SRD5A polymorphisms and biochemical failure after radical prostatectomy.

BACKGROUND: The relationship between inherited germ-line variations in the 5α-reductase pathways of androgen biosynthesis and the risk of biochemical recurrence (BCR) after radical prostatectomy (RP) remains an unexplored area. OBJECTIVE: To determine the link between germ-line variations in the steroid-5α-reductase, α-polypeptide 1 (SRD5A1) and steroid-5α-reductase, α-polypeptide 2 (SRD5A2) genes and BCR. DESIGN, SETTINGS, AND PARTICIPANTS: We studied retrospectively two independent cohorts composed of 526 white (25% BCR) and 320 Asian men (36% BCR) with pathologically organ-confined prostate cancer who had a median follow-up of 88.8 and 30.8 mo after surgery, respectively. MEASUREMENTS: Patients were genotyped for 19 haplotype-tagging single nucleotide polymorphisms (htSNPs) in SRD5A1 and SRD5A2 genes, and their prognostic significance on prostate-specific antigen recurrence was assessed using Kaplan-Meier analysis and the Cox regression model. RESULTS AND LIMITATIONS: After adjusting for all clinicopathologic risk factors, four SNPs (rs2208532, rs12470143, rs523349, and rs4952197) were associated with BCR in both whites and Asians. The strongest effect was conferred by the SRD5A2 V89L nonsynonymous SNP (rs523349C) with a hazard ratio (HR) of 2.87 (95% confidence interval [CI], 2.07-4.00; p = 4 × 10⁻¹°; 48% BCR). In addition, in whites, the combination of two SNPs, rs518673T in SRD5A1 and rs12470143A in SRD5A2, was associated with a reduced BCR rate for carriers of three or four alleles (HR: 0.37; 95% CI, 0.19-0.71; p=0.003;16% BCR) compared with noncarriers (38% BCR), whereas the SRD5A2 rs12470143A was significant in Asians (HR: 0.46; 95% CI, 0.28-0.73; p=0.001). Limitations of our study include few events of androgen-deprivation resistance or cancer-specific death. CONCLUSIONS: Our study is the first to show positive associations of several SRD5A1 and SRD5A2 variations as independent predictors of BCR after RP.

UGT1A1 and UGT1A9 functional variants, meat intake, and colon cancer, among Caucasians and African-Americans.

Glucuronidation by the UDP-glucuronosyltransferase enzymes (UGTs) is one of the primary detoxification pathways of dietary heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). In a population-based case-control study of 537 cases and 866 controls, we investigated whether colon cancer was associated with genetic variations in UGT1A1 and UGT1A9 genes and we determined if those variations modify the association between colon cancer and dietary HCA and PAH exposure. We measured functional UGT1A1 polymorphisms at positions -53 (28; A(TA)6TAA to A(TA)7TAA), -3156 (G>A), -3279 (T>G) and the UGT1A9-275(T>A) polymorphism, and found no association with colon cancer overall. However, when stratified by race, the UGT1A1-3279 GG/TG intermediate/low activity genotypes were associated with an increased risk of colon cancer (odds ratio (OR)=1.5, 95% confidence interval (CI)=1.1-2.0) in Caucasians. This finding is also supported by haplotype analyses where the UGT1A1-3279G-allele-bearing haplotype is overrepresented in case group. Overall, UGT1A1-53 and -3156 genotypes modified the association between dietary benzo(a)pyrene (BaP) and colon cancer (P for interaction=0.02 and 0.03, respectively). The strongest association was observed for those with <7.7 ng/day BaP exposure and the low activity genotypes, for both UGT1A1 28/28 (OR=1.8, 95% CI=1.1-2.9) and -3156AA (OR=1.7, 95% CI=1.0-3.0), compared to >or=7.7 ng/day and combined high/intermediate genotypes. These data support a hypothesis that UGTs modify the association between meat-derived PAH exposure and colon cancer by their role in the elimination of dietary carcinogens.

Regulation of the UGT1A1 bilirubin-conjugating pathway: role of a new splicing event at the UGT1A locus.

UNLABELLED: UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3' end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99-amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that isoform 2 is differentially expressed in liver, kidney, colon, and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions. CONCLUSION: Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway.

The novel UGT1A9 intronic I399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver.

Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP-glucuronosyltransferase (UGT) protein expression, glucuronidation activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (-2152C>T, -275T>A, and -118T(9>10)), three novel UGT1A9 variants (-5366G>T, -4549T>C, and I399C>T), and UGT1A1 (-53TA(6>7), -3156G>A, and -3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p = 0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r(2) < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 -118T(9/10) was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p = 0.03). However, UGT1A9 -118T(10) appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the -118T(10) allele in HepG2 cells (p = 0.004). In contrast, the UGT1A9 -5366T, -4549C, -2152T, and -275A, associated with higher UGT1A9 protein (2-fold; p < 0.05), have no influence on SN-38G. Despite limitations resulting from sample size, results indicate that UGT1A9 I399 and -118T(9/10) may represent additional candidates in combination with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo.

UGT1A1 polymorphisms are important determinants of dietary carcinogen detoxification in the liver.

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine), the most abundant heterocyclic amine in diet, is involved in the etiology of cancer. PhIP and its carcinogenic metabolite N-hydroxy-PhIP (N-OH-PhIP) are extensively conjugated by UDP-glucuronosyltransferase (UGTs) with wide variability. This study aimed to determine the genetic influence of UGTs on the hepatic detoxification of this carcinogen. The formation of N-OH-PhIP glucuronides was studied in 48 human liver samples by mass spectrometry. Liver samples were genotyped for common polymorphisms and correlated with UGT protein levels and N-OH-PhIP glucuronidation activities. The formation of four different N-OH-PhIP glucuronide metabolites was observed in all livers. The major metabolite was N-OH-PhIP-N(2)-glucuronide (N(2)G), which is the primary metabolite found in human urine, and showed a high interindividual variability (up to 28-fold). Using an heterologous expression system, the bilirubin-conjugating UGT1A1 enzyme was identified among all known UGTs (n = 16) as the predominant enzyme involved. The significant correlation between UGT1A1 protein content and formation of N(2)G (Rs = 0.87; P < .0001) suggests a critical role for UGT1A1 in the hepatic metabolism of this carcinogen. UGT1A1 expression was strongly determined by the presence of the common promoter polymorphisms, UGT1A1*28 (TATA box polymorphism) (P = .0031), -3156G/A (P = .0006) and -3279G/T (P = .0017), and rates of N(2)G were indeed correlated with these polymorphisms (P < .05), whether analyzed individually or in combination (haplotypes). In conclusion, UGT1A1 polymorphisms modulate the hepatic metabolism of the carcinogenic intermediate of PhIP and may determine the level of its exposure and potentially influence the risk of cancer through dietary exposure to HCAs.

Hepatic expression of the UGT1A9 gene is governed by hepatocyte nuclear factor 4alpha.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation reaction, which is a major pathway in the catabolism and elimination of numerous endo- and xenobiotics. Among the UGT enzyme family members, the UGT1A7, UGT1A8, UGT1A9, and UGT1A10 isoforms are issued from a single gene through differential splicing. However, these enzymes display distinct tissue-specific expression patterns. Indeed, UGT1A7, UGT1A8, and UGT1A10 are exclusively expressed in extrahepatic tissues, whereas UGT1A9 transcripts are found at high concentrations in liver. In the present study, we report that the liver-enriched hepatocyte nuclear factor 4 (HNF4)-alpha controls the hepatic expression of the UGT1A9 enzyme. Liver-specific disruption of the HNF4alpha gene in mice drastically decreases liver UGT1A9 mRNA levels. Furthermore, an HNF4alpha response element (HNF4alpha RE) was identified in the promoter of human UGT1A9 at position -372 to -360 base pairs by transient transfection, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments. It is interesting that this response element is absent in the proximal UGT1A7, UGT1A8, and UGT1A10 gene promoters. In conclusion, the present study identifies HNF4alpha as a major factor for the control of UGT1A9 hepatic expression and suggests that the absence of UGT1A7, UGT1A8, and UGT1A10 expression in the liver is caused by, at least in part, a few base pair changes in their promoter sequences in the region corresponding to the HNF4alpha RE of the UGT1A9 gene.

Identification of common polymorphisms in the promoter of the UGT1A9 gene: evidence that UGT1A9 protein and activity levels are strongly genetically controlled in the liver.

OBJECTIVES: Polymorphisms in UDP-glucuronosyltransferases (UGTs) can influence detoxifying capacities and have considerable therapeutic implications in addition to influence various (patho)physiological processes. UGT1A9 plays a central role in the metabolism of various classes of therapeutic drugs in addition to carcinogens and steroids. The great interindividual variability of UGT1A9-mediated glucuronidation remains poorly explained, while evidence for its genetic origin exists. METHODS: The proximal UGT1A9 promoter was screened for polymorphisms by sequencing and, the contribution of single nucleotide polymorphisms (SNPs) to the variability of UGT1A9 protein levels and activity was evaluated. RESULTS: We confirmed the presence of the -109 to -98 T10 polymorphism and found ten novel SNPs that generated a diversity of haplotypes in two independent populations. In a panel of 48 human liver microsomes, the UGT1A9 expression varied by 17-fold and was significantly correlated with SNPs -275, -331/-440, -665 and -2152. The base insertion T10 reported to increase reporter gene expression in HepG2 cells [] was not linked to -275 and -2152 SNPs and was not associated with changes in UGT1A9 protein levels. Compared to wild-type individuals, there were statistically significant higher glucuronidating activities in livers with the -275 and -2152 using mycophenolic acid and propofol as UGT1A9 substrates, indicating an extensive glucuronidator phenotype associated with these variants. CONCLUSIONS: This is the first study to demonstrate that naturally occurring sequence variations in the UGT1A9 promoter are informative in predicting the levels of protein and glucuronidating activity, providing a potential mechanism for interindividual variation in UGT1A9-mediated metabolism.

Novel functional polymorphisms in the UGT1A7 and UGT1A9 glucuronidating enzymes in Caucasian and African-American subjects and their impact on the metabolism of 7-ethyl-10-hydroxycamptothecin and flavopiridol anticancer drugs.

In vitro metabolic studies revealed that along with UDP-glucuronosyltransferase (UGT) 1A1, the hepatic UGT1A9 and the extrahepatic UGT1A7 are involved in the biotransformation of the active and toxic metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38). Variant UGT1A1 and UGT1A7 alleles have been reported but the polymorphic nature of the UGT1A9 gene has not been revealed yet. To further clarify the molecular determinants of irinotecan-induced toxicity, we have identified and characterized the functionality of novel UGT1A9 polymorphisms and determined whether additional missense polymorphisms exist in UGT1A7. Using direct DNA sequencing, four single nucleotide polymorphisms (SNPs) were identified in the first exons of UGT1A7 and UGT1A9. One of the two amino acid substitutions found in the UGT1A9 gene, UGT1A9*3 (M33T), results in a dramatic decrease in SN-38 glucuronide formation, with 3.8% of the activity of the UGT1A9*1 allele. In turn, the glucuronidation of flavopiridol, an anticancer drug biotransformed predominantly by UGT1A9, remains unaffected, indicating a substrate-dependent impact of this variant. UGT1A9*3 is detected only in Caucasians and 4.4% of the population tested was found heterozygous (*1/*3). Two additional UGT1A7 SNPs were found exclusively in African-American subjects and generate five alleles (UGT1A7*5 to *9) when combined to the four known SNPs present in UGT1A7*2, *3, and *4. Upon functional analysis with SN-38, five out of nine UGT1A7 allozymes exhibited much lower SN-38 glucuronidation activities compared with UGT1A7*1, all having in common the mutational changes at codons 115 or 208. Results suggest that these low SN-38 glucuronidating alleles may represent additional molecular determinants of irinotecan-induced toxicity and warrant further investigations.