Abstract
Aim: Metalloproteinases (MMPs) are a family of multifunctional proteins which have been shown to be up-regulated in various types of cancer. However, the contribution of MMP1 genotype to oral cancer has not been elucidated. This study aimed to evaluate the contribution of MMP1 promoter 1607 genotype to the risk of oral cancer. Materials and Methods: In this case–control study, MMP1 genotype and its interaction with consumption of areca, cigarettes, and alcohol in determining oral cancer risk were investigated in 788 patients with oral cancer and 956 gender-matched healthy controls. Results: The distribution of 2G/2G, 1G/2G and 1G/1G for MMP1 promoter 1607 genotype was 36.8%, 40.2% and 23.0% in the oral cancer group and 34.3%, 44.9% and 20.8% in the non-cancer control group, respectively (p for trend=0.1454). We also analyzed the allelic frequency distributions and found that the variant 1G allele of MMP1 promoter 1607 conferred similar oral cancer susceptibility as the wild-type 2G allele (odds ratio=0.99, 95% confidence interval=0.87-1.14, p=0.9199). As for the gene–lifestyle interaction, there was an obvious protective effect of MMP1 promoter 1607 1G/2G genotype on the risk of oral cancer among smokers (odds ratio=0.71, 95% confidence interval=0.55-0.91, p=0.0076), but not non-smokers. There was no interaction between MMP1 promoter 1607 genotype and areca chewing or alcohol drinking habits. Conclusion: The 1G/2G genotype of MMP1 promoter 1607 may have a protective effect on oral cancer risk for smokers. The detailed mechanisms involved in this require further investigation.
- Genotype
- MMP1
- oral cancer
- polymorphism
- Taiwan
Statistically, oral cancer, the tenth most commonly diagnosed cancer worldwide, has the highest incidence density in Taiwan (1). According to the government annual report, oral cancer is the fourth cause of cancer-related death among males in Taiwan, and has been reported to be closely associated with tobacco, alcohol and betel nut consumption habits (2-5). Although several biomarkers for oral cancer in Taiwan have been revealed (6-12), the genomic etiology of oral cancer and the interactions among the genetic and lifestyle factors are of great interest but largely unknown.
Extracellular matrix (ECM) structures contribute to microenvironmental remodeling during the initial morphogenesis, angiogenesis, inflammation, would healing and tumorigenesis (13). The matrix metalloproteinases (MMPs) are a family of endopeptidases that play a key role in ECM remodeling, and control degradation of the components of connective tissue matrices (13, 14). They are also related to the regulation of oral cancer invasion and metastasis (15). The homeostasis of each MMP is also under the control of a complex network at several levels, including through their interactions with specific inhibitors, e.g., the tissue inhibitors of metalloproteinases (TIMPs) (14). MMP1, also known as collagenase-1, is most abundant among the MMPs and under the control of activator protein-1 (AP1) which binds to the promoter region of mitogen-activated kinase through polyomavirus-enhancing activity-3 (16, 17). A polymorphic site was found in the MMP1 promoter region at upstream position of 1607 bp, which was reported to control the transcriptional activity of the MMP1 gene and was also correlated with the incidence and progression of several cancer types (18). Studies of brain tumors suggest that there is a close relationship between the polymorphism of the MMP1 promoter region and an increase in tumor grade in astrocytoma, glioblastoma and pituitary adenoma (17, 19-22). In an animal model, exposure to side-stream cigarette smoke 5 days per week for 1 month will induced an increase in MMP1 mRNA levels in the lung tissue of male Wistar rats (23). Since smoking is a main lifestyle factor contributing to oral cancer, it is possible that abnormal expression of MMP1 may play a role in the carcinogenesis of smoking-related oral cancer.
