Abstract
Background/Aim: This study aimed to investigate the involvement of matrix metalloproteinase-8 (MMP-8) genotypes in the development of colorectal cancer (CRC). Materials and Methods: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to analyze the genotypes of MMP-8 C-799T (rs11225395), Val436Ala (rs34009635), and Lys460Thr (rs35866072) in 362 patients with CRC and 362 controls. Additionally, the potential associations between these genotypes and factors such as age, sex, smoking, alcohol consumption, and body mass index (BMI) status in relation to CRC risk were also assessed. Results: No significant differences in the distribution of MMP-8 rs11225395 genotypes were found between the control and case groups (p for trend=0.3836). Logistic regression analysis demonstrated that individuals with the MMP-8 rs11225395 variant CT and TT genotypes had a 0.83 and 0.77-fold risk of CRC, respectively. Moreover, carriers of the rs11225395 CT+TT genotypes were not associated with CRC risk either (p=0.2063). Furthermore, individuals with the MMP-8 rs11225395 TT genotype exhibited significantly lower odds of CRC risk compared to those with the CC genotype among non-smokers (p=0.0379). No significant associations were observed with respect to MMP-8 rs34009635 or rs35866072. Conclusion: The analyzed genotypes of MMP-8 play a minor role in determining individual susceptibility to CRC risk.
Colorectal cancer (CRC) accounts for approximately 11% of all newly diagnosed cancer cases and is the third most common cancer worldwide, causing the second highest number of cancer-related deaths (1, 2). The pathogenesis of CRC involves various factors that contribute to complex genetic and epigenetic processes, ultimately leading to the transformation of normal colonic mucosa into cancerous tissue (3). Numerous molecular signaling networks implicated in CRC initiation and progression have been reported in the literature, including the ERK/MAPK, TGF-β, PI3K/Akt, Src/FAK, and β-catenin related signal transduction pathways. These pathways are associated with the hallmarks of cancer, such as inflammation, angiogenesis, metastasis, and invasion. Notably, activation and over-expression of matrix metalloproteinases (MMPs) have been linked to these signal transduction pathways, making MMPs potential prognostic factors for CRC (4, 5). Thus, MMPs have been suggested to be potential prognostic factors for CRC.
MMP-8 is an intriguing matrix metalloproteinase (MMP) that has been found to possess antitumor activity and immune-regulatory properties, although its role in CRC has not been extensively studied. MMP-8 is commonly expressed by neutrophils and is responsible for cleaving various substrates, including type I, II, and III collagen. A study conducted by Väyrynen and his colleagues in 2012 revealed that preoperative serum MMP-8 levels were higher in 148 patients with CRC compared to 83 controls. Furthermore, the levels of preoperative serum MMP-8 were positively correlated with disease stage, the extent of primary tumor necrosis, and blood neutrophil count (6). In 2018, Sirnio and his colleagues found that elevated serum MMP-8 levels were associated with decreased survival and systemic inflammation in patients with CRC (7). Additionally, in 2021, Reijonen et al. reported that high levels of MMP-8, both preoperatively and postoperatively, were associated with worse 10-year overall survival rates (8). However, the genetic role of MMP-8 in CRC remains undisclosed.
In 2020, Tai et al. conducted a study to investigate the association between MMP-8 rs11225395 polymorphism and CRC risk. The study included 551 CRC cases and 623 controls from a Han population (9). They found that individuals carrying the variant TT genotype had a 1.76-fold increased risk of CRC compared to those carrying the CC genotype. Furthermore, individuals carrying the rs11225395 TT genotype not only had a higher CRC risk but also exhibited poorer overall survival compared to those carrying the CC genotype (9). However, no other literature is available to validate their findings. Therefore, our aim was to examine the role of MMP-8 rs11225395 in determining CRC risk in a Taiwanese population consisting of 362 patients with CRC and 362 controls.
Materials and Methods
Investigated CRC population. The recruitment of CRC cases and healthy controls followed the methodology outlined in our previous publications (10, 11). Briefly, CRC cases were recruited from patients visiting the Department of General Surgery at China Medical University Hospital (CMUH), and comprehensive pathological data were recorded for each case. The control subjects were carefully matched 1:1 to the cases based on age and sex. All participants provided informed consent and donated blood samples for the study. The study protocols were approved and overseen by the Institutional Review Board of CMUH (approval code: DMR99-IRB-108) and conducted in accordance with the principles of the Declaration of Helsinki.
Genotyping methodology of MMP-8 polymorphisms. Genomic DNA was extracted from the blood samples using a Qiagen kit (Qiagen, Chatsworth, CA, USA) according to the protocols described in our previous publication (12, 13). The genotyping of MMP-8 rs11225395, rs34009635, and rs35866072 was carried out using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, as previously described (14, 15).
PCR amplification was conducted using a PCR Thermocycler (Bio-RAD, Hercules, CA, USA) with the following cycling conditions: initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 64°C for 40 s, and extension at 72°C for 45 s. This was repeated for 35 cycles, followed by a final extension step at 72°C for 10 min. The amplified PCR products for MMP-8 rs11225395, rs34009635, and rs35866072 were visualized using 3% agarose gel electrophoresis to confirm the success of PCR amplification. Subsequently, the PCR products of MMP-8 rs11225395, rs34009635, and rs35866072 were subjected to digestion using Sfc I, Nla III, and Bbs I, respectively. The resulting digestion fragments were then analyzed by 4% agarose gel electrophoresis to identify the specific genotypes.
Statistical analysis. To compare the ages between the case and control groups, we conducted an unpaired Student’s t-test. The distributions of sex, personal habits, BMI, different MMP-8 genotypes, and alleles among the subgroups were assessed using either Pearson’s chi-square test (when n>5) or Fisher’s exact test (when n≤5). The associations between different MMP-8 genotypes and the risk of CRC were evaluated by calculating individual odds ratios (ORs) with their corresponding 95% confidence intervals (Cis). Statistical significance was indicated when p-value <0.05.
Results
Characteristics of study population. The demographic characteristics of the 362 patients with CRC and the matched 362 controls are shown in Table I. Since the controls were matched 1:1 to the cases based on age and sex, there were no significant differences in the distribution of these two variables between the case and control groups. Additionally, there were no significant differences in the distribution frequencies of smokers (p=0.543), alcohol drinkers (p=0.441), and individuals with lower (<24) or higher (≥24) BMI (p=0.181) between the case and control groups.
Selected characteristics of the 362 patients with colorectal cancer and 362 non-cancer controls.
Association of MMP-8 rs11225395, rs34009635, and rs35866072 with CRC risk in Taiwan. The genotypes of MMP-8 rs11225395 and rs34009635 in the control groups were consistent with the expected frequencies based on the Hardy-Weinberg equation (p=0.1401 and 0.7899, respectively). All individuals examined had the same TT genotype at the rs35866072 polymorphic site (Table II). There appeared to be no significant association between MMP-8 rs11225395 genotypes and the risk of CRC (p for trend=0.3836). Specifically, compared to the wild-type CC genotype, individuals carrying the heterozygous variant genotype CT had an OR of 0.83 (95%CI=0.61-1.14, p=0.2963), whereas those carrying the homozygous variant TT genotype had a 0.77-fold increased risk of CRC (95%CI=0.48-1.23, p=0.3260). Individuals carrying the variant genotypes (CT+TT) had a 0.82-fold increased risk of CRC compared to those carrying the wild-type CC genotype (95%CI=0.61-1.10, p=0.2063) (Table II). Similarly, no significant association was found between MMP-8 rs34009635 genotypes and the risk of CRC. Specifically, compared to the wild-type AA genotype, individuals carrying the heterozygous variant genotype AC had an OR of 1.10 (95%CI=0.46-2.63, p=0.8247). No subjects were found to carry the MMP-8 rs34009635 CC genotype (Table II).
Associations between MMP-8 genotypes and colorectal cancer risk in Taiwan.
Association of MMP-8 rs11225395, rs34009635, and rs35866072 allelic frequencies with CRC risk in Taiwan. The results of allelic frequency tests for MMP-8 rs11225395, rs34009635, and rs35866072 polymorphic sites in relation to CRC risk are presented in Table III. Consistent with the findings in Table II, the frequency of the variant T allele in rs11225395 was slightly lower in the CRC patient group (29.7%) compared to the control group (33.1%) but did not reach statistical significance (p=0.1742). Individuals carrying the variant T allele had a 0.85-fold increased risk of CRC (95%CI=0.68-1.06). Similarly, there was no significant difference in the allelic frequency of MMP-8 rs34009635 between the case and control groups (OR=1.10, 95%CI=0.46-2.61, p=0.8260) (Table III).
Associations of MMP-8 alleles with colorectal cancer risk.
Stratified analyses of MMP-8 rs11225395 genotypes by age, sex, smoking, alcohol drinking, and BMI status. We conducted stratified analyses to examine the association between MMP-8 rs11225395 genotype and CRC risk based on age, sex, smoking, alcohol drinking, and BMI status, and the results are presented in Table IV. Overall, significant associations between MMP-8 rs11225395 genotype and CRC risk were observed in all the strata, except for the non-smoker subgroup. Notably, in the non-smoker subgroup, the risk associated with the homozygous variant TT genotype reached statistical significance (OR=0.53, 95%CI=0.30-0.93, p=0.0379). Even after adjusting for age, sex, BMI, and alcohol drinking behavior, the significance persisted (OR=0.57, 95%CI=0.28-0.88) (Table IV). However, no significant associations were found for MMP-8 rs34009635 and rs35866072 polymorphic sites in any of the analyzed subgroups (data not shown).
Associations between MMP-8 rs11225395 genotypes and colorectal cancer risk in stratified analyses.
Discussion
The precise role of MMP-8 in CRC carcinogenesis remains unclear. Previous studies have shown conflicting results regarding the correlation between MMP-8 protein levels and tumor malignancy in CRC. Verspaget and his colleagues reported a step-wise increase in MMP-8 levels correlating with tumor malignancy as early as 1999 (16). However, Koskensalo and Takeha did not observe a similar correlation in their studies (17, 18). Furthermore, increased serum levels of MMP-8 have been reported in patients with CRC compared to healthy controls, and high levels of MMP-8 have been associated with increased malignancy, reduced survival rates, and systemic inflammation (6, 7, 19). In this current study, we investigated the potential contribution of MMP-8 rs11225395, rs34009635, and rs35866072 genotypes to the risk of CRC in Taiwan. Our results indicate that none of these three SNPs, rs11225395, rs34009635, or rs35866072, were associated with an increased risk of CRC in the Taiwanese population (Table II and Table III).
Our findings are inconsistent with a previous study conducted by Tai and colleagues, which reported a significant association between the MMP-8 rs11225395 TT genotype and increased CRC risk in a Chinese population (9). Their study had a representative sample size, including 551 CRC cases and 623 controls. However, they did not investigate other SNPs in their study.
In recent years, several studies have examined the association of MMP-8 rs11225395 genotypes with various types of cancer in different populations. Kubben and colleagues found no association between MMP-8 rs11225395 genotypes and gastric cancer risk or survival rate in the Dutch population (20). Qiu et al. reported no association between MMP-8 rs11225395 genotypes and hepatocellular carcinoma risk in a subpopulation of Han Chinese consisting of 434 cases and 480 controls (21). Debniak et al. reported that MMP-8 rs11225395 TT genotypes were associated with an increased risk of malignant melanoma in a Polish population (22). Nor Hashim and colleagues suggested that MMP-8 rs11225395 polymorphism was a protective factor for nasopharyngeal carcinoma susceptibility in a Southeast Asian population, including 24 Chinese, 24 Malaysians, and 48 controls (23). It is worth noting that although the sample size was small, this study was a genome-wide association study examining a panel of 768 SNPs. In an Indian study with 200 cases and 200 age-matched controls, MMP-8 rs11225395 TT genotypes were found to decrease the risk of bladder cancer (24). Arechavaleta-Velasco et al. reported that MMP-8 rs11225395 TT genotypes were associated with an increased risk of ovarian cancer in Mexican women (25). Their DNA samples were extracted from 35 malignant ovarian tumors, 51 benign tumors, and 37 normal ovary tissues, not from blood. Debniak and his colleagues also found no association between MMP-8 rs11225395 genotypes and breast cancer risk (22). This is consistent with Hsiao’s study investigating 1,232 breast cancer cases and 1,232 age-matched non-cancer controls in Taiwanese women (26). On the contrary, Wang et al. reported that MMP-8 rs11225395 TT genotypes increased the risk of breast cancer in a subpopulation of East Asians comprising 571 cases and 578 controls (27). In addition to the findings in breast cancer (26), other studies aimed at the Taiwanese population consistently showed no association between MMP-8 rs11225395 genotypes and childhood leukemia (14), lung cancer (28), oral cancer (15), and bladder cancer risk (29). To date, there is only one study investigating the association of MMP-8 rs34009635 and rs35866072 with cancer, which found no association with lung cancer risk (28).
In 2019, a meta-analysis was conducted by Feng et al. to address the inconsistent results from various studies investigating the association of MMP-8 rs11225395 genotype with different types of cancer, including those mentioned previously. The meta-analysis revealed no association between MMP-8 rs11225395 genotype and overall cancer risk worldwide (30). Based on the current available evidence, it is tentatively concluded that there may be an elevated cancer risk associated with MMP-8 rs11225395 genotype in non-Asian populations, while no association has been found in Asian populations (30). This finding is consistent with the present study, which found no association between MMP-8 rs11225395 genotype and CRC risk. Further studies are necessary to validate the role of MMP-8 genotypes in determining individual susceptibility to different types of cancer, particularly CRC.
In the present study, we investigated the association between MMP-8 genotypes, specifically MMP-8 rs11225395 (C-799T at promoter region), rs34009635 (Val436Ala), and rs35866072 (Lys460Thr), and the risk of CRC in the Taiwanese population. Our findings, as summarized in Table II and Table III, indicate that none of these genotypes were significantly associated with CRC risk. Furthermore, when we performed stratified analyses based on age, sex, smoking status, drinking status, and BMI of patients with CRC, no significant joint effects between these subgroups and MMP-8 rs11225395 genotype were observed, except in the case of non-smokers (Table IV). However, the underlying mechanisms by which the MMP-8 rs11225395 TT genotype exerts a protective effect on non-smokers with respect to CRC risk remain unknown. Further investigations are warranted to provide a comprehensive understanding of these mechanisms. Interestingly, a previous study has suggested that the MMP-8 rs11225395 TT genotype may be associated with higher expression levels of MMP-8 in the serum of CRC patients (9).
In conclusion, our study findings suggest that the genotypes of MMP-8 rs11225395, rs34009635, and rs35866072 are not associated with a modified risk of CRC in the Taiwanese population. However, we observed a potential protective effect of the TT genotype of MMP-8 rs11225395 in non-smokers. Further investigations are required to validate and elucidate the role of MMP-8 genotypes in determining individual susceptibility to various types of cancer, particularly CRC.
Acknowledgements
The Authors would like to acknowledge the Tissue-Bank of China Medical University Hospital for their invaluable technical support. Furthermore, the Authors would like to extend their gratitude to all the study participants, as well as the doctors, nurses, and colleagues who contributed to the study. The technical assistances from Yu-Hsin Lin, Yi-Wen Hung, and Hou-Yu Shih are appreciated by all the Authors.
Footnotes
Authors’ Contributions
Conceptualization: D.Y., D.T.B., C.W.T. and W.S.C.; Collection: T.W.K. and Y.C.H.; Data curation: M.C.M. and C.W.T.; Genotyping: Y.C.W., Y.T.C. and W.S.C.; Statistics: Y.C.Y. and C.W.T.; Phenotyping: D.T.B. and W.S.C.; Project administration: T.C.Y. and D.T.B.; Supervision: D.T.B., W.S.C. and C.W.T.; Validation: T.W.K. and W.S.C.; Writing – original draft: D.Y., and C.W.T.; Writing – review and editing, D.T.B., D,Y. and W.S.C.; All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no competing interests in relation to this study.
Funding
This study received significant support from China Medical University Hospital (DMR-112-022). The funders had no involvement in the study design, data collection, statistical analysis, decision to publish, or manuscript preparation.
- Received June 13, 2023.
- Revision received July 13, 2023.
- Accepted July 14, 2023.
- Copyright © 2023 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).
