, Mohammad Saud Alanazi

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism (SNP) at -1306, which disrupts a Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. In the present study, we investigate whether this MMP-2 SNP is associated with susceptibility to breast cancer in the Saudi population. Ninety breast cancer patients and 92 age matched controls were included in this study. TaqMan Allele Discrimination assay and DNA sequencing techniques were used for genotyping. The results showed that, the frequency of MMP-2 CC wild genotype was lower in breast cancer patients when compared with healthy controls (0.65 versus 0.79). The homozygous CC (OR=2, χ(2)=5.36, p=0.02) and heterozygous CT (OR=1.98, χ(2)=4.1, p=0.04) showing significantly high risk of breast cancer in the investigated group. In conclusion our data suggest that the MMP-2 C(-1306)T polymorphism may be associated with increased breast cancer risk in the Saudi population.


INTRODUCTION
Breast cancer is one of the most common malignancies among women worldwide, in both developed and undeveloped countries.It is also leading cause of death in women in the 35-59 years age group (Strnad et al., 2007).The annual worldwide incidence of breast cancer is about 1.2 million (Cheema et al., 2008).One in nine women will suffer from breast carcinoma during her life and over 130 thousand women die from this disease each year (Tyczynski et al., 2002).Breast cancer is a leading malignancy among Saudi women comprising about 26% of all malignancies and is the foremost cause of cancer related death (Saudi Cancer Registry 2011).Moreover, carcinoma of the breast that developed before the age of 40 was significantly more common in Saudi women compared with patients in the United States (Elkum et al., 2007).This difference in the early onset of the disease in Saudi females could be due to considerable molecular differences both genetic and epigenetic between the ethnic groups.Breast cancer in young patients is often associated with more aggressive disease and poorer prognosis (Anders et al., 2008).One of the biological markers of interest in breast cancer is the matrix metalloproteinases (MMPs).Metastasis of breast carcinoma cells depends on some important factors such as proteolysis, cellular attachment, angiogenesis, migration through the barrier into secondary sites and colonization and proliferation in distant organs (Liotta & Kohn, 1990).Proteolytic degradation of the basement membrane is a fundamental aspect of cancer development and a key event in the regulation of tumor proliferation and metastasis (Schwartz, 1996).Matrix degradation in the basement membrane is closely related to activities of various subtypes of MMPs and corresponding tissue inhibitors of matrix metalloproteinase, TIMP (Shim, 2007;Zhang 2012).Genetic alterations in breast cancer are those that change the DNA sequence such as single nucleotide polymorphisms (SNPs), insertion-deletion mutations and rearrangements.These types of mutations typically alter the gene product by changing the amino acid sequence of the protein or by altering the quantity of the produced protein.The matrix metalloproteinases, MMPs, constitute a family of secreted and membrane-associated zinc-dependent endopeptidases that are capable of selectively degrading a wide spectrum of extracellular matrix and non-matrix proteins (Simon et al., 2001).The MMP family has expanded to include 23 zinc-dependent endopeptidases, many of which were first identified by their over-expression in tumor cells (Laurie et al., 2012).They can be categorized by substrate specificity to give collagenases, stromelysins, gelatinases and membrane type MMPs.The broad range of substrates conveys their pivotal role during both normal physiological processes such as embryonic development, bone remodeling, angiogenesis and nerve growth and pathological states as in arthritis, atherosclerosis, liver fibrosis and cancer (Woessner 1998).The MMP-2 (gelatinase A) has type IV collagenolytic activity and is constitutively expressed by most connective tissue cells including endothelial cells, osteoblasts, fibroblasts and myoblasts (Strongin et al., 1995).Numerous investigators have demonstrated that MMP-2 is one of the essential players in promoting tumor invasiveness and metastasis and the levels of MMP-2 expression can be correlated with tumor grade (Poulsom et al., 1992;Boag & Young, 1994).The MMP-2 gene is located on chromosome 16 at q13-21 and spans 27 049 bp with thirteen exons.There were 283 entries of SNPs for the MMP-2 gene in the public NCBI Single Nucleotide Polymorphism database (dbSNP; build 125:http:// www.ncbi.nlm.nih.gov/SNP/)(Yihong et al., 2009).A SNP in the promoter region of the MMP-2 (the C -1306 T/ rs 243865) which disrupts an Sp1-type promoter site (CCACC box) affects MMP-2 expression or activity and may predispose to disease conditions, especially in those individuals carrying the MMP-2 variants associated with increased MMP-2 concentrations (Flavia et al., 2013).However, there are no reports about the association between MMP-2 C -1306 T gene SNP and breast cancer in Saudi population.In this study, we investigated whether this MMP-2 genetic polymorphism was associated with susceptibility to breast cancer in the Saudi population.

Chemicals.
All chemicals used were of analytical reagent, molecular biology, or chromatographic grade as appropriate.Water was deionized and distilled.
Samples collection.This study was conducted after review and approval of the Institutional Review Board of the Ethics Committee at King Khalid University Hospital in Riyadh, Kingdom of Saudi Arabia.Blood samples were collected from 90 confirmed breast cancer patients (age range, 33-77 years; mean age, 49 years) and 92 agematched healthy controls.The diagnosis of cancer was based on standard clinical, endoscopic, radiological, and histological criteria.Clinical and demographic characteristics were recorded, including age at diagnosis, estrogen receptor, progesterone receptor, HER status, family history, smoking habits, disease behavior, