Single-Nucleotide Polymorphisms in p53 Pathway and Aggressiveness of Prostate Cancer in a Caucasian Population

Tumor suppressor p53 lies at critical point of a complex signaling network for response to stress. The normal functioning p53 protein is involved in cell cycle arrest, DNA repair, apoptosis, and maintenance of genetic integrity (1). Therefore, it is regarded as a potent barrier to cancer. Malfunction of the p53 pathway is an almost universal hallmark of human tumors (2). p53 is also found as one of the most commonly mutated genes in all types of human cancer. Mutations involving p53 are considered as late events during multistep prostate carcinogenesis (3). Apparently, loss of the wild-type p53 function can contribute to the hormone resistance of prostate cancer cells (4).

The Mdm2 oncoprotein is an established regulator of p53 via effects on p53 degradation and negative feedback inhibition (5). The level of the Mdm2 protein in a cell or organism has a significant effect on cancer formation. Mdm2 can also reduce androgen receptor signaling in prostate cancer via p53 inhibition and is implicated in ubiquitination and degradation of androgen receptor by the Akt pathway (6). Mdm2 protein is overexpressed in ∼30% to 45% of analyzed prostate cancers (7, 8). This overexpression was associated with advanced tumor stage or increased cell proliferation (7).

Mdm4, which is a structural homologue of Mdm2, binds to the amino terminus of p53, functioning as a major inhibitor of p53 activity (9, 10). Hausp (herpes virus–associated ubiquitin-specific protease) stabilizes Mdm2, Mdm4, and p53 via its specific deubiquitinase activity (11). The Mdm2, Mdm4, and Hausp proteins thus maintain p53 level and activity.

In humans, functional single-nucleotide polymorphisms (SNP) have been identified in both p53 and its negative regulators. The p53 SNP rs1042522, which is located at codon 72 in the putative Src homology 3 binding domain, results in G to C change and an Arg to Pro amino acid substitution, influencing binding capacity and thereby functional properties of p53. The p53 Pro is a stronger inducer of target gene transcription than p53 Arg, whereas the p53 Arg seems to induce apoptosis with faster kinetics and suppresses transformation more efficiently than the p53 Pro variant (12). The relationship of p53 Arg72Pro with cancer susceptibility, reproduction, and aging has been well studied (13–16).

The pivotal role of Mdm2, Mdm4, and Hausp in the control of p53 function argues that polymorphisms at these loci may be important for the modulation of p53 function. A SNP (309 T > G) (rs2279744) in the promoter region of Mdm2 increases affinity for binding stimulatory protein 1, leading to increased Mdm2 expression and the subsequent attenuation of the p53 pathway (17). This SNP has been associated with susceptibility of certain types of cancer, including breast, lung, gastric, and colon cancer (18–20).

Recent studies on the haplotype SNP structure on Mdm4 and Hausp indicated the presence of candidate SNPs that influence p53 function and subsequently confer cancer risk. Mdm4 gene SNP (rs2279744) was reported under positive evolutionary selection and associated with risk of breast and ovarian cancers or human fertility in Caucasian population (21, 22). Hausp gene SNP (rs1529916) was also associated with human fertility (21).

Several studies have investigated the association of p53 and Mdm2 polymorphisms with prostate cancer susceptibility (23–30). However, results among these studies are inconsistent and inconclusive. Moreover, Mdm4 and Hausp polymorphisms have never been studied in prostate cancer. One of the most important questions in prostate cancer research is to identify people at risk of developing aggressive forms of the disease. Therefore, we are interested in the study of genetic variants in p53 pathway that are associated with more aggressive and/or with a better response to current therapies. By studying a large, well-defined homogenous ethnic background patient cohort, we evaluated the association between candidate p53 pathway SNPs and prostate cancer aggressiveness in Caucasian Americans.

The details of the Dana-Farber Harvard Cancer Center Specialized Programs of Research Excellence (Gelb Center) Prostate Cancer cohort have been previously described (31). Briefly, all patients seen at Dana-Farber Cancer Institute and Brigham and Women's Hospital with a diagnosis of prostate cancer are approached to participate. The consent rate for patients is 86%. A total of 4,073 prostate cancer patients diagnosed between 1976 and 2007, who had been consented during 1993 to 2007 to provide information and tissue and had blood collected for research purposes, were included in this study cohort.

To control the quality of the ethnicity information from the self-reported data, we sampled 3% of self-reported Caucasian (n = 180) and performed genotyping using 26 SNPs, which can distinguish Caucasian population from non-Caucasian populations (32); the genotyping data showed that none of the tested samples were in discordance. This confirmed the reliability of self-reported Caucasian ethnicity. For all individuals who ambiguously reported their ethnicity, such as reported as “American,” or who do not have the ethnicity information, their Caucasian identity was determined by genotyping using the same set of 26 SNPs. Only reliably self-reported or SNP-confirmed Caucasians were eligible for this study. Age at diagnosis was calculated from the date of the first positive biopsy. Using the D'Amico risk classification criteria, prostate cancer patients were identified as at low, intermediate, or high risk of clinical recurrence after primary therapy (33). Because original D'Amico risk classification was set to predict biochemical outcome of localized patients, in this study, patients who diagnosed with N1 or M1 diseases were regarded as high D'Amico risk class. Within the entire cohort, 1,716 of 4,073 patients received radical prostatectomy (RP) as the primary treatment. RP Gleason scores and pathologic stages of RP specimen were acquired by reviewing pathology reports.

Two well-studied SNPs, p53 Arg72Pro (rs1042522) and Mdm2 SNP309 (rs2279744), which have been shown to modify the activity or the levels of the p53 protein and influence cancer susceptibility, were chosen for this study (12, 17). For Mdm4 gene, we chose to study SNP rs1380576, which is located in Mdm4 gene intron 1 region and is in complete linkage disequilibrium (r² = 1) with a previously described SNP (rs2279744), which was reported under positive evolutionary selection and associated with risk of certain cancer (21, 22). We also included the reported Hausp SNP (rs1529916), which is under evolutionary selection pressure and associated with human fertility in Caucasian (21).

All DNA samples were extracted from peripheral whole blood using QIAamp DNA Blood mini kit (QIAGEN, Inc.). Genotyping was done with Sequenom iPLEX matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. For quality control, ∼5% random selected duplicates were included. No discrepancy between duplicates was observed in the genotyping data of all four SNPs. All SNPs had >99% genotype passing rates.

We analyzed each SNP as a categorical variable with a common homozygote, a rare homozygote, and a heterozygote. Observed genotype distributions were tested for departure from Hardy-Weinberg equilibrium using Pearson's goodness-of-fit test. No SNP violated Hardy-Weinberg equilibrium (all P values > 0.10).

To investigate the association between genotypes and early onset prostate cancer (≤60 years), we estimate odds ratios (OR) and their 95% confidence intervals (95% CI) using unconditional logistic regression. Prostate cancer aggressiveness at diagnosis was categorized using D'Amico risk classes (low, intermediate, or high risk) with criteria described previously. Generalized logistic regression was used to evaluate its relationship with SNPs with the low-risk group as reference for comparison. In a subcohort of patients who received RP, we also examined the association between p53 pathway SNPs and RP Gleason score or pathologic stages with unconditional logistic regression. The analyses, with the exception of those for early onset prostate cancer, were adjusted for age at diagnosis.

All statistical tests were done using SAS version 9.1 (SAS Institute, Inc.), and P < 0.05 (two sided) was considered statistically significant.

Selected clinical characteristics of study participants are described in Table 1. Briefly, all participants are Caucasian, and their mean age at diagnosis is 61.3 years (range, 42-91 years). Among patients with sufficient information for modified D'Amico risk classification, 1,004 (30%) patients were low risk, 1,357 (40%) patients were intermediate risk, and 986 (30%) patients were high risk. In patients who received RP, 1,161 (68%) men had organ-confined (T1/T2) disease at the time of surgery, whereas 475 (28%) men had extraprostatic tumor (T3/T4) and 80 (4%) men had metastatic tumor (N1 or M1). The post-RP Gleason score was <7 in 652 (39%) patients, 7 in 769 (45%) patients, and ≥7 in 271 (16%) patients.

