families containing germline mutations in one allele of p53 revealed that individuals who carried the G allele of SNP309, in either the heterozygous or homozygous state, showed a signiﬁcantly earlier age of onset for all tumor types (a minimum of 9 years earlier). Additionally, individuals homozygous for the G allele of SNP309 also had an increased occurrence of independent subsequent cancers (Bond et al., 2004). Interestingly, a recent report conﬁrms a 10 year earlier age of onset of cancer in Li–Fraumeni carriers for individuals with the G allele of SNP309; this report also suggests that the codon 72 polymorphism of p53 may amplify this effect, with individuals with a combined G allele of SNP309,

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and the Arg variant of codon 72 of p53, demonstrating an over 20 year increase in age of onset (Bougeard et al., 2005). These ﬁndings reiterate the importance of analysis of polymorphisms in multiple genes in the same pathway. Conclusions, future prospects Clearly a take-home message from this chapter is that functionally signiﬁcant polymorphisms in the p53 path-

way exist which impair the function of this pathway. In some cases, these variants are clearly associated with altered age of onset or prognosis of cancer. However, whether these variants are associated with altered cancer risk is not currently clear. Such associations await combined analyses of multiple variants in this pathway, along with more precise functional studies. Finally, the generation of mouse models for variants in the p53 pathway may be the single most important next step toward elucidation of their contribution to cancer risk and treatment efﬁcacy.

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Oncogene

Cooperation between p53 and p130(Rb2) in induction of ... - Nature