Abstract
Background/Aim: Matrix metalloproteinases (MMPs) are a family of proteins which are involved in breakdown of the extracellular matrix in embryonic development, tissue remodeling and in some diseases. MMP8 has both cancer-promoting and anticancer properties. However, the contribution of MMP8 to laryngeal squamous cell carcinoma (LSCC) has not been elucidated. In this study we aimed to test the contribution of two MMP8 polymorphisms, located in the gene promoter region, to the development of LSCC. Materials and Methods: This case–control study involved 569 DNA samples which were genotyped for two single nucleotide polymorphisms using real-time polymerase chain reaction method. Statistical analysis was performed with SPSS Statistics 20 software. Results: Regression analysis adjusted by age showed that for MMP8 rs11225395 each minor A allele copy significantly reduced the odds for LSCC development (odds ratio=0.49, 95% confidence intervaI=0.04-2.19, p=0.048). MMP8 rs11225395 AA genotype was associated with smaller laryngeal tumour size (p=0.023). Smoking habit also correlated with laryngeal tumor size. Conclusion: MMP8 rs11225395 and smoking habits have a prominent interface with LSCC tumour size.
- Laryngeal squamous cell carcinoma
- matrix metalloproteinases
- matrix metalloproteinase 8
- MMR8
- single nucleotide polymorphism
- rs11225395
- rs2155052
Laryngeal squamous cell carcinoma (LSCC) is the second-most common type of head and neck cancer worldwide. It comprised 1.0% of all new cancer cases and 1.0% deaths in 2018 (1). In terms of histopathology, the dominant cancer form in this region is invasive laryngeal squamous cell carcinoma (2). The disease is typically diagnosed in the male patients, usually between the fifth and seventh decade of life (3), and is extremely rare in adolescents (4, 5).
Tobacco smoking and alcohol consumption are the major risk factors for LSCC in developed countries (6). Both factors affect epigenetic reprogramming and genetic instability which induce oncogenesis (7). Tobacco smoking has a major impact on the onset of the disease and current-smokers have around a 20-fold higher risk of LSCC than never-smokers (6, 8). Heavy alcohol consumption is also an independent risk factor for LSCC, however, it has a weaker effect than smoking (increasing disease risk by 2- to 5-fold) (9). Both of these factors are considered to act synergistically. Combined alcohol and tobacco consumption confer multiplicative risk for heavy drinkers and smokers (6, 7). Other risk factors such as radiation, low vegetable intake, gastroesophageal reflux disease, viral infections with human papilomavirus oncogenes mutations and genomic instability are related in the mechanisms of development of LSCC (8-10).
Matrix metalloproteinases (MMPs), also known as matrixins, are a family of zinc-dependent proteolytic enzymes that can degrade all protein components of the extracellular matrix (ECM) and basement membranes (11, 12). MMPs are involved in various physiological processes e.g. in tissue morphogenesis and repair, ovulation, and angiogenesis (13, 14). MMPs also play role in pathological conditions caused by excessive degradation of the ECM such as rheumatoid arthritis, periodontitis, cardiovascular disease, as well as tumor invasion and metastasis (13, 15, 16). The role of MMP2 and MMP9 in LSCC tumorigenesis has been investigated in several studies and overexpression of these MMPs has been associated with the development of LSCC (17-20). The results of a recent study revealed that combination of smoking and 6A/6A genotype of MMP3 (-1171 5A/6A) polymorphism are associated with significantly increased odds of LSCC, thus confirming a presumably important role of MMP3 (-1171) 6A/6A genotype in LSCC carcinogenesis (21).
MMP8 (neutrophil collagenase II) is produced mainly by neutrophils, in the course of inflammatory conditions (13, 14). In recent years, several studies showed that MMP8 might play suppressive roles in cancer and metastasis (22, 23). Firstly, knockout of Mmp8 caused increased aggressive and undifferentiated grade III fibrosarcoma formation in a male mouse model (23). Overexpression of MMP8 in melanoma (B16F10) and Lewis lung carcinoma cell lines reduced metastasis and invasive capacity, and increased cell adhesion (22). The single nucleotide polymorphisms (SNPs) at the MMP8 gene promoter region rs11225395 C-799T and rs2155052 C+17G, which are in strong linkage disequilibrium with the entire MMP8 coding region, provided higher promoter activity and were associated with early-stage diseases (14, 24-27).
Regarding head and neck squamous cell carcinomas, MMP8 expression is linked with prolonged survival in human tongue cancer and reduced susceptibility to tongue squamous cell carcinoma in mouse models (28). However, to the best our knowledge, the associations between MMP8 and LSCC have never been investigated before.
In this study we aimed to reveal the relationship of MMP8 rs11225395 and rs215052 polymorphisms on the risk of LSCC development and its clinical and morphological manifestation.
Materials and Methods
Study population. A group of 226 of patients diagnosed with LSCC at the Department of Otorhinolaryngology of the Lithuanian University of Health Sciences (LUHS) was recruited to this study. Participants completed a questionnaire including questions on history of alcohol consumption, smoking habit and body mass index (BMI). We also included information about tumor stage, tumor differentiation grade, size of the primary tumor and the degree of spread to regional lymph nodes from pathologist's records according to the TNM classification of malignant tumours (29).
The reference group consisted of 343 random research sample of Kaunas city (Lithuania) residents who were selected during the international Health, Alcohol and Psychosocial Factors in Eastern Europe project (HAPPIE) study (30). The sample was collected by the Population Study Laboratory at the Institute of Cardiology of LUHS by carrying out a selection based on the lists of residents of Kaunas city. The LSCC patient and reference groups were matched by age and gender (p>0.05) (Table I).
The present study was approved by Kaunas Regional Biomedical Research Ethics Committee (Protocol No. BE-2-34) and informed consent was obtained from all the participants prior to inclusion in the study.
MMP8 genotyping. Genotyping of MMP8 rs11225395 and rs2155052 was carried out at the Laboratory of Genetics of the Institute of Biology Systems and Genetic Research of LUHS. Venous blood samples for DNA extraction were collected in ethylenediaminetetra-acetic acid tubes. Genomic DNA from peripheral blood leucocytes was extracted using genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommendations. SNP rs11225395 in MMP8 gene was estimated by using a commercially available genotyping kits C_1366493_10 (rs11225395) and C_16139909_10 (rs2155052) (Applied Biosystems, Foster City, CA, USA). Applied Biosystems 7900HT Real-Time Polymerase Chain Reaction System (Applied Biosystems, Foster City, CA, USA) was used for SNP detection. The cycling program started with heating at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Finally, allelic discrimination was performed using SDS 2.3 software provided by Applied Biosystems.
Statistical analysis. Statistical analysis was performed using IBM SPSS Statistics 20 software (IBM Corp., Armonk, NY, USA). The results are presented as total number, percentages, mean and standard deviation (SD). Student's t-test was used to compare mean values between two groups when data values were normally distributed. Distribution of SNP genotypes in patients and reference groups was evaluated by Hardy–Weinberg equilibrium (HWE) using chi-squared test. The association between the MMP8 polymorphisms and LSCC was estimated by computing odds ratios (ORs) and their 95% confidence intervals (Cls) from logistic regression in five inheritance models: Recessive (wild-type homozygous with heterozygous vs. minor allele homozygous), dominant (wild-type homozygous vs. heterozygous with minor allele homozygous), overdominant (wild-type homozygous with minor allele homozygous vs. heterozygous) and additive inheritance model. Akaike information criterion was used to choose the model that best fitted the data.
Polymorphisms rs11225395 and rs2155052 are both located on chromosome 15, therefore haplotype analysis was carried out. Estimation of haplotype frequencies and haplotype association with frequencies of at least 5% were carried out using PLINK software version 1.07 (31). Results were considered statistically significant when the p-value was less than 0.05.
Results
LSCC patient characteristics according to smoking and alcohol consumption habits, BMI, cancer stage, grade, T-stage and N-stage are given in Table II.
MMP8 rs11225395 and rs2155052 genotypes and allelic frequencies among the reference group and patients with LSCC are presented in Table III. The distribution of both SNPs in LSCC and control groups was consistent with the HWE. The analysis of MMP8 gene polymorphisms did not show any statistically significant differences in the distribution of genotypes between the patients with LSCC and the controls (MMP8 rs11225395 genotypes GG, GA and AA: 32.7%, 51.8% and 15.5% vs. 29.2%, 50.4% and 20.4%, respectively; p=0.300; and MMP8 rs2155052 genotypes GG, GC and CC: 85.0%, 14.1% and 0.9% vs. 85.7%, 13.4% and 0.9%, respectively; p=0.172) (Table III). Moreover, no statistically significant differences were found between the distribution of the MMP8 rs2155052 and rs11225395 genotypes according to tumor grade.
Associations between MMP8 rs11225395 polymorphism and LSCC according to the inheritance models are presented in Table IV. The lowest Akaike information criterion (762.613) was for the additive model of MMP8 rs11225395. Regression analysis demonstrated that the additive model was statistically significant in the group when age was 60 years or less (OR=0.54, 95% CI=0.05-2.26, p=0.026).
Inheritance models were also calculated to determine the link between LSCC and MMP8 rs2155052 genotype (Table IV). The lowest Akaike information criterion (764.314) was found for the dominant model. However, the binominal logistic regression analysis adjusted by age in patient and control groups did not indicate any significant variables.
Association analysis between the risk of LSCC and haplotypes for MMP8 rs11225395-r2155052 are shown in Table V. The linkage disequilibrium between these two polymorphisms (D’ value) was 0.662. However, analysis did not shown any statistical significant results.
Analysis of the size of primary tumor and MMP8 gene SNP rs11225395 genotypes showed that patients with wild-type GG genotype had larger primary tumors than AA genotype carriers (p=0.023; Table VI). However, no relation was found for MMP8 rs2155052 genotypes. We also found that larger primary laryngeal tumor size was also significantly associated with smoking habit (p=0.002) and male gender (p=0.027) (Table VII).