We first estimate associations between the genotypes of p53 Arg72Pro, Mdm2 SNP309, Mdm4 SNP (rs1380576), and Hausp SNP (rs152 9916) and the risk of developing an early onset prostate cancer (Table 2). Compared with the p53-72 Pro/Pro genotype, patients carrying one 72 Arg allele or two 72 Arg alleles had an OR of 1.42 (95% CI, 1.10-1.85) or 1.29 (95% CI, 1.00-1.66), respectively, of developing prostate cancer at or before 60 years. This result indicates that the p53 SNP had a borderline association with the age at diagnosis (P = 0.021). Compared with Mdm2 GG genotype, a moderate increase of OR was found in the association of the Mdm2 309 GT heterozygous genotype (OR, 1.23; 95% CI, 1.02-1.49) with the age at diagnosis. The Mdm2 309 TT genotype increased the likelihood early onset prostate cancer (OR, 1.38; 95% CI, 1.14-1.68). However, we did not find a significant association of polymorphisms in the Mdm4 or Hausp genes with age at diagnosis.

In this analysis, we found that Mdm4 intron 1 SNP (rs1380576) was associated with D'Amico category (P = 0.023; Table 3). Comparing with Mdm4 GG genotype, CG genotype, or CC genotyping had an OR of 1.47 (95% CI, 1.11-1.95) or 1.31 (95% CI, 0.99-1.74) for developing intermediate-risk prostate cancer and an OR of 1.38 (95% CI, 1.01-1.89) or 1.50 (95% CI, 1.10-2.04) for developing high-risk disease, respectively. We also observed that Hausp gene SNP (rs1529916) minor allele T allele conferred an increased risk of developing intermediate- or high-risk prostate cancer in a recessive manner. The Hausp AA genotype exhibited an OR of 1.44 (95% CI, 1.06-1.97) for developing intermediate-risk prostate cancer and an OR of 1.39 (95% CI, 0.99-1.94) for developing high-risk prostate cancer comparing with GG genotype. We did not observe any statistically significant association between p53 or Mdm2 genotypes and disease aggressiveness.

In patients who underwent RP, cases with RP Gleason score of ≥7 were compared with cases with RP Gleason score of <7 (Table 4). The Mdm2 309 TT genotype had an OR of 1.51 (95% CI, 1.11-2.05), having tumor RP Gleason score of ≥7 at the time of surgery compared with GG genotype. Similarly, Mdm4 GG genotype had an OR of 1.50 (95% CI, 1.07-2.09), developing higher RP Gleason score prostate cancer by the time of surgery. The p53 and Hausp SNPs had no statistically significant association with the RP Gleason score.

We also classified men as either having evidence of extraprostatic (T3/T4) or metastatic (N1 or M1) disease or localized disease (T1/T2) at prostatectomy. In this analysis (Table 5), we found that Mdm2 309 TT genotype was correlated with the development of extraprostatic or metastatic prostate cancer (OR, 1.53; 95% CI, 1.10-2.12) compared with GG genotype.

The p53 Arg72Pro (rs1042522) polymorphism has been well characterized in both functional analyses and association studies (12–16). In a limited number of studies in prostate cancer, no consistent conclusion has been made about the association between p53 polymorphism and prostate cancer risk or clinicopathologic variables (23–26, 28). This may be due to small sample sizes (<200 prostate cancer cases) and/or mixed ethnic populations. In our present large homogeneous case-case analysis, we did not observe any statistically significant association between p53 Arg72Pro polymorphism and prostate cancer aggressiveness or pathologic variables.

The Mdm2 SNP309 T > G (rs2279744) is a functional SNP that increases Mdm2 expression levels and attenuates the p53 pathway (17). Only a few studies have addressed an association of Mdm2 SNP309 with prostate cancer in relatively small cohorts (27–30). Intriguingly, previous studies have implicated that G allele of Mdm2 SNP309 was the high-risk allele in many other kinds of carcinoma (17, 19, 20). Our current study found that the T allele was associated with earlier onset prostate cancer (≤60 years) in the entire cohort, higher RP Gleason score (≥7), and advanced pathologic stages in RP patients. Our data are not the first to note that the T allele is associated with an increased risk or aggressiveness of prostate cancer. Kibel et al. (29) examined the Mdm2 SNP309 polymorphism in a European-American cohort of 186 patients with advanced prostate cancer and 220 cancer-free controls and found that the T allele of Mdm2 SNP309 was associated with an increased risk of advanced prostate cancer. In addition, studies from Germany (27) or Japan (30) suggested no correlation between a certain allelic variant of Mdm2 and an increased prostate cancer risk.

Although speculative, these conflicting observations in different cancers raise the possibility that each variant (T or G) may be associated with increased risk for a particular disease (17–20). Furthermore, increased cancer risk could be associated with a specific geographic or genetic background (18). The effect of variance may also be influenced by the hormonal environment. Bond et al. showed that the 309 G variant is bound more efficiently by the transcriptional factor stimulatory protein 1 (a coactivator for many hormone receptors) than 309 T allele. The estrogen receptor also binds the Mdm2 promoter in the region of SNP309 (34). Hu et al. determined that, in estrogen-responsive cells, estrogen preferentially induced the transcription of Mdm2 from the SNP309 promoter and that the levels of Mdm2 in SNP309 GG cells were higher than in heterozygous GT or TT cells (35). Bond et al. hypothesize that women carrying a G allele would preferentially benefit from lower estrogen level, which could retard the progression of their disease. They further proposed that Mdm2 SNP309 G allele accelerates tumor formation in a gender-specific and hormone-dependent manner (16). Although no evidence to date on androgen signaling was presented, given the importance of androgen axis in prostate tumorigenesis and progression, our observation suggests the possibility that the effect of Mdm2 309 G allele may also be dependent on hormonal signaling.

Mdm4 and Hausp are two important p53 regulators (11). A recently described haplotype structure analysis indicates the presence of candidate functional SNPs in Mdm4 and Hausp (21, 22). Kulkarni et al. did a case-only study of breast cancer patients and found that SNP in Mdm4 haplotype analysis (rs1563828) was associated with early age at diagnosis of estrogen receptor–negative, but not estrogen receptor–positive, breast cancers (36). Subsequently, they hypothesized that Mdm4 SNP may influence expression of different isoforms of Mdm4 or affect hormone signaling pathway in a cooperative way with Mdm2 (36). In our study, we evaluated the association between prostate cancer clinicopathologic variables and another intronic SNP rs1380576, which was located in intron 1 regulatory region of Mdm4 gene and in complete linkage disequilibrium with rs1563828. We also included the reported candidate SNP in Hausp (rs1529916). Interestingly, both Mdm4 and Hausp SNPs were found to be associated with increased risk of intermediate or high D'Amico category prostate cancer at the time of diagnosis, indicating that they are associated with more aggressive prostate cancer. Furthermore, Mdm4 dominant genotype GG was also found to be associated with higher RP Gleason score in subset RP patients. These results implicated that Mdm4 and Hausp SNPs contribute to the risk of aggressive prostate cancer, probably through influencing their crucial roles in p53 network or affecting hormonal signaling.

In summary, we found that alleles in p53 gene regulators, Mdm2, Mdm4, and Hausp, instead of SNPs in p53 itself, are associated with earlier age at diagnosis or more aggressive prostate cancer patients in a Caucasian population. These results suggest the importance of these p53 regulators in prostate cancer development, whereas the biological functions of these SNPs need further exploration.

No potential conflicts of interest were disclosed.

We thank Dr. Arnold J. Levine for his kind advice on this work.

Grant Support: Specialized Programs of Research Excellence in Prostate Cancer 2 P50 CA090381-06 and Prostate Cancer Foundation.

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.