RGS proteins negatively regulate heterotrimeric G protein signaling. Recent reports have shown that RGS proteins modulate neuronal, cardiovascular, and lymphocytic activity, yet their role in carcinogenesis has not been explored. In an epidemiologic study of 477 bladder cancer patients and 446 matched controls, three noncoding single-nucleotide polymorphisms (SNPs) in RGS2 and RGS6 were each associated with a statistically significant reduction in bladder cancer risk. The risk of bladder cancer was reduced by 74% in those individuals with the variant genotype at all three SNPs (odds ratio, 0.26; 95% confidence interval, 0.09–0.71). When the SNPs were analyzed separately, the RGS6-rs2074647 (C→T) polymorphism conferred the greatest overall reduction in risk of bladder cancer (odds ratio, 0.66; 95% confidence interval, 0.46–0.95). These reductions in risk were more pronounced in ever smokers, suggesting a gene-environment interaction. In transfection assays, the RGS6-rs2074647 (C→T) polymorphism increased the activity of a luciferase-RGS fusion protein by 2.9-fold, suggesting that this SNP is functionally significant. Finally, we demonstrate that RGS2 transcripts and several splice variants of RGS6 are expressed in bladder cancer cells. These data provide the first evidence that RGS proteins may be important modulators of cancer risk and validate RGS6 as a target for further study.

The RGS (regulators of G protein signaling) family of proteins participates in a wide range of signal transduction pathways. All 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 integrating G protein pathways with a diverse range of other cellular signaling events (2). Recent reports have shown that RGS proteins modulate neuronal, cardiovascular, and lymphocytic activities, yet their role in carcinogenesis has not been explored in any depth

Heterotrimeric G proteins have transforming potential, and this alone would make RGS proteins a relevant target for analysis (3). However, there is growing but indirect evidence that supports the notion that RGS proteins also regulate other key pathways of carcinogenesis. For example, RGS14 binds to the Ras-related G protein, Rap1/2 (4). RGS12 is a transcriptional repressor, and RGS12 overexpression in select cell lines inhibits DNA synthesis (5). RGS-RhoGEFs activate Rho G proteins, key regulators of the cytoskeleton (6). RGS16, which is induced by genotoxic shock in a p53-dependent fashion, inhibits G protein activation of the mitogen-activated protein kinase cascade (7). RGS-axin regulates the activity of the β-catenin transcription factor (8, 9). Several RGS proteins regulate the sorting of intracellular vesicles (reviewed in ref. 2). Finally, RGS3 induces apoptosis (10).

Two well-characterized RGS proteins, RGS2 and RGS6, are of particular interest. RGS2 mRNA is highly expressed in acute leukemia but not in the chronic form or in normal bone marrow (11, 12, 13). RGS6 binds to the DNA methylating protein, DNMT1, and de-represses transcriptional inhibition by the DNMT1-associated protein, DMAP1 (14). Both RGS2 and RGS6 undergo nucleocytoplasmic shuttling, suggesting that these proteins may provide a link between the regulation of cytoplasmic events and DNA or RNA metabolism (15, 16). Finally, both of these proteins are heat shock responsive, a characteristic of some regulators of apoptosis (17, 18).

There are polymorphisms in genes that may modulate protein function and produce downstream effects contributing to variation in cancer risk. Therefore, in an ongoing hospital-based case-control study, we explored the association between 12 noncoding single-nucleotide polymorphisms (SNPs) in five RGS genes identified in the National Center for Biotechnology Information (NCBI) database (dbSNP) and the risk of bladder carcinoma, a cause of over 12,000 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 to identify functional changes in transcript levels, alternative splicing events, and protein translation efficiency that may result from the presence of the variant alleles.

Study Population.

Available for study were 477 Caucasian patients presenting with urinary bladder cancer cases and accrued from the Departments of Urology at The University of Texas M. D. Anderson Cancer Center and the Baylor College of Medicine. The case patients, who had histologically confirmed incident bladder cancer, had received no previous chemotherapy or radiotherapy and were enrolled in an ongoing case-control study described previously (19). There were no recruitment restrictions on age, gender, or cancer stage. A brief eligibility questionnaire assessing prior cancer therapy and willingness to participate in the epidemiologic study was administered to assess eligibility.

A total of 446 Caucasian healthy control subjects without a prior history of cancer (except nonmelanoma skin cancer) were recruited as controls into the study from the Kelsey-Seybold clinics, a large private multispecialty physician group in the Houston metropolitan area. Control subjects were frequency matched to the cases on the basis of age (±5 years), gender, and ethnicity.

All study participants completed a structured 1.5-hour personal interview 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.

Genotyping RGS Polymorphisms.

DNA was isolated from peripheral blood lymphocytes using a nonphenol method. RFLP-polymerase chain reaction (PCR) was used to detect SNPs for RGS5, rs15049 (A/C); RGS6, rs2238280 (G/A); RGS6, rs2074647 (C/T); RGS11, rs3743878 (C/T); RGS11, rs3743879 (C/G); RGS17, rs2295232 (G/T); and RGS17, rs3870366 (G/A). No polymorphism was detected at RGS11, rs3743878. The TaqMan assay was used to detect SNPs for RGS2, rs4606 (G/C); RGS6, rs2238284 (T/G); RGS6, rs3784058 (T/C); RGS6, rs2238285 (C/T); and RGS17, rs2295231 (A/G).

For genotyping by RFLP-PCR, the genomic DNA regions containing the polymorphisms were amplified by PCR. The primers used for these polymorphisms are listed as an online supplementary data. The PCR assay was performed in a 25-μL reaction mixture containing 100 ng of genomic DNA, 1× PCR buffer (Promega, Madison WI), 2 mmol/L MgCl2 (3 mmol/L for rs3870366), 0.2 mmol/L deoxynucleotide triphosphates, 1.5 unit of Taq polymerase (Promega), and 0.2 μmol/L primers. The PCR thermal cycling conditions were 5 minutes at 94°C followed by 35 cycles of 30 seconds at 94°C; 30 seconds at 55°C (rs15049), 48°C (rs2238280), 57°C (rs2074647), 57°C (rs3743878), 59°C (rs3743879), 58°C (rs2295232), or 45°C (rs3870366), respectively; and 45 seconds at 72°C; and, finally, extension for 5 minutes at 72°C. The PCR product was digested with restriction enzymes (New England Biolabs, Beverly, MA) overnight at 37°C, and the fragments were separated on agarose gel stained with ethidium bromide. The gene respective enzyme, genotype, and expected bands are as follows: (a) RGS5 (rs15049), HaeIII digestion, A/A (212 bp), A/C (212 + 193 + 19 bp), and C/C (193 + 19 bp); (b) RGS6 (rs2238280), AseI digestion, G/G (142 bp), G/A (142 + 125 + 17 bp), and A/A (125 + 17 bp); (c) RGS6 (rs2074647), HpyCH4 IV digestion, C/C (177 + 19 bp), C/T (196 + 177 + 19 bp), and T/T (196 bp); (d) RGS11 (rs3743878), MspI digestion, C/C (94 + 20 bp), C/T (114 + 94 + 20 bp), and T/T (114 bp); (e) RGS11 (rs3743879), SpeI digestion, C/C (126 + 17 bp), C/G (143 + 126 + 17 bp), and G/G (143 bp); (f) RGS17 (rs2295232), MboI digestion, G/G (151 + 146 bp), G/T (297 + 151 + 146 bp), and T/T (297 bp); and (g) RGS17 (rs3870366), NsiI digestion, G/G (132 bp), G/A (132 + 111 + 21 bp), and A/A (111 + 21 bp).

Genotyping using TaqMan 5′ nuclease assay for allelic discrimination was carried out on the 384-well ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and probes were designed using the Primer Express software (Applied Biosystems). The probes were fluorescence labeled with either 6-FAM or VIC on the 5′-end and a nonfluorescent minor groove binder (MGB) quencher on the 3′ end (Applied Biosystems). The sequences of primers and probes are listed as an online supplementary data. Typical amplification mixes (5 μL) contained sample DNA (5 ng), 1× TaqMan buffer A, 200 μmol/L deoxynucleotide triphosphates, 5 mmol/L MgCl2, 0.65 unit of AmpliTaq Gold, 900 nmol/L each primer, and 200 nmol/L each probe. The thermal cycling conditions consisted of 1 cycle for 10 minutes at 95°C and 40 cycles for 15 seconds at 95°C and for 1 minute at 60°C. SDS version 2.1 software (Applied Biosystems) was used to analyze end point fluorescence. Water control, ample internal controls, and previously genotyped samples were included in each plate to ensure accuracy of genotyping.

Statistical Analyses.

Differences between the cases and the controls in the distribution of smoking status, gender, and RGS genotypes were tested using the χ2 test statistic. The Wilcoxon rank-sum test was used to test for differences between the cases and controls for mean age and select cigarette smoking variables. Unconditional logistic regression was used to calculate odds ratios (ORs) as estimates of the relative risk associated with the RGS polymorphisms. Multivariate logistic regression was performed to adjust for the potential confounding effects of age, gender, and years smoked, where appropriate. To assess for interaction between variables, we fitted a logistic model including product terms of the variables in the model. All statistical 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 defined as an ever smoker. Ever smokers include former smokers, current smokers, and recent quitters (who quit smoking within the previous year). A former smoker had quit smoking at least 1 year before diagnosis of cancer (case patients) or at least 1 year before the interview (control subjects).

Cell Lines and Tumor Tissue.

Human bladder carcinoma cell lines (5637, T24, and UMVC) were kindly provided by Dr. Jonathan Coleman (National Cancer Institute, National Institutes of Health, Bethesda, MD). Lymphoblastoid cell lines were derived from eight subjects with known RGS genotypes. These cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. Anonymized frozen transitional cell carcinoma tissue was obtained from the Laboratory of Pathology frozen tissue bank with appropriate National Institutes of Health institutional review board approval.

RNA Isolation and Polymerase Chain Reaction.

Cultured cells were solubilized with Trizol (Invitrogen, Carlsbad, CA), the supernatant was buffered with 50 mmol/L Tris (pH 7.5), and total RNA was purified using the RNAeasy kit (Qiagen Valencia, CA). Five hundred nanograms of total RNA were converted into cDNA using the Superscript III kit (Invitrogen, Valencia, CA). Frozen transitional cell carcinoma was microdissected by AutoPix laser capture microdissection, and RNA was isolated and linearly amplified using the PicoPure and RiboAmp kits, respectively (Arcturus, Mountain View, CA). For traditional PCR, equivalent amounts of cDNA substrate were introduced to puReTaq Ready-to-go PCR beads (Amersham, Piscataway, NJ), and PCR was performed with the following cycling parameters: 95°C for 5 minutes; followed by 35 to 40 cycles of 95°C for 20 seconds, 57°C for 30 seconds, and 72°C for 30 seconds; with a final extension of 72°C for 7 minutes. Real-time PCR using splice site-spanning primers for RGS2, RGS6, and β-actin was performed using the Lightcycler (Roche, Indianapolis, IN) with the following parameters: 95°C for 10 minutes, followed by 40 cycles of 95°C for 8 seconds, 57°C for 6 seconds, and 72°C for 6 seconds. Crossing threshold (Ct) values were calculated using the Roche Lightcycler software. The Ct values for the RGS reactions are derived from at least three experiments. Experiments were also performed with a 10-fold dilution of template to ensure that measurements were in the linear range (data not shown). The probes and primers for this experiment are listed as an online supplementary data.

Luciferase Reporter Constructs and Luciferase Assays.

Two primers (5′-AGCTATGAGATAACCAGTCAA-3′ and 5′-GATCAGGGCCTCTTAGCGAGT-3′) were designed to amplify a fragment of RGS6 encompassing the SNP RGS6 rs2074647 (C/T). The cDNA substrate was derived from lymphoblastoid cell lines with known RGS genotypes as described above. The target fragment contains 258 bases of COOH-terminal coding sequence of RGS6 as well as 285 bases of the 3′-untranslated region (UTR). The amplified fragments were sequenced to confirm that there were no other SNPs and then reamplified again using primers containing XbaI restriction sites at both termini followed by cloning into the pCR2.1-TOPO vector (Invitrogen). The amplified fragments were then cloned into the XbaI site located between the luciferase gene and the SV40 late poly(A) signal in the pGL3-Promoter vector (Promega). The resulting luciferase constructs were digested with SacII and HpaI to confirm correct orientation.

NIH3T3 cells, cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, were transfected using LipofectAMINE 2000 (Invitrogen) with the above-mentioned pGL3-Promoter vector harboring either the wild-type or variant allele at rs2074647. Unmodified pGL3 promoter vector was transfected in parallel. Luciferase activity in transfected cells was assayed 40 hours after transfection using a luciferase assay kit (Promega). Luciferase activity was normalized for transfection efficiency by cotransfection with a β-galactosidase reporter gene for which activity was assayed with the Galacto chemiluminescence assay kit (Tropix, Bedford, MA). The data are expressed as ratios of activity of the fusion protein to that of the unmodified luciferase protein.

This study is based on data from 477 Caucasian bladder cancer case patients and 446 Caucasian control subjects (Table 1). We restricted our analysis to Caucasians because of the small numbers of African-American and Mexican-American subjects available for analysis. Because the study is still on going, perfect matching between case patients and control subjects has not been achieved.

Overall, there was no statistically significant difference in sex distribution between the cases and the controls (P = 0.098). The mean age was 64.0 years for the case patients, compared with 63.1 years for the control subjects (P = 0.19). As expected, case patients were significantly more likely than the control subjects to be current smokers (22.0% versus 8.1%) and, conversely, were less likely to be never smokers (26.0% versus 47.3%; P < 0.001). Moreover, mean self-reported number of cigarettes smoked per day and mean pack-years smoked were also statistically significantly higher in the cases than in the controls (P < 0.001 for both). However, the potential bias due to the above-mentioned differences is not realized because, as demonstrated below, the calculated ORs are even more significant when the data are controlled for smoking status.

Twelve SNPs in five RGS genes (RGS2, RGS5, RGS6, RGS11, and RGS17) were identified in the NCBI dbSNP database. All SNPs were located in untranslated regions and had estimated population frequencies greater than 5%. No nonsynonymous SNPs were found in any of the RGS genes. By sequencing, we confirmed that 11 of the 12 SNPs were indeed real SNPs (Table 2). The observed frequencies of all of the RGS genotypes among controls did not differ from those expected under the Hardy-Weinberg equilibrium (online supplementary data).

ORs were calculated to estimate bladder cancer risk associated with these polymorphisms. For this analysis, genotypes were combined into the following two classes due to the small number of carriers of the homozygous variant genotypes: (a) homozygous wild-type allele (i.e., C/C; referred to as W) and (b) the variant genotypes (i.e., C/T plus T/T; referred to as V). Of the 11 SNPS, only 3 were associated with significant reductions in risk of bladder cancer (Table 3). Overall, the most protective variant allele was located at RGS6-2074647 (C→T; abbreviated as RGS6-2) and was associated with a 34% reduction in bladder cancer risk [OR, 0.66; 95% confidence interval (CI), 0.46–0.95]. On stratified analysis, protective effects were more evident in ever smokers (OR, 0.60; 95% CI, 0.39–0.94), individuals who started smoking before age 17 years (OR, 0.42; 95% CI, 0.20–0.86), and those who had a younger age at diagnosis of bladder cancer (OR, 0.45; 95% CI, 0.23–0.90; Table 3).

Similar trends were noted for two other SNPS: RGS2-4606 (C→G; abbreviated as RGS2-1) and RGS6-3784058 (C→T; abbreviated as RGS6-3; Table 3). The protective effect of RGS2-1V was of borderline statistical significance in the overall analysis (OR, 0.85; 95% CI, 0.64–1.11) but with significant reductions noted for ever smokers (OR, 0.71), individuals who started smoking before age 17 years (OR, 0.61), and patients with younger age at diagnosis (OR, 0.53). The variant genotype of RGS6-3 was similarly associated with reduced bladder cancer risk (OR, 0.81; 95% CI, 0.60–1.09). We did not find any significant effects for the nine other polymorphisms (in RGS5, RGS11, and RGS17) studied either in the overall analysis or in the subsequent stratified analysis (data not shown).

To further characterize interactions between these three polymorphisms, we analyzed combinations of the three genotypes (Table 4). The combinations are reported in the following order: RGS2-1/RGS6-3/RGS6-2. Compared with the reference group (i.e., individuals with wild-type genotypes at the three loci), the presence of the variant genotype at all three sites (V/V/V) was associated with a substantial reduction in the OR to 0.26 (95% CI, 0.09–0.71), although this risk estimate was based on only 6 cases and 17 controls (Table 4). The small number of subjects in this stratum reflects the low frequency of the RGS6-2V allele (11%). A similar protective effect was evident in the larger combination genotype group (57 subjects) with the genotype W/W/V (OR, 0.34; 95% CI, 0.17–0.65). Overall, individuals with the genotype W/W/V or V/V/V (a total of 80 subjects) exhibited a 66% to 74% decreased risk of bladder carcinoma relative to those subjects with a wild-type genotype.

Because ever smokers consistently showed reduced cancer risk in the analysis of single polymorphisms, we also examined the joint effects of the three polymorphisms in ever smokers, specifically. Significantly decreased risk was apparent in the combinations of V/W/W, V/V/W, and W/W/V, with ORs of 0.54 (95% CI, 0.33–0.88), 0.51 (95% CI, 0.27–0.95), and 0.35 (95% CI, 0.16–0.76), respectively (Table 4). As in the overall analysis, the presence of the variant genotype at all three sites further lowered the OR to 0.19 (95% CI, 0.06–0.64).

Expression of RGS2 and RGS6 in Bladder Cancer.

Whereas RGS2 and RGS6 transcripts are present in a wide range of normal tissues (20, 21), their expression in tumor tissue has not been investigated. To confirm that these RGS genes are expressed in tumor tissue, the levels of β-actin, RGS2, and RGS6 transcripts in microdissected frozen bladder carcinoma and three bladder cancer cell lines were analyzed by PCR. Because this PCR reaction is nonquantitative, the results are scored as either transcript present or absent. The “long” and “short” forms of RGS6, RGS6L and RGS6S, as well as RGS2 were all present in microdissected bladder carcinoma and in the three bladder cancer cell lines (Fig. 1 A). RGS6L is the full-length RGS6 protein, whereas RGS6S results from an alternative transcription start site (22, 23). A control reaction for β-actin was positive in each sample.

Expression of RGS2 and RGS6 as a Function of SNP Status.

Two of the three most significant SNPs, RGS2-1 and RGS6-2, are located in the 3′-UTR, whereas the third, RGS6-3, is in the first intron. Such noncoding SNPs may functionally affect protein activity by influencing alternative splicing events, mRNA stability, or protein translation (24, 25, 26). Unfortunately, bladder cancer cell lines with the requisite genotypes were not available. Therefore, we investigated whether these three SNPs functionally regulate mRNA splicing or steady-state transcript levels in lymphoblastoid cell lines derived from individuals with known RGS genotypes.

We measured the steady-state levels of RGS transcripts in lymphoblastoid cell lines derived from individuals with one of the following four RGS genotypes: wild-type, RGS2-1V, RGS6-2V, and RGS6-3V. The relative levels of RGS2 or RGS6L transcripts from each sample were quantified by real-time PCR, whereby the calculated Ct, or cycle at which a set signal threshold is crossed, reflects the transcript abundance. The Ct values for the internal control β-actin were similar in all samples, confirming that equivalent amounts of input cDNA were used (Table 5). The Ct values for RGS6L were equivalent among cell lines with the wild-type alleles (W/W/W) and those with the variant RGS6 genotypes, W/V/W and W/W/V. Likewise, the Ct values for RGS2 were similar between cell lines with the wild-type (W/W/W) and variant (V/W/W) genotypes. This assay cannot exclude minor effects on transcript levels or changes in both the rate of mRNA synthesis and degradation. However, we can conclude that the variant alleles do not significantly influence steady-state RGS mRNA levels.

Whereas only one transcript of RGS2 is known, the RGS6 transcript pattern is complicated by two separate transcription start sites (short and long forms) and several 3′ splice events that result in six separate COOH termini (identified by letters α–η; ref. 22). We investigated whether the presence of the variant RGS6 SNP alleles results in a change in the splicing profile of RGS6. RNA was isolated from exponentially growing lymphoblastoid cell lines derived from individuals homozygous for either the wild-type or variant alleles. By PCR, the α and γ but not δ or η 3′ splice variants were identified in all cell lines (Fig. 1,B). Furthermore, both RGS6L (Table 5) and RGS6S transcripts (data not shown) were expressed in all cell lines, suggesting no correlation between SNP status and mRNA splicing. Specific PCR products could not be produced for the other 3′ splice variants. Similar results were observed with normal human whole brain cDNA (data not shown). Thus, the presence of the two variant RGS6 SNP alleles does not alter the mRNA splicing profile.

Of the three SNPs studied, only RGS6-2, located in the 3′-UTR, is not excised by RNA splicing. Because the 3′-UTR is known to regulate protein translation (26, 27), we investigated whether the protective profile of RGS6-2V derives from its ability to regulate protein translation. Because commercial antibodies were neither sensitive nor specific (data not shown), we measured the effects of this SNP on protein translation by creating a fusion protein between a luciferase reporter protein and the extreme COOH terminus of the RGS6 gene (including the 3′-UTR with either the wild-type or variant allele). These constructs were transfected into NIH3T3 cells, and luciferase activity was assayed. In the presence of the variant SNP, the luciferase activity was 2.9-fold higher relative to that measured for the wild-type allele (Fig. 1 C). 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.

Heterotrimeric G proteins are central regulators of cellular homeostasis. 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 consistent 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 3′-UTR, a region known to regulate protein translation (26, 27). This difference in luciferase activity may result from an increase in translation efficiency, a slight (but difficult to assay) increase in steady-state transcript levels, or a combination of both effects.

A 3-fold change in the cellular concentration of signaling proteins can have dramatic effects on intracellular signal transduction. Indeed, when induced by physiologic stimulus, the yeast RGS protein Sst2p increased by only 2.5-fold, and this was sufficient to terminate G protein activation of the mitogen-activated protein kinase cascade (29). In this same study, 2-fold overexpression of Sst2p reduced G protein signaling by almost 30%. Furthermore, recent advances in proteomics technology have confirmed that slight changes in the levels of select proteins correlate with histologic progression from benign to preinvasive to invasive tumors (30). We speculate that an increase in RGS6 protein may be the mechanism by which RGS6-2V confers its protective effect, and we are currently developing antibodies to RGS6 to confirm this hypothesis.

RGS6 mRNA splicing is complex and yields two distinct transcription start sites and six distinct COOH termini (22, 23). The actual splice variants present in human tissues have not yet been examined. We report that both start sites (RGS6S and RGS6L) and at least two alternative 3′ splice variants (α and γ) are expressed in bladder cancer, normal human brain, and lymphocytes. Whereas alternative splicing does not appear to be modified by the presence of the variant SNP genotype, the significance of alternative splicing of RGS6 transcripts remains an area of obvious interest.

RGS2 regulates lymphocytic function and cardiovascular status. Deletion of the RGS2 gene in knockout mice results in hypertension, decreased production of interleukin 2, and hyporesponsive T lymphocytes (31, 32, 33). In bladder carcinoma, T lymphocytes and interleukin 2 are thought to be central components of immune surveillance and antitumor response (34). Because they are present in both bladder carcinoma and lymphocytes, RGS2 and RGS6 may be important modulators of carcinogenesis through their action in the tumor tissue and/or via modulation of lymphocytic activity.

In addition to negatively regulating G proteins, RGS6 inhibits stimulation of phospholipase β2, promotes neuronal differentiation (via the neuronal protein SGC10), and de-represses transcriptional inhibition of DMAP-1 (14, 23, 35). This latter protein complex also includes DNMT-1, a protein that regulates chromatin structure and gene silencing. Promoter-specific methylation is recognized as a key mechanism of gene silencing in many tumors (36). It is tempting to speculate that the presumed elevation in RGS6 protein (e.g., by RGS6-2V) confers a protective effect through either RNA transcription (via DMAP-1) or by preventing aberrant DNA methylation (via DNMT1), and we are currently investigating these hypotheses.

Although this report is the first to link SNPs in the G protein-coupled receptor (GPCR) signaling pathway to cancer, numerous epidemiologic studies have provided linkages between this pathway and other diseases. For example, SNPs in the noncoding regions of GPCRs and the G protein Gβ3 are associated with clinically significant differences in cardiovascular performance (37). In contrast to these RGS SNPs, GPCR SNPs are found in both coding and noncoding regions, which likely reflects a greater tolerance for amino acid changes in GPCRs (37, 38, 39, 40, 41). Recently, four noncoding SNPs in RGS4 were reported to be linked with the development of schizophrenia in a family-based study (42).

The results presented here suggest that RGS proteins may be important modulators of cancer risk. The presence of variant SNP alleles in RGS2 and RGS6 is associated with a significant reduction in the risk of bladder cancer. The singularly most protective SNP, RGS6-2, appears to modulate protein translation and may contribute to the protective phenotype by increasing the level of RGS6 protein. The large number of RGS genes, their differential tissue expression, and their ability to robustly regulate signaling pathways make them attractive for further study in other cancers and as targets for chemotherapeutic intervention (43, 44).

Fig. 1.

A. RGS2 and RGS6 transcripts are expressed in bladder carcinoma. PCR for β-actin, RGS2, RGS6S, and RGS6L was performed on cDNA prepared from microdissected bladder carcinoma (Lane 1) and three bladder carcinoma cell lines, 5637, T24, and UMVC (Lanes 2–4). These results are representative of three experiments. B. The α and γ 3′ splice variants of RGS6 are expressed in lymphoblastoid cell lines independently of genotype status. PCR using primers specific for 3′ splice variants (α, γ, δ, and μ) was performed on cDNA prepared from three lymphoblastoid cell lines with the following genotypes (RGS6-3/RGS6-2): W/W, V/W, and W/V. These results are representative of three experiments. C. The RGS6-2 polymorphism (C→T) 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 efficiency by cotransfection with β-galactosidase and represent the mean ± SD for three replicates. The experiment is representative of three independent experiments.

Fig. 1.

A. RGS2 and RGS6 transcripts are expressed in bladder carcinoma. PCR for β-actin, RGS2, RGS6S, and RGS6L was performed on cDNA prepared from microdissected bladder carcinoma (Lane 1) and three bladder carcinoma cell lines, 5637, T24, and UMVC (Lanes 2–4). These results are representative of three experiments. B. The α and γ 3′ splice variants of RGS6 are expressed in lymphoblastoid cell lines independently of genotype status. PCR using primers specific for 3′ splice variants (α, γ, δ, and μ) was performed on cDNA prepared from three lymphoblastoid cell lines with the following genotypes (RGS6-3/RGS6-2): W/W, V/W, and W/V. These results are representative of three experiments. C. The RGS6-2 polymorphism (C→T) 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 efficiency by cotransfection with β-galactosidase and represent the mean ± SD for three replicates. The experiment is representative of three independent experiments.

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Grant support: R. Fisher is supported by National Institutes of Health grant GM067881. X. Wu is supported by National Cancer Institute grants CA 74880 and CA 91846.

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.

Note: Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org).

Requests for reprints: David M. Berman, Laboratory of Pathology, National Cancer Institute, Building 10-2N212, 10 Center Drive, Bethesda, MD 20892. Phone: 301-496-1888, Fax: 301-480-9488, E-mail: bermand@mail.nih.gov

Table 1

Distribution of select characteristics in the case subjects and control patients

VariablesNo. of cases (N = 477)No. of controls (N = 446)P
Sex    
 Male 367 (76.9%) 322 (72.0%)  
 Female 110 (23.1%) 124 (27.0%) 0.098 
Age (y)    
 Mean (SD) 63.96 (11.1) 63.10 (10.9) 0.19 
Smoking status    
 Never 124 (26.0%) 211 (47.3%)  
 Former 248 (52.0%) 199 (44.6%)  
 Current 105 (22.0%) 36 (8.1%) <0.001 
No. of cigarettes per day*    
 Mean (SD) 26.0 (14.2) 22.8 (16.0) <0.001 
Years smoked*    
 Mean (SD) 31.3 (14.0) 23.2 (14.2) <0.001 
Pack-years*    
 Mean (SD) 42.7 (31.3) 29.6 (29.8) <0.001 
Age started smoking (y)*    
 Mean (SD) 17.9 (4.4) 17.4 (4.2) 0.078 
VariablesNo. of cases (N = 477)No. of controls (N = 446)P
Sex    
 Male 367 (76.9%) 322 (72.0%)  
 Female 110 (23.1%) 124 (27.0%) 0.098 
Age (y)    
 Mean (SD) 63.96 (11.1) 63.10 (10.9) 0.19 
Smoking status    
 Never 124 (26.0%) 211 (47.3%)  
 Former 248 (52.0%) 199 (44.6%)  
 Current 105 (22.0%) 36 (8.1%) <0.001 
No. of cigarettes per day*    
 Mean (SD) 26.0 (14.2) 22.8 (16.0) <0.001 
Years smoked*    
 Mean (SD) 31.3 (14.0) 23.2 (14.2) <0.001 
Pack-years*    
 Mean (SD) 42.7 (31.3) 29.6 (29.8) <0.001 
Age started smoking (y)*    
 Mean (SD) 17.9 (4.4) 17.4 (4.2) 0.078 
*

Ever smokers only.

Table 2

Summary of RGS SNPs

GeneNCBI SNP cluster IDChromosomeContig accession no.Abbreviation in textPosition in geneAllele frequency
RGS2 rs4606 NT_004671 RGS2–1 3′-UTR C: 0.715 G: 0.285 
RGS5 rs15049 NT_004668 None 3′-UTR A: 0.697 C: 0.303 
RGS6 rs2074647 14 NT_026437 RGS6–2 3′-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 3′-UTR C: 0.901 T: 0.099 
RGS11 rs3743879 16 NT_037887 None 3′-UTR C: 0.570 G: 0.430 
RGS17 rs2295231 NT_023451 None Intron* A: 0.572 G: 0.428 
RGS17 rs2295232 NT_023451 None Intron* G: 0.572 T: 0.428 
RGS17 rs3870366 NT_023451 None Intron* A: 0.390 G: 0.610 
GeneNCBI SNP cluster IDChromosomeContig accession no.Abbreviation in textPosition in geneAllele frequency
RGS2 rs4606 NT_004671 RGS2–1 3′-UTR C: 0.715 G: 0.285 
RGS5 rs15049 NT_004668 None 3′-UTR A: 0.697 C: 0.303 
RGS6 rs2074647 14 NT_026437 RGS6–2 3′-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 3′-UTR C: 0.901 T: 0.099 
RGS11 rs3743879 16 NT_037887 None 3′-UTR C: 0.570 G: 0.430 
RGS17 rs2295231 NT_023451 None Intron* A: 0.572 G: 0.428 
RGS17 rs2295232 NT_023451 None Intron* G: 0.572 T: 0.428 
RGS17 rs3870366 NT_023451 None Intron* A: 0.390 G: 0.610 
*

These SNPs are located in the 5′-UTR.

No polymorphisms were identified in 300 samples.

Table 3

Risk estimates for select RGS6 and RGS2 SNPs in bladder cancer cases and control subjects

GenotypeAdjusted OR (95% CI)*
RGS2–1 (RGS2-rs4606)RGS6–3 (RGS6-rs3784058)RGS6–2 (RGS6-rs2074647)
Overall    
 Wild-type Reference Reference Reference 
 Variant 0.85 (0.64–1.1) 0.81 (0.60–1.09) 0.66 (0.46–0.95) 
Never smokers    
 Wild–type Reference Reference Reference 
 Variant 1.2 (0.73–1.8) 0.93 (0.57–1.5) 0.78 (0.43–1.4) 
Ever smokers    
 Wild–type Reference Reference Reference 
 Variant 0.71 (0.48–1.0) 0.74 (0.51–1.1) 0.60 (0.39–0.94) 
Age started smoking < 17 y    
 Wild-type Reference Reference Reference 
 Variant 0.61 (0.35–1.07) 0.70 (0.39–1.3) 0.42 (0.20–0.86) 
Age started smoking ≥ 17 y    
 Wild-type Reference Reference Reference 
 Variant 0.81 (0.51–1.3) 0.73 (0.45–1.2) 0.74 (0.41–1.3) 
Age < 64 y    
 Wild-type Reference Reference Reference 
 Variant 0.53 (0.31–0.91) 0.72 (0.40–1.3) 0.45 (0.23–0.90) 
Age ≥ 64 y    
 Wild-type Reference Reference Reference 
 Variant 0.89 (0.56–1.4) 0.73 (0.45–1.2) 0.75 (0.41–1.4) 
GenotypeAdjusted OR (95% CI)*
RGS2–1 (RGS2-rs4606)RGS6–3 (RGS6-rs3784058)RGS6–2 (RGS6-rs2074647)
Overall    
 Wild-type Reference Reference Reference 
 Variant 0.85 (0.64–1.1) 0.81 (0.60–1.09) 0.66 (0.46–0.95) 
Never smokers    
 Wild–type Reference Reference Reference 
 Variant 1.2 (0.73–1.8) 0.93 (0.57–1.5) 0.78 (0.43–1.4) 
Ever smokers    
 Wild–type Reference Reference Reference 
 Variant 0.71 (0.48–1.0) 0.74 (0.51–1.1) 0.60 (0.39–0.94) 
Age started smoking < 17 y    
 Wild-type Reference Reference Reference 
 Variant 0.61 (0.35–1.07) 0.70 (0.39–1.3) 0.42 (0.20–0.86) 
Age started smoking ≥ 17 y    
 Wild-type Reference Reference Reference 
 Variant 0.81 (0.51–1.3) 0.73 (0.45–1.2) 0.74 (0.41–1.3) 
Age < 64 y    
 Wild-type Reference Reference Reference 
 Variant 0.53 (0.31–0.91) 0.72 (0.40–1.3) 0.45 (0.23–0.90) 
Age ≥ 64 y    
 Wild-type Reference Reference Reference 
 Variant 0.89 (0.56–1.4) 0.73 (0.45–1.2) 0.75 (0.41–1.4) 
*

Adjusted for age, gender, and years smoked and adjusted for gender and years smoked in age strata.

Wild-type, homozygous wild-type genotype; Variant, homozygous + heterozygous variant genotype.

Age at diagnosis of bladder carcinoma.

Table 4

Risk estimates for combinations of RGS genotypes

GenotypesNo. of casesNo. of controlsOR (95% CI)Adjusted OR (95% CI)*
RGS2-1RGS6-3RGS6-2
Overall       
 CC CC CC 154 100 Reference Reference 
 CC CC CT+TT 21 36 0.38 (0.21–0.69) 0.34 (0.17–0.65) 
 CG+GG CC CC 113 112 0.66 (0.46–0.9) 0.68 (0.46–01.0) 
 CC CT+TT CC 69 65 0.69 (0.45–1.1) 0.70 (0.44–1.10) 
 CG+GG CC CT+TT 31 24 0.84 (0.47–1.5) 0.77 (0.41–1.5) 
 CC CT+TT CT+TT 10 13 0.50 (0.21–1.2) 0.37 (0.14–1.0) 
 CG+GG CT+TT CC 48 47 0.66 (0.41–1.1) 0.66 (0.39–1.1) 
 CG+GG CT+TT CT+TT 17 0.23 (0.09–0.60) 0.26 (0.09–0.71) 
Ever smokers       
 CC CC CC 122 50 Reference Reference 
 CC CC CT+TT 17 18 0.39 (0.18–0.81) 0.35 (0.16–0.76) 
 CG+GG CC CC 79 58 0.56 (0.35–0.90) 0.54 (0.33–0.88) 
 CC CT+TT CC 50 32 0.64 (0.37–1.11) 0.61 (0.34–1.1) 
 CG+GG CC CT+TT 23 15 0.63 (0.30–1.30) 0.60 (0.28–1.3) 
 CC CT+TT CT+TT 0.48 (0.15–1.49) 0.31 (0.09–1.0) 
 CG+GG CT+TT CC 34 26 0.54 (0.29–0.98) 0.51 (0.27–0.95) 
 CG+GG CT+TT CT+TT 11 0.15 (0.05–0.49) 0.19 (0.06–0.64) 
GenotypesNo. of casesNo. of controlsOR (95% CI)Adjusted OR (95% CI)*
RGS2-1RGS6-3RGS6-2
Overall       
 CC CC CC 154 100 Reference Reference 
 CC CC CT+TT 21 36 0.38 (0.21–0.69) 0.34 (0.17–0.65) 
 CG+GG CC CC 113 112 0.66 (0.46–0.9) 0.68 (0.46–01.0) 
 CC CT+TT CC 69 65 0.69 (0.45–1.1) 0.70 (0.44–1.10) 
 CG+GG CC CT+TT 31 24 0.84 (0.47–1.5) 0.77 (0.41–1.5) 
 CC CT+TT CT+TT 10 13 0.50 (0.21–1.2) 0.37 (0.14–1.0) 
 CG+GG CT+TT CC 48 47 0.66 (0.41–1.1) 0.66 (0.39–1.1) 
 CG+GG CT+TT CT+TT 17 0.23 (0.09–0.60) 0.26 (0.09–0.71) 
Ever smokers       
 CC CC CC 122 50 Reference Reference 
 CC CC CT+TT 17 18 0.39 (0.18–0.81) 0.35 (0.16–0.76) 
 CG+GG CC CC 79 58 0.56 (0.35–0.90) 0.54 (0.33–0.88) 
 CC CT+TT CC 50 32 0.64 (0.37–1.11) 0.61 (0.34–1.1) 
 CG+GG CC CT+TT 23 15 0.63 (0.30–1.30) 0.60 (0.28–1.3) 
 CC CT+TT CT+TT 0.48 (0.15–1.49) 0.31 (0.09–1.0) 
 CG+GG CT+TT CC 34 26 0.54 (0.29–0.98) 0.51 (0.27–0.95) 
 CG+GG CT+TT CT+TT 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.

Table 5

RGS transcript levels as a function of genotype status

Lymphoblastoid cell line genotypeCt
Actin (SD)RGS2 (SD)RGS6* (SD)
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) 
RGS6–3W/RGS6–2V/RGS2–1W 23.9 (0.7) ND 29.1 (1.1) 
RGS6–3W/RGS6–2W/RGS2–1V 24.8 (0.3) 29.3 (0.6) ND 
Lymphoblastoid cell line genotypeCt
Actin (SD)RGS2 (SD)RGS6* (SD)
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) 
RGS6–3W/RGS6–2V/RGS2–1W 23.9 (0.7) ND 29.1 (1.1) 
RGS6–3W/RGS6–2W/RGS2–1V 24.8 (0.3) 29.3 (0.6) ND 

NOTE. Real-time PCR was performed using the Lightcycler (Roche), and Ct value was calculated using Roche Lightcycler software. Results are the mean of at least three experiments.

Abbreviation: ND, no data.

*

Primers designed for full-length RGS6L protein.

The authors would like to thank Dr. Jonathan Coleman for the gift of the bladder cancer cell lines.

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