A Functional Polymorphism in RGS6 Modulates the Risk of Bladder Cancer

[CANCER RESEARCH 64, 6820–6826, September 15, 2004] A Functional Polymorphism in RGS6 Modulates the Risk of Bladder Cancer

David M. Berman,1 Yunfei Wang,2 Zhengyu Liu,3 Qiong Dong,2 Lorri-Anne Burke,1 Lance A. Liotta,1 Rory Fisher,3 and Xifeng Wu2 1Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland; 2Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, Houston Texas; and 3Department of Pharmacology, University of Iowa, Iowa City, Iowa

ABSTRACT intracellular vesicles (reviewed in ref. 2). Finally, RGS3 induces apoptosis (10). RGS proteins negatively regulate heterotrimeric G protein signaling. Two well-characterized RGS proteins, RGS2 and RGS6, are of Recent reports have shown that RGS proteins modulate neuronal, car- particular interest. RGS2 mRNA is highly expressed in acute leuke- diovascular, and lymphocytic activity, yet their role in carcinogenesis has not been explored. In an epidemiologic study of 477 bladder cancer mia but not in the chronic form or in normal bone marrow (11–13). patients and 446 matched controls, three noncoding single-nucleotide RGS6 binds to the DNA methylating protein, DNMT1, and de- polymorphisms (SNPs) in RGS2 and RGS6 were each associated with a represses transcriptional inhibition by the DNMT1-associated protein, statistically significant reduction in bladder cancer risk. The risk of DMAP1 (14). Both RGS2 and RGS6 undergo nucleocytoplasmic bladder cancer was reduced by 74% in those individuals with the variant shuttling, suggesting that these proteins may provide a link between genotype at all three SNPs (odds ratio, 0.26; 95% confidence interval, the regulation of cytoplasmic events and DNA or RNA metabolism 0.09–0.71). When the SNPs were analyzed separately, the RGS6- (15, 16). Finally, both of these proteins are heat shock responsive, a rs2074647 (C3T) polymorphism conferred the greatest overall reduction characteristic of some regulators of apoptosis (17, 18). in risk of bladder cancer (odds ratio, 0.66; 95% confidence interval, There are polymorphisms in genes that may modulate protein 0.46–0.95). These reductions in risk were more pronounced in ever smok- function and produce downstream effects contributing to variation in ers, suggesting a gene-environment interaction. In transfection assays, the RGS6-rs2074647 (C3T) polymorphism increased the activity of a lucif- cancer risk. Therefore, in an ongoing hospital-based case-control erase-RGS fusion protein by 2.9-fold, suggesting that this SNP is func- study, we explored the association between 12 noncoding single- tionally significant. Finally, we demonstrate that RGS2 transcripts and nucleotide polymorphisms (SNPs) in five RGS genes identified in several splice variants of RGS6 are expressed in bladder cancer cells. the National Center for Biotechnology Information (NCBI) database These data provide the first evidence that RGS proteins may be important (dbSNP) and the risk of bladder carcinoma, a cause of over 12,000 modulators of cancer risk and validate RGS6 as a target for further study. deaths per annum in the United States. Our results suggest that selected RGS variant SNPs may be important modifiers of cancer risk. To validate the biological significance of these SNPs, we also sought INTRODUCTION to identify functional changes in transcript levels, alternative splicing The RGS (regulators of G protein signaling) family of proteins events, and protein translation efficiency that may result from the participates in a wide range of signal transduction pathways. All presence of the variant alleles. family members possess an RGS domain responsible for accelerating 1,000-fold the deactivation of heterotrimeric G proteins (1). Many RGS proteins also possess additional protein domains responsible for MATERIALS AND METHODS integrating G protein pathways with a diverse range of other cellular Study Population. Available for study were 477 Caucasian patients pre- signaling events (2). Recent reports have shown that RGS proteins senting with urinary bladder cancer cases and accrued from the Departments of modulate neuronal, cardiovascular, and lymphocytic activities, yet Urology at The University of Texas M. D. Anderson Cancer Center and the their role in carcinogenesis has not been explored in any depth Baylor College of Medicine. The case patients, who had histologically con- Heterotrimeric G proteins have transforming potential, and this firmed incident bladder cancer, had received no previous chemotherapy or alone would make RGS proteins a relevant target for analysis (3). radiotherapy and were enrolled in an ongoing case-control study described However, there is growing but indirect evidence that supports the previously (19). There were no recruitment restrictions on age, gender, or notion that RGS proteins also regulate other key pathways of carci- cancer stage. A brief eligibility questionnaire assessing prior cancer therapy nogenesis. For example, RGS14 binds to the Ras-related G protein, and willingness to participate in the epidemiologic study was administered to assess eligibility. Rap1/2 (4). RGS12 is a transcriptional repressor, and RGS12 over- A total of 446 Caucasian healthy control subjects without a prior history of expression in select cell lines inhibits DNA synthesis (5). RGS- cancer (except nonmelanoma skin cancer) were recruited as controls into the RhoGEFs activate Rho G proteins, key regulators of the cytoskeleton study from the Kelsey-Seybold clinics, a large private multispecialty physician (6). RGS16, which is induced by genotoxic shock in a p53-dependent group in the Houston metropolitan area. Control subjects were frequency fashion, inhibits G protein activation of the mitogen-activated protein matched to the cases on the basis of age (Ϯ5 years), gender, and ethnicity. kinase cascade (7). RGS-axin regulates the activity of the ␤-catenin All study participants completed a structured 1.5-hour personal interview transcription factor (8, 9). Several RGS proteins regulate the sorting of that was administered by trained M. D. Anderson staff interviewers after written informed consent was obtained. In addition, a 40-mL blood sample was drawn into coded heparinized tubes for analysis. Received 6/1/04; revised 7/19/04; accepted 7/21/04. Grant support: R. Fisher is supported by National Institutes of Health grant Genotyping RGS Polymorphisms. DNA was isolated from peripheral GM067881. X. Wu is supported by National Cancer Institute grants CA 74880 and CA blood lymphocytes using a nonphenol method. RFLP-polymerase chain reac- 91846. tion (PCR) was used to detect SNPs for RGS5, rs15049 (A/C); RGS6, The costs of publication of this article were defrayed in part by the payment of page rs2238280 (G/A); RGS6, rs2074647 (C/T); RGS11, rs3743878 (C/T); RGS11, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rs3743879 (C/G); RGS17, rs2295232 (G/T); and RGS17, rs3870366 (G/A). No Note: Supplementary data for this article can be found at Cancer Research Online polymorphism was detected at RGS11, rs3743878. The TaqMan assay was (http://cancerres.aacrjournals.org). used to detect SNPs for RGS2, rs4606 (G/C); RGS6, rs2238284 (T/G); RGS6, Requests for reprints: David M. Berman, Laboratory of Pathology, National Cancer rs3784058 (T/C); RGS6, rs2238285 (C/T); and RGS17, rs2295231 (A/G). Institute, Building 10-2N212, 10 Center Drive, Bethesda, MD 20892. Phone: 301-496- 1888, Fax: 301-480-9488, E-mail: [email protected]. For genotyping by RFLP-PCR, the genomic DNA regions containing the ©2004 American Association for Cancer Research. polymorphisms were amplified by PCR. The primers used for these polymor- 6820

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. RGS PROTEINS AND CANCER phisms are listed as an online supplementary data. The PCR assay was CA). Frozen transitional cell carcinoma was microdissected by AutoPix laser performed in a 25-␮L reaction mixture containing 100 ng of genomic DNA, capture microdissection, and RNA was isolated and linearly amplified using ϫ 1 PCR buffer (Promega, Madison WI), 2 mmol/L MgCl2 (3 mmol/L for the PicoPure and RiboAmp kits, respectively (Arcturus, Mountain View, CA). rs3870366), 0.2 mmol/L deoxynucleotide triphosphates, 1.5 unit of Taq poly- For traditional PCR, equivalent amounts of cDNA substrate were introduced to merase (Promega), and 0.2 ␮mol/L primers. The PCR thermal cycling condi- puReTaq Ready-to-go PCR beads (Amersham, Piscataway, NJ), and PCR was tions were 5 minutes at 94°C followed by 35 cycles of 30 seconds at 94°C; 30 performed with the following cycling parameters: 95°C for 5 minutes; fol- seconds at 55°C (rs15049), 48°C (rs2238280), 57°C (rs2074647), 57°C lowed by 35 to 40 cycles of 95°C for 20 seconds, 57°C for 30 seconds, and (rs3743878), 59°C (rs3743879), 58°C (rs2295232), or 45°C (rs3870366), 72°C for 30 seconds; with a final extension of 72°C for 7 minutes. Real-time respectively; and 45 seconds at 72°C; and, finally, extension for 5 minutes at PCR using splice site-spanning primers for RGS2, RGS6, and ␤-actin was 72°C. The PCR product was digested with restriction enzymes (New England performed using the Lightcycler (Roche, Indianapolis, IN) with the following Biolabs, Beverly, MA) overnight at 37°C, and the fragments were separated on parameters: 95°C for 10 minutes, followed by 40 cycles of 95°C for 8 seconds, agarose gel stained with ethidium bromide. The gene respective enzyme, 57°C for 6 seconds, and 72°C for 6 seconds. Crossing threshold (Ct) values genotype, and expected bands are as follows: (a) RGS5 (rs15049), HaeIII were calculated using the Roche Lightcycler software. The Ct values for the digestion, A/A (212 bp), A/C (212 ϩ 193 ϩ 19 bp), and C/C (193 ϩ 19 bp); RGS reactions are derived from at least three experiments. Experiments were (b) RGS6 (rs2238280), AseI digestion, G/G (142 bp), G/A (142 ϩ 125 ϩ 17 also performed with a 10-fold dilution of template to ensure that measurements bp), and A/A (125 ϩ 17 bp); (c) RGS6 (rs2074647), HpyCH4 IV digestion, were in the linear range (data not shown). The probes and primers for this C/C (177 ϩ 19 bp), C/T (196 ϩ 177 ϩ 19 bp), and T/T (196 bp); (d) RGS11 experiment are listed as an online supplementary data. (rs3743878), MspI digestion, C/C (94 ϩ 20 bp), C/T (114 ϩ 94 ϩ 20 bp), and Luciferase Reporter Constructs and Luciferase Assays. Two primers T/T (114 bp); (e) RGS11 (rs3743879), SpeI digestion, C/C (126 ϩ 17 bp), C/G (5Ј-AGCTATGAGATAACCAGTCAA-3Ј and 5Ј-GATCAGGGCCTCTTAG- (143 ϩ 126 ϩ 17 bp), and G/G (143 bp); (f) RGS17 (rs2295232), MboI CGAGT-3Ј) were designed to amplify a fragment of RGS6 encompassing the digestion, G/G (151 ϩ 146 bp), G/T (297 ϩ 151 ϩ 146 bp), and T/T (297 bp); SNP RGS6 rs2074647 (C/T). The cDNA substrate was derived from lympho- and (g) RGS17 (rs3870366), NsiI digestion, G/G (132 bp), G/A blastoid cell lines with known RGS genotypes as described above. The target (132 ϩ 111 ϩ 21 bp), and A/A (111 ϩ 21 bp). fragment contains 258 bases of COOH-terminal coding sequence of RGS6 as Genotyping using TaqMan 5Ј nuclease assay for allelic discrimination was well as 285 bases of the 3Ј-untranslated region (UTR). The amplified frag- carried out on the 384-well ABI 7900HT Sequence Detection System (Applied ments were sequenced to confirm that there were no other SNPs and then Biosystems, Foster City, CA). Primers and probes were designed using the reamplified again using primers containing XbaI restriction sites at both Primer Express software (Applied Biosystems). The probes were fluorescence termini followed by cloning into the pCR2.1-TOPO vector (Invitrogen). The labeled with either 6-FAM or VIC on the 5Ј-end and a nonfluorescent minor amplified fragments were then cloned into the XbaI site located between the groove binder (MGB) quencher on the 3Ј end (Applied Biosystems). The luciferase gene and the SV40 late poly(A) signal in the pGL3-Promoter vector sequences of primers and probes are listed as an online supplementary data. (Promega). The resulting luciferase constructs were digested with SacII and Typical amplification mixes (5 ␮L) contained sample DNA (5 ng), 1ϫ HpaI to confirm correct orientation. TaqMan buffer A, 200 ␮mol/L deoxynucleotide triphosphates, 5 mmol/L NIH3T3 cells, cultured in Dulbecco’s modified Eagle’s medium supple-

MgCl2, 0.65 unit of AmpliTaq Gold, 900 nmol/L each primer, and 200 nmol/L mented with 10% fetal bovine serum, were transfected using LipofectAMINE each probe. The thermal cycling conditions consisted of 1 cycle for 10 minutes 2000 (Invitrogen) with the above-mentioned pGL3-Promoter vector harboring at 95°C and 40 cycles for 15 seconds at 95°C and for 1 minute at 60°C. SDS either the wild-type or variant allele at rs2074647. Unmodified pGL3 promoter version 2.1 software (Applied Biosystems) was used to analyze end point vector was transfected in parallel. Luciferase activity in transfected cells was fluorescence. Water control, ample internal controls, and previously genotyped assayed 40 hours after transfection using a luciferase assay kit (Promega). samples were included in each plate to ensure accuracy of genotyping. Luciferase activity was normalized for