A Six-Nucleotide Insertion-Deletion Polymorphism in the CASP8 Promoter Associated with Risk and Progression of Bladder Cancer

Apoptosis is an essential genetic program necessary for the proper development of an organism (1). Thus far, two major apoptosis signaling pathways have been described: extrinsic and intrinsic pathways. In both pathways, the initiator caspase, caspase-8 encoded by CASP8 (MIM: 601763) located at 2q33, plays an important role in transducing the death signal to more downstream death effectors such as caspase-3 and caspase-7 (2). In the extrinsic apoptosis pathway, caspase-8 triggers apoptosis caused mainly by death receptor-induced apoptotic signaling and mediated by Fas and Fas ligand (3, 4). Studies have reported that two common polymorphic variants of the CASP8 gene, D302H (rs1045485) and 6N ins/del (rs3834129), are associated with risk of multiple cancers (5–11). However, the D302H polymorphism with unknown functionality seems extremely rare (minor allele frequency <1%) in Asian populations (based on the HapMap Project and the Environmental Genome Project databases). Recently, Sun et al. (8) identified a 6-bp ins/del polymorphism (-652 6N ins/del) in the CASP8 promoter that may abolish the Sp1 transcription factor binding site and was associated with the decreased RNA expression in lymphocytes and lower caspase-8 and T lymphocyte apoptotic activities. This deletion variant was found to have a protection against several types of cancer (lung, esophageal, stomach, colorectal, breast, and cervical cancers) in Chinese populations. To the best of our knowledge, no published studies have investigated the role of CASP8 polymorphisms in the development of bladder cancer.

Bladder cancer, the ninth most frequent cancer throughout the world, is an important health problem. A 2005 analysis of the worldwide incidence of and mortality from cancer showed that 357,000 cases of bladder cancer occurred in 2002 and that 145,000 patients die of this disease annually (12). Tobacco smoking is a predominant risk factor for bladder cancer, responsible for nearly half of the cases in men and for one third in women, whereas occupational exposure to carcinogens is the second major risk factor (13). However, only a part of the exposed individuals develop bladder cancer in their lifetime, suggesting that genetic susceptibility also play a role in bladder carcinogenesis.

In the present study, we hypothesized that the CASP8 -652 6N ins/del polymorphism is associated with risk of bladder cancer. To test this hypothesis, we genotyped the polymorphism and assessed its association with risk of bladder cancer in our ongoing, hospital-based, case-control study in a Chinese population.

Study subjects. The analysis included 365 histologically confirmed transitional cell carcinoma of bladder cases and 368 cancer-free control subjects. All subjects were recruited in an ongoing study starting in January 2003. The detailed diagnosis of patients was described previously (14). Approximately 95% of the eligible patients contacted chose to participate. The cancer-free control subjects were genetically unrelated to the cases and had no individual history of cancer. The controls were recruited from healthy subjects who were seeking health care in the outpatient departments at the hospital. All controls of appropriate age and sex for frequency matching with the cases were recruited and included if they gave their informed consent. As for the cases, exclusion criteria for the controls were significant mental impairment or blood transfusion in the past months; controls were also excluded if they had symptoms suggestive of bladder cancer, such as hematuria. The response rate of those control subjects we approached for participation in the study was >85%. Before recruitment, informed consent was obtained from each of the eligible subjects. Information about individual demographics as well as data on smoking status were obtained through face-to-face interviews. Those subjects who smoked daily for >1 year were defined as ever smokers. Ever smokers who had quit smoking for >1 year were defined as former smokers, and the other smokers as current smokers. Pack-years [(cigarettes per day / 20) × years smoked] were calculated to indicate the cumulative smoking dose, and the smokers were further categorized into two groups: light smokers (pack-years ≤20) and heavy smokers (pack-years >20) according to the median pack-year value of ever smokers. All patients were classified according to the 1997 International Union Against Cancer tumor-node-metastasis classification for the stage and OMS 2004 for the grade (15). The research protocol was approved by the institutional review board of Nanjing Medical University.

Genotyping. The CASP8 -652 6N ins/del polymorphism was determined using the PCR-restriction fragment length polymorphism method. The primers, lengths, and restriction enzymes have been described previously (8). The polymorphism analysis was done by two persons independently in a blind fashion. More than 15% of the samples were randomly selected for confirmation, and the results were 100% concordant.

Statistical analysis. χ² test was used to compare the differences in frequency distributions of selected demographic variables, the known risk factors, such as tobacco smoking, each allele and genotype of the CASP8 polymorphism between cases and controls. Hardy-Weinberg equilibrium of the controls' genotype distributions was tested by a goodness-of-fit χ² test. Unconditional univariate and multivariate logistic regression analyses were done to obtain crude and adjusted odds ratios (OR) for risk of bladder cancer and their 95% confidence intervals (95% CI). Tumor stage analyses were done by comparing superficial tumors (pT_a-pT₁) versus invasive tumors (pT₂-pT₄) as well as grading analyses including low-grade tumors (grades 1 and 2) versus high-grade tumors (grade 3) as reported previously (16). To explore potential interaction between the polymorphism and tobacco smoking, we tested the null hypotheses of multiplicative gene-environment interaction by evaluating the departure from multiplicative interaction model that included main-effect variables and their product terms in the logistic regression model (17). When the test for multiplicative interaction was not rejected, the additive interaction was further assessed by a bootstrapping test by using the Stata software (version 8.2; StataCorp). All tests were two-sided by using the SAS software (version 9.1; SAS Institute) unless indicated otherwise.

Characteristics of the study subjects. The frequency distributions of selected characteristics of the cases and controls are presented in Table 1. There was no significant difference in the distribution of age and sex between cases and controls (P = 0.503 for age and 0.608 for sex), but there was a significant difference in smoking status between cases and controls. There were more ever smokers among the cases (56.1%) than among the controls (40.5%; P < 0.001). Specifically, light smokers (≤20 pack-years) had a 2.08-fold (95% CI, 1.45-2.98) and heavy smokers (>20 pack-years) had a 1.69-fold (95% CI, 1.18-2.44) increased risk compared with nonsmokers. In addition, of the 365 bladder cancer patients, 303 (83.0%) had a low-grade tumor and the remaining 62 (17.0%) had a high-grade tumor. Furthermore, 231 (63.3%) had a superficial tumor and the remaining 134 (36.7%) had an invasive tumor.

Association between CASP8 polymorphism and bladder cancer risk. As shown in Table 2, the frequencies of the ins/ins, ins/del, and del/del genotypes were 65.2%, 31.5%, and 3.3%, respectively, among the case, and 55.7%, 37.5%, and 6.8%, respectively, among the controls (P = 0.0105). Furthermore, the CASP8 del allele frequency was 0.190 among the cases and 0.255 among the controls, and the difference was statistically significant (P = 0.0034). The observed genotype frequencies among the control subjects were in agreement with the Hardy-Weinberg equilibrium (χ² = 0.074; P = 0.786).

In the present study, when we used the CASP8 -652 6N ins/ins genotype as the reference, we found that both ins/del and del/del genotypes were associated with a statistically significantly decreased risk of bladder cancer (adjusted OR, 0.72; 95% CI, 0.53-0.99 for ins/del and OR, 0.37; 95% CI, 0.18-0.77 for del/del; Table 2) and the del allele was associated with the decreased risk of bladder cancer in a dose-response manner (P_trend = 0.0030). Furthermore, a significant decreased risk of bladder cancer was found in the combined variant genotypes ins/del + del/del compared with the ins/ins genotype (adjusted OR, 0.67; 95% CI, 0.49-0.90; Table 3).

Association between CASP8 polymorphism and progression of bladder cancer. In this study, we further evaluated the association between CASP8 -652 6N ins/del polymorphism and T stage and grade of bladder cancer. As shown in Table 3, a statistically significant deceased risk was found in bladder cancer with both low and high grades (adjusted OR, 0.70; 95% CI, 0.51-0.96; P = 0.025 for low grade and OR, 0.49; 95% CI, 0.27-0.90; P = 0.021 for high grade). However, in the stratification of stage, we found that a significantly decreased risk was only significant in superficial bladder cancer (OR, 0.61; 95% CI, 0.43-0.87; P = 0.006), but no significant associations between the genotypes and invasive bladder cancer were observed (OR, 0.74; 95% CI, 0.49-1.11; P = 0.147), which is likely due to the reduced number of subjects.

Interaction between CASP8 polymorphism and tobacco smoking. We evaluated whether there exist an interaction between CASP8 -652 6N ins/del polymorphism and tobacco smoking on bladder cancer risk. To reflect the risk from the ins genotypes, we used the ins genotype (ins/ins) as the risk genotype for the comparison. As shown in Table 4, compared with those who were both nonsmokers and del/del carriers, significantly increased risk was observed only in those who were smokers and ins/del carriers (adjusted OR, 4.40; 95% CI, 1.18-16.45) and those who were smokers and ins/ins carriers (adjusted OR, 8.23; 95% CI, 2.24-30.19). We further observed an additive interaction between tobacco smoking and CASP8 -652 6N genotypes (ins/ins, ins/del, and del/del genotypes; P = 0.041). Because relatively few subjects were del/del carriers in both nonsmokers and smokers, we combined del/del with ins/del as one reference group. As a result, smokers with the ins/ins genotype had a statistically significantly increased risk of bladder cancer (adjusted OR, 2.92; 95% CI, 1.86-4.58) compared with nonsmokers who carried the combined (ins/del + del/del) genotypes. Further interaction analysis suggested that the CASP8 -652 6N ins/del polymorphism appeared to have an additive interaction with smoking status (P = 0.032; Fig. 1), although there was only a suggestive evidence for a multiplicative interaction between this CASP8 polymorphism and tobacco smoking (P = 0.086), which is also likely due to a limited study power of the reduced sample size in the stratum. Besides, we also evaluated the interaction between CASP8 -652 6N ins/del genotypes (ins/ins and ins/del + del/del genotypes) and increasing dose of smoking such as pack-years on bladder cancer risk, a significant additive (P = 0.023), but not multiplicative (P = 0.053), interaction was observed (Table 4).

In the present study, we found that the CASP8 -652 6N ins/del and del/del genotypes were associated with a significantly decreased bladder cancer risk compared with the ins/ins genotype. In addition, the lower bladder cancer risk was more significant in superficial bladder cancer. Furthermore, statistical evidence was observed for an additive interaction between CASP8 -652 6N ins/del polymorphism and tobacco smoking. To the best of our knowledge, this is the first study of the association of CASP8 polymorphism with bladder cancer risk.

Caspase-8 is a regulator of apoptosis, which is an essential defense mechanism against hyperproliferation and malignancy (18). Two common polymorphic variations in the CASP8 gene, D302H and -652 6N ins/del, have been well studied. The CASP8 D302H, located in the external surface of the expressed protein, may influence autoprocessing of procaspase-8 molecules or caspase-8 interactions with the antiapoptotic FADD-like apoptosis regulator (19). The CASP8 302H has been shown to be a common low penetrance susceptibility allele for breast cancer (5, 9–11), glioma (7), and meningioma risk (19). Because the minor H allele frequency of the CASP8 D302H polymorphism was very low in Asians, we could not perform an adequate analysis for risk of bladder cancer in this study. The CASP8 promoter -652 6N ins/del polymorphism removes a Sp1 binding site and was shown to be associated with the reduced CASP8 transcription in lymphocytes (8). Furthermore, the T lymphocytes with the del variant allele had lower caspase-8 activity and lower cancer cell antigen-induced cell death (8). Studies reported that the -652 6N ins/del polymorphism could influence the risk of multiple cancers, including cancer of the lung, esophagus, stomach, colorectum, breast, and cervix in Chinese populations (8) and cutaneous melanoma in Caucasians (20). However, recent studies of the breast cancer in Europeans (21) and colorectal cancer in a United Kingdom population (22) failed to confirm this association with cancer risk. Haiman et al. (23) also failed to replicate the findings of the CASP8 polymorphism associated with cancer risk of the breast, prostate, and colorectum in multiple U.S. populations. In the present study, however, we found that the CASP8 -652 6N ins/del polymorphism had a reverse association with bladder cancer risk in a Chinese population, which was consistent with the findings reported by Sun et al. (8) in Chinese populations.

The reported discrepancy of in the association studies may be due to different genetic backgrounds and population-specific differences. Because the CASP8 -652 6N del variant shows lower expression of caspase-8, which reduces the activation-induced cell death of T lymphocytes involved in the immune response to malignant cells. Therefore, one possible mechanism underlying the CASP8 polymorphism associated in the decreased risk in bladder cancer in our study is that this polymorphism may reduce apoptotic potential in the T lymphocyte and make malignant cells to less likely escape from CTL killing, ultimately protecting against bladder cancer (8).

Subgroup analysis according to tumor stage and grade may help in identifying prognostic factors involved in different bladder cancer progression pathways (24). After stratification analysis by tumor grade and T stage, it appeared that the CASP8 -652 6N del allele was associated with an obviously reduced risk of developing superficial bladder cancer. It has been postulated that superficial and invasive bladder carcinomas may have a different etiology involving different genetic and epigenetic defects (25). However, this finding from our relatively small study needs validation by other larger studies.

Tobacco smoke contains hundreds of chemicals, some of which are carcinogens, such as polycyclic aromatic hydrocarbons and N-nitroso compounds, as evidenced in animal studies (26). A positive dose-response relationship in increased risk associated with both increased number of cigarettes smoked daily and increased number of years of smoking has been implicated in the etiology of bladder cancer and has been reported (24). Our results suggested that an additive joint effect between CASP8 polymorphism and risk of bladder cancer among smokers may reflect the additional effect from increased levels of reactive oxygen species production and DNA adducts resulting from smoking (26). It is noted that the bladder cancer risk is slightly higher in light smokers than in heavy smokers. This counterintuitive finding may be explained that low-dose smokers may inhale more deeply, decrease the time between puffs, or cover the air holes in the cigarette that otherwise dilute the smoke delivered to the smoker (27).

The limitation of our study is its hospital-based study design, because we cannot rule out the possibility of selection bias of subjects that may have been associated with a particular genotype. However, the genotype distributions in our study population were similar to that reported in published data for Chinese populations. For instance, the frequencies of the ins/ins, ins/del, and del/del genotypes among our 368 southern Chinese controls were 55.7%, 37.5%, and 6.8%, respectively, compared with 56.3%, 37.2%, and 6.5% in northern Chinese populations in the study by Sun et al. (8). Li et al. (20) reported that the frequency of the -652 6N del allele was 48.9% in cancer-free Caucasians, which may represent an ethnic difference in frequency distribution of the CASP8 -652 6N ins/del genotypes. The other limitation was our relatively small sample size. However, our post hoc power calculation suggested that we had an 83% power to detect a minimal OR of 0.55 with an exposure frequency of 20% under the current sample size.

In conclusion, the present study indicated that genetic variants of the CASP8 gene may modulate the risk of bladder cancer, particularly among smokers. These findings further supported that the CASP8 -652 6N ins/del polymorphism may be a marker for genetic susceptibility to the smoking-related bladder cancer. These findings, after validation by larger studies, may help identify at-risk populations for primary cancer prevention.

No potential conflicts of interest were disclosed.

We thank Qingyi Wei (Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center) for critical comments and scientific editing.