Abstract
Background/Aim: Programmed cell death 6 (PDCD6) is up-regulated and highly expressed in early apoptotic cells. In several types of cancer, such as cervical, breast and lung cancers, the association of PDCD6 genotypes have been investigated. However, the contribution of PDCD6 variant genotypes to oral cancer has never been examined. The current study aimed to evaluate the contribution of the PDCD6 rs4957014 and rs3756712 genotypes to the risk of oral cancer in Taiwan. Patients and Methods: The contribution of PDCD6 genotypes to oral cancer risk was examined among 958 patients with lung cancer and 958 age- and sex-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP). Results: The data showed that the hetero-variant GT and homo-variant GG genotypes of PDCD6 rs4957014 were associated with a decreased risk of oral cancer [odds ratio (OR)=0.81 and 0.39, 95% confidence interval (CI)=0.67-0.97 and 0.27-0.56, respectively]. The recessive and dominant models also showed that G carriers have protective effects (OR=0.43 and 0.72, 95% CI=0.30-0.61 and 0.61-0.87, respectively). The analysis of allelic frequency distributions showed that the G allele of PDCD6 rs4957014 was associated with reduced oral cancer risk (OR=0.71, 95% CI=0.62-0.82). There was no significant association between any PDCD6 rs3756712 genotype and oral cancer risk. In addition, the GG genotype at PDCD6 rs4957014 significantly decreased the risk of oral cancer among both males (adjusted OR=0.31, 95%CI=0.24-0.56) and females (adjusted OR=0.44, 95% CI=0.22-0.91). Furthermore, the GG genotype at PDCD6 rs4957014 significantly decreased the risk of oral cancer among smokers (adjusted OR=0.35, 95% CI=0.22-0.58), alcohol drinkers (adjusted OR=0.33, 95% CI=0.18-0.49), non-betel quid chewers (adjusted OR=0.33, 95% CI=0.17- 0.81), betel quid chewers (adjusted OR=0.34, 95% CI=0.21- 0.59), but not among never-smokers and non-alcohol drinkers. Conclusion: The G allele carriers of PDCD6 rs4957014 may have protective effects on oral cancer risk and serve as a practical marker for early detection of oral cancer in Taiwan.
Oral cancer is the fourth most common male cancer and the fourth leading cause of male cancer death in Taiwan, where the density of oral cancer is the top of the world (1-3). There are already several environmental factors known to contribute to the development of oral cancer, including alcohol, tobacco and betel quid consumption, bad brushing, dental implanting habits, and viral infectious status (4, 5). From the molecular epidemiological viewpoint, several studies have provided evidence that specific genotypes may also contribute to oral cancer risk (6-11). A combined analysis of genomic, environmental, and personal behavioral aspects to oral cancer development may greatly help in precise and personalized therapy for oral cancer patients.
PDCD6 (also known as apoptosis-linked gene 2, ALG-2), is located on the human chromosome 5p15.33 encoding for a 22- kD calcium-binding protein (12, 13). PDCD6 is a multiple binding protein that is capable of interacting with Alix/AIP1 (14), ASK1 (15), and Sec31A (16), participating in the T cell receptor-, Fas-, glucocorticoid- and endoplasmic reticulum stress-induced programmed cell death (12, 17). PDCD6 does not seem to be essential for T-cell maturation, since PDCD6 knockout mice exhibit normal T-cell development and function (18). On the contrary, PDCD6 deficiency cells were defective in blocking the apoptosis signals induced by TCR, FAS and dexamethasone (18). In the literature, PDCD6 expression levels and PDCD6 genotypes are reported to be associated with cancer risk (19-21). As early as 2003, it was found that PDCD6 was expressed significantly higher in the tumor sites compared to the non-tumor sites among patients of hepatoma and lung cancer (20). In 2012, the G allele at PDCD6 rs4957014 was reported to be associated with decreased lung cancer risk (21). In 2013, the GG genotype at PDCD6 rs4957014 together with the GT and GG genotypes at PDCD6 rs3756712 were associated with decreased leiomyoma risk (19). In 2014, GT genotype at PDCD6 rs4957014 was reported that it may contribute to decreased bladder cancer risk (22). One year later, the GG genotype at PDCD6 rs4957014 was found to be associated with increased cervical cancer risk (23). In 2017, the GT genotype at PDCD6 rs4957014 was found to decrease endometrial cancer risk (23) while in 2019, Shen and colleagues found that the GG genotype at PDCD6 rs4957014 contributed to lower risk of lung cancer (24). So far, there hasn’t been any report examining the contribution of PDCD6 genotypes to oral cancer susceptibility up. Thus, in the current study, we aimed at investigating the contribution of PDCD6 rs3756712 and rs4957014 single-nucleotide polymorphic (SNP) genotypes to the risk of oral cancer and examining the joint effect of environmental factors and PDCD6 genotypes on oral cancer risk in Taiwan.
Patients and Methods
Population collecting methodology. Originally, cases diagnosed with oral cancer were recruited at the Outpatient Clinics of General Surgery in China Medical University Hospital in Taichung, Taiwan. Characteristics of the cases, including histological details, were all graded and defined by experienced clinical-pathological experts. Patients with history of other cancers were excluded, and a final of 958 cases were recruited. All participants were Taiwanese, providing 3-5 ml of blood sample for further genotyping studies. The same amounts of healthy volunteers as controls were selected by matching for age, sex, and behavioral factors such as alcohol, tobacco and areca-nut consumption, 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, any genetic or familial diseases, and other nationalities. The study was approved by the Institutional Review Board of the China Medical University Hospital (DMR101-IRB1-306). Selected characteristics of all participants are summarized in Table I.
Characteristics of the investigated 958 oral cancer patients and 958 non-cancer healthy subjects.
PDCD6 genotyping. Total DNA from the blood of each participant was extracted using the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC) and further processed as previously published (25-27). Briefly, the polymerase chain reaction (PCR) programs were set as: 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 elongation at 72°C for 10 min. The sequences of paired primers for PDCD6 rs4957014 were 5’-TGGTGTTTCATACCATTGACACTTGC-3’ and 5’-CTCAGAA CCAAGCAGGTTCCTTCA-3’, respectively. For PDCD6 rs3756712, the sequences of paired primers were 5’- TACAGTGG CAAAGGACCACA-3’ and 5’-CACATTCCAGCACTCACCAC-3’, respectively. Following the PCR reaction, the products for PDCD6 rs4957014 and rs3756712 were subject to Hph I and Rsa I (New England BioLabs, Ipswich, MA, USA) digestion, respectively. As for PDCD6 rs4957014, the digestable G allele was cut into 113 and 13 base pair products, while the indigestable T allele was 126 base pairs long. As for PDCD6 rs3756712, the digestable G allele was cut into 113 and 13 base pair products, while the indigestable T allele was 126 base pairs long. Overall, the genotypic process was conducted by two researchers independently. Three percent of the PCR products were selected for direct sequencing and the results from PCR-RFLP and sequencing were 100% concordant with each other.
Statistical analysis. The sample sizes of the case and control groups were both 958. To examine the controls for being representative of the general population, the distribution of PDCD6 genotype frequencies among the controls under the Hardy-Weinberg equilibrium was checked by applying the goodness-of-fit test. At the same time, the Student’s t-test was applied to compare the distribution of age between case and control groups. In addition, Pearson’s Chi-square test was applied to compare the distributions of the PDCD6 genotypes between the case and control groups in Table II and Table III, or between other counterpart subgroups in Table IV, Table V, Table VI, and Table VII. Further, the associations between the PDCD6 genotypes and oral cancer risk are estimated by odds ratios (ORs) and corresponding 95% confidence intervals (CIs) before and after adjusting for confounding factors, such as age, sex, smoking, alcohol drinking and betel quid chewing behaviors, as indicated. A test for trend was based on chi-square test without Yates’ correction checking the relationships of adding a variant genotype on the oral cancer risk. Results with p-Value less than 0.05 were considered statistically significant.
Distribution of PDCD6 rs4957014 genotypes among the oral cancer patients and non-cancer healthy subjects.
Distribution of PDCD6 rs3756712 genotypes among the oral cancer patients and non-cancer healthy subjects.
Distribution of PDCD6 rs4957014 and rs3756712 allelic frequencies among the oral cancer patients and non-cancer healthy subjects.
Odds ratios for PDCD6 rs4957014 genotype and oral cancer after stratified by gender.
Odds ratios for PDCD6 rs4957014 genotype and oral cancer after stratified by smoking status.
Odds ratios for PDCD6 rs4957014 genotype and oral cancer after stratification by alcohol drinking status.
Results
The characteristics such as age, sex, smoking, alcohol drinking and betel quid chewing habits of the 958 oral cancer patients and the 958 controls are listed and compared in Table I. Additionally, the primary tumor sites of the oral cancer patients were summarized in the bottom of Table I. Firstly, no difference was found concerning age and sex between the case and control groups (p=0.3755 and 1.0000, respectively) (Table I, top part). Secondly, there are more smokers, alcohol drinkers and betel quid chewers in the oral cancer than the control group (p=0.0107, 0.0377 and 0.0001, respectively), indicating that these behaviors are oral cancer risk factors for Taiwanese (Table I, middle part). Lastly, tongue (41.4%) and buccal mucosa (37.2%) are the most prevalent primary tumor sites among the Taiwan oral cancer population (Table I, bottom part).
The distribution of genotypic frequencies of PDCD6 rs4957014 and rs3756712 are compared in codominant, recessive, and dominant models in Table II and Table III, respectively. Firstly, the genotypic frequencies of the PDCD6 rs4957014 and rs3756712 both fit well with the Hardy–Weinberg equilibrium in the control group (p=0.7237 and 0.1181, respectively). Secondly, there is a significant difference in the distribution of PDCD6 rs4957014 genotypes between the oral cancer and control groups (p for trend=0.0001). In detail, the frequencies of the heterozygous variant GT and homozygous variant GG of PDCD6 rs4957014 are 41.4 and 5.0% in the oral cancer group, significantly lower than those (43.5 and 11.0%, respectively) in the control group (p= 0.0268 and 0.0001, respectively). These results indicate that GT (OR=0.81,95% CI=0.67-0.97) and GG (OR=0.39, 95% CI=0.27-0.56) genotypes at PDCD6 rs4957014 can serve as a protective predictor of oral cancer (Table II, top panel). In the recessive model, GG genotype at PDCD6 rs4957014 is also associated with a decreased oral cancer risk, compared to the TT+GT genotypes (OR=0.43, 95% CI=0.30-0.61, p=0.0001) (Table II, middle panel). In the dominant model, GT+GG genotypes at PDCD6 rs4957014 are also associated with a decreased oral cancer risk, compared with the wild-type TT genotype (OR=0.72, 95% CI=0.61-0.87, p=0.0004) (Table II, bottom panel). Overall, PDCD6 rs4957014 genotypes can serve as a novel biomarker for oral cancer in the Taiwanese population.
Similarly, the PDCD6 rs3756712 genotypic frequency was examined about its contribution to oral cancer in each model. However, no significant differences were found in codominant, recessive, or dominant models (Table III).
To validate our findings in PDCD6 rs4957014 and rs3756712 genotypes, we also conducted allelic frequency analysis. Consistent with the findings in Table II, the G allele of PDCD6 rs4957014 is associated with a decreased risk of oral cancer, compared with the wild-type T allele (OR=0.71, 95% CI=0.62-0.82, p=0.0001) (Table IV, top panel). On the contrary, the variant G allele of PDCD6 rs3756712 is not associated with any significantly altered risk for oral cancer (Table IV, bottom panel).
The oral cancer prevalence among Taiwanese is extremely high and there is a sex difference, the male versus female is about 9 to 1. In Table V, we have stratified the distribution of PDCD6 rs4957014 among oral cancer patients and controls according to sex. The PDCD6 rs4957014 genotype is differentially distributed among oral cancer patients and controls both in males (adjusted OR=0.31, 95% CI=0.24- 0.56) and females (adjusted OR=0.44, 95% CI=0.22-0.91) (Table V).
Since betel quid chewing behavior together with smoking and alcohol drinking behaviors are well accepted as contributors to oral cancer risk, we are also interested in accessing the combinative effects of the PDCD6 genotype and these personal behaviors on oral cancer susceptibility. The joint effects of PDCD6 rs4957014 with smoking status are presented in Table VI. For never-smokers, the genotypes of PDCD6 rs4957014 are not associated with oral cancer. On the contrary, the GG genotype of PDCD6 rs4957014 is associated with decreased oral cancer risk (adjusted OR=0.35, 95% CI=0.22-0.58) (Table VI). The joint effects of PDCD6 rs4957014 with alcohol drinking status are presented in Table VII. For the non-alcohol drinkers, the genotypes of PDCD6 rs4957014 are not associated with oral cancer. On the contrary, the GG genotype of PDCD6 rs4957014 is associated with decreased oral cancer risk (adjusted OR=0.33, 95% CI=0.18-0.49) (Table VII). The joint effects of PDCD6 rs4957014 with betel quid status are presented in Table VIII. For the non-betel quid chewers, the GG genotype of PDCD6 rs4957014 is associated with decreased oral cancer risk (adjusted OR=0.33, 95% CI=0.17- 0.81). Similarly, the GG genotype of PDCD6 rs4957014 is also associated with decreased oral cancer risk (adjusted OR=0.34, 95% CI=0.21-0.59) (Table VIII).
Odds ratios for PDCD6 rs4957014 genotype and oral cancer after stratified by betel quid chewing status.
Discussion
PDCD6 is an apoptosis-linked gene (12) and its encoded protein plays a role in T cell receptor-induced programmed cell death in a Ca2+-dependent manner as a pro-apoptotic factor (28). On the contrary, PDCD6 also has an anti-apoptotic capacity via promoting HeLa cell passing through G2/M checkpoints, resulting in enhanced proliferation (29). This contrasting character of PDCD6 may be explained by its multiple binding capacity with various Pro-rich proteins, such as Alix (ALG-2-interacting protein X) (30), annexins VII/XI (31), Sec31A (SEC31 homolog A) (16), and TSG101 (tumor susceptibility gene 101) (30). In mice, knockout of PDCD6 did not hinder apoptotic processes (18). In the literature, there have only been a few studies examining the association of PDCD6 genotypes with several types of cancer, including lung (21, 24), ovarian (32), endometrial (33), and cervical cancer (23). We have summarized all of the studies reporting PDCD6 rs4957014 genotypes with cancer types, for the overview of its association with several types of cancer (Table IX). Up to now, there is neither investigation of PDCD6 genotype contribution to oral cancer determination, nor the conclusive role of PDCD6 genotypes in carcinogenesis.
Summary of findings about PDCD6 rs4957014 genotype with cancer risk.
In the current study, the contribution of PDCD6 genotypes, in addition to smoking, alcohol drinking, and betel quid chewing status to oral cancer risk are firstly examined. The highlights of the study include that the heterozygous GT and homozygous GG genotypes at PDCD6 rs4957014 are significantly associated with a decreased risk of oral cancer in codominant, recessive, and dominant models (Table II). In addition, the variant G allele of PDCD6 rs4957014 was associated with a decreased oral cancer risk (Table IV). Further, the protective effects of PDCD6 rs4957014 GG genotype on oral cancer risk is obvious among both males and females (Table V), smokers (Table VI), alcohol drinkers, and non-betel quid chewers and betel quid chewers (Table VII). The protective effect of rs4957014 GG genotype on oral cancer risk is also in line with He’s and Zhou’s findings in lung and cervical cancer, respectively (21, 23). On the other hand, it has been reported that PDCD6 rs3756712 and rs4957014 polymorphisms are associated with increased endometrial cancer risk (33). Further studies in various and larger populations are needed for understanding the role of PDCD6 in oral carcinogenesis.
Sex preference is a potential risk factor for oral cancer. The ratio of male to female is about 9 to 1 in Taiwan. Although the possible mechanisms are still largely unknown, it is thought that endocrine-related factors play a critical part in the sex difference for oral cancer risk. While our oral cancer patients were stratified by sex, PDCD6 genotypes significantly associate with oral cancer risk among both men and women, thus no sex difference was found (Table V).
As mentioned above, cigarette smoking, alcohol drinking and betel quid chewing are well-known behavioral risk factors for oral cancer. For instance, tobacco carcinogen- induced DNA adducts are reported to greatly enhance the probability of cancer (34). In this regard, the interaction of the genotype of PDCD6 with cigarette smoking, alcohol drinking, and betel quid chewing status were concisely discussed. Firstly, concerning smoking, the results showed that the genotypic distribution of the variant genotypes of PDCD6 rs4957014 were significant among those who were ever smokers, but not among never-smokers (Table VI). There was no literature reporting that PDCD6 rs4957014 T allele attenuated cells’ DNA repair capacity with too many DNA adducts left unrepaired as those cells with G allele. Secondly, concerning alcohol drinking, the results showed that the genotypic distribution of the variant genotypes of PDCD6 rs4957014 were significantly different among those who were ever alcohol drinkers, but not that for never drinkers (non-alcohol drinkers) (Table VII). Lastly, concerning betel quid chewing, the results showed that the genotypic distribution of the variant genotypes of PDCD6 rs4957014 was significantly different among both ever chewers and non-chewer subgroups (Table VIII). Further investigations using cells from patients with different genotypes of PDCD6 rs4957014 should be conducted to explore the joint effects of tobacco smoking, alcohol drinking, and betel quid chewing with PDCD6 rs4957014 genotypes on cancer risk.
There has been no phenotypic evidence up to now showing that the PDCD6 rs4957014 T allele attenuates the capacity to promote those cells with high DNA damage or leave too many DNA adducts unrepaired as those cells with G allele. Further investigations using cells from patients with different genotypes of PDCD6 rs4957014 should be conducted to explore the joint effects of gender, personal smoking, drinking, betel quid chewing status, and other oral cancer risk factors. From the molecular viewpoint, the methylation status (35) and cooperation with C-Raf (36) may alter some oncogenic signaling and promote carcinogenesis. More recently, a novel Circ-Calm4/miR-124-3p/PDCD6 axis has been reported to regulate pyroptosis, another type of cell death, in hypoxia-Induced pulmonary arterial smooth muscle cells (37).
In conclusion, the current study provides solid evidence that the G allele of PDCD6 rs4957014 can serve as a protective biomarker, specifically interacting with smoking, alcohol drinking and betel quid chewing behaviors for oral cancer risk among Taiwanese. These novel findings should be validated in larger and different populations to understand the role of PDCD6 in carcinogenesis.
Acknowledgements
We are grateful to Tissue-bank of China Medical University Hospital and doctors/nurses for their excellent sample collection and technical assistance. The technical assistance from Yu-Chen Hsiau, Yi-Ru Huang, Yu-Hsin Lin were very critical in manuscript preparation. This study was supported mainly by China Medical University and Asia University (CMU110-ASIA-01).
Footnotes
↵* These Authors contributed equally to this study.
Authors’ Contributions
Research design was done by LCS, CWT and JLH. Patient and questionnaire collections were conducted by LCS and CLH. Experimental work was done by WSC, JSY, CLH, and YCW. Statistical analysis was done by TCH, WSC and JLH, while DTB and CWT wrote the manuscript, whereas DTB, CWS, and WSC reviewed it and are responsible for the revision.
Conflicts of Interest
The Authors declare no conflicts of interest with any company or person.
- Received March 7, 2022.
- Revision received April 20, 2022.
- Accepted April 26, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