transfection efficiency by cotransfec- Statistical Analyses. Differences between the cases and the controls in the tion with a ␤-galactosidase reporter gene for which activity was assayed with distribution of smoking status, gender, and RGS genotypes were tested using the Galacto chemiluminescence assay kit (Tropix, Bedford, MA). The data are the ␹2 test statistic. The Wilcoxon rank-sum test was used to test for differ- expressed as ratios of activity of the fusion protein to that of the unmodified ences between the cases and controls for mean age and select cigarette luciferase protein. smoking variables. Unconditional logistic regression was used to calculate odds ratios (ORs) as estimates of the relative risk associated with the RGS RESULTS polymorphisms. Multivariate logistic regression was performed to adjust for the potential confounding effects of age, gender, and years smoked, where This study is based on data from 477 Caucasian bladder cancer case appropriate. To assess for interaction between variables, we fitted a logistic patients and 446 Caucasian control subjects (Table 1). We restricted model including product terms of the variables in the model. All statistical our analysis to Caucasians because of the small numbers of African- analyses were performed using the SAS 8.2 statistical software package (SAS Institute, Inc., Cary, NC). An individual who had never smoked or had smoked less than 100 cigarettes in his or her lifetime was defined as a never smoker. An individual who had smoked at least 100 cigarettes in his or her lifetime was Table 1 Distribution of select characteristics in the case subjects and control patients defined as an ever smoker. Ever smokers include former smokers, current No. of cases No. of controls smokers, and recent quitters (who quit smoking within the previous year). A Variables (N ϭ 477) (N ϭ 446) P former smoker had quit smoking at least 1 year before diagnosis of cancer Sex (case patients) or at least 1 year before the interview (control subjects). Male 367 (76.9%) 322 (72.0%) Cell Lines and Tumor Tissue. Human bladder carcinoma cell lines (5637, Female 110 (23.1%) 124 (27.0%) 0.098 Age (y) T24, and UMVC) were kindly provided by Dr. Jonathan Coleman (National Mean (SD) 63.96 (11.1) 63.10 (10.9) 0.19 Cancer Institute, National Institutes of Health, Bethesda, MD). Lymphoblas- Smoking status toid cell lines were derived from eight subjects with known RGS genotypes. Never 124 (26.0%) 211 (47.3%) These cell lines were grown in RPMI 1640 supplemented with 10% fetal Former 248 (52.0%) 199 (44.6%) Current 105 (22.0%) 36 (8.1%) Ͻ0.001 bovine serum and antibiotics. Anonymized frozen transitional cell carcinoma No. of cigarettes per day* tissue was obtained from the Laboratory of Pathology frozen tissue bank with Mean (SD) 26.0 (14.2) 22.8 (16.0) Ͻ0.001 appropriate National Institutes of Health institutional review board approval. Years smoked* Ͻ RNA Isolation and Polymerase Chain Reaction. Cultured cells were Mean (SD) 31.3 (14.0) 23.2 (14.2) 0.001 Pack-years* solubilized with Trizol (Invitrogen, Carlsbad, CA), the supernatant was buff- Mean (SD) 42.7 (31.3) 29.6 (29.8) Ͻ0.001 ered with 50 mmol/L Tris (pH 7.5), and total RNA was purified using the Age started smoking (y)* RNAeasy kit (Qiagen Valencia, CA). Five hundred nanograms of total RNA Mean (SD) 17.9 (4.4) 17.4 (4.2) 0.078 were converted into cDNA using the Superscript III kit (Invitrogen, Valencia, * Ever smokers only. 6821

Table 2 Summary of RGS SNPs Gene NCBI SNP cluster ID Chromosome Contig accession no. Abbreviation in text Position in gene Allele frequency RGS2 rs4606 1 NT_004671 RGS2–1 3Ј-UTR C: 0.715 G: 0.285 RGS5 rs15049 1 NT_004668 None 3Ј-UTR A: 0.697 C: 0.303 RGS6 rs2074647 14 NT_026437 RGS6–2 3Ј-UTR C: 0.894 T: 0.106 RGS6 rs2238280 14 NT_026437 None Intron 1* A: 0.162 G: 0.838 RGS6 rs2238284 14 NT_026437 None Intron 1* G: 0.668 T: 0.332 RGS6 rs2238285 14 NT_026437 None Intron 1* C: 0.165 T: 0.835 RGS6 rs3784058 14 NT_026437 RGS6–3 Intron 1* C: 0.849 T:0.151 RGS11† rs3743878 16 NT_037887 None 3Ј-UTR C: 0.901 T: 0.099 RGS11 rs3743879 16 NT_037887 None 3Ј-UTR C: 0.570 G: 0.430 RGS17 rs2295231 6 NT_023451 None Intron* A: 0.572 G: 0.428 RGS17 rs2295232 6 NT_023451 None Intron* G: 0.572 T: 0.428 RGS17 rs3870366 6 NT_023451 None Intron* A: 0.390 G: 0.610 * These SNPs are located in the 5Ј-UTR. † No polymorphisms were identified in 300 samples.

American and Mexican-American subjects available for analysis. Similar trends were noted for two other SNPS: RGS2-4606 (C3G; Because the study is still on going, perfect matching between case abbreviated as RGS2-1) and RGS6-3784058 (C3T; abbreviated as patients and control subjects has not been achieved. RGS6-3; Table 3). The protective effect of RGS2-1V was of border- Overall, there was no statistically significant difference in sex line statistical significance in the overall analysis (OR, 0.85; 95% CI, distribution between the cases and the controls (P ϭ 0.098). The mean 0.64–1.11) but with significant reductions noted for ever smokers age was 64.0 years for the case patients, compared with 63.1 years for (OR, 0.71), individuals who started smoking before age 17 years (OR, the control subjects (P ϭ 0.19). As expected, case patients were 0.61), and patients with younger age at diagnosis (OR, 0.53). The significantly more likely than the control subjects to be current smok- variant genotype of RGS6-3 was similarly associated with reduced ers (22.0% versus 8.1%) and, conversely, were less likely to be never bladder cancer risk (OR, 0.81; 95% CI, 0.60–1.09). We did not find smokers (26.0% versus 47.3%; P Ͻ 0.001). Moreover, mean self- any significant effects for the nine other polymorphisms (in RGS5, reported number of cigarettes smoked per day and mean pack-years RGS11, and RGS17) studied either in the overall analysis or in the smoked were also statistically significantly higher in the cases than in subsequent stratified analysis (data not shown). the controls (P Ͻ 0.001 for both). However, the potential bias due to the above-mentioned differences is not realized because, as demon- Table 3 Risk estimates for select RGS6 and RGS2 SNPs in bladder cancer cases and strated below, the calculated ORs are even more significant when the control subjects data are controlled for smoking status. Adjusted OR (95% CI)* Twelve SNPs in five RGS genes (RGS2, RGS5, RGS6, RGS11, and RGS2–1 RGS6–3 RGS6–2 RGS17) were identified in the NCBI dbSNP database. All SNPs were Genotype† (RGS2-rs4606) (RGS6-rs3784058) (RGS6-rs2074647) located in untranslated regions and had estimated population frequen- Overall cies greater than 5%. No nonsynonymous SNPs were found in any of Wild-type Reference Reference Reference Variant 0.85 (0.64–1.1) 0.81 (0.60–1.09) 0.66 (0.46–0.95) the RGS genes. By sequencing, we confirmed that 11 of the 12 SNPs Never smokers were indeed real SNPs (Table 2). The observed frequencies of all of Wild –type Reference Reference Reference the RGS genotypes among controls did not differ from those expected Variant 1.2 (0.73–1.8) 0.93 (0.57–1.5) 0.78 (0.43–1.4) Ever smokers under the Hardy-Weinberg equilibrium (online supplementary data). Wild –type Reference Reference Reference ORs were calculated to estimate bladder cancer risk associated with Variant 0.71 (0.48–1.0) 0.74 (0.51–1.1) 0.60 (0.39–0.94) Age started these polymorphisms. For this analysis, genotypes were combined smoking Ͻ 17 y into the following two classes due to the small number of carriers of Wild-type Reference Reference Reference the homozygous variant genotypes: (a) homozygous wild-type allele Variant 0.61 (0.35–1.07) 0.70 (0.39–1.3) 0.42 (0.20–0.86) Age started (i.e., C/C; referred to as W) and (b) the variant genotypes (i.e., C/T smoking Ն 17 y plus T/T; referred to as V). Of the 11 SNPS, only 3 were associated Wild-type Reference Reference Reference with significant reductions in risk of bladder cancer (Table 3). Over- Variant 0.81 (0.51–1.3) 0.73 (0.45–1.2) 0.74 (0.41–1.3) Age Ͻ 64 y‡ all, the most protective variant allele was located at RGS6-2074647 Wild-type Reference Reference Reference (C3T; abbreviated as RGS6-2) and was associated with a 34% Variant 0.53 (0.31–0.91) 0.72 (0.40–1.3) 0.45 (0.23–0.90) Age Ն 64 y reduction in bladder cancer risk [OR, 0.66; 95% confidence interval Wild-type Reference Reference Reference (CI), 0.46–0.95]. On stratified analysis, protective effects were more Variant 0.89 (0.56–1.4) 0.73 (0.45–1.2) 0.75 (0.41–1.4) evident in ever smokers (OR, 0.60; 95% CI, 0.39–0.94), individuals * Adjusted for age, gender, and years smoked and adjusted for gender and years who started smoking before age 17 years (OR, 0.42; 95% CI, 0.20– smoked in age strata. † Wild-type, homozygous wild-type genotype; Variant, homozygous ϩ heterozygous 0.86), and those who had a younger age at diagnosis of bladder cancer variant genotype. (OR, 0.45; 95% CI, 0.23–0.90; Table 3). ‡ Age at diagnosis of bladder carcinoma. 6822

To further characterize interactions between these three polymor- influencing alternative splicing events, mRNA stability, or protein phisms, we analyzed combinations of the three genotypes (Table 4). translation (24–26). Unfortunately, bladder cancer cell lines with the The combinations are reported in the following order: RGS2-1/RGS6- requisite genotypes were not available. Therefore, we investigated 3/RGS6-2. Compared with the reference group (i.e., individuals with whether these three SNPs functionally regulate mRNA splicing or wild-type genotypes at the three loci), the presence of the variant steady-state transcript levels in lymphoblastoid cell lines derived from genotype at all three sites (V/V/V) was associated with a substantial individuals with known RGS genotypes. reduction in the OR to 0.26 (95% CI, 0.09–0.71), although this risk We measured the steady-state levels of RGS transcripts in lympho- estimate was based on only 6 cases and 17 controls (Table 4). The blastoid cell lines derived from individuals with one of the following small number of subjects in this stratum reflects the low frequency of four RGS genotypes: wild-type, RGS2-1V, RGS6-2V, and RGS6-3V. the RGS6-2V allele (11%). A similar protective effect was evident in The relative levels of RGS2 or RGS6L transcripts from each sample the larger combination genotype group (57 subjects) with the geno- were quantified by real-time PCR, whereby the calculated Ct, or cycle type W/W/V (OR, 0.34; 95% CI, 0.17–0.65). Overall, individuals at which a set signal threshold is crossed, reflects the transcript with the genotype W/W/V or V/V/V (a total of 80 subjects) exhibited abundance. The Ct values for the internal control ␤-actin were similar a 66% to 74% decreased risk of bladder carcinoma relative to those in all samples, confirming that equivalent amounts of input cDNA subjects with a wild-type genotype. were used (Table 5). The Ct values for RGS6L were equivalent among Because ever smokers consistently showed reduced cancer risk in cell lines with the wild-type alleles (W/W/W) and those with the the analysis of single polymorphisms, we also examined the joint variant RGS6 genotypes, W/V/W and W/W/V. Likewise, the Ct effects of the three polymorphisms in ever smokers, specifically. values for RGS2 were similar between cell lines with the wild-type Significantly decreased risk was apparent in the combinations of (W/W/W) and variant (V/W/W) genotypes. This assay cannot exclude V/W/W, V/V/W, and W/W/V, with ORs of 0.54 (95% CI, 0.33–0.88), minor effects on transcript levels or changes in both the rate of mRNA 0.51 (95% CI, 0.27–0.95), and 0.35 (95% CI, 0.16–0.76), respec- synthesis and degradation. However, we can conclude that the variant tively (Table 4). As in the overall analysis, the presence of the variant alleles do not significantly influence steady-state RGS mRNA levels. genotype at all three sites further lowered the OR to 0.19 (95% CI, Whereas only one transcript of RGS2 is known, the RGS6 tran- 0.06–0.64). script pattern is complicated by two separate transcription start sites Expression of RGS2 and RGS6 in Bladder Cancer. Whereas (short and