Novel associations of UDP-glucuronosyltransferase 2B gene variants with prostate cancer risk in a multiethnic study

Prostate cancer is the second leading cause of cancer-related deaths in men, after lung cancer [1]. The incidence of prostate cancer has increased over the past twenty years and African American men have been disproportionally affected compared to other racial/ethnic groups [2‐7]. In the U.S., the incidence of prostate cancer among African Americans is more than 60% higher than in Caucasians, and the mortality rate in African Americans is twice that of Caucasian men [8, 9]. Although differences in incidence and mortality rates may be due, in part, to race/ethnicity, socioeconomic conditions and availability of health care [10], familial aggregation studies suggest that genetic factors may also be contributing to prostate cancer demographic disparity. Candidate gene approaches involving hormone metabolic pathways have been examined in prostate cancer association studies, however results from these studies have not been replicated [11, 12]. Nonetheless, current therapies are primarily targeted at specific androgen biosynthetic pathways [13], thus, improved knowledge on genetic variants associated with both androgen metabolism and prostate cancer risk is important.

The UDP-glucuronosyltransferase (UGT) genes code for enzymes that convert a diverse group of xenobiotic and endobiotic substances into lipophilic compounds, facilitating clearance from the body as part of the phase II liver detoxification system [14]. These enzymes are subdivided into families according to similarities in amino acid sequence and target substrates dictated by the specificity of their amino-terminal ends [15]. A specific subfamily, UGT2B, includes two enzymes, UGT2B17 and UGT2B15, which are also expressed extrahepatically in the basal and luminal epithelium of the prostate, respectively. These enzymes exhibit specificity for androgen metabolites such as testosterone, dihydrotestosterone (DHT), androsterone (ADT), and androstane-3α,17β-diol (3β-diol) in prostate tissue and cell lines [16, 17].

Functional polymorphisms of the UGT2B gene family such as the copy number variant (CNV) of UGT2B17, and the aspartic acid (D or nucleotide G) to tyrosine substitution (Y or nucleotide T) found in codon 85 of the UGT2B15 gene (UGT2B15 ^D85Y , rs1902023) have been identified [18, 19]. Studies suggest that men with 0 copies of the UGT2B17 gene are unable to break down testosterone through the UGT2B pathway and subsequently secrete negligible amounts of urinary testosterone compared to men with at least one copy of UGT2B17[18, 20]. Experimental evidence showed that the minor allele (T) of the UGT2B15 variant, UGT2B15 ^D85Y , causes the enzyme to have an increased V_max activity when compared to the presence of the major allele (G) [19]. Subsequently, the resulting phenotype of this UGT2B15 polymorphism is a quicker androgen metabolite clearance which may raise the “effective” amount of steroids within the prostate and decrease risk for prostate cancer [21]. These two major UGT2B variants (the CNV in UGT2B17 and the polymorphism in UGT2B15) have been evaluated in relation to prostate cancer risk, with inconsistent findings [22‐32]. Discrepancies could have been due to the genetic heterogeneity of the populations studied, as well as variable sample sizes of these populations. We have recently shown that individuals with a major allele (G) of the UGT2B15 ^D85Y polymorphism (rs1902023) have higher risk of prostate cancer when compared to individuals homozygous for the “rapid clearance” minor allele (Y) [33]. In the same study, the UGT2B17 CNV showed no association with prostate cancer risk [33]. Recently, an additional 7 novel UGT2B15 SNPs that are in strong linkage disequilibrium (LD) with the UGT2B15 ^D85Y gene variant, have been identified by re-sequencing the promoter and exon one regions of the UGT2B15 gene, using DNA samples from Yoruba (YRI), CEPH/European (CEU), and Japanese/Chinese (ASN) populations [34]. Most of these UGT2B variants have not been evaluated in relation to prostate cancer risk in population-based studies or in studies that included African American men. In this present work, we examined associations between functional SNPs of UGT2B17, UGT2B15 and three other related UGT2B SNPs, and prostate cancer risk among African American and Caucasian men.

Table 2 describes the clinical characteristics of the study participants. Prostate cancer cases (n = 233) and controls (n = 342) were comparable in height (p = 0.31), however controls (mean BMI = 30.77, SD = 6.05) had slightly higher BMI than cases (mean BMI = 29.25, SD = 5.60, p = 0.002) and were slighter younger than cases (p = 0.005). African American men comprised 63% of the cases and 41% of the controls (p <0.0001). A family history of prostate cancer was reported by 14% of cases and 10% of the controls. (p = 0.16). Most cases and controls reported tobacco use (p = 0.43). Ninety-one percent of the cases had low-grade prostate cancer (Gleason sum < 8) vs. 9% of cases with high grade prostate cancer (Gleason sum ≥ 8).

The functional variants of the UGT2B15 and UGT2B17 SNPs were assessed for associations with prostate cancer risk and summarized in Table 3. After adjusting for age, race and BMI, four UGT2B15 SNPs, rs9994887, rs13112099, rs7686914 and rs7696472, were associated with increased risk of prostate cancer, when compared to the minor allele (Log additive model OR = 1.49, 95% CI = 1.09, 2.04; OR = 1.48, 95% CI = 1.08, 2.03, OR = 1.48, 95% CI = 1.08, 2.03, OR = 1.49, 95% CI = 1.06, 2.00, respectively). UGT2B15 SNPs rs4148269 and rs3100, were also associated with increased risk for prostate cancer (Log additive model OR = 1.45, 95% CI = 1.03, 2.03; OR = 1.46, 95% CI = 1.08, 1.98, respectively). These data suggest that these associations were allele dose-dependent (Table 3). None of the UGT2B17 SNPs studied were associated with prostate cancer risk, however the UGT2B17 cis-acting SNP, rs17147338, showed borderline significance for increased risk of prostate cancer in an allele dose dependent fashion (OR = 1.65, 95% CI = 1.00, 2.70), after adjusting for age, race and BMI (Table 3). SNPs in high LD with UGT2B15 ^D85Y , rs9994887, rs13112099, rs7686914, and rs796472 [34] were associated with increased risk of prostate cancer in an allele dose-dependent manner for both high-grade (OR = 2.34, 95% CI = 1.22, 4.49; OR = 2.31, 95% CI = 1.20, 4.44; OR = 2.31, 95% CI = 1.20, 4.44; OR = 2.26, 95% CI = 1.18, 4.33, respectively) and low-grade prostate cancer lesions (OR = 1.94, 95% CI = 1.06, 3.53; OR = 1.93, 95% CI = 1.06, 3.52; OR = 1.94, 95% CI = 1.06, 3.53; OR = 1.99, 95% CI = 1.10, 3.63, respectively) after adjusting for age, race, and BMI (Table 4). Intriguingly, SNPs rs4148269 and rs3100 were associated with increased PC risk in high grade lesions only (OR = 1.96, 95% CI = 1.01, 3.82; OR = 2.16, 95% CI = 1.18, 3.95).

Restricting analyses by race/ethnicity, and after adjusting for age and BMI, further revealed that these associations were present in African American men (rs9994887, OR = 1.66, 95% CI = 1.05, 2.63; rs13112099, OR = 1.66, 95% CI = 1.03, 2.63; rs7686914, OR = 1.64, 95% CI = 1.03, 2.63; rs7696472, OR = 1.64, 95% CI = 1.03, 2.63; rs4148269, OR = 2.04, 95% CI = 1.19, 3.44; and rs3100, OR = 1.89, 95% CI = 1.22, 2.94) (Table 5). However, the interaction terms for these SNPs and race were not statistically significant (p > 0.4) for all six SNPs. For both groups, carrying the UGT2B17 or cis-acting SNPs was not associated with prostate cancer risk (Table 5).

To investigate the extent to which these SNPs were associated in our subjects we calculated correlation coefficients among the controls as shown in Table 6. The r² values for the UGT2B15 variants rs1580083, rs1960773, rs13112099, rs7686914, rs7696472 and rs9994887 ranged from 0.62 to 0.99/1.00 (p < 0.0001). UGT2B15 variants rs4148269 and rs3100 were also strongly correlated (r² = 0.85, p < 0.0001). Including both SNPs into the same regression model to evaluate whether one SNP would have a stronger association with PC risk did not yield additional insights. Notably, these two SNPs showed no correlation with the other SNPs that were highly associated with each other. UGT2B17 SNP rs7435827 was moderately correlated with UGT2B15 SNP rs2168047 (r² = 0.36, p < 0.0001) and UGT2B17 SNP rs7671342 (r² = 0.32, p < 0.0001), and strongly correlated with UGT2B17 SNP rs59678213 (r² = 0.83, p < 0.0001); however these SNPs showed no association with prostate cancer risk. UGT2B17 SNP rs59678213 was inversely associated with UGT2B17 SNPs rs2168047 (r² = −0.47, p < 0.0001), rs7686008 (r² = −0.29, p < 0.0001) and rs7671342 (r² = −0.49, p < 0.0001). No correlations were observed for the cis-acting UGT2B SNPs that were identified through the recent UGT2B SNPs discovery study [38]. All SNPs that showed associations with prostate cancer risk were in Hardy-Weinberg equilibrium in control samples, except for UGT2B15 SNP rs3100.

In this study we examined associations between functional SNPs of the UDP-glucuronosyltransferase 2B (UGT2B) genes and prostate cancer risk. After adjusting for age and BMI, we found that two UGT2B15 SNPs, rs4148269 and rs3100, were associated with a two-fold increase risk of prostate cancer and the association was more apparent in African American men. In addition, SNPs rs9994887, rs13112099, rs7686914 and rs7696472 were also associated with increased risk of prostate cancer. These associations persisted either in low or high-grade prostate cancer lesions for SNPs rs9994887, rs13112099, rs7686914, and rs7696472. However, SNPs rs4148269 and rs3100 were only associated with increased risk of prostate cancer in high-grade prostate cancer lesions. These findings are novel and support the hypothesis that these SNPs may affect UDP-glucuronosyltransferase enzyme function, presumably by increasing the efficiency of androgen metabolite clearance compared to enzyme products of the UGT2B wild type genotype, as has been shown for UGT2B15 SNP rs1902023 [21].

Associations between functional SNPs of the UGT2B15 and UGT2B17 genes and prostate cancer risk have been previously examined and have rendered conflicting results [22‐32]. Discrepancies could be due in part to racial/ethnic differences in the populations studied but also to sample size and allele dosage. Comparison of two studies with different sample sizes revealed contradictory results: no associations between SNP UGT2B15 ^D85Y and prostate cancer risk were found in a study with a large sample size [9], whereas a study with a smaller sample size found an increased risk of prostate cancer associated with the UGT2B15 ^D85Y variant in Caucasian subjects [32]. In reference to allele dosage, we previously concluded [33], concordant with previous reports [31, 32, 39], that homozygous carriers of the dominant UGT2B15 ^D85Y (G) allele missense polymorphism, (rs1902023), had an almost three-fold higher risk of prostate cancer. Our current results further confirm that associations with UGT2B15 polymorphisms are allele dose-dependent and may also be dependent on race/ethnicity. Furthermore, our study includes a large and multiethnic sample size in comparison to previous reports.

Some of our stratified analyses revealed that UGT2B15 wild type alleles were significantly associated with increased risk of prostate cancer in African American men, but not in Caucasian men, suggesting that the wild type genotype may confer protection against prostate cancer in Caucasian men. While these findings require replication in larger studies, we provide new evidence that wild type genotypes for the UGT2B15 gene more frequently found in African American men may increase the risk of prostate cancer, and thus may contribute to the present racial/ethnical disparities seen in prostate cancer incidence. Few studies have assessed the association of UGT2B genes with prostate cancer risk in African Americans. Park et al. [25] were one of the first to report that associations between UGT2B17 polymorphisms and increased risk of prostate cancer were linked to race/ethnicity; they showed that Caucasian men carrying a UGT2B17 deletion polymorphism had a two-fold increased risk of prostate cancer, but not Black men who carry the same deletion, compared to non-carriers [25]. Subsequent work supported these findings [22, 26], which were further confirmed by others [23, 24]. Our results show no significant associations of UGT2B17 polymorphisms with prostate cancer risk in neither Caucasian nor African American men. Our findings that UGT2B17 SNP rs59678213 was not associated with prostate cancer risk are consistent with those of others [22, 24].

Four of the UGT2B15 SNPs that conferred prostate cancer risk in this study are among seven SNPs (rs1580083, rs1960773, rs9994887, rs13112099, rs7686914, rs7696472, and rs1120265 [not in study]) that are in high linkage disequilibrium (LD) with UGT2B15 ^D85Y SNP rs1902023 [34]. The phenotype of the variations, established with reporter gene assays in the liver cancer cell line HepG2, showed that transcriptional activity was higher for constructs containing the major alleles when compared to constructs containing the minor alleles. When a construct with the minor allele of rs9994887 was transfected into a prostate cancer cell line, LNCaP, transcriptional activity was decreased by approximately 14%, compared to constructs containing the major allele [34], which suggested that rs9994887 may influence gene expression in the prostate [34]. Further experiments would have to be done to determine the impact of these variants on the kinetics and efficacy of UGT2B enzymes. Interestingly, the other three SNPs that showed associations with increased prostate cancer risk, rs13112099, rs7686914, and rs7696472 were highly correlated with rs9994887 (r² = 0.99, 0.99, and 0.97, respectively). In addition, the three SNPs are all located upstream of rs9994887 (−1139 bp relative to translation start) while the other two SNPs are located downstream. While the impact of these variants on UGT2B15 enzyme kinetics has not been reported, the associations suggest that the phenotype of the enzyme may be similar to that of the UGT2B15 ^85Y variant or rs1902023 [19]. SNPs rs4148269 and rs3100 also demonstrated strong associations with decreased risk of PC, but did not show any correlation with the promoter SNPs in this study population. SNP rs4148269 has been characterized as a T523K amino acid substitution [40], however a comprehensive analysis of the enzyme phenotype has not been reported. This suggests that rs3100, which is highly correlated (r² = 0.85) with rs4148269, may be important in the etiology of PC. Results from reporter gene assays showed that the major allele of rs3100 is associated with significantly higher gene expression in LNCaP cells when compared to constructs made with the minor allele [36].

The lack of association observed for the UGT2B17 SNPs and PC risk was unexpected, as previous reports suggested that the UGT2B17 enzyme plays a major role in testosterone glucuronidation [20]. However results from a recent report comparing UGT2B17 and UGT2B15 mRNA and protein expression, suggest that UGT2B15 may be negatively regulated in naïve and castration-resistant tumors while undetectable in lymph node metastases [41]. More in vitro analyses are required to understand the functional impact of these genetic variations. Nonetheless this is the first time these SNPs are reported in relation to prostate cancer risk.

The main limitation of this study is the sample size which limited our ability to simultaneously examine race/ethnicity in the associations with SNPs and prostate cancer grade. The low numbers of Caucasians may have limited our ability to detect significant associations within this group given that they were less likely to present a high grade PC tumor. The small sample size could also have been a contributing factor in the lack of significance for the cross-product term for the SNPs and race, however race/ethnicity was self-reported and we did not measure ancestral markers, which could indicate the percentage of African American ancestry of each subject. Also, we cannot exclude the possibility of potential confounding due to differences in MAFs between races. Nonetheless our study was conducted at the DVAMC which provides medical care for all former military personnel. This type of setting has important advantages. The Veterans Affairs Medical Centers (VAMC) are considered an equal-access health care settings that may control for health care availability among various U.S. ethnicities [42]. Moreover, the VAMCs provide a setting for PC case–control studies as this is the leading cancer reported in Veterans Affairs Central Registry (VACCR) [43].