Abstract
Aim: Bladder cancer is the sixth most common cancer worldwide and its incidence is particularly high in many developed regions including southwestern Taiwan. However, the genetic contribution to the etiology of bladder cancer is not well-understood. The aim of this study was to evaluate the association of the enhancer of zeste homolog 2 (EZH2) genotypes with Taiwan bladder cancer risk. Materials and Methods: Three polymorphic variants of EZH2 were analyzed regarding their association with bladder cancer risk, and three hundred and seventy-five patients with bladder cancer and same number of age- and gender-matched healthy controls recruited were genotyped by the PCR-RFLP method. Results: Among the three polymorphic sites examined, the genotypes of EZH2 rs887569 (C to T), but not rs41277434 (A to C) or rs3757441 (T to C), were positively associated with bladder cancer risk (p for trend =0.0146). Individuals with the EZH2 rs887569 TT genotypes were associated with decreased cancer risk than those with wild-type CC genotype. The stratified analyses showed that EZH2 rs887569 TT genotypes had protective effects on non-smokers but obviously not on smokers. Conclusion: Our findings provide evidence that the T allele of EZH2 rs887569 may be associated with the lower risk of bladder cancer development, especially among non-smokers.
Bladder cancer is the most serious urinary neoplasm worldwide, with high incidence rates of approximately 10/100,000 and 3/100,000 in more and less developed regions, respectively (1). According to the statistical data provided by the International Agency for Research on Cancer, there were an estimated 429,800 new cases of bladder cancer and 165,100 deaths occurred in 2012 worldwide (1, 2). In Taiwan, bladder cancer ranks seventh in incidence and mortality among common carcinomas (3). Carcinogenesis of cancer is a complex, multistep and multifactorial process being the result of interactions of lifestyle, environmental and genetic factors (3-8).
The enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 homolog (SUZ12) are polycomb group proteins which are important epigenetic chromatin modifiers, cell-cycle regulators, and believed to be involved in carcinogenesis. EZH2 is reported to serve as a histone methyl transferase, playing a crucial role in methylation of H3K27 and recruiting of protein regulator of cytokinesis 1 (PRC1), leading to initiation of gene silencing (9-11). In the early twentieth century, EZH2 overexpression was firstly reported in hematological malignancies (12, 13). In recent years, an increasing number of studies have reported overexpression of EZH2 to be associated with poor prognosis in several types of cancers including oesophageal (14), breast (15), endometrial (16), gastric (17-21), colorectal (22, 23), hepatocellular (24), pancreatic (25) and oral (26) cancer.
In bladder cancer, the mRNA levels of EZH2 were found to be higher in bladder cancer specimens than non-tumor bladder specimens, and EZH2 mRNA overexpression was detected in 100% of high-grade and invasive bladder tumors (27). However, the contribution of EZH2 genotypes to susceptibility for abnormal expression of EZH2 and bladder carcinogenesis is unknown. The current work focused on revealing the association of genotypes of EZH2 at rs887569 (C to T), rs41277434 (A to C) and rs3757441 (T to C) among 375 bladder cancer and 375 healthy controls in Taiwan, a highly genetically-conserved population.
Materials and Methods
Study population and sample collection. Our study was approved by the Institutional Review Board of China Medical University Hospital (DMR104-IRB-158), and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Three hundred and seventy-five patients diagnosed with bladder cancer were enrolled at the outpatient clinics of general surgery between 2003 to 2009 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. All patients who voluntarily participated completed a self-administered questionnaire and provided their peripheral blood samples. The clinical characteristics of patients including histological details were all graded and defined by expert surgeons. An equal number of non-cancer healthy controls were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital. The exclusion criteria of the control group included previous malignancy, metastasized cancer from other or unknown origin, and any familial or genetic diseases. Both groups completed a short questionnaire which included personal habits. Smokers were defined as daily or almost daily smokers who had smoked at least five packs of cigarettes per year in their lifetime. Age of smoking initiation, whether they were currently smoking or had already quit, and if so, when they had quit, and on average, how many cigarettes they smoked or had smoked daily were recorded for smokers.
Genotyping conditions. Each participant provided 3-5 ml venous blood. Genomic DNA was prepared within two days from their peripheral blood leucocytes with the protocol of a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and further processed and stored according to our regular methodology (28-32). The polymerase chain reaction (PCR) cycling conditions for all the EZH2 genotyping work were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min using BIO-RAD Mycycler PCR machine (BIO-RAD, Hercules, CA, USA). The primers for rs887569 and rs41277434 genotyping were all designed by our team, and the PCR primer sequences in addition to each responsive restriction enzyme for each DNA product are listed in Table I. Amplified and digested DNA products for rs887569 and rs41277434 were monitored by electrophoresis on 3% agarose gels, stained with ethidium bromide and imaged under UV irradiation. The sequencing for rs3757441 were 100% consistent between the results using forward and reverse primers.
Statistical analyses. Pearson's Chi-square test or Fisher's exact test (when the expected number in any cell was less than five) was used to compare the distribution of the EZH2 genotypic and allelic frequencies between case and control groups. The odds ratios (OR) together with 95% confidence intervals (CI) were calculated to assess the relative risk conferred by a particular allele and genotype. Demographic and clinical data between groups were compared by Chi-square test and by Student's t-test. Data was assumed as significant at a statistical level when the p-value was less than 0.05.
Results
The demographic characteristics of patients with bladder cancer and the non-cancer controls are summarized in Table II. There were no significant differences between groups regarding their age, gender, cigarette smoking and alcohol drinking status (Table II).
The analysis for the distribution frequencies of the genotypes and alleles of EZH2 rs887569 in the bladder cancer and control groups are summarized in Table III. Firstly, there was a significant difference between bladder cancer and control groups in the distribution of genotypic frequency (p for trend=0.0146), and the ORs for those with CT and TT genotype were 0.78 (95% CI=0.58-1.06) and 0.47 (95% CI=0.27-0.80) compared to that for those with the CC wild-type genotype. Secondly, we performed dominant and recessive comparisons, finding that the ORs of the CC+CT versus TT and CC versus CT+TT were 0.53 (95% CI=0.31-0.89, p=0.0212) and 0.72 (95% CI=0.54-0.96, p=0.0329), respectively. Lastly, from the results of EZH2 rs887569 allelic frequency analysis, we found that people carrying the T allele appear to have a protective effect, compared to those carrying an A allele (OR=0.74, 95% CI=0.60-0.92, p=0.0069) (Table III). For EZH2 rs41277434 (Table IV) and rs3757441 (Table V), there was no difference in the distribution of either genotypic or allelic frequencies between patient and control groups. The conclusive finding deduced from the results in Tables III, IV and V is that the T allele of EZH2 rs887569 or TT genotype may serve as a protective biomarker for bladder cancer detection and prediction.
After finding that T-bearing genotypes of EZH2 rs887569 were associated with bladder cancer risk, we examined the interaction between the genotype of EZH2 rs887569 and cigarette smoking and alcohol drinking habits. The results showed that the genotypic distribution of CC, CT and TT at EZH2 rs887569 was significantly different between bladder cancer and control non-smokers (p for trend=0.0164) (Table VI). Consistent with the findings in Table III, the frequency of TT genotype was still significantly lower (5.8%) in non-smoking patients with bladder cancer than those for control non-smokers (13.2%). On the contrary, there was no such distribution difference in the smoking subgroups (p for trend=0.9196). There was no obvious interaction between the genotype of EZH2 rs887569 and personal alcohol drinking habits (data not shown). For EZH2 rs41277434 and rs3757441, there was no distribution difference in the smoking, non-smoking, alcohol drinking, and non-alcohol drinking sub-groups (data not shown).
Discussion
EZH2 plays a role not only in cell-cycle regulation and proliferation, but also in tissue maturation and differentiation (33, 34). In the current study, we selected three polymorphic sites of the EZH2 gene, rs887569 (C to T), rs41277434 (A to C) and rs3757441 (T to C), and clarified their associations with susceptibility for bladder cancer risk. EZH2 rs887569 is located in the 19th intron of the EZH2 gene, which was reported to bind with the PPAR-α/RXR-α complex. The PPAR-α/RXR-α complex can down-regulate EZH2 expression and trigger the cell to undergo programmed cell death and suppress cell proliferation (35). The significant findings showed that homozygous TT genotype, but not heterozygous CT of EZH2 rs887569 was associated with decreased susceptibility for bladder cancer in the investigated Taiwanese population, comparing with those with CC wild-type genotype (Table III). This novel finding in bladder cancer is supported by previous findings that the T allele of EZH2 rs887569 is associated with decreased susceptibility for colorectal cancer in another Han population in Shandong, an eastern province of China (23). In addition, TT genotype of EZH2 rs887569 was associated with smaller tumor size and less differentiation of colorectal cancer (23). However, the authors also found that the C allele of rs3757441 was also associated with colorectal cancer risk, but similar results were not found in this study for bladder cancer (Table V). In another supporting report for the current study, the TT genotype of EZH2 rs887569 was also found to be associated with longer overall survival and lower mortality among patients with lung cancer (35).
To investigate the joint effects of genotypic and lifestyle factors, we firstly analyzed the interactions of EZH2 rs887569 genotype and with factors such as smoking and alcohol drinking. The genotypes of EZH2 rs887569 indeed had joint effects with individual smoking habits on bladder cancer susceptibility (Table VI): the protective T allele at EZH2 rs887569 may contribute to lower susceptibility for non-smokers, but not for smokers (Table VI). At the same time, no obvious joint effect of EZH2 rs887569 genotype with alcohol drinking habits on bladder cancer risk was observed in this population.
To sum up, to our knowledge, this is the first study to focus on EZH2 genotype and its synergistic effects with smoking or alcohol drinking habit on bladder cancer risk. The TT genotype of EZH2 rs887569 appears to be protective against bladder carcinogenesis, especially in non-smokers. The findings in bladder cancer should be validated in larger samples and other ethnicities. Molecular mechanisms and functional studies are also required.
Acknowledgements
The Authors declare no conflict of interest in regard to this study. This study was supported mainly by Taichung Armed Forces General Hospital to Dr. Liao (supporting number 105-A14) and partially by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-113019). We thank Hsin-Ting Li, Shiou-Ting Yen and colleagues in Tissue bank of China Medical University Hospital for their technical assistance.
Footnotes
↵* These Authors contributed equally to this study.
- Received June 25, 2016.
- Revision received July 18, 2016.
- Accepted July 25, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved