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

Abstract

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, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (P = 0.031) and a history of smoking (P = 0.015). RUNX3 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 RUNX3 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 RUNX3 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.

Fig. 1

Methylation in bladder samples at nine different loci using quantitative methylation-sensitive real-time PCR and shown as percent of a fully methylated reference (PMR), with PMR > 10% in grey, PMR ≤ 10% in black, and unavailable data in white. A Ten normal urothelium samples from cancer-free bladders were obtained during prostatectomies from age-matched patients. Forty-six matched sets of bladder tumor tissue and corresponding tissue were obtained during cystectomies. B Methylation data for 342 bladder cancer samples from paraffin-embedded tissues.

Fig. 2

Average percent methylation of two CpG sites located in the promoter of RUNX3 in 7 cases of matched bladder tumors (black bars) and corresponding tissues (grey bars) measured by Ms-SNuPE.

Fig. 3

Estimated probability of RUNX3 methylation (PMR ≥ 10%) is plotted as a function of age at diagnosis for current or former smokers (N=265, dashed grey line) and for never or irregular smokers (N=38, dashed black line). These fitted curves are superimposed over the raw data that has been smoothed using a LOESS procedure, with the smokers represented by the solid grey line and the nonsmokers by the solid black line. The logistic regression analysis was performed on tumors from 304 patients and yielded a statistically significant association between RUNX3 methylation and smoking history (p=0.009, likelihood ratio test), adjusted by age at diagnosis, tumor grade, and tumor stage.