Carla J. Gallagher

The UDP-Glucuronosyltransferase 2B17 Gene Deletion Polymorphism: Sex-Specific Association with Urinary 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol Glucuronidation Phenotype and Risk for Lung Cancer

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent and abundant procarcinogen found in tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), by UDP-glucuronosyltransferases (UGT) including UGT2B17 is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Both copies of the UGT2B17 gene are deleted in ∼10% of Whites and the deletion is associated with a reduction in NNAL glucuronidation activity in vitro. In this study, we examined the effects of the UGT2B17 deletion (0/0) on NNAL glucuronidation rates in a sample of 82 healthy cigarette smokers and further examined its effects on lung cancer risk in a separate case-control study. In the healthy smokers study, a lower urinary ratio of NNAL-glucuronide to NNAL was observed in women with the UGT2B17 deletion (0/0) as compared with women with either the wild-type or heterozygous genotypes (P = 0.058). There were no significant differences in this ratio by genotype in men (P = 0.597). In the case-control study of 398 lung cancer patients and 697 community controls, the UGT2B17 deletion (0/0) was associated with a significant increase in risk of lung cancer in women (odds ratio, 2.0; 95% confidence interval, 1.01-4.0). The risk for the subset of women with lung adenocarcinoma was 2.8 (95% confidence interval, 1.2-6.3). The deletion was not associated with other lung histologic types in women and was not associated with the risk for any lung histologic types in men. The association of the UGT2B17 deletion with increased lung adenocarcinoma in women is consistent with its association with decreased NNAL glucuronidation rates in women and with studies showing that NNAL is a selective inducer of lung adenocarcinoma in experimental animals. (Cancer Epidemiol Biomarkers Prev 2007;16(4):823–8)

Association of the Estrogen Receptor-α Gene With the Metabolic Syndrome and Its Component Traits in African-American Families: The Insulin Resistance Atherosclerosis Family Study

OBJECTIVE— We previously detected an association between a region of the estrogen receptor-α (ESR1) gene and type 2 diabetes in an African-American case-control study; thus, we investigated this region for associations with the metabolic syndrome and its component traits in African-American families from the Insulin Resistance Atherosclerosis Family Study. RESEARCH DESIGN AND METHODS— A total of 17 single nucleotide polymorphisms (SNPs) from a contiguous 41-kb intron 1–intron 2 region of the ESR1 gene were genotyped in 548 individuals from 42 African-American pedigrees. Generalized estimating equations were computed using a sandwich estimator of the variance and exchangeable correlation to account for familial correlation. RESULTS— Significant associations were detected between ESR1 SNPs and the metabolic syndrome (P = 0.005 to P = 0.029), type 2 diabetes (P = 0.001), insulin sensitivity (P = 0.0005 to P = 0.023), fasting insulin (P = 0.022 to P = 0.033), triglycerides (P = 0.021), LDL (P = 0.016 to P = 0.034), cholesterol (P = 0.046), BMI (P = 0.016 to P = 0.035), waist circumference (P = 0.012 to P = 0.023), and subcutaneous adipose tissue area (P = 0.016). CONCLUSIONS— It appears likely that ESR1 contributes to type 2 diabetes and CVD risk via pleiotropic effects, leading to insulin resistance, a poor lipid profile, and obesity.

Functional Significance of UDP-Glucuronosyltransferase Variants in the Metabolism of Active Tamoxifen Metabolites

Tamoxifen (TAM) is a selective estrogen receptor modulator widely used in the prevention and treatment of breast cancer. A major mode of metabolism of the major active metabolites of TAM, 4-OH-TAM and endoxifen, is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To examine whether polymorphisms in the UGT enzymes responsible for the glucuronidation of active TAM metabolites play an important role in interindividual differences in TAM metabolism, cell lines overexpressing wild-type or variant UGTs were examined for their activities against TAM metabolites in vitro. For variants of active extrahepatic UGTs, the UGT1A8(173Ala/277Tyr) variant exhibited no detectable glucuronidation activity against the trans isomers of either 4-OH-TAM or endoxifen. Little or no difference in TAM glucuronidating activity was observed for the UGT1A8(173Gly/277Cys) or UGT1A10(139Lys) variants compared with their wild-type counterparts. For active hepatic UGTs, the UGT2B7(268Tyr) variant exhibited significant (P < 0.01) 2- and 5-fold decreases in activity against the trans isomers of 4-OH-TAM and endoxifen, respectively, compared with wild-type UGT2B7(268His). In studies of 111 human liver microsomal specimens, the rate of O-glucuronidation against trans-4-OH-TAM and trans-endoxifen was 28% (P < 0.001) and 27% (P = 0.002) lower, respectively, in individuals homozygous for the UGT2B7 Tyr(268)Tyr genotype compared with subjects with the UGT2B7 His(268)His genotype, with a significant (P < 0.01) trend of decreasing activity against both substrates with increasing numbers of the UGT2B7(268His) allele. These results suggest that functional polymorphisms in TAM-metabolizing UGTs, including UGT2B7 and potentially UGT1A8, may be important in interindividual variability in TAM metabolism and response to TAM therapy.

Characterization of UGTs Active against SAHA and Association between SAHA Glucuronidation Activity Phenotype with UGT Genotype

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple other cancers. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To characterize the UGTs active against SAHA, homogenates from HEK293 cell lines overexpressing UGT wild-type or variant UGT were used. The hepatic UGTs 2B17 and 1A9 and the extrahepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by V(max)/K(M) (16+/-6.5, 7.1+/-2.2, 33+/-6.3, and 24+/-2.4 nL x min(-1) x microg UGT protein(-1), respectively), with UGT2B17 exhibiting the lowest K(M) (300 micromol/L) against SAHA of any UGT in vitro. Whereas the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P<0.005) decrease in glucuronidation activity against SAHA compared with wild-type UGT1A8, the UGT1A8p.Cys277Tyr variant exhibited no detectable glucuronidation activity; a similar lack of detectable glucuronidation activity was observed for the UGT1A10p.Gly139Lys variant. To analyze the effects of the UGT2B17 gene deletion variant (UGT2B17*2) on SAHA glucuronidation phenotype, human liver microsomes (HLM) were analyzed for glucuronidation activity against SAHA and compared with UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P<0.01) decrease in glucuronidation activity and a 75% (P<0.002) increase in K(M) compared with HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Overall, these results suggest that several UGTs play an important role in the metabolism of SAHA and that UGT2B17-null individuals could potentially exhibit altered SAHA clearance rates with differences in overall response.

International Lung Cancer Consortium: coordinated association study of 10 potential lung cancer susceptibility variants.

BACKGROUND Analysis of candidate genes in individual studies has had only limited success in identifying particular gene variants that are conclusively associated with lung cancer risk. In the International Lung Cancer Consortium (ILCCO), we conducted a coordinated genotyping study of 10 common variants selected because of their prior evidence of an association with lung cancer. These variants belonged to candidate genes from different cancer-related pathways including inflammation (IL1B), folate metabolism (MTHFR), regulatory function (AKAP9 and CAMKK1), cell adhesion (SEZL6) and apoptosis (FAS, FASL, TP53, TP53BP1 and BAT3). METHODS Genotype data from 15 ILCCO case-control studies were available for a total of 8431 lung cancer cases and 11 072 controls of European descent and Asian ethnic groups. Unconditional logistic regression was used to model the association between each variant and lung cancer risk. RESULTS Only the association between a non-synonymous variant of TP53BP1 (rs560191) and lung cancer risk was significant (OR = 0.91, P = 0.002). This association was more striking for squamous cell carcinoma (OR = 0.86, P = 6 x 10(-4)). No heterogeneity by center, ethnicity, smoking status, age group or sex was observed. In order to confirm this association, we included results for this variant from a set of independent studies (9966 cases/11,722 controls) and we reported similar results. When combining all these studies together, we reported an overall OR = 0.93 (0.89-0.97) (P = 0.001). This association was significant only for squamous cell carcinoma [OR = 0.89 (0.85-0.95), P = 1 x 10(-4)]. CONCLUSION This study suggests that rs560191 is associated to lung cancer risk and further highlights the value of consortia in replicating or refuting published genetic associations.

Sex Differences in UDP-Glucuronosyltransferase 2B17 Expression and Activity

UDP-glucuronosyltransferases (UGTs) are enzymes involved in the metabolism of steroid hormones, carcinogens, cancer chemotherapy agents, and addictive agents from cigarettes. Because the UGT2B family of genes has been linked to hormonal regulation in human cell lines in vitro, we hypothesized that there may be sex-related differences in the expression and activity of these genes in human tissues. To evaluate whether there are sex differences in UGT2B expression and activity, we examined 103 normal human liver specimens for UGT2B expression by real-time polymerase chain reaction and in vitro glucuronidation activities in human liver microsomes (HLM). Men exhibited an approximately 4-fold higher level of expression of UGT2B17 than women (p = 0.007). Consistent with the increased expression of UGT2B17 in men, HLM from men also had a higher level of glucuronidation activity than HLM from women against three UGT2B17 substrates: 3-fold higher for 17-dihydroexemestane (p = 0.002); 3-fold higher for 3-hydroxycotinine (p < 0.001); and 1.5-fold higher for suberoylanilide hydroxamic acid (p = 0.014). When we stratified by UGT2B17 gene deletion genotype, similar patterns were observed for all three substrates, with HLM from men with the UGT2B17 (+/+) or (+/0) genotypes exhibiting significantly higher levels of glucuronidation activity against all three substrates compared with HLM from women. These data suggest that men have a higher amount of UGT2B17 glucuronidation activity then women. This sex difference in UGT2B17 gene expression and corresponding protein activity could potentially result in different levels of carcinogen detoxification or drug elimination in men versus women.

Identification of a prevalent functional missense polymorphism in the UGT2B10 gene and its association with UGT2B10 inactivation against tobacco-specific nitrosamines.

Objective To study the potential association between UDP-glucuronosyltransferase (UGT)2B10 genotypes and [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol] NNAL-N-glucuronidation activity in human liver microsomes (HLM) and to identify potential functional polymorphisms. Methods A total of 77 subjects were genotyped for three UGT2B10 tagging single nucleotide polymorphisms NNAL-N-glucuronidation activity in HLM was determined by high-pressure liquid chromatography and analyzed by UGT2B10 haplotypes. Results Four common UGT2B10 haplotypes (termed A through D) were identified. Haplotype C was found to be significantly (P<0.001) associated with lower NNAL-N-glucuronidation in HLM. A 1.8-fold and 12-fold reduction in NNAL-N-glucuronidation levels and a 1.7-fold and 11-fold reduction in the ratio of NNAL-N-Gluc: NNAL-O-Gluc, were observed in HLM from subjects with one and two copies of UGT2B10 haplotype C, respectively. A novel polymorphism resulting in an aspartic acid to tyrosine amino acid change at codon 67 of the UGT2B10 complementary DNA was identified exclusively in subjects with a haplotype C. Unlike the high activity observed in microsomes from HEK293 cells over expressing the wild-type UGT2B1067Asp variant, microsomes from HEK293 cells over expressing the UGT2B1067Tyr variant exhibited minimal glucuronide formation activity against NNAL or other tobacco-specific nitrosamines tested in vitro. Conclusions The UGT2B1067Tyr variant corresponding to the UGT2B10 haplotype C is a functional single nucleotide polymorphism that may be responsible for inter individual variation in NNAL-N-glucuronidation activity and may increase susceptibility to smoking-related cancers.

The effect of copy number variation in the phase II detoxification genes UGT2B17 and UGT2B28 on colorectal cancer risk

Genetic polymorphisms in combination with the Western‐style diet, physical inactivity, smoking, excessive alcohol consumption, and obesity have been hypothesized to affect colorectal cancer (CRC) risk. Metabolizers of environmental carcinogenic and endogenous compounds affecting CRC risk include the phase II detoxification UDP‐glucuronosyltransferase (UGT) enzymes UGT2B17 and UGT2B28, which are 2 of the most commonly deleted genes in the genome.

Investigation of the Estrogen Receptor-α Gene With Type 2 Diabetes and/or Nephropathy in African-American and European-American Populations

The estrogen receptor-α gene (ESR1) was selected as a positional candidate under a type 2 diabetes linkage peak at 6q24-27. A total of 42 ESR1 single nucleotide polymorphisms (SNPs) were genotyped in 380 African-American type 2 diabetic case subjects with end-stage renal disease (ESRD) and 276 African-American control subjects. A total of 22 ancestry informative markers were also genotyped, and the program Admixmap was used to adjust allelic and haplotypic association tests for individual estimates of admixture. The most significant association with type 2 diabetes–ESRD was with rs1033182 in intron 2 (P = 0.013, admixture-adjusted Pa = 0.021). Genotyping 17 SNPs across a region of ESR1 intron 1–intron 2 in an expanded population of 851 case and 635 control subjects supported association with rs1033182 (P = 0.004, Pa = 0.027) and with an independent six-SNP haplotype of high linkage disequilibrium spanning 6.4 kb (P < 0.0001, Pa < 0.0001). The same 17 ESR1 SNPs were genotyped in 300 European-American type 2 diabetes–ESRD case subjects and 310 European-American control subjects. Two intron 2 SNPs, rs2431260 (P = 0.015) and rs1709183 (P = 0.019), and a four-SNP haplotype containing these SNPs (P = 0.033) were associated with type 2 diabetes and/or ESRD. Results suggest that intron 1 and intron 2 of the ESR1 gene may contain functionally important regions related to type 2 diabetes or ESRD risk.

FOXO3 Encodes a Carcinogen-Activated Transcription Factor Frequently Deleted in Early-Stage Lung Adenocarcinoma

The FOXO family of transcription factors elicits cell cycle arrest, apoptosis, and resistance to various physiologic and pathologic stresses relevant to sporadic cancer, such as DNA damage and oxidative stress. Although implicated as tumor suppressors, FOXO genetic inactivation has not been observed in human cancer. In an investigation of the two major types of non-small cell lung cancer, here, we identify the FOXO3 gene as a novel target of deletion in human lung adenocarcinoma (LAC). Biallelic or homozygous deletion (HD) of FOXO3 was detected in 8 of 33 (24.2%) mostly early-stage LAC of smokers. Another 60.6% of these tumors had losses of FOXO3 not reaching the level of HD (hereafter referred to as sub-HD). In contrast, no HD of FOXO3 was observed in 19 lung squamous cell carcinoma. Consistent with the deletion of FOXO3 were corresponding decreases in its mRNA and protein levels in LAC. The potential role of FOXO3 loss in LAC was also investigated. The carcinogen (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is strongly implicated as a cause of human lung cancer. Here, we show that FOXO3a is functionally activated and augments the level of caspase-dependent apoptosis in cells exposed to this DNA-damaging carcinogen. These results implicate FOXO3 as a suppressor of LAC carcinogenesis, a role frequently lost through gene deletion.