The genomic contribution of MMP1 to cancer has not been well elucidated. For oral cancer, Cao and colleagues reported that the 2G allele of MMP1 promoter 1607 polymorphism was associated with higher risk of oral squamous cell carcinoma, compared with the 1G allele in a Chinese population (24). However, their investigation was limited to 96 cases and 120 controls. In the current study, we aimed to reveal the contribution of MMP1 genotype at the promoter 1607 site to the risk of oral cancer in Taiwanese.
Materials and Methods
Investigated population. Our study was approved by the Institutional Review Board of the China Medical University Hospital (DMR101-IRB1-306) and written-informed consent was obtained from all the participants. Seven hundred and eighty-eight patients diagnosed with oral cancer were recruited at the China Medical University Hospital in central Taiwan. All patients voluntarily participated, completed a self-administered questionnaire and willingly provided 5 ml of their peripheral blood. The questionnaire administered to participants included questions on history and frequency of alcohol consumption, areca chewing and smoking habit. Self-reported alcohol consumption, areca chewing and smoking habits were evaluated and classified as categorical variables. Information on these factors was obtained as more than twice a week for years as “ever”. A total of 956 non-cancer healthy individuals as controls were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital. The male versus female ratio was 76% to 24% in each group. The mean age of the patients and the controls was 55.8 (SD=9.9) and 56.6 (SD=8.7) years, respectively. More detailed information is summarized in Table I.
Genotyping conditions. The genomic DNA from the peripheral blood leucocytes of each patient and control was prepared applying the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) and stored at −80°C until processed as per our previous articles (6-8). The sequences of primers and the restriction enzymes for MMP1 promoter 1607 genotyping are modified from a previous publication (25). The forward and reverse primers were 5’-TGACTTTTAAAACATAGTCTATGT-3’ and 5’-GATTGATTTGAGATAAGTCATAGC-3’, respectively. The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. After amplification, the PCR products were subject to the digestion by AluI restriction endonuclease for 2 h at 37°C and separation using 3% agarose gel electrophoresis. The genotypes were identified as homozygous 2G/2G with 269-bp product, heterozygous 1G/2G with 269-, 241- and 28-bp products, and homozygous 1G/1G with 241- and 28-bp products. All the genotypic processing was repeated by two researchers independently, and blindly, and the results were 100% concordant.
Statistical analyses. Student's t-test was used for the comparison of ages between the case and the control groups. Pearson's Chi-square test was used to compare the distribution of the MMP1 promoter 1607 genotypes among the subgroups. The associations between the MMP1 promoter 1607 genotypes and oral cancer risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from logistic regression analysis. Any difference at p<0.05 was considered statistically significant, and all statistical tests were two-sided.
Results
The frequency distributions of selected characters including age, gender, personal habits and primary tumor sites for the 788 patients with oral cancer and 956 non-cancer controls are summarized and compared in Table I. Since we applied frequency matching to recruit the non-cancer healthy controls, there was no difference in the distributions of age and gender between the control and case groups (Table I). For these investigated individuals, betel quid chewers and smokers were more freqent in patients with oral cancer than in the controls (Table I). Most primary tumors occurred in the tongue and buccal mucosa.
The distributions of the MMP1 promoter 1607 genotype among the non-cancer controls and the oral cancer patients are presented and statistically analyzed in Table II. The genotypes of MMP1 promoter 1607 were not differently distributed between oral cancer and non-cancer control groups (p for trend=0.1454) (Table II). In detail, the MMP1 promoter 1607 heterozygous 1G/2G and homozygous 1G/1G were not associated with oral cancer risk (p=0.1011 and 0.8283, respectively; Table II). In the recessive and dominant models, there was still no association between the genotype of MMP1 promoter 1607 and oral cancer risk (p=0.2782 and 0.2788, respectively; Table II).
To confirm the findings in Table II, the analysis of allelic frequency distribution for the MMP1 promoter 1607 was also conducted and the results are summarized in Table III. Supporting the findings that neither heterozygous 1G/2G nor homozygous 1G/1G genotype of MMP1 promoter 1607 was associated with oral cancer risk, the variant allele 1G was found at 43.1% in the patient group, non-significantly different from that of 43.3% in the control group (p=0.9199). To sum up, there was no significant difference in the allelic frequencies of MMP1 promoter 1607 between the control and oral cancer groups (Table III).
Next, we examined the interactions among the genotype of MMP1 promoter 1607 and lifestyle factors, such as personal cigarette smoking, betel quid chewing, and alcohol drinking habits. Among smokers, those with genotype of 1G/2G at MMP1 promoter 1607 were at 0.71-fold odds of having oral cancer (95% CI=0.55-0.91, p=0.0076), seemingly conferring a protective effect, but this was not the case among non-smokers (Table IV). There was no interaction of MMP1 promoter 1607 genotype with areca chewing and alcohol drinking habits (Table V and VI).
Discussion
In the current case–control association study, the contribution of MMP1 promoter 1607 to oral cancer risk and its interaction with alcohol drinking, cigarette smoking and areca chewing was firstly evaluated among Taiwanese. The results showed that although neither the genotypic nor the allelic frequencies of MMP1 promoter 1607 were differentially distributed among the patients and non-cancer healthy controls (Table II), the heterogeneous 1G/2G genotype was associated with increased risk of oral cancer for the smokers (Table IV).
This is the first study to reveal an interaction between MMP1 1607 genotype and cigarette smoking on the susceptibility to oral cancer. Long-term tobacco smoking and areca chewing have been shown to contribute to etiology of oral cancer development (6, 7, 26-33). However, the mechanisms are very complex and need more investigations. In Table I, it can be seen that a higher proportion of individuals had consumed areca, cigarettes and alcohol in the group of patients with oral cancer than the controls.
The MMP1 protein is involved in the degradation of the native fibrillar collagen, hence it is called collagenase-1. In normal conditions, MMP1 is expressed at a relatively low level under the suppressive regulation of TIMP1 protein (34, 35). MMP1 has been reported to play an important role in tumor cells undergoing invasion and migration (36, 37). Mounting evidence indicates that activated MMP1 is observed in the borders of solid tumors, such as breast and oral cancer (38-40). MMP1 is thought to promote invasion and metastasis through the degradation of the ECM as the main component of connective tissue, especially in the tumorigenesis of oral mucosal cancer (41-44). In 2016, MMP1 and TIMP1, together with MMP1 and TIMP2, were shown to gradually increase with progression of tongue cancer in a hamster model of tongue cancer (45). In 2012, Liu and colleagues performed a meta-analysis exploring the association between MMP1 promoter 1607 1G/2G polymorphism and risk of several types of cancer, and the results showed that an elevated cancer risk was found regarding breast, colorectal, genitourinary neoplasm but not oral cancer (46). The dynamic balance between MMP1 and TIMP1 play a pivotal role in the maintenance of normal physiological conditions for cells, but it seems that the balance between MMP1 and TIMP1 in oral tissues is not as simple as a ‘see-saw’ relationship. In the near future, an overall analysis of MMP1 and TIMP1 genotype/phenotype may provide further evidence for evaluating the contribution of these genotypes to oral carcinogenesis.
In conclusion, our pilot study provides evidence that the heterozygous 1G/2G genotype at MMP1 promoter 1607 may interact with personal smoking status to determine the susceptibility to oral cancer, and this should be confirmed in multicenter and multi-population studies.
Acknowledgements
The Authors declare no conflict of interest in regard to this study. They also appreciate the Tissue-bank of China Medical University Hospital for their excellent technical assistance. This study was supported mainly by the Taiwan Ministry of Science and Technology to Professor Bau (MOST-104-2320-B-039-036) and partially by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-133019) and China Medical University Hospital (CMU-102-031) to Dr. Tsai.
Footnotes
* These Authors contributed equally to this study.
- Received March 15, 2016.
- Revision received April 12, 2016.
- Accepted April 15, 2016.
- Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved