Abstract
Aim: Hepatocellular carcinoma (HCC), the fifth most common cancer worldwide, has high prevalence and mortality rates in Taiwan. Tumor necrosis factor-α (TNFα), an important proinflammatory cytokine, is involved in multiple physiological and pathogenic phenomena that lead to the destruction and dysregulation of tissues. The present study aimed to evaluate the contribution of TNFA genotype, together with cigarette smoking and alcohol drinking lifestyle to the risk of HCC. Materials and Methods: In this hospital-based case–control study, association of TNFA single-nucleotide polymorphisms −1031T/C, −863C/A, −857T/C, −308G/A and +489A/G, with HCC risk were examined in 298 patients with HCC and 889 age- and gender-matched healthy controls. Results: The percentages of AA, AG and GG TNFA −308G/A were 6.4%, 18.1% and 75.5% in the HCC patient group and 2.0%, 16.0% and 82.0% in the non-cancer control group, respectively. The AA and AG genotypes were associated with 3.42- and 1.23-fold higher odds of HCC than the GG genotype (95% confidence interval=1.76-6.63 and 0.87-1.74, respectively). No such significant difference was found for other polymorphic sites. We further stratified the populations by gender, cigarette smoking and alcohol drinking status to investigate their combined contributions with TNFA −308G/A genotype to HCC risk. The results showed that the AA and AG genotypes of TNFA −308G/A increased HCC susceptibility which was obvious among males, smokers, and alcohol drinkers, but not females, non-smokers, or non-drinkers (p=0.0003, 0.0003, 0.0014, 0.6127, 0.7442 and 0.3010, respectively). Conclusion: Our results suggest that the AA and AG polymorphism of TNFA −308G/A genotypes associated with HCC risk in Taiwan, particularly among males, smokers and alcohol drinkers.
Liver cancer, the fifth rated cancer worldwide and the third most common cause of cancer mortality, is prevalent in countries of low- or middle-income (over 80%), especially in the Chinese Han population (about 50%) (1). Clinically, hepatocellular carcinoma (HCC) accounts for over 90% of cases and is characterized by frequent local recurrence but relatively fewer metastases than other types of cancer (2, 3). Well-known factors for HCC include chronic infection with hepatitis B and C viruses, alcohol consumption and alcohol-related cirrhosis, tobacco consumption, overweight, diabetes and contamination of cereal foodstuff with aflatoxin in selected countries (4-6). In spite of great advances in HCC diagnosis and treatment (7), approximately 70% of patients with HCC receiving curative therapies had local or extrahepatic tumor recurrence (8). Therefore, useful biomarkers for identifying high-risk populations, as well as novel early detection and prediction tools in addition to preventive care are urgently needed.
One of the proinflammatory cytokines, tumor necrosis factor (TNF)-α, exerts a variety of physiological and pathogenic effects on the activation of a cascade of inflammatory cytokines and tissue dysfunction and destruction (9, 10). TNFα has two receptors, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2) and in liver physiology, TNF-R1 plays a predominant role. The functions of TNF can be pro-inflammatory via activating of NFκB-related pathways, or apoptotic via activating caspase-8 (11).
The studies on the contributions of genetic variations in TNFA to human diseases have focused on the polymorphisms in regulatory regions which directly control its transcription levels, and subsequently influence the circulating levels of TNFα and increase susceptibility to human diseases, such as cancer (12-14). Among the promoter polymorphic sites, the -308G/A polymorphism has been the most frequently studied TNFA genotype in human disease susceptibility (15). However, the results of studies in this area are inconsistent among different populations and diseases. For instance, it was found that the TNFA -308G/A polymorphism increased the risk of breast cancer, cervical cancer and gastric carcinoma (16-18). However, certain studies have reported that no statistically significant association existed between the TNFA -308G/A polymorphism and cancer risk (19, 20). Therefore, more information on other polymorphic sites and on more populations are beneficial for revealing the contribution of TNFA polymorphisms and their possible influence on transcription of the TNFA gene.
In this study, we aimed to reveal the frequencies of genotypes of −1031T/C, −863C/A, −857T/C, −308G/A and +489A/G polymorphisms of TNFA, focusing on the combined effects of TNFA genotypes with factors such as smoking and drinking status on HCC susceptibility among Taiwanese people.
Materials and Methods
Investigated population and sample collection. Two hundred and ninety-eight patients diagnosed with HCC were recruited at the Departments of Surgery at the China Medical University Hospital, Taiwan, during 2004-2010. Three-times as many non-cancer healthy volunteers, as controls, were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of China Medical University Hospital. The exclusion criteria of the controls included previous malignancy, metastasized cancer from other or unknown origin, and any genetic or familial diseases. The included control population was 894 individuals. Each patient and non-cancerous healthy person completed a self-administered questionnaire and provided their peripheral blood samples. Our study was approved by the Institutional Review Board of the China Medical University Hospital (DMR103-IRB-094) and written-informed consent was obtained from all participants. Those who have the habit of consuming cigarette and alcohol for at least half year were defined as ever smokers and drinkers.
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping conditions. Genomic DNA was extracted from peripheral blood leucocytes using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and stored as previously published (21-23). The paired primers and restriction enzymes applied for TNFA genotyping are summarized in Table I. The PCR conditions 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. After the PCR process, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The genotype analysis was performed by two researchers independently and blindly. During this process, five of the control samples were excluded since their PCR products were not sufficient for clear genotyping recognition after PCR-RFLP.
Statistical analyses. Finally, 889 of the controls and 298 cases with genotypic and clinical data were analyzed and the data are shown in Tables III, IV, V and VI. Pearson's Chi-square and Fisher's exact (when one or more cells were less than five) tests were used to compare the distribution of the genotypes between case and control groups. Differences between data were recognized as significant when the statistical p-value was less than 0.05. The HCC risk associated with the genotypes was estimated as an odds ratio (ORs) and 95% confidence intervals (CIs) by logistic regression.
Results
The characteristics for the investigated 298 patients with HCC and 889 non-cancer controls are summarized in Table II. Since we applied frequency matching methodology to select the non-cancer healthy controls, the distributions of age and gender were comparable among the cases and the controls (Table II). Regarding personal behavior, the HCC cases had a significantly higher percentage of smokers (75.2% vs. 65.1%, p=0.0017) and drinkers (69.1% vs. 58.3%, p=0.0011) than the non-cancerous controls (Table II).
The distributions of TNFA −1031T/C, −863C/A, −857T/C, −308G/A, +489A/G genotypes together with their frequencies among the controls and the patients with HCC are shown in Table III. After the statistical analysis, it was found that the genotypic frequencies of −1031T/C, −863C/A, −857T/C and +489A/G were not significantly different between the control and patient groups. For the −308G/A, the GG, AG and AA genotypic frequencies were 82.0%, 16.0% and 2.0% for the controls and 75.5%, 18.1% and 6.4% for the patients, respectively (Table III). The p-value for trend was 0.0008, and p-values for AG versus GG, AA versus GG, AG+AA versus GG and AA versus GG+AG at −308G/A were 0.2358, 0.0003, 0.0187 and 0.0007, respectively (Table III). The ORs for AG versus GG, AA versus GG, AG+AA versus GG and AA versus GG+AG at −308G/A were 1.23 (95% CI=0.87-1.74), 3.42 (95% CI=1.76-6.63), 1.48 (95% CI=1.08-2.02) and 3.42 (95% CI=1.77-6.61), respectively. Statistically speaking, the heterozygous AG and homozygous AA genotypes of TNFA −308G/A contributed to increased risk of HCC.
According to the records during the past decade, there were more males than females with HCC in Taiwan. We were interested in the genotypic contribution of TNFA −308G/A to gender difference in HCC susceptibility. After stratification by gender, the results showed that the genotypes of TNFA −308G/A were differently distributed among males (p=0.0003) but not females (p=0.6127) (Table IV).
The interaction of the genotype of TNFA −308G/A with cigarette smoking and alcohol drinking of the participants was also analyzed since HCC has been reported to be related smoking and alcohol consumption. The results in Table V show that the genotypic distribution of the variant TNFA −308G/A genotypes was differently distributed between HCC and control groups who were ever smokers (p=0.0003), but not for those who were non-smokers (p=0.7442) (Table V). Similarly, the results in Table VI showed that the genotypic distribution of the variant TNFA −308G/A genotypes was different between HCC and control groups who were ever drinkers (p=0.0014), but not different among the non-drinkers (p=0.3010) (Table VI). Overall, it seems that there exists an interaction between genetic (TNFA −308G/A genotype) and lifestyle (smoking and alcohol drinking behaviors) factors for HCC susceptibility.
Discussion
Liver cancer is a complex disease of uncertain etiological carcinogenesis. In recent years, the genomic contribution to liver cancer susceptibility in Taiwan has been investigated (24-27). TNFA gene is located in the class III region of human major histocompatibility complex (MHC) on chromosome 6p21 (28). Its encoded protein is an important inflammatory factor that acts as a master switch in the interaction between extracellular inflammation stimulation and intracellular signal transduction during carcinogenesis. A wide variety of evidence has pointed to a critical role of TNFα in tumor proliferation, migration, invasion and angiogenesis. Among the studied SNPs of TNFA, −1031T/C, −863C/A, −857T/C, −308G/A and +489A/G, the −308G/A is the one most commonly studied. A few studies have shown conflicting evidence for and against association of HCC with TNFA −308G/A among different populations (13, 29-32). Several of the studies found that the A allele was associated with increased risk in Turkish (13), Shandong (30) and Shanghai (32) people; while others found no association in Beijing and Italian people (29, 31). A recent meta-analysis of the TNFA −308G/A promoter polymorphism in HCC showed evidence of association for the A allele among Asians (33). In accordance with this, our results strongly suggest that the A allele and genotypic frequencies of the polymorphism at −308G/A were significantly different in Taiwanese HCC patients and controls. The functional analysis of polymorphism in the promoter region of TNFA −308G/A position also yielded conflicting observations. Some studies have suggested that the protein encoded by A allele of TNFA −308G/A has higher transcriptional activity, whereas other studies showed that this polymorphism appeared not to influence TNFα production (34, 35). The genotypic-phenotypic correlation between the TNFA −308G/A genotype and circulation concentration of TNFα remains to be characterized.
In the present study, we improved our analytical power via enrolling a larger population of controls than our previous genotypic investigations in HCC (25-27). We have also further examined the interaction between genetic and lifestyle factors. In Table II, cigarette smoking and alcohol drinking lifestyles were found to be risk factors for HCC (Table II). After finding that the AG and AA genotypes of TNFA −308G/A were associated with HCC risk in the whole population (Table III), we aimed at revealing the contribution of TNFA −308G/A genotypes to Taiwanese in different groups by gender, and smoking and alcohol drinking behaviors. After stratifying both the controls and cases according to their gender, it was found that AG and AA genotypes of TNFA −308G/A were associated with increased risk of HCC for Taiwanese males (Table IV). For the females, the frequency of wild-type GG genotype was lower in the case group (77.7%) than the control group (81.4%), but the difference did not reach the statistically significant level (p=0.6127) (Table IV). As liver cancer is recognized to be one of the tobacco- and alcohol-related cancer types (6, 36), the interactions of TNFA −308G/A genotypes with smoking and drinking habits on HCC risk in Taiwanese were also of interest. For the smoking status, we found that AG and AA genotypes of TNFA −308G/A were associated with increased risk of HCC for the smokers but not the non-smokers (Table V). Similarly as for the alcohol drinking status, the association between TNFA −308G/A genotypes with HCC risk was obvious among ever drinkers but not non-drinkers (Table VI). Overall, males carrying variant A allele at TNFA −308G/A site may have higher HCC risk than those carrying wild-type G allele, especially for smoking- and alcohol-induced HCC development.
In conclusion, our findings suggest that the A allele of TNFA −308G/A is associated with increased HCC risk, especially among those who are male, smokers and alcohol drinkers in Taiwan. Further prospective studies in different ethnic populations will be necessary to confirm our findings and to elucidate the underlying genomic–lifestyle interactions for the carcinogenesis of HCC.
Acknowledgements
The Authors appreciate Chia-En Miao, Lin-Lin Hou and Tissue-bank of China Medical University Hospital for their excellent technical assistance. This study was supported by research grants from Taichung Armed Forces General Hospital (supporting number 103B03) and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002).
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
The Authors declare that they have no conflict of interest in regard to this study.
- Received June 1, 2015.
- Revision received July 8, 2015.
- Accepted July 9, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved