Abstract
Background/Aim: Upregulation of matrix metallo-proteinase-8 (MMP-8) serves as a protein-based indicator for predicting nasopharyngeal carcinoma (NPC) metastasis. Nevertheless, the role of MMP-8 genotypes in NPC has never been investigated. This study aimed to explore the involvement of MMP-8 genotypes in NPC development. Materials and Methods: We employed the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to analyze MMP-8 genotypes, specifically C-799T (rs11225395), Val436Ala (rs34009635), and Lys460Thr (rs35866072), in a Taiwanese cohort comprising 208 NPC cases and 416 healthy controls. Results: Individuals with either heterozygous or homozygous variant genotypes of MMP-8 rs11225395 showed no significant change in NPC risk compared to those with the wild-type genotype [odds ratio (OR)=0.97 and 0.79, 95% confidence intervals (95%CI)=0.68-1.38 and 0.46-1.36; p=0.9304 and 0.4736, respectively]. Similarly, there was no significant association between the heterozygous genotypes of MMP-8 rs34009635 and NPC risk (OR=0.66, 95%CI=0.24-1.84; p=0.5738). For MMP-8 rs35866072, all individuals in the study were of the TT genotype. Furthermore, the presence of variant alleles at MMP-8 rs11225395 or rs34009635 did not result in altered NPC risk (OR=0.91 and 0.66, 95%CI=0.71-1.16 and 0.24-1.84, p=0.4876 and 0.5769, respectively). Additionally, no significant association was observed between MMP-8 rs11225395 variant genotypes and NPC risk among individuals regardless of smoking, alcohol consumption, or betel quid chewing habits (all p>0.05). Conclusion: There was no association between the MMP-8 genotypes rs11225395, rs34009635, or rs35866072 and NPC risk among Taiwanese individuals. Moreover, no combined effects of MMP-8 genotype with smoking, alcohol consumption, or betel quid chewing habits on NPC risk were observed.
Nasopharyngeal carcinoma (NPC) is a malignancy originating from nasopharynx epithelial cells and is associated with Epstein-Barr virus (EBV) infection, dietary practices, environmental exposures, and genetic predispositions (1-3). The molecular diagnosis of NPC is essential for early detection, choosing appropriate treatments, monitoring disease progression, and predicting outcomes. Despite the identification of several genome-wide biomarkers as potential NPC indicators (4), there is an ongoing need to uncover clinically relevant markers specifically applicable to certain populations, such as the Taiwanese (5-7).
Matrix metalloproteinase-8 (MMP-8), also referred to as neutrophil collagenase, is involved in extracellular matrix modulation through collagen degradation and is encoded by the MMP-8 gene on chromosome 11q22.3 of human genome (8, 9). This enzyme is produced not only by neutrophils, but also by various tumor cells (10, 11). Elevated levels of MMP-8 have been detected in the cyst fluids from ovarian cancer tissues compared to those of respective control tissues (12). Certain single nucleotide polymorphisms (SNPs) of MMP-8 can modify gene expression by affecting promoter activity. For instance, the MMP-8 rs11225395 T allele has been linked to increased breast cancer susceptibility and metastasis severity in both Asian and Caucasian populations (13). Over the past decades, numerous studies have explored the relationship between MMP-8 genetic variants, such as rs11225395, rs35866072, and rs1940475, and cancer risk. These investigations have spanned multiple cancer types, including bladder (14-17), skin (18), liver (19), melanoma (20), gastric (21), colorectal (22, 23), ovarian (24), oral (25), leukemia (26), lung (27), breast (28), and kidney cancer (29). However, the results regarding MMP-8 variations and cancer risk have been inconsistent across different case-control studies, and there is a notable lack of investigation on NPC.
Building on the aforementioned context, this study aims to evaluate the role of MMP-8 rs11225395, rs34009635, and rs35866072 genotypes in determining the risk of NPC in the Taiwanese population, the physical location of MMP-8 genotypes is illustrated in Figure 1. Furthermore, the investigation includes examining potential interactions between MMP-8 genotypes and lifestyle factors, such as cigarette smoking, alcohol consumption, and betel quid chewing.
Location of the MMP-8 polymorphic sites, rs11225395, rs34009635, and rs35866072, together with neighboring DNA sequences.
Materials and Methods
Recruitment of Taiwan non-cancer controls and NPC cases. A cohort of 208 NPC patients was recruited from the Department of General Surgery at China Medical University Hospital in Taichung. These participants volunteered for the study, completed a self-administered questionnaire, and provided their peripheral blood samples ranging from 3 to 5 ml. Non-cancer controls were selected in a 2:1 ratio to the cases, matched precisely by sex, age (within a 5-year range), and personal behaviors, such as cigarette smoking, alcohol consumption, and betel quid chewing. Controls were excluded if they had a history of malignancies, metastatic tumors of unknown or different origins, or any known genetic or hereditary disorders. Information on smoking, alcohol use, and betel quid chewing was gathered using the same self-reported questionnaires as those for the NPC cases. “Ever” use was defined as engaging in these behaviors more than twice a week for longer one year. These behaviors were quantified and categorized as discrete variables. The study protocols received approval and oversight from the Institutional Review Board of China Medical University Hospital (DMR101-IRB1-306). All clinical procedures and documentation strictly adhered to the principles outlined in the Declaration of Helsinki. The demographic and clinical characteristics of the NPC cases and the control group are detailed in Table I.
Demographic characteristics of the 416 control subjects and 208 NPC patients.
Genotyping methodologies for MMP-8 polymorphisms of the NPC cases and non-cancer controls. DNA was extracted from peripheral leukocytes obtained from each participant within 24 hours of collection using the QIAamp Blood Mini Kit (Qiagen, Valencia, CA, USA) (30, 31). The extracted DNA samples were stored at −80°C for long-term preservation and also diluted for use as a working stock for MMP-8 genotyping (32, 33). The genotyping of MMP-8 followed our previously published methodologies (22, 29). In brief, amplification of MMP-8 rs11225395, rs34009635, and rs35866072 was performed using a T100 Thermal Cycler 96-well system (Biorad, San Jose, CA, USA). Subsequently, restriction fragment length polymorphism analysis was conducted with the respective restriction endonucleases from New England Biolabs (Taipei, Taiwan, ROC).
Statistical analytical methodology. To ensure the control group’s representativeness within the general population, the Hardy-Weinberg equilibrium was evaluated using the goodness-of-fit test to identify any deviations in genotype frequencies. The unpaired Student’s t-test was used to compare the mean ages between NPC cases and non-cancer controls. The distribution of genotypes among different subgroups was assessed using Pearson’s Chi-square test with Yates’ correction according to our previously published papers (34, 35). A significance threshold of p less than 0.05 was applied to all statistical analyses. Logistic regression analysis was conducted to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for NPC risk associated with specific genotypes.
Results
Demographic and clinical characteristics of NPC cases and non-cancer control groups. Table I presents the frequency distributions of demographic and clinical characteristics for the 208 NPC cases and 416 non-cancer controls. Since the controls were frequency-matched to the cases by sex and age, no significant difference between the two groups was found (p=0.4639). Additionally, the histological details are shown in Table I. It is important to note that the higher proportions of smokers (38.0%), alcohol drinkers (40.4%), and betel quid chewers (37.5%) among the non-cancer controls are a result of the frequency-matching strategy and may not represent the general Taiwanese population accurately. Among the NPC cases, 3.8% were classified as keratinizing squamous cell carcinoma (WHO type I), and 96.2% as non-keratinizing carcinoma (WHO type II). The type II NPC cases were further divided into 16.0% non-keratinizing differentiated carcinoma (WHO type IIa) and 84.0% non-keratinizing undifferentiated carcinoma (WHO type IIb) (Table I).
Association of MMP-8 genotypes with NPC risk. Table II details the genotypic distributions of MMP-8 rs11225395, rs34009635, and rs35866072 among the 416 non-cancer controls and 208 NPC cases. Firstly, the genotypic frequencies of MMP-8 rs11225395 and rs34009635 in the control group adhered to the Hardy-Weinberg equilibrium (p=0.1938 and 0.7081, respectively). Secondly, there was no significant difference in the genotypic frequencies of MMP-8 rs11225395 between the NPC cases and the controls (p for trend=0.6928). Specifically, the heterozygous CT and homozygous TT genotypes of MMP-8 rs11225395 seemed to provide a protective effect against NPC risk; however, these findings were not statistically significant (OR=0.97 and 0.79, 95%CI=0.68-1.38 and 0.46-1.36, p=0.9304 and p=0.4736, respectively). This lack of significant association was further confirmed when comparing the combined CT and TT genotypes with the wild-type CC genotype (OR=0.92, 95%CI=0.66-1.29, p=0.7108). Thirdly, the heterozygous AC genotype of MMP-8 rs34009635 did not show a significant association with NPC risk (OR=0.66, 95%CI=0.24-1.84, p=0.5738). Notably, there were no individuals with the homozygous CC genotype in either the NPC or control groups. Lastly, all participants in both the NPC and control groups were found to carry the TT genotype at MMP-8 rs35866072 (Table II).
Distribution of matrix metalloproteinase-8 rs11225395, rs34009635, and rs35866072 variant genotypes among the controls and patients with NPC.
Association of MMP-8 allelic frequencies with NPC risk. To further validate the pilot findings in Table II, an allelic frequency analysis was performed to investigate the roles of MMP-8 rs11225395 and rs34009635 in NPC susceptibility. In line with the observations of Table II, the data showed no significant difference in the presence of the variant T alleles for MMP-8 rs11225395 between the NPC cases and controls (p=0.4876), with an OR of 0.91 (95%CI=0.71-1.16) for NPC risk compared to those with the wild-type C allele (Table III). Similarly, the variant C alleles for MMP-8 rs34009635 also did not show a significant difference between the two groups (p=0.5769), presenting an OR of 0.66 (95%CI=0.24-1.84) for NPC susceptibility relative to the wild-type A allele (Table III). As a result, the combined results from Table II and Table III indicate that the variant genotypes of MMP-8 rs11225395 and rs34009635 may not significantly affect the susceptibility to NPC among Taiwanese individuals.
Allelic frequencies for matrix metalloproteinase-8 rs11225395, and rs34009635 in the control and NPC patient groups.
Stratified analysis of MMP-8 rs11225395 genotypes based on personal behaviors. Our next step focused on examining the combined impact of MMP-8 rs11225395 genotype with cigarette smoking, alcohol consumption, and betel quid chewing habits on NPC risk (Table IV, Table V, and Table VI). No significant interaction was observed between MMP-8 rs11225395 genotype and cigarette smoking status, regardless of smoking status (all p>0.05) (Table IV). Similarly, there was no significant interaction between genotype and alcohol consumption status, whether individuals were non-drinkers or drinkers (Table V). Likewise, no interaction was found regarding betel quid chewing status (Table VI). We adjusted the data for age, sex, and corresponding behavioral status and re-evaluated the interactions, but still found no significance in each individual analysis (Table IV, Table V, and Table VI).
Distribution of matrix metalloproteinase-8 rs11225395 genotypes among NPC cases and controls after stratification by smoking status.
Distribution of matrix metalloproteinase-8 rs11225395 genotypes among NPC cases and controls after stratification by alcoholism status.
Distribution of matrix metalloproteinase-8 rs11225395 genotypes among NPC cases and controls after stratification by betel quid chewing status.
Discussion
MMP-8 functions as one of the most effective collagenases, playing a pivotal role in carcinoma invasion and metastasis. Existing literature highlights MMP-8’s role in promoting the invasion of colorectal cancer cells and its potential as a prognostic marker (36, 37). However, in the context of NPC, there is a paucity of literature suggesting MMP-8 alone as a biomarker. Intriguingly, the upregulation of MMP-8, along with SLAM family member 5 (SLAMF5), endothelial cell-specific molecule-1 (ESM-1), insulin receptor (INSR), and Serpin Family A Member 5 (Serpin A5), has been associated with a protein-based signature for distant metastasis in a cohort of 226 NPC patients (38). Moreover, the mRNA expression levels of MMP-8 in peripheral blood mononuclear cells and granulocytes have been reported to function as biological markers to assess disease severity in viral lower respiratory tract infections among Netherlands children (39). MMP-8 and MMP-9 The limited research on MMP-8 in NPC may be attributed to the typically small NPC sample sizes, which are usually unsuitable for proteomic analysis. This limitation may prompt the exploration of markers from a genomic perspective rather than a protein-based approach for precise medication.
The rationale behind hypothesizing MMP-8’s potential role in NPC etiology stems from several investigations into MMP-8’s involvement in head and neck disorders. Notably, MMP-8 expression was found to be notably elevated in the serum of patients with squamous cell carcinomas of the head and neck as early as 2002 (40, 41), a finding subsequently validated in multiple studies (42-47). However, limited literature has explored the association of MMP-8 genotypes with head and neck cancer, let alone NPC. In our current study, we found that the variant genotypes of MMP-8 rs11225395 and rs34009635 are not significantly associated with altered NPC risk (Table II). At another SNP site, rs35866072, there appeared to be no polymorphism observed among Taiwanese individuals (Table II). While our pilot investigation did not unveil any clinically useful MMP-8 marker for NPC, it is noteworthy that the etiological factors contributing to NPC may vary among different populations due to differences in viral infection status, dietary habits, and genetic backgrounds, necessitating further investigations across diverse populations to validate our hypothesis. Notably, in 2019, Zhang and colleagues analyzed the association of the three MMP-8 SNPs with various malignant carcinomas, including breast, bladder, lung, and oral cancer, albeit not NPC, utilizing a database of 10,529 healthy individuals and 8,140 patients. They tentatively concluded that MMP-8 rs11225395, rs34009635, and rs35866072 variant genotypes are not associated with cancer susceptibility (48).
In addition to evaluating the impact of MMP-8 genotypes on NPC risk, our study has expanded to explore potential interactions between MMP-8 rs11225395 genotypes and behavioral factors, such as cigarette smoking, alcohol consumption, and betel quid chewing habits, related to NPC susceptibility (Table IV, Table V, and Table VI). We observed no significant association among subgroups stratified by age or sex (data not shown). Furthermore, there was no noticeable difference in susceptibility among individuals with variant MMP-8 rs11225395 genotypes, regardless of cigarette smoking, alcohol consumption, or betel quid chewing behaviors, in relation to NPC risk (Table IV, Table V, and Table VI). Previous studies have shown no combined impact of MMP-8 genotypes and cigarette smoking or alcohol consumption on renal cell carcinoma risk (29) or lung cancer risk (27). However, an interaction between cigarette smoking or alcohol consumption and the MMP-8 rs11225395 genotype has been implicated in increased colorectal cancer risk (23). The precise mechanisms by which MMP-8 genotypes influence altered NPC risk remain unclear, not to mention in combination with behavioral factors. Further investigations with larger sample sizes are urgently needed to gain deeper insight into the involvement of MMP-8 genotypes in NPC carcinogenesis.
In summary, our results indicate that variant MMP-8 genotypes may exert a modest impact on NPC susceptibility. Particularly, the MMP-8 rs11225395 genotype appears to show no significant combined effects with cigarette smoking, alcohol consumption, or betel quid chewing behaviors on individual NPC risk. Further investigation using a larger sample size and diverse population groups is essential to validate and extend our findings.
Acknowledgements
The Authors are grateful to Hou-Yu Shih for their excellent technical assistance. This study was supported by Taichung Armed Forces General Hospital (TCAFGH-D-112023), in addition to Asia and China Medical University (grand number: CMU112-ASIA-04 and ASIA-112-CMUH-17). The funders had not involved in the study design, data collection, analysis, or annotation of the manuscript.
Footnotes
Authors’ Contributions
Research design: Chen CH, Hsu SW, and Chang WS; patient and questionnaire summaries: Shih LC, Tien HC, and Liu YF; experimental work: Yu-Ting Chin, Wang YC, Chang WS, and Tsai CW; data clearance and identification: Chen CH, Hsu SW, Hsu SW, and Yu-Ting Chin; statistical analysis: Yu-Ting Chin, Hsu SW, and Chang WS; literature review and manuscript writing: Chen CH, Shih LC, Yu-Ting Chin, Chang WS and Bau DT; review and revision: Hsu SW, Chang WS and Bau DT.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received June 12, 2024.
- Revision received July 4, 2024.
- Accepted July 10, 2024.
- Copyright © 2024 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).