long forms) and several 3Ј splice events that result in six RGS2 and RGS6 transcripts are present in a wide range of normal separate COOH termini (identified by letters ␣–␩; ref. 22). We tissues (20, 21), their expression in tumor tissue has not been inves- investigated whether the presence of the variant RGS6 SNP alleles tigated. To confirm that these RGS genes are expressed in tumor results in a change in the splicing profile of RGS6. RNA was isolated tissue, the levels of ␤-actin, RGS2, and RGS6 transcripts in micro- from exponentially growing lymphoblastoid cell lines derived from dissected frozen bladder carcinoma and three bladder cancer cell lines individuals homozygous for either the wild-type or variant alleles. By were analyzed by PCR. Because this PCR reaction is nonquantitative, PCR, the ␣ and ␥ but not ␦ or ␩ 3Ј splice variants were identified in the results are scored as either transcript present or absent. The “long” all cell lines (Fig. 1B). Furthermore, both RGS6L (Table 5) and and “short” forms of RGS6, RGS6L and RGS6S, as well as RGS2 RGS6S transcripts (data not shown) were expressed in all cell lines, were all present in microdissected bladder carcinoma and in the three suggesting no correlation between SNP status and mRNA splicing. bladder cancer cell lines (Fig. 1A). RGS6L is the full-length RGS6 Specific PCR products could not be produced for the other 3Ј splice protein, whereas RGS6S results from an alternative transcription start variants. Similar results were observed with normal human whole site (22, 23). A control reaction for ␤-actin was positive in each brain cDNA (data not shown). Thus, the presence of the two variant sample. RGS6 SNP alleles does not alter the mRNA splicing profile. Expression of RGS2 and RGS6 as a Function of SNP Status. Of the three SNPs studied, only RGS6-2, located in the 3Ј-UTR, Two of the three most significant SNPs, RGS2-1 and RGS6-2, are is not excised by RNA splicing. Because the 3Ј-UTR is known to located in the 3Ј-UTR, whereas the third, RGS6-3, is in the first intron. regulate protein translation (26, 27), we investigated whether the Such noncoding SNPs may functionally affect protein activity by protective profile of RGS6-2V derives from its ability to regulate

Table 4 Risk estimates for combinations of RGS genotypes Genotypes No. of No. of Adjusted OR RGS2-1 RGS6-3 RGS6-2 cases controls OR (95% CI) (95% CI)* Overall CC CC CC 154 100 Reference Reference CC CC CTϩTT 21 36 0.38 (0.21–0.69) 0.34 (0.17–0.65) CGϩGG CC CC 113 112 0.66 (0.46–0.9) 0.68 (0.46–01.0) CC CTϩTT CC 69 65 0.69 (0.45–1.1) 0.70 (0.44–1.10) CGϩGG CC CTϩTT 31 24 0.84 (0.47–1.5) 0.77 (0.41–1.5) CC CTϩTT CTϩTT 10 13 0.50 (0.21–1.2) 0.37 (0.14–1.0) CGϩGG CTϩTT CC 48 47 0.66 (0.41–1.1) 0.66 (0.39–1.1) CGϩGG CTϩTT CTϩTT 6 17 0.23 (0.09–0.60) 0.26 (0.09–0.71) Ever smokers CC CC CC 122 50 Reference Reference CC CC CTϩTT 17 18 0.39 (0.18–0.81) 0.35 (0.16–0.76) CGϩGG CC CC 79 58 0.56 (0.35–0.90) 0.54 (0.33–0.88) CC CTϩTT CC 50 32 0.64 (0.37–1.11) 0.61 (0.34–1.1) CGϩGG CC CTϩTT 23 15 0.63 (0.30–1.30) 0.60 (0.28–1.3) CC CTϩTT CTϩTT 7 6 0.48 (0.15–1.49) 0.31 (0.09–1.0) CGϩGG CTϩTT CC 34 26 0.54 (0.29–0.98) 0.51 (0.27–0.95) CGϩGG CTϩTT CTϩTT 4 11 0.15 (0.05–0.49) 0.19 (0.06–0.64) Abbreviations: RGS2-1, RGS2-rs4606; RGS6-3, RGS6-rs3784058; RGS6-2, RGS6-rs2074647. * Adjusted for age, sex, and years smoked. 6823

of the variant SNP, the luciferase activity was 2.9-fold higher relative to that measured for the wild-type allele (Fig. 1C). This is remarkable because the only difference between these two constructs is the single nucleotide at RGS6-2. Once again, RGS2-1 and RGS6-3 are removed during RNA splicing and therefore cannot modulate protein translation. We conclude that RGS6-2 is a functional SNP and that the variant RGS6-2 allele may increase the steady-state levels of RGS6 protein.

DISCUSSION

Heterotrimeric G proteins are central regulators of cellular home- ostasis. All RGS proteins negatively regulate these G proteins; however, a subset of RGS proteins participates in other diverse cellular functions (1, 2). Because of the importance of RGS proteins in regulating numerous signaling pathways, we investigated whether RGS genes modulate the risk of developing bladder cancer. Twelve informative SNPS in five RGS genes were identified in the NCBI dbSNP database; however, only 11 were confirmed to be real SNPs. All were located in noncoding regions. In this molecular epidemiologic study, we found that variant genotypes of the RGS2-1, RGS6-2, and RGS6-3 were underrepresented in bladder cancer cases. Furthermore, the protective effects were more evident among ever smokers, people who started smoking at an earlier age, and those who were diagnosed with bladder cancer at a younger age. The pronounced reduction in risk of cancer seen for ever smokers is suggestive of a gene-environment interaction. When the three SNPs were analyzed separately and in combination, the RGS6-2 variant genotype was associated with the greatest reduction in risk. Our results are consist- ent with the notion that genetic factors play an especially relevant role in patients with younger age at onset (28). Although the total number of subjects in the two combination groups with the greatest reduction in risk was only 80, the data are indeed intriguing and warrant further study. To validate the biological significance of these SNPs, we sought to identify functional changes that may result from the presence of the variant alleles. SNPs in RGS2 and RGS6 do not appear to regulate splicing or steady-state mRNA levels. However, when expressed as a fusion with the luciferase reporter protein, RGS6-2V was associated with a 2.9-fold increase in luciferase activity relative to the fusion reporter with the wild-type allele. Indeed, RGS6-2 is located in the 3Ј-UTR, a region known to regulate protein translation (26, 27). This Fig. 1. A. RGS2 and RGS6 transcripts are expressed in bladder carcinoma. PCR for ␤-actin, RGS2, RGS6S, and RGS6L was performed on cDNA prepared from microdis- difference in luciferase activity may result from an increase in trans- sected bladder carcinoma (Lane 1) and three bladder carcinoma cell lines, 5637, T24, and lation efficiency, a slight (but difficult to assay) increase in steady- UMVC (Lanes 2–4). These results are representative of three experiments. B. The ␣ and ␥ 3Ј splice variants of RGS6 are expressed in lymphoblastoid cell lines independently of state transcript levels, or a combination of both effects. genotype status. PCR using primers specific for 3Ј splice variants (␣, ␥, ␦, and ␮) was A 3-fold change in the cellular concentration of signaling proteins performed on cDNA prepared from three lymphoblastoid cell lines with the following can have dramatic effects on intracellular signal transduction. Indeed, genotypes (RGS6-3/RGS6-2): W/W, V/W, and W/V. These results are representative of three experiments. C. The RGS6-2 polymorphism (C3T) increases the activity of a luciferase-RGS6 fusion protein. A luciferase-RGS6 fusion gene containing either the wild-type allele (C) or variant RGS6-2 allele (T) is expressed in NIH3T3 cells. The data shown represent the luciferase activity for each fusion protein normalized to that measured for the unmodified luciferase protein. Data were also normalized for transfection effi- Table 5 RGS transcript levels as a function of genotype status ciency by cotransfection with ␤-galactosidase and represent the mean Ϯ SD for three Ct replicates. The experiment is representative of three independent experiments. Actin RGS2 RGS6* Lymphoblastoid cell line genotype (SD) (SD) (SD) protein translation. Because commercial antibodies were neither RGS6–3W/RGS6–2W/RGS2–1W 24.2 (0.8) 31.2 (2.0) 30.4 (1.7) RGS6–3V/RGS6–2W/RGS2–1W 24.8 (0.9) ND 30.7 (2.4) sensitive nor specific (data not shown), we measured the effects of RGS6–3W/RGS6–2V/RGS2–1W 23.9 (0.7) ND 29.1 (1.1) this SNP on protein translation by creating a fusion protein be- RGS6–3W/RGS6–2W/RGS2–1V 24.8 (0.3) 29.3 (0.6) ND tween a luciferase reporter protein and the extreme COOH termi- NOTE. Real-time PCR was performed using the Lightcycler (Roche), and Ct value was nus of the RGS6 gene (including the 3Ј-UTR with either the calculated using Roche Lightcycler software. Results are the mean of at least three experiments. wild-type or variant allele). These constructs were transfected into Abbreviation: ND, no data. NIH3T3 cells, and luciferase activity was assayed. In the presence * Primers designed for full-length RGS6L protein. 6824

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