The MMP-2 -735 C Allele is a Risk Factor for Susceptibility to Breast Cancer

Breast cancer is the most common cancer in women that is associated with an enormous amount of morbidity and mortality throughout the world (Hughes et al., 2007). Its incidence is increasing in developing countries in a rate of 3-4% (Babu et al., 2011). Due to high incidence rate of breast cancer, it is considered as one of the common health problems among various populations. Interaction between genetic background and different environmental risk factors is involved in the pathogenesis of the disease. Reports from different populations suggest that genetic factors such as single nucleotide polymorphisms, insertion or deletion


Introduction
Breast cancer is the most common cancer in women that is associated with an enormous amount of morbidity and mortality throughout the world (Hughes et al., 2007). Its incidence is increasing in developing countries in a rate of 3-4% (Babu et al., 2011).
Due to high incidence rate of breast cancer, it is considered as one of the common health problems among various populations. Interaction between genetic background and different environmental risk factors is involved in the pathogenesis of the disease. Reports from different populations suggest that genetic factors such as single nucleotide polymorphisms, insertion or deletion on individual's susceptibility to breast cancer (Boroujeni et al., 2013).
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidase that degrade and restructure the extracellular matrix components and basal lamina, the two physical barriers for cancer cells (Rahimi et al., 2013, Rahimi et al., 2014. The extracellular proteins such as collagen

The MMP-2 -735 C Allele is a Risk Factor for Susceptibility to Breast Cancer
Kheirollah Yari 1 , Ziba Rahimi 1 , Mohamad Taher Moradi 1 , Zohreh Rahimi 1,2 * type I-VIII, gelatin, elastin, laminin and also myelin basic protein and several growth factors are substrates of MMPs (Ke et al., 2013). These enzymes are generally present at lower level in healthy individuals, but their upexpression has been reported in human cancers in all stages of carcinogenesis (Zhou et al., 2011). Over-expression with tumor grade and tumor cell invasion for a variety of cancers. So, it might the high expression of MMPs genes allow the remodeling of extracellular matrix to increase the invasive potential of cancer cells (Gonzalez-Arriaga et al., 2012).
MMP-2 is known as gelatinase A that degrades the type IV collagen and gelatin, which are the main constituents of basal lamina and might affect early events in carcinogenesis and tumor invasion and/or metastasis (Sanii et al., 2012). The MMP-2 gene locates on chromosome 16 at q13-21 and has thirteen exons (Saeed et al., 2013). Numerous studies have demonstrated that breast cancer disease is associated with elevated level of MMP-2 gene expression (Zhou et al., 2011;Srivastava et al., 2013a). Alteration in the gene transcription could be an susceptibility to breast cancer (Gonzalez-Arriaga et al., 2012;Srivastava et al., 2013b). A functional single nucleotide polymorphism in the cis-acting regulatory the expression of this gene (Wang et al., 2011;Zhang et al., 2013). A C T transition at nucleotide -735 (rs2285053) transcription (Wang et al., 2011).
According to literature there is no available report to examine the possible association between MMP-2 C-735T polymorphism with breast cancer susceptibility. The aim of present study was to investigate the frequency of MMP-2 C-735T variants and their possible association with breast cancer risk in an Iranian population with Kurdish ethnic background.

Sample
In a case-control study 98 breast cancer patients including 97 females and 1 male with the mean age of 49.5±10.2 years (29-79 years) and 135 healthy individuals consisted of 129 females and 6 males with the mean age of 38.7±9.4 years (22-68 years) as controls were investigated. The breast cancer diagnosis was according to standard clinical, radiological and histological parameters. All patients and controls were from Kermanshah and Ilam provinces of Iran with Kurdish ethnic background who admitted to the Kermanshah University of Medical Sciences clinics. Demographic, histopathology and medical characteristics of patients including age, sex, family history of cancer, tumor stage, lymph node metastasis and the status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2), P53 and KI67 were obtained were approved by the Ethics Committee of Kermanshah University of Medical Sciences, Iran. All individuals contributed in this research agreed to participate in the study and signed an informed consent in accordance with the principles of the Helsinki II declarations. All subjects were informed about their disease and the aim and procedures of the research.

Genotyping
From each subject 5 ml whole blood sample was collected into tubes containing EDTA. Genomic DNA was extracted from peripheral blood leukocytes according to a standard phenol-chloroform method as previously described (Rahimi et al., 2010;Rahimi et al., 2006). agarose gel. The concentration and purity of DNA was assessed with a Nanodrop spectrophotometer (Thermo).
T h e M M P -2 C -7 3 5 T p o l y m o r p h i s m w a s fragment length polymorphism (PCR-RFLP). The PCR was conducted by the forward primer of 5'-ATAGGGTAAACCTCCCCACAT T-3' and the reverse primer of 5'-GGTAAAATGAGGCTGAGACCT G-3' using Master Cycler Gradient-Eppendorf (Germany). The PCR reagents consisted of 20 pmol of each primer, 300 ng were: 1 cycle at 94 o C for 5 min, 35 cycles by 94 o C for 60s, 62 o C for 60s and 72 o extension for 10 min at 72 o C. The obtained 300-bp PCR product was digested with HinfI restriction enzyme. In the presence of C allele, the PCR product remained intact while in the presence of T allele two fragments of 254-and 46-bp were produced (Gai et al., 2010).