Discussion
MMP8 is a major collagenase that cleaves collagen type I and can suppress tumor growth and invasion (32). It was thought that by using MMPs malignant cells are able to break down the basement membrane and degrade interstitial stroma, thus leading to tumor invasion or metastases (33). However, some new studies showed rather controversial results, i.e. MMP8 was associated with antitumor properties in tongue squamous cell carcinoma and a reduced number of lymph node metastases in human breast cancer (22, 32). Moreover, SNP of MMP8 rs2155052 was associated with decreased risk of lung cancer, especially in high-risk patients such as ever-smokers (34). Three independent studies showed that MMP8 rs11225395 was associated with better breast cancer survival, however, opposite findings were presented in studies with ovarian cancer and melanoma (32, 34).
It is known that increased expression of MMP2 and MMP9 is associated with glottis squamous cell carcinoma grade and reduced survival (35, 36). However, as far as we are aware, there are no studies yet to show the impact of polymorphisms in MMP8 gene on laryngeal cancer development. In this study, we examined MMP8 gene polymorphisms rs11225395 and rs2155052 in patients with LSCC.
Our results showed no statistically significant genotypic differences between LSCC patient and reference groups. These results concur with those of Hung et al., where MMP8 gene polymorphism rs11225395 was analyzed in patients with oral cancer; it was found this SNP may not play a major role in mediating personal risk of oral cancer (37). However, González-Arriaga et al. demonstrated that polymorphism +17 C/G in MMP8 was associated with reduced lung cancer risk (38). To the best of our knowledge, the present study is the first to investigate MMP8 rs2155052 polymorphism in LSCC.
In Lithuania, LSCC is usually diagnosed in males above 60 years old (39). For this reason, we divided the study subjects into groups by age (analysis by gender would be inappropriate because female groups were smaller than those of men (n=16 vs. n=210 and n=5 vs. n=163). MMP8 gene allelic frequencies were not significantly different between the two groups divided by age (<60 vs. ≥60 years old); however, logistic regression analysis revealed that for the MMP8 rs11225395 SNP, each copy of the A allele reduced the risk of LSCC in group of those 60 years old or less (OR=0.49, 95% CI=0.04-2.19; p=0.048). To the best of our knowledge, the association between MMP8 and LSCC has never been investigated before, therefore we cannot compare our results with similar studies. However, MMP8 gene polymorphism rs11225395 was analyzed in other disorders and obtained opposite results. Chen et al. showed that in the additive inheritance model, minor A allele increased the risk of osteonecrosis in Chinese males (40). Debniak et al. also showed minor allele homozygous (AA) and heterozygous (GA) genotypes for MMP8 rs11225395 polymorphism to be related to increased risk of malignant melanoma (41). Majumder et al. similarly showed that the minor A allele increased susceptibility to chronic periodontitis (42). However, Decock et al. showed that MMP8 rs1122395 minor A allele was related to reduced risk of breast cancer and better patient survival (25).
Interestingly, in some studies, MMP8 gene activity was related to gender. Koroi and coworkers found that MMP8 antitumor mechanisms in tongue squamous cell carcinoma may be estrogen related (32). Balbin et al. showed that ovarian elimination increased susceptibility to skin cancer in Mmp8-null mice (18). According to these studies, it would be interesting to use a larger group of females with LSCC and compare MMP8 gene polymorphism distributions between male and female groups and calculate inheritance models according to gender.
It was shown in previous studies that MMP8 expression levels correlated with tumor size and invasive properties (22). Decock et al. demonstrated that in Mmp8-wild-type and heterozygotic mice, mammary glands tumors were significantly smaller than in Mmp8-null mice (43). Results of our study are in concordance with these data, and show that MMP8 rs11225395 AA genotype, with its higher promoter activity (44), are associated with smaller laryngeal tumor size than the wild-type GG genotype (p=0.023).
In addition to analysis of the impact of this genetic factor on laryngeal cancer development, an influence of the modifiable factors, smoking habit, alcohol consumption and BMI, were evaluated in the present study. It is known that all these factors increase laryngeal cancer risk and affect the course of the disease (6, 32, 45). In a study by Chen et al., patients who never smoked had significantly smaller tumors of non-small cell lung cancer than did smokers (46). In another study, O'Donnell et al. reported that smokers trended to have larger size germ-cell tumors than non-smokers (47). Our data agreed with these results and showed that smoking to be significantly related to the size of LSCC (p=0.002).
Our findings suggest that MMP8 gene polymorphism rs11225395 and smoking habit have a prominent association with LSCC tumor size. The interaction between the effect of gene polymorphism on the growth of laryngeal cancer and gender should be further investigated.
Footnotes
Authors' Contributions
The Author(s) made the following declaration about their contributions: Study design: Alina Smalinskiene. Assembled study population: Vykintas Liutkevicius and Virgilijus Uloza. Performed experiments: Ruta Insodaite and Rosita Solovejutė. Analysed the data: Ruta Insodaite. Wrote the article: Ruta Insodaite and Alina Smalinskiene. Critical revision of the article: Vykintas Liutkevicius and Virgilijus Uloza.
Conflicts of Interest
None of the Authors had any conflicts of interest to declare.
- Received February 11, 2020.
- Revision received February 21, 2020.
- Accepted February 25, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved