DNA Methyltransferase 1(DNMT1) Represses Regulator of G Protein Signaling 6 (RGS6) Expression in BBN‐induced Bladder Carcinogenesis

Abstract

Urinary bladder cancer, the fifth most common cancer in the United States, is largely caused by exposure to toxic chemicals including those from smoking. Polymorphisms in the RGS6 gene leading to increased RGS6 expression are associated with a pronounced reduction in the risk of bladder cancer, especially among smokers. Recent studies in our laboratory indicate that RGS6 functions as a tumor suppressor in bladder as RGS6−/− mice exhibit a dramatic acceleration of bladder carcinogenesis induced by 4‐Hydroxybutyl(butyl)nitrosamine (BBN), a model that closely resembles human invasive bladder cancer. We also found that BBN‐induced bladder carcinogenesis in wild‐type (WT) mice is associated with loss of RGS6 expression. Given the tumor suppressor role of RGS6 in bladder we undertook studies to investigate the mechanisms underlying its loss during bladder carcinogenesis. In silico analyses of the RGS6 gene promoter revealed a highly conserved CpG island, located at −491/+398 or −751/+299 in the human and mouse RGS6 gene, respectively. Given that CpGs are substrates of DNMT1, whose increased expression is correlated with human bladder cancer progression, we investigated the role of DNMT1 in modulating RGS6 expression during bladder carcinogenesis. BBN treatment of mice led to a reciprocal change in expression of DNMT1 and RGS6 in bladder epithelium, where transitional carcinoma originates. The normally high level of RGS6 and low level of DNMT1 expression in bladder epithelium was reversed within 4 days of BBN treatment. Genomic DNA isolated from BBN‐treated mouse bladder epithelium revealed a time‐dependent methylation of the RGS6 gene promoter, consistent with the observed upregulation of DNMT1 and downregulation of RGS6 during carcinogenesis. Given that DNMT1 possesses transcriptional repressor activity independent of its methylation activity, we next examined whether DNMT1 repressed RGS6 promoter activity using a mouse RGS6 promoter driven luciferase (Rgs6‐luc) construct. In PC6 cells which exhibit a high level of RGS6 promoter activity, consistent with high neuronal expression of RGS6, transient transfection with DNMT1 markedly repressed Rgs6‐luc reporter activity. In contrast, RGS6 promoter activity was barely detectable in T24 bladder cancer cells, possibly reflecting DNMT1 mediated silencing. To test this possibility we examined the effects of treating cells with DNMT1 inhibitor 5‐Aza‐2′‐deoxycytidine (5‐Aza) on RGS6 promoter activity. Consistently, we observed that RGS6 promoter activity in T24 stable cell lines was induced by treatment with 5‐Aza. In conclusion, our data demonstrates that DNMT1 negatively regulates RGS6 gene promoter activity during bladder carcinogenesis, likely via a combination of DNMT1 effects on the RGS6 promoter including direct repression and methylation of a highly conserved CpG island. Given our findings that RGS6 promotes Tip60‐mediated DNMT1 degradation, silencing RGS6 by DNMT1 would represent a feed forward loop in which loss of RGS6 further promotes cellular accumulation of DNMT1 and bladder carcinogenesis.Support or Funding InformationSupported by AHA 11SDG7580008 (JY) and NIH CA161882 (RAF)