Statistics
The allelic frequencies were calculated by the difference of alleles and genotype frequencies between the groups was tested using the chi-square method. Data on quantitative characteristics are expressed as means±standard deviations. Odds ratios (OR) were calculated as estimates of relative risk for disease and regression using SPSS software. A two-tailed Student's t test analysis was used to compare quantitative data.
All of the statistical analyses were performed using SPSS statistical software package version 16.0.
Agarose gel electrophoresis pattern of some of RFLP products of the MMP-2 gene digested with HinfI is demonstrated in Figure 1.
The frequencies of MMP-2 genotypes were in Hardy-Weinberg equilibrium in both patients and controls 2 =2.72, p>0.1). Distribution of MMP2 genotypes and alleles in breast cancer patients and controls is depicted in Table  2. All three genotypes of CC (59.3%), CT (38.5%) and TT (2.2%) were observed in healthy individuals, but in breast cancer patients, only CC genotype with a frequency of 71.4% and CT genotype with a frequency of 28.6% distribution of MMP-2 C-735T genotypes was observed between controls and breast cancer patients (p=0.077). In higher (85.7%) compared to that in controls (78.5 %, p=0.048). Also, the presence of C allele of MMP-2 increased the risk of breast cancer by 1.64 fold [OR=1.64 (95%CI: 1.01 -2.7, p=0.049)].
A statistical analysis was carried out to investigate any possible association between MMP-2 alleles and clinical parameters such as family history of cancer, breast cancer stage, lymph node metastasis, histological type and immunohistochemical data of the cancer. As indicated in Table 3, the frequency of MMP-2 C allele was tended to be higher in patients with a family history that without a family history of cancer (67.3%, p=0.31). The frequency of this allele was the highest in patients with the stage III of the disease (80%), was the modest in patients with the stage II of the disease (75%) and was the lowest in patients with the stage I of the disease (69.2%, p=0.83). Also, a higher frequency of MMP-2 C allele was detected in ER-(77.3%) than ER+ (69.5%), in PR-(75%) compared to PR+ (70.5%), and in P53-(73.5%) than P53+ (60%) patients that did not reach to a statistical Furthermore, the frequency of MMP-2 C allele was

Discussion
The present study in a homogenous population of breast cancer patients from Western Iran reports a compared to that in healthy individuals that increased the risk of breast cancer 1.64 times. Also, the frequency of this allele tended to be higher in patients with a family history of the disease than those without a family history of the cancer and there was a higher prevalence of MMP-2 C allele in higher stages than lower stages of the disease. The MMP-2 C allele was more prevalent in young breast cancer patients than older breast cancer patients. Furthermore, the frequency of this allele was tended to be higher in ER-, PR-and P53-breast cancer patients than those patients that were positive for ER, PR, and P53.
Breast cancer is the most frequent malignancy diagnosed in women worldwide and it is the third cause of death among Iranians after coronary heart diseases and accidents (Taghavi et al., 2012;Zheng et al., 2014). Epidemiologic studies have reported numerous risk factors for breast cancer, including genetic and environmental factors (Zhou et al., 2011;Yadav et al., 2014). A number of polymorphisms have been reported in the promoter regions of MMPs genes including MMP-2 gene that some of them are associated with the increased risk of breast cancer (Shagisultanova et al., 2004). Some of regulation of MMP-2 gene expression and are associated with the development and progression of many diseases including cancers, coronary heart disease, ischemic stroke and adenomyosis (Wang et al., 2011;Saeed et al., 2013). Because the human MMP-2 promoter contains a number of cis-acting regulatory elements, the constitutive and   , 2004;Srivastava et al., 2013a). In contrast, the MMP-2 TT genotype increased the risk of bladder cancer when combined with CT genotype in Northern Indian population (Srivastava et al., 2013a). However, in a study from Indian population Srivastava C-735T gene polymorphism and the risk of developing cervical cancer (Srivastava et al., 2013b). The absence of Sp1 consensus sequence results in a lower promoter activity and decreased MMP-2 expression in individuals carrying the TT than CC genotype. The other extracellular matrix components and development of breast cancer (Zhou et al., 2004;Wang et al., 2011). So, it seems the higher expression of MMP-2 C allele than the T allele that was more prevalent in our studied patients compared to healthy individuals not only increased the risk of breast cancer but also might be a risk factor for younger onset of the disease, higher stages of the tumor and familial breast cancer disease.
In conclusion, the present study indicates that the C allele of MMP-2 is a risk factor for susceptibility to breast cancer. Also, the MMP-2 C allele might increase the risk of young onset breast cancer in population of Western sample size.