RUNX3 methylation reveals that bladder tumors are older in patients with a history of smoking

Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma tumors at BCL2, PTGS2 (COX2)' DAPK, CDHI (ECAD), EDAWB, RASSFIA, RUAX3, TERT, and TIMP3. The prevalence of methylation at RUAX3, a polycomb target gene, increased as a function of age at diagnosis (P = 0.031) and a history of smoking (P = 0.015). RUAX3 methylation also preceded methylation at the other eight genes (P < 0.001). It has been proposed that DNA methylation patterns constitute a "molecular clock"and can be used to determine the "age" of normal tissues (i.e., the number of times the cells have divided). Because RLTAX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are "older" than tumors from nonsmokers (P = 0.009) due to tumors in smokers either initiating earlier or undergoing more rapid cell divisions. Because RUAX3 methylation is acquired early on in tumorigenesis, then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present.