Abstract
Aim: Metalloproteinases (MMPs) are a family of enzymes involved in many physiological processes, such as skeletal development, wound healing, and scar formation, as well as carcinogenesis. However, the contribution of MMP1 genotype to breast cancer has not been elucidated. This study aimed to evaluate the contribution of commonly studied MMP1 promoter 1607 genotype to breast cancer risk. Materials and Methods: In this hospital-based case–control study, contribution of MMP1 genotype to breast cancer risk was evaluated among 1,232 patients with breast cancer and 1,232 gender-matched healthy controls. Results: The distribution of 2G/2G, 1G/2G and 1G/1G for MMP1 promoter 1607 genotype was 36.0%, 41.3% and 22.7% in the breast cancer group and 34.2%, 44.5% and 21.3% in the non-cancer group, respectively (p for trend=0.2820). We also analyzed the allelic frequency distributions and found that the variant 1G allele of MMP1 promoter 1607 conferred similar breast cancer susceptibility as the wild-type 2G allele (odds ratio=0.99, 95% confidence interval=0.89-1.11, p=0.8858). There was no interaction between MMP1 promoter 1607 genotype and cigarette smoking or alcohol drinking habits. Conclusion: The genotype of MMP1 promoter 1607 may not be a major determining factor for breast cancer risk. The contribution of MMP1 promoter 1607 genotype to prognosis and subtypes of breast cancer needs further investigation.
Statistically, breast cancer is one of the most commonly diagnosed types of cancer in females worldwide, and the incidence of early-onset breast cancer has rapidly increased in Taiwan and other Asian areas compared to Western countries. Although several predictive biomarkers for breast cancer in Taiwanese have been reported in recent years (1-6), the genomic etiology of breast cancer is of great interest but largely unknown.
Matrix metalloproteinases (MMPs), a family of enzymes that can selectively digest individual components of the extracellular matrix in many tissues (7), regulate the cell migration, differentiation, and regeneration in processes of both normal physiological and pathological states (8-11). MMPs are also closely related to regulation of invasion and metastatic capacities of cancer cells (12). Many studies have shown that single nucleotide polymorphisms of MMP genes may be associated with breast cancer risk, but the relationships remaining controversial. Collagenase 1 (also called MMP1) 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 (13, 14). MMP1 expression was significantly higher in atypical ductal hyperplasia than in benign breast disease and in invasive breast cancer compared to in situ breast cancer (15, 16). The most well-known polymorphic site in the MMP1 promoter region is at the upstream position of 1607 bp (rs1799750), and was reported to control the transcriptional activity of MMP1 (17). No significant difference in genotype distribution between breast cancer and non-cancer groups was observed in a US population (15), in mixed ethnicity with Caucasian patients comprising 65% (18), Polish population (19), nor in meta-analysis (20, 21). To date, as far as we are aware, there is neither any study of MMP1 genotype in Taiwanese nor a single representative MMP1 genotype study with more than one thousand cancer patients anywhere else. In the current study, we aimed to reveal the contribution of MMP1 genotype at the promoter 1607 site to the risk of breast cancer among 1,232 patients with breast cancer and 1,232 non-cancer healthy people in Taiwan.
Demographics and lifestyle factors of the investigated patients with breast cancer and the control women.
Materials and Methods
Investigated population. Our study was approved by the Institutional Review Board of the China Medical University Hospital (DMR96-IRB-240) and written-informed consent was obtained from all participants. One thousand two hundred and thirty-two patients diagnosed with breast cancer were recruited at the outpatient clinics of general surgery at the China Medical University Hospital in Taiwan. The clinical characteristics of patients, including histological details, were all defined by expert surgeons, Dr. Wang, Dr. Liu and their teammates. The slides were reviewed and scored by at least two independent pathologists. All patients voluntarily participated and provided peripheral blood samples. A total of 1,232 age-matched healthy volunteers were selected after initial random sampling from the Health Examination Cohort of the hospital as the controls of this study. The exclusion criteria of the control group included previous malignancy, metastasized cancer from other or unknown origin, and any familial or genetic diseases. The ages of the patients and the controls and other detailed information are summarized in Table I.
Genotyping conditions. The genomic DNA from the peripheral blood leucocytes of each patient and control was prepared and stored at −80°C until processed as per our previous articles (22-24). The primers for MMP1 genotype at the promoter 1607 site were 5’-TGACTTTTAAAACATAGTCTATGT-3’ (forward) and 5’-GATTGATTTGAGATAAGTCATAGC-3’ (reverse), 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 Alu I 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 breast 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 at diagnosis, age at menarche, age at first birth of child, age at menopause, cancer site and personal behavioral habits for the 1232 patients with breast cancer and 1232 matched 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 between the control and case groups (Table I). From the viewpoint of epidemiology, the analysis of behavioral habits showed that cigarette smoking and alcohol consumption were more frequent in the breast cancer population studied (p<0.0001) (Table I).
Distribution of matrix metalloproteinase-1 (MMP1) promoter 1607 genotypes among controls and patients with breast cancer.
Distribution of allelic frequencies for matrix metalloproteinase-1 (MMP1) promoter 1607 among controls and patients with breast cancer.
The distributions of the MMP1 promoter 1607 genotype among the non-cancer controls and the patients with breast cancer are presented and statistically analyzed in Table II. The genotypes of MMP1 promoter 1607 were not differently distributed between breast cancer and non-cancer control groups (p for trend=0.2820) (Table II). In detail, the MMP1 promoter 1607 heterozygous 1G/2G and homozygous 1G/1G were not associated with increased or decreased breast cancer risk (p=0.1739 and 0.9105, respectively; Table II). In the recessive and dominant models, there was still no association between the genotype of MMP1 promoter 1607 and breast cancer risk (p=0.4087 and 0.3530, respectively; Table II).
To confirm the genotypic findings in Table II, the analyses of allelic frequency distribution for the MMP1 promoter 1607 among the investigated groups was also performed and the results are presented in Table III. Consistent with the findings that neither heterozygous 1G/2G nor homozygous 1G/1G genotype of MMP1 promoter 1607 was associated with breast cancer risk, the variant allele 1G was found at 43.4% in the breast cancer group, similar to that of 43.6% in the non-cancer control group (OR=0.99, 95% CI=0.89-1.11, p=0.8858). To sum up, there was no significant difference in the genotypic or allelic frequencies of MMP1 promoter 1607 between the control and breast cancer patient groups (Tables II and III).
Discussion
In the current case–control association study, the contribution of MMP1 promoter 1607 genotypes to breast cancer risk was firstly evaluated in a Taiwanese population. The results showed that neither the genotypic nor the allelic frequencies of MMP1 promoter 1607 were differentially distributed among the breast cancer patients and non-cancer healthy controls (Tables II and III). The sample sizes of previous studies investigating the association of MMP1 genotypes with breast cancer risk were all less than the current breast cancer patient collection (15, 18, 19). The findings are consistent that genotypes of MMP1 promoter 1607 may not be a high-penetrant determinant factor for breast cancer susceptibility. In Table I, we found that cigarette smoking and alcohol consumption were more frequent in the breast cancer population investigated (Table I). However, no obvious interaction between MMP1 1607 genotype and cigarette smoking or alcohol consumption on the susceptibility to breast cancer was observed after our performing the stratification analysis (data not shown).
The MMP1 protein is involved in the degradation of native fibrillar collagen, hence it is also called collagenase-1. MMP1 is thought to promote invasion and metastasis through the degradation of the extracellular matrix as the main component of connective tissue (25-27). In 2004, Przybylowska and colleagues explored the association between MMP1 promoter 1607 1G/2G genotypes and the risk of breast cancer, and the results showed that the frequency of the 2G allele was higher in patients with lymph node metastasis than in the group without metastasis (p<0.001) (19). In 2007, Hughes and colleagues found that the genotypes of MMP1 and MMP3, but not those of MMP3, MMP7, MMP12 or MMP13, were associated with lymph node-positive breast cancer (18). In addition, MMP1 2G/2G genotype was associated with reduced survival (hazard ratio=3.1, 95% CI=1.1-8.7) (18). A possible mechanism has been proposed by Tower and his colleagues, providing evidence showing that MMP1 2G/2G genotype contributed to higher expression of MMP1 in MCF-7/ADR breast cancer cells, and by inhibiting ERK signaling, MMP1 expression both blocks the degradation of type I collagen and reduces the invasive capacity of MCF-7/ADR cells (17). In the near future, we will investigate the gene–gene interactions among MMP1 and other genes, for instance other MMP members or tissue inhibitors of MMPs, in order to reveal the possible contribution of MMP1 genotypes to subtypes of breast cancer, such as triple-negative breast cancer.
In conclusion, our study indicated that the heterozygous 1G/2G or homozygous 1G/1G genotype at MMP1 promoter 1607 does not seem to serve as a predictive marker for breast cancer susceptibility.
Acknowledgements
The Authors declare no conflicts of interest with regard to this study. They appreciate the Tissue-bank of China Medical University Hospital and Min-Chun Chao for their excellent data collection and technical assistance. This study was supported partially by research grant from Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-133019).
Footnotes
↵* These Authors contributed equally to this study.
- Received May 2, 2016.
- Revision received June 2, 2016.
- Accepted June 3, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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