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Cancer Research 68, 6208, August 1, 2008. doi: 10.1158/0008-5472.CAN-07-6616
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

RUNX3 Methylation Reveals that Bladder Tumors Are Older in Patients with a History of Smoking

Erika M. Wolff1, Gangning Liang1, Connie C. Cortez1, Yvonne C. Tsai1, J. Esteban Castelao2, Victoria K. Cortessis2, Denice D. Tsao-Wei2, Susan Groshen2 and Peter A. Jones1

Departments of 1 Urology and 2 Preventive Medicine, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California

Requests for reprints: Peter A. Jones, University of Southern California/Norris Comprehensive Cancer Center, Room 8302L, 1441 Eastlake Avenue, MC-9181, Los Angeles, CA 90089. Phone: 323-865-0816; Fax: 323-865-0102; E-mail: jones_p{at}ccnt.hsc.usc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
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. [Cancer Res 2008;68(15):6208–14]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Cancer etiology involves interactions between the environment, the genome, and the epigenome (1, 2). The epigenome consists of several layers of heritable transcriptional regulation imposed on the genome, including DNA methylation, histone modifications, and nucleosome positioning (3). DNA becomes methylated at CpG dinucleotides and is associated with transcriptional repression when it occurs in CpG-rich regions (CpG islands) located in gene promoters (4). During carcinogenesis, CpG islands become aberrantly hypermethylated (5). Alterations in the epigenome also accumulate during the aging process (6). For instance, normal epithelium in the colon acquires methylation in an age-dependent manner at genes that subsequently become hypermethylated in colon cancer (7). Therefore, 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; ref. 8). It may also be possible to use methylation as a molecular clock to determine the age of tumor tissues by using a locus that is specifically methylated in tumor cells and does not acquire methylation in normal tissues as a function of aging. Information about the age of a tumor may give insight into when tumorigenesis initiated and have important implications for early detection.

Numerous types of environmental exposures, such as tobacco smoke, arsenic, cadmium, and nickel, are associated with aberrant DNA methylation (2). Specifically, tobacco-derived carcinogens are associated with DNA methylation at p16 (9), CYP1A1 (10), and RASSF1A (11) in lung tumors and p16 in bladder tumors (12). Marsit and colleagues (13) recently measured promoter hypermethylation of 16 different genes in bladder tumors and found a correlation between overall methylation and age, gender, and smoking history. However, their analysis combined the methylation at all the genes into one measure based on the premise that hypermethylation of promoters is not a targeted event. Whereas the precise mechanism resulting in de novo methylation of specific CpG islands in cancer is currently unknown, recent work has shown that genes targeted by polycomb complexes in embryonic stem cells are more likely than other genes to undergo promoter hypermethylation during carcinogenesis (14–16).

Bladder cancer is the fifth most commonly diagnosed cancer in the United States, where the majority of tumors are transitional cell carcinoma (17). Long-term cigarette smokers are 2.5 times more likely to develop bladder cancer than nonsmokers (18). In this study, we took advantage of a large number of transitional cell carcinoma samples from patients with known age, gender, and exposure to tobacco smoke to address the possibility that these factors are associated with epigenetic alterations. We used the real-time methylation-sensitive PCR assay MethyLight, developed by Eads and colleagues (19), to specifically and sensitively detect DNA methylation at nine different genes [RUNX3, BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, TERT, and TIMP3] in bladder tumor DNA samples from 342 patients. RUNX3 is a tumor suppressor involved in apoptosis and is both frequently silenced by methylation in bladder cancer (20, 21) and a confirmed polycomb target (22–24). RUNX3 methylation is associated with bladder tumor grade, invasiveness (20), stage, recurrence, and progression (21). Our results revealed interactions between environmental exposure, aging, and the epigenome. In particular, we have shown for the first time that DNA methylation, in addition to being used as a molecular clock to determine the age of normal tissues, can determine the age of tumors, and our RUNX3 methylation data suggest that bladder tumors from smokers are "older" than those from nonsmokers. Therefore, bladder tumorigenesis may initiate early in a smoker's life, whereas tumors from nonsmokers initiate at a later age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Study population. From 1987 to 1996, a population-based case-control study of bladder cancer was conducted in Los Angeles County as previously described (18). Eligibility criteria for cases included histologically confirmed transitional cell carcinoma diagnosed between January 1, 1987 and April 30, 1996 among non-Asian patients of ages 25 to 65 years. In total, 2,098 cases were identified through the Los Angeles County Cancer Surveillance Program, and of these 1,582 patients were included as part of case-control pairs.

Tissue collection. Hospitals and pathology laboratories provided tumor tissue blocks to the Los Angeles County Surveillance, Epidemiology and End Results Registry Slide Retrieval Program, a component of the Tissue Procurement Core Resource of the University of Southern California/Norris Comprehensive Cancer Center. Of the specimens retrieved, 342 cases had sufficient tumors available to permit the analysis of DNA methylation (Table 1 ). Frozen blocks of matched bladder tumors and corresponding mucosa have previously been described (25) and urothelia from cancer-free bladders were obtained from age-matched patients undergoing prostatectomies. All study subjects had signed informed consent forms approved by the Human Subjects Committee at the University of Southern California Keck School of Medicine.


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  		Table 1. Clinicopathologic characteristics of study population

 
DNA isolation and sodium bisulfite modification. DNA extraction from frozen tissues has previously been described (25). Microdissection was done on one to three consecutive 5-µm sections (H&E stained) composed mainly of tumor tissues from 342 transitional cell carcinoma patients. DNA was extracted and bisulfite converted as previously described (26, 27).

Quantitative methylation-sensitive real-time PCR. Methylation analysis was done as previously described (26). Briefly, the proportion of bisulfite-converted DNA in each sample was controlled for by a collagen IIA (COL2A1) reaction, which only amplifies bisulfite-converted DNA and is located in a region with no CpG sites to be independent of the methylation status. TNFRSF25 (DR3) was used as a positive control and FADD as a negative control for methylation. Genomic DNA (Promega) was treated with SssI DNA methyltransferase (New England Biolabs) and used as a fully methylated reference with which all samples were compared to yield the percentage of fully methylated DNA (PMR; ref. 26). Primer sequences and locations have previously been described for BCL2, DAPK, EDNRB, FADD, RASSF1A, TERT, TNFRSF25 (DR3), COL2A1 (26), CDH1 (ECAD; ref. 28), PTGS2 (COX2), and TIMP3 (29). RUNX3 (NM_004350, –326/–258) forward primer, probe, and reverse primer sequences were as follows: 5'-CGTTTTAGCGTTAGGGAGTTACG-3'; 6FAM, 5'-TTTGAGAGAGGGCGGTAAGGGCG-3'; BHQ1, 5'-AACGTCCGAATCCCACGA-3'.

Quantification of DNA methylation by methylation-sensitive single nucleotide primer extension. Quantification of methylation at specific CpG sites using methylation-sensitive single nucleotide primer extension, a method developed in our lab, has previously been described (30). The promoter region of RUNX3 was amplified with primers specific for bisulfite-converted DNA (forward, 5'-GGGGTTGTAGAAGTTATAGGT-3'; reverse, 5'-CCAATACCACAACCCAAAAC-3') and with an annealing temperature of 58°C, and two specific CpG sites were assayed using primers for single nucleotide primer extension (5'-GGGGTTGTAGAAGTTATAGGTT-3' and 5'-TAGTAAGAGTTGGGGAAGTT-3') with an annealing temperature of 56°C. RUNX3 methylation was calculated as the average percent methylation of two CpG sites.

Statistical analysis. For each gene (BCL2, COX2, DAPK, ECAD, EDNRB, RASSF1A, RUNX3, TERT, and TIMP3) the methylation, measured as the percent of the fully methylated reference (PMR; ref. 31), was scored in two ways: as the rank among study samples of all PMR values for that gene and as methylated (PMR ≥10%) or unmethylated (PMR <10%), using a biologically determined cutoff value. For each patient, the following information was also included in the analysis: age at diagnosis, sex, smoking history, tumor stage, and tumor grade (Table 1). To assess the overall association between each of these characteristics and the PMR ranking for each gene, patient and tumor characteristics were evaluated using either the Wilcoxon test for the dichotomous characteristics of sex (male and female), grade (low grade and high grade), and smoking history (never/irregular smokers and former/current smokers) or the Kruskal-Wallis test for the characteristics with three or more categories of tumor stage [Ta, carcinoma in situ (CIS), T1, and T2–T4] and patient's age at diagnosis (≤40, 41–50, 51–60, and 61–65 y; Table 2 ).


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  		Table 2. Association of demographics with gene methylation (PMR)

 
A logistic regression model was used to evaluate the association between RUNX3 methylation, classified as positive (PMR ≥10%) or negative, and clinicopathologic characteristics (Table 3 ). The model used in the logistic regression included the following variables: age at diagnosis (1-y increase), smoking history (never/irregular versus former/current), sex, and tumor stage and grade (Ta low grade, Ta high grade, CIS, T1, and T2–T4). The interaction term for smoking status and age was not significant (P = 0.23) and was omitted from the final model; similarly, a quadratic term for age, to capture a nonlinear relationship between age and the log odds of methylation, was also not significant and was omitted in the final model. P values were based on a likelihood ratio test. The curves fit to these data display the association between age and probability of methylation for smokers and nonsmokers separately; the raw data were smoothed using the LOESS procedure (32), and the estimated probability of methylation was based on the logistic regression model with age as the only covariate. All P values were reported as two-sided.


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  		Table 3. Effect of sex, age, tumor stage, tumor grade, and smoking history on RUNX3 methylation (PMR ≥10%; N = 304)

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
RUNX3 is frequently and specifically methylated in bladder tumors. Because the methylation at nine genes in 10 urothelium samples from cancer-free bladders were all <10% PMR, this value was used as a biological cutoff for the presence or absence of methylation (Fig. 1A ). In matched sets of bladder tumors and corresponding tissues, EDNRB, RASSF1A, and BCL2 were frequently methylated in both the tumors (65%, 60%, and 39%) and the corresponding tissues (15%, 11%, and 9%; Fig. 1A). Whereas TERT was methylated in 30% of tumors and only 4% of corresponding tissues, RUNX3 was the most specific marker for bladder cancer, with frequent methylation in the tumor samples (23 of 41, 56%) and infrequent methylation in the corresponding tissues (2 of 46, 4%). The two corresponding tissues with RUNX3 methylation have nearly identical methylation patterns as their matched high-grade and invasive tumors, suggesting that these bladders had epigenetic aberrancies throughout or that the invasive tumor had spread across the bladder. The pathologic reports for these two cases indicated that the first case had multiple foci of carcinoma in situ and severe urothelial atypia and the second case had diffuse lesions throughout the bladder. Therefore, the two corresponding tissue samples with RUNX3 methylation were atypical and are not considered "normal." Of the other 44 cases, 2 had areas of urothelial hyperplasia and 1 had moderate chronic cystitis, although there was no detectable methylation in the corresponding tissues, and 1 case had areas of squamous metaplasia with methylation at TIMP3, COX2, and EDNRB in the corresponding tissue.


Figure 1
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  		Figure 1. Methylation in bladder samples at nine different loci using quantitative methylation-sensitive real-time PCR and shown as PMR. Black, PMR >10%; gray, PMR <10%; white, unavailable data. 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.

 
Because the matched tumor and corresponding tissue sets were taken from patients undergoing cystectomies, most of the tumors were highly invasive. Therefore, we extended our study to include more noninvasive and low-grade tumor samples obtained by transurethral resections. When we assayed the methylation status of nine genes in these additional 342 bladder tumor samples, RUNX3 was still the most frequently methylated gene (39%), with the next most frequently methylated genes being RASSF1A (37%), EDNRB (30%), and BCL2 (28%; Fig. 1B; Table 2).

To confirm our methylation results, we used another methylation assay, methylation-sensitive single-nucleotide primer extension, which was developed in our lab (30) and is able to quantify the methylation at specific CpG sites. When we examined two CpG sites just downstream of the region assayed using MethyLight, we found that five of seven bladder tumors had high levels of RUNX3 methylation and only one of seven corresponding tissues had a high level of RUNX3 methylation in an independent cohort of seven matched sets (Fig. 2 ). On examination of the pathologic reports for these patients, it was noted that in the patient with RUNX3 methylation in the corresponding tissue (case 5), the entire bladder showed signs of cystitis cystica, a type of proliferative cystitis, with acute and chronic inflammation and had multifocal carcinoma in situ. Of the other six cases, only one had reported areas of urothelial atypia.


Figure 2
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  		Figure 2. Average percent methylation of two CpG sites located in the promoter of RUNX3 in seven cases of matched bladder tumors (black columns) and corresponding tissues (gray columns) measured by methylation-sensitive single-nucleotide primer extension.

 
RUNX3 methylation precedes methylation at the other eight genes. Methylation at all genes except COX2 and TERT was significantly associated with tumor grade, and methylation at EDNRB, RASSF1A, RUNX3, and TIMP3 was associated with tumor stage, revealing that methylation was more likely in tumors of higher grade or stage (Table 2). Based on the assumption that tumors of the lowest stage and grade are the precursors of tumors of higher stage and grade, we compared the methylation patterns of the four most frequently methylated genes in Ta low grade tumors to tumors of higher stage and grade to determine whether RUNX3 methylation precedes methylation at the other genes (Fig. 1B). An analysis of the discordance of RUNX3 methylation revealed that of the 20 tumors that had only RUNX3 methylated, 17 (85%) cases were Ta low grade tumors, compared with 32 of 89 (36%) cases with methylation at any of the other genes besides RUNX3 (P < 0.001, Fisher's exact test). In contrast, methylation of EDNRB (35% compared with 31%, P = 0.80) and BCL2 (33% compared with 28%, P = 0.67) showed no discordance of methylation whereas RASSF1A showed the opposite pattern of RUNX3, with significantly more methylation in tumors of higher grade and stage (24% compared with 44%, P = 0.042). The observed discordance is compatible with the hypothesis that RUNX3 methylation occurs early in tumorigenesis and before methylation of any of the other genes examined.

RUNX3 methylation increases with age and a history of smoking. Only methylation at RUNX3 was significantly associated with the age at which diagnosis of transitional cell carcinoma was made (P = 0.027) and a history of tobacco smoking (P = 0.004) based on univariable analyses (Table 2). Using a logistic regression model controlling for age, sex, tumor stage, and grade, we found methylation of RUNX3 more frequently in tumors of smokers versus those of nonsmokers [P = 0.015; odds ratio, 2.76 (95% confidence interval, 1.15, 6.66); Table 3]. There was no effect of the duration of smoking when added to the above logistic regression model (P = 0.88). Methylation at RUNX3 was significantly associated with the age at diagnosis (P = 0.031) based on a logistic regression adjusting for sex, smoking history, tumor stage, and grade.

To examine the joint association between smoking, age, and RUNX3 methylation, we used a logistic regression model with age, smoking history, tumor stage, and grade (Fig. 3 ). We found a statistically significant association between RUNX3 methylation and both age (P = 0.004) and smoking status (P = 0.009). The probability of RUNX3 methylation is higher in smokers and increases as a function of age in both nonsmokers and smokers (Fig. 3). Because RUNX3 methylation increases with age, is not present in the normal urothelium, and occurs early in tumorigenesis, it can be used as a molecular clock to determine the age of a bladder tumor, and doing so reveals that tumors from smokers are older than tumors from nonsmokers.


Figure 3
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  		Figure 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 gray line) and for never or irregular smokers (n = 38; dashed black line). These fitted curves are superimposed over the raw data that have been smoothed using the LOESS procedure. Solid gray line, smokers; solid black line, nonsmokers. The logistic regression analysis was done 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this study, we evaluated the methylation of nine genes in transitional cell carcinoma samples from 342 patients about whom detailed demographic, clinicopathologic, and smoking information had previously been collected (18). We found several genes to be more frequently methylated in tumors of higher stage and grade. The four most commonly methylated genes in our cohort of bladder tumors are involved in apoptotic pathways, including RUNX3, BCL2, EDNRB, and RASSF1A (Fig. 1), consistent with our previous study (26). In addition, we found that RUNX3 is not methylated in normal-appearing tissues corresponding to tumors or tissues from cancer-free bladders. However, because quantitative methylation-sensitive real-time PCR detects individual strands of DNA that are simultaneously methylated at all sites, a cutoff value of 10% PMR cannot rule out the presence of sporadic sites of methylation. Therefore, we measured methylation of two specific CpG sites in the RUNX3 promoter in a small independent cohort of matched tumors and corresponding tissues. We found significant levels of methylation in five of the seven tumors and only one of the corresponding tissues (Fig. 2). However, this specific corresponding tissue is not considered to be nonneoplastic because it was taken from a bladder with cystitis cystica with acute and chronic inflammation and multifocal carcinoma in situ. In addition, other studies have also found that RUNX3 is not methylated in normal bladder (21), prostate (20, 33, 34), lung (20), breast (20), and colon (34) tissues using a variety of methods and is also never or infrequently methylated in nonneoplastic tissues corresponding to bladder tumors (20), gastric tumors (34, 35), breast tumors (36), non–small cell lung tumors (37), and hepatocellular carcinomas (38). Because RUNX3 is not methylated in normal urothelia or normal-appearing tissues corresponding to tumors, we can assume that RUNX3 methylation was not initially present in the normal urothelia of these patients, is specific to bladder tumors, and is not acquired as a function of aging.

We have shown that RUNX3 methylation precedes methylation at the other eight genes we examined, indicating that it is an early event in bladder tumorigenesis. RUNX3 methylation seems to occur early in tumorigenesis in a variety of cancer types. Methylation of RUNX3 has been found in invasive ductal breast carcinoma and its precursor lesion of ductal carcinoma in situ (39), prostate cancer and its precursor prostatic intraepithelial neoplasia (33), and gastric cancer and its precursor lesions of chronic gastritis and intestinal metaplasia (34). RUNX3 methylation also occurs in Barrett's esophagus, a precursor of esophageal adenocarcinoma, and is associated with progression to esophageal adenocarcinoma (40). In addition, we found methylation in the corresponding tissues from three bladders, two of which had multifocal carcinoma in situ and acute and chronic inflammation with cystitis cystica, a proliferative cystitis, or severe urothelial atypia, and one of which with diffuse lesions throughout the bladder. Other groups have shown that similar inflammatory lesions in the bladder express telomerase (41, 42), indicating that these lesions are likely to be undergoing immortalization or malignant transformation. RUNX3 is a polycomb target (24) and several groups have found that polycomb targets become preferentially methylated during cancer (14–16), lending support to the theory that cancer is derived from stem cells. Several preinitiation events occur in stem cells before they are initiated and then clonally expand during promotion (43). Initiating events are irreversible, and if they occur in stem cells then that event will remain in the asymmetrically dividing stem cell. Therefore, our results showing that corresponding tissue from a bladder with widespread inflammatory lesions has elevated RUNX3 methylation indicate that RUNX3 methylation occurs earlier than tumorigenesis and accumulates in the bladder stem cells that will eventually develop into a tumor.

According to a multivariable logistic regression model, the probability of RUNX3 methylation increases with age in both smokers and nonsmokers. Biological age is a surrogate marker for the number of times a cell has divided, and the more times a cell divides the more opportunities for aberrant methylation to accumulate (44). Because the degree of hypermethylation in normal colonic epithelium is related to age (7), it has been suggested that methylation can be used as a molecular clock to predict the age of a tissue (8). In addition, previous work has shown that tumors from older patients can have more methylation than tumors from younger patients (45). Therefore, we are able to use RUNX3 methylation to show for the first time that methylation in tumor cells might be useful as a molecular clock. We have shown that RUNX3 methylation is present early in tumorigenesis and that the level increases with age (Fig. 3). When we apply our RUNX3 molecular clock to tumors from smokers versus nonsmokers, our data suggest that bladder tumors in smokers are older than those in nonsmokers (i.e., have undergone more cell divisions before diagnosis) because RUNX3 is more prevalent in tumors from smokers. There are at least two possible explanations for such an observation: either tumors in smokers age or divide more quickly than tumors in nonsmokers, resulting in faster accumulation of methylation errors over a similar amount of time, or tumors in smokers were initiated at an earlier age compared with tumors of nonsmokers and therefore have had more time to divide and accumulate RUNX3 methylation. Because RUNX3 methylation seems to be an early event in bladder tumorigenesis and increases over time, then detection of RUNX3 methylation in biopsy or urine specimens could provide a marker to screen the at-risk population of cigarette smokers long before any symptoms are present.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Cancer Institute grant P01 CA86871.

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.

Received 12/11/07. Revised 5/20/08. Accepted 5/24/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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