[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).