Abstract
Background/Aim: In gastric cancer (GCa) tissues, mRNA expression of matrix metalloproteinase-8 (MMP-8) is notably reduced compared to healthy tissues. Furthermore, abnormally low or elevated serum levels of MMP-8 have been linked to a significantly poor prognosis. The involvement of MMP-8 genotypes in susceptibility to GCa remains underexplored. We aimed to assess the influence of MMP-8 genotypes on GCa susceptibility and their potential interactions with smoking, alcohol consumption, and Helicobacter pylori (H. pylori) infection. Patients and Methods: The study utilized polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) to analyze MMP-8 rs11225395, rs34009635, and rs35866072 genotypes in 161 GCa patients and 483 controls. Results: No statistically significant difference was detected in the distribution of genotypic (p for trend=0.3635) or allelic (p=0.1954) frequencies of MMP-8 rs11225395. Under a dominant model, combined CT+TT genotypes showed no association with GCa risk [odds ratio (OR)=0.77, 95% confidence interval (95%CI)=0.54-1.10, p=0.1852]. Similarly, no association was observed for MMP-8 rs34009635 or rs35866072. Importantly, individuals with the MMP-8 rs11225395 CC genotype demonstrated a significant increase in GCa risk when exposed to smoking (OR=4.04, 95%CI=2.28-7.16, p=0.0001), alcohol consumption (OR=2.83, 95%CI=1.64-4.89, p=0.0002), and H. pylori infection (OR=3.53, 95%CI=2.12-5.90, p=0.0001). Conclusion: The findings indicate that individuals carrying the MMP-8 rs11225395 CC genotype have increased susceptibility to GCa, especially when combined with risk factors, such as smoking, alcohol consumption, and H. pylori infection. These results suggest that MMP-8 genotype-based preventive strategies, including lifestyle alterations and targeted infection treatments, may be valuable in mitigating GCa development.
Gastric cancer (GCa) ranks as the fifth most prevalent malignancy and stands as the third leading cause of cancer-related mortality worldwide. According to global statistics, approximately 19.3 million new cancer cases and 10 million cancer-related deaths occurred, with GCa accounting for 5.6% of all new cancer cases (1, 2). In the United States alone, it is estimated that 26,890 new cases of GCa and 10,880 related deaths will occur in 2024 (3). Despite a notable decline in GCa morbidity and mortality in recent years, it remains a significant global public health issue due to its intricate pathogenesis and poor prognosis (4, 5). Although the precise mechanisms driving gastric carcinogenesis are not fully understood, the incidence of GCa is influenced by geographic factors, age-related differences, and the involvement of E-cadherin in epithelial-mesenchymal transition (6, 7). Generally, the incidence and mortality rates of GCa are significantly higher in Asian countries, particularly in Eastern Asia, which accounts for over 60% of recent cases (8, 9). Major risk factors associated with GCa include Helicobacter pylori (H. pylori) infection, genetic mutations, high-salt diets, smoking, and alcohol consumption (10-13). Nonetheless, the contribution of inherited genetic variations to GCa susceptibility remains largely unexplored (14, 15). In recent years, with advancements in human genome research, numerous studies have investigated the links between specific genetic variants and the risk of GCa (16-18).
MMP-8 is a matrix metalloproteinase (MMP) that has drawn attention due to its antitumor and immune-regulatory functions, though its role in GCa has not been widely explored. Predominantly expressed by neutrophils, MMP-8 is involved in the degradation of various substrates, including type I, II, and III collagen. In 2006, Kubben and his colleagues conducted a comprehensive study on the expression levels of MMPs and their inhibitors, TIMP-1 and -2, in 81 patients with GCa (19). Their findings indicated that the expression levels of MMP-7, -8, and -9 in tumor tissues were associated with clinicopathologic parameters, such as TNM staging, WHO, and Lauren classifications. However, there was no correlation between the levels of MMP-7, -8, or -9 and patient survival (19). A subsequent study by Laitinen and his colleagues in 2018 reported that GCa patients with either low or high serum MMP-8 levels had a poor prognosis, and the MMP-8/TIMP-1 ratio was identified as a reliable predictor of prognosis in Finnish patients with GCa (20). Nevertheless, the literature did not specify which subgroups of GCa patients exhibited low or high MMP-8 expression levels, nor did it provide detailed insights into the underlying mechanisms, which require further investigation. Additionally, the role of MMP-8 genotypes in GCa susceptibility remains largely unexplored.
As highlighted above, the role of MMP-8 genotypes in GCa has been scarcely investigated. Consequently, this study aimed to address two key objectives. The first objective was to determine whether the genotypes of MMP-8 rs11225395, rs34009635, and rs35866072 are linked to the risk of developing GCa in a well-defined Taiwanese cohort comprising 161 patients with GCa and 483 healthy controls. The second objective was to explore the combined influence of smoking, alcohol consumption, H. pylori infection, and MMP-8 genotypes on GCa susceptibility.
Patients and Methods
Recruiting of GCa cases and healthy controls. A cohort of 161 patients with GCa was recruited from the general surgery outpatient clinics at China Medical University Hospital (CMUH), as previously documented in the literature (15, 16, 21). Each participant voluntarily provided a 5 ml sample of peripheral blood. For comparative analysis, 483 healthy, cancer-free individuals with matching age and sex were selected from the CMUH Health Examination Cohort. The study design and protocols received approval from the Institutional Review Board of the hospital (IRB number: DMR100-IRB-107), with written informed consent obtained with assistance from the Tissue Bank of China Medical University Hospital. Table I summarizes the demographic characteristics of the study population, including age, sex, body mass index (BMI), smoking and alcohol consumption habits, H. pylori infection status, and histological classifications.
Selected characteristics of the control and gastric cancer groups.
Genotyping methodology of MMP-8 polymorphisms. Genomic DNA was isolated from the collected blood samples using a Qiagen kit (Qiagen, Chatsworth, CA, USA), following the procedures outlined in our earlier studies (22, 23). The genotyping of MMP-8 polymorphisms rs11225395, rs34009635, and rs35866072 was conducted using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, consistent with methods detailed in prior publications (23-25).
PCR amplification was performed using a Bio-RAD PCR Thermocycler (Hercules, CA, USA) under the following conditions: an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 40 s, and extension at 72°C for 45 s. A final extension step was carried out at 72°C for 10 min. The PCR products for MMP-8 rs11225395, rs34009635, and rs35866072 were confirmed using 3% agarose gel electrophoresis. Subsequently, these amplified products were digested with Sfc I, Nla III, and Bbs I enzymes, respectively. The digestion fragments were then resolved on 4% agarose gels to determine the specific genotypes. For accuracy, the genotyping was conducted independently by three researchers in a blinded manner, with all results showing 100% concordance.
Statistical analyses. An unpaired Student’s t-test was used to compare the ages between the GCa and control groups. Pearson’s chi-square test with Yates’ correction was applied to evaluate the distributions of sex, personal habits, BMI, and various MMP-8 genotypes and alleles among the subgroups. The relationship between different MMP-8 genotypes and GCa risk was determined by calculating the odds ratios (ORs) along with their 95% confidence intervals (CIs). A p-value less than 0.05 was considered statistically significant.
Results
Table I summarizes the demographic data of the 644 participants in this study, consisting of 161 patients with GCa and 483 healthy controls. The groups were matched for age and sex, with no significant differences observed in age (p=0.3519) or sex (p=1.0000) between them. However, there were significant differences in the distributions of smoking habits (p<0.0001), alcohol consumption (p<0.0001), and H. pylori infection status (p<0.0001) between the patients with GCa and the control group, suggesting that these factors could be potential risk factors for GCa in the Taiwanese population. Regarding tumor localization, the majority of GCa cases (60.9%) had tumors situated in the upper (14.2%), middle (42.9%), and lower (42.9%) regions (Table I).
Table II provides an overview of the genotypic frequencies for MMP-8 polymorphisms rs11225395, rs34009635, and rs35866072 among the study participants. First, the control group’s genotypic distributions for MMP-8 rs11225395 and rs34009635 conformed to Hardy-Weinberg equilibrium (p=0.2901 and 0.9050, respectively). Second, for MMP-8 rs11225395, no significant differences in genotype frequencies were detected (p for trend=0.3635). Specifically, the heterozygous CT and homozygous TT variants were observed in 38.5% and 11.2% of patients with GCa, respectively, compared to 43.1% and 13.0% in the control group (OR=0.78 and 0.75, 95%CI=0.53-1.14 and 0.42-1.34, p=0.2387 and 0.4027, respectively). When analyzed under a dominant model, the combined CT+TT genotypes of MMP-8 rs11225395 were not associated with an altered risk of GCa (OR=0.77, 95%CI=0.54-1.10, p=0.1852, Table III, top panel). Third, a significant difference in the distribution of MMP-8 rs34009635 genotypes between patients with GCa and controls was noted (OR=0.69, 95%CI=0.19-2.44, p=0.7700), with no individuals in either group carrying the homozygous GG genotype (Table III, middle panel). Finally, no carriers of the MMP-8 rs35866072 variant were identified within the Taiwanese population (Table III, bottom panel).
Associations between MMP-8 genotypes and gastric cancer risk.
Distributions of MMP-8 rs11225395, rs34009635 and rs35866072 allelic frequencies among the patients with gastric cancer and the control subjects.
To further investigate the findings presented in Table II, allelic frequency analyses of MMP-8 rs11225395, rs34009635, and rs35866072 were performed. The variant T allele of MMP-8 rs11225395 and the variant G allele of MMP-8 rs34009635 did not exhibit any significant association with altered risk of GCa (Table III). Additionally, no variant alleles for MMP-8 rs35866072 were observed in the Taiwanese population (Table III). Given that GCa is associated with smoking, we examined the interaction between the MMP-8 rs11225395 genotype and smoking status. When comparing non-smokers who were carriers of the MMP-8 rs11225395 CT+TT genotypes with non-smokers carrying the CC genotype, the latter group showed a slight but non-significant increase in GCa risk (OR=1.14, 95%CI=0.74-1.76, p=0.6283) (Table IV). In contrast, among ever-smokers, both CT+TT and CC genotype carriers demonstrated significantly higher GCa risks (OR=2.25 and 4.04, 95%CI=1.29-3.93 and 2.28-7.16, p=0.0059 and 0.0001, respectively). Notably, ever-smokers with the CC genotype at MMP-8 rs11225395 exhibited the highest risk of GCa (Table IV). No significant combined effect of MMP-8 rs34009635 genotype and smoking behavior on GCa risk was observed (data not shown).
Combined effects of MMP-8 rs11225395 genotype and cigarette consumption on gastric cancer risk.
Table I suggests that alcohol consumption may be a risk factor for GCa in the Taiwanese population. Therefore, we investigated the combined impact of the MMP-8 rs11225395 genotype and alcohol drinking on GCa risk. When non-drinkers carrying the MMP-8 rs11225395 CT+TT genotypes were used as a reference group, non-drinkers with the CC genotype showed only a modest increase in GCa risk (OR=1.07, 95%CI=0.70-1.65, p=0.8373) (Table V). For ever-drinkers with the CT+TT genotype at MMP-8 rs11225395, there was a slight, though not significant, elevation in GCa risk (OR=1.44, 95%CI=0.82-2.55, p=0.2518) (Table V). In contrast, ever-drinkers with the CC genotype at MMP-8 rs11225395 demonstrated a significant increase in GCa risk compared to non-cancer controls (OR=2.83, 95%CI=1.64-4.89, p=0.0002) (Table V). No significant interaction was observed between the MMP-8 rs34009635 genotype and alcohol consumption regarding GCa risk (data not shown).
Combined effects of MMP-8 rs11225395 genotype and alcohol consumption on gastric cancer risk.
As indicated in Table I, H. pylori infection is a risk factor for GCa in our population like smoking. Therefore, we investigated the combined effect of H. pylori infection status and MMP-8 rs11225395 genotypes on GCa risk. Using non-infected individuals with the MMP-8 rs11225395 CT+TT genotypes as a reference, we found no significant association between the MMP-8 rs11225395 CC genotype and GCa risk among those without H. pylori infection (OR=0.87, 95%CI=0.47-1.62, p=0.7807). However, among individuals with H. pylori infection, the risk of GCa was notably higher in those carrying the CT+TT genotypes (OR=1.97, 95%CI=1.19-3.30, p=0.0119). Furthermore, H. pylori-infected individuals with the CC genotype at MMP-8 rs11225395 faced an even greater risk, with a 3.53-fold increase in GCa risk (OR=3.53, 95%CI=2.12-5.90, p=0.0001). No significant interaction was found between the MMP-8 rs34009635 genotype and H. pylori infection in relation to GCa risk (data not shown).
Discussion
In this study, we investigated the relationship between MMP-8 genotypes and GCa risk in a Taiwanese cohort comprising 161 patients with GCa and 483 healthy controls (Table I). The genotyping analysis indicated that carriers of the variant genotypes MMP-8 rs11225395 and rs34009635 did not exhibit a modified risk for GCa (Table III and Table IV). Notably, there were no carriers of the variant genotype at MMP-8 rs35866072 within the Taiwanese population. Despite the lack of association between the variant genotypes of MMP-8 rs11225395 and rs34009635 and GCa risk, our findings provided significant evidence that the wild-type CC genotype of MMP-8 rs11225395 was associated with increased GCa risk in specific subgroups, including smokers, alcohol drinkers, and individuals with H. pylori infection (Table IV, Table V, and Table VI). Notably, among these subgroups, smokers with the MMP-8 rs11225395 CC genotype faced the highest GCa risk, showing a 4.04-fold increase (Table IV).
Combined effects of MMP-8 rs11225395 genotype and H. pylori infection on gastric cancer risk.
The MMP-8 rs11225395 polymorphism, resulting from the merging of rs60704849 and rs17292466, represents a promoter variant that introduces a C to T single-nucleotide change in the promoter region (Figure 1). Evidence indicates that the T allele of MMP-8 rs11225395 exhibits significantly higher transcriptional and translational activity compared to the C allele (26-28). Clinically, the T allele of MMP-8 rs11225395 has been associated with improved overall survival in early-stage breast cancer patients (26, 29), but with poorer survival outcomes in patients with ovarian cancer (30). It should be noticed that the prevalence of the T allele at MMP-8 rs11225395 varies by ethnicity. In East Asians (n=3126), the T allele frequency is 0.3708, higher than in Africans (0.2447, n=41984) but lower than in Europeans (0.4344, n=75876) and Americans (0.4136, n=13652). In our study, the frequency of the T allele in the control group is 0.3458, which is comparable to that of East Asians (p=0.1431) but significantly different from frequencies observed in Africans (p=0.0003), Europeans (p=0.0001), and Americans (p=0.0001). Regarding MMP-8 rs34009635, this exonic polymorphism shows a low frequency of the G allele, with a prevalence of 0.0423 in East Asians (n=4518) and less than 0.01 in Europeans (n=185,144), Africans (n=6,504), and African Americans (n=6,260). Further large-scale case-control and prospective studies are needed to elucidate the role of MMP-8 genotypes, particularly rs11225395, in GCa risk across different ethnic groups.
Physical map for MMP-8 rs11225395, rs34009635, and rs35866072 polymorphic sites.
Several studies provide valuable insights into the role of MMP-8 genotypes in cancer risk. First, in 2017, Lin and his colleagues investigated the association between the MMP-8 rs1940475 TT genotype and cancer recurrence and survival in 254 patients with GCa. Their findings suggested that the TT genotype was linked to higher recurrence risk and reduced survival (31). However, this study did not include a control group, limiting the ability to assess the genotype’s association with GCa risk comprehensively. Additionally, the study focused solely on a single SNP, MMP-8 rs1940475, which is an exonic polymorphism. Notably, the same study did not find significant differences in mRNA expression among different MMP-8 rs1940475 genotypes (31). Second, in 2006, Kubben and his colleagues found no association between the MMP-8 C-799T or C+17G genotypes and GCa risk (32). Given their negative results and the small sample size with GCa patients less than 100, which has not been validated in subsequent studies, we chose not to replicate their research design.
In the context of this study, the association between smoking behavior and GCa risk has been well-documented in prior research (33, 34). Our investigation extended this by exploring how MMP-8 genotypes interact with smoking status to influence GCa susceptibility. The findings indicate that smokers with the CC genotype for MMP-8 rs11225395 face a significantly increased risk of GCa (Table IV). This elevated risk was similarly observed in alcohol drinkers and individuals infected with H. pylori (Table V and Table VI). Particularly, smokers carrying the CC genotype of MMP-8 rs11225395 demonstrated a markedly higher odds ratio (4.04-fold) for GCa risk (Table IV). While the underlying mechanisms remain unclear, this data can aid clinicians in guiding patient management. Specifically, GCa patients with the CC genotype should consider eliminating smoking and alcohol consumption to mitigate their risk. Further research is needed to elucidate the role of MMP-8 genotypes in GCa among Taiwanese individuals. Additionally, it would be valuable to investigate how MMP-8 genotypes interact with other risk factors, such as environmental pollution, occupational exposure, sanitation conditions, and dietary habits, if such data becomes available.
In 2017, our team investigated the role of MMP-1 rs1799750 (promoter −1607 1G/2G) genotypes in GCa risk, involving 121 patients with GCa and 363 controls in Taiwan. Our results suggested that the variant 1G allele in MMP-1 rs1799750 might be linked to GCa development and could be beneficial for early detection and prevention when combined with cigarette smoking and H. pylori infection status (21). That same year, we shifted focus to MMP-7 promoter polymorphisms (A-181G and C-153T) in the same cohort. The GG genotype at MMP-7 A-181G was identified as a risk factor for GCa, particularly among smokers, whereas no individuals with the variant genotype at MMP-7 C-153T were observed in this Taiwanese population (15). In 2021, we examined the MMP-9 rs3918242 promoter polymorphism in the same Taiwanese population. The TT genotype of MMP-9 rs3918242 was associated with an increased risk of GCa compared to the wild-type CC genotype. This was the first MMP SNP positively linked with GCa. Additionally, CT/TT genotypes at MMP-9 rs3918242 were correlated with a higher risk of metastasis. However, no association was found between MMP-9 rs3918242 genotypes and GCa risk concerning smoking, alcohol consumption, or H. pylori infection status (16). Regarding MMP-2, two polymorphic sites, MMP-2 promoter −1306 (rs243865) and −735 (rs2285053), were studied for their contributions to GCa risk. Analysis of both genotypic and allelic frequencies revealed that MMP-2 rs243865 genotypes were associated with a decreased GCa risk. Further stratification showed that these genotypes interact with smoking, alcohol consumption, and H. pylori infection status, influencing individual susceptibility to GCa. No such association was observed for MMP-2 rs2285053 genotypes. MMP-2 rs243865 may serve as a novel predictive marker for GCa susceptibility among Taiwanese (18).
In conclusion, our study offers robust evidence indicating that the variant TT genotype of MMP-8 rs243865 is linked to a lower risk of GCa in Taiwanese individuals, especially among those who smoke, consume alcohol, or are infected with H. pylori. Further research is needed to fully understand the protective effects of the MMP-8 rs243865 TT genotype.
Acknowledgements
The Authors are grateful to the Tissue-bank of China Medical University Hospital and doctors/nurses under Prof. Yang’s leadership for their excellent sample collection and technical assistance. The technical assistance from Yun-Chi Wang and Hou-Yu Shih are appreciated. This study was supported by Taichung Armed Forces General Hospital (TCAFGH-D-111012), Chang Bing Show Chwan Memorial Hospital (BRD-112044), China Medical University Hospital (DMR-113-101) and Taichung Veterans General Hospital (TCVGH-1125302B). None of the funders had taken part in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Footnotes
Authors’ Contributions
Conceptualization: CKF, WCC and YJC; Data curation: CKF, YJC and MDY; Formal analysis: WCC, JCC and TWK; Funding acquisition: CKF, WCC, YJC and DTB; Investigation: TWK, CWT and WSC; Methodology: CWT, WSC and DTB; Project administration: YCH and DTB; Resources: CKF and MDY; Supervision: DTB; Validation: CWT and DTB; Writing – original draft: CKF, YCH and DTB; Writing – review & editing: DTB.
Conflicts of Interest
All the Authors declare no conflicts of interest regarding this study.
- Received August 12, 2024.
- Revision received August 29, 2024.
- Accepted August 30, 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).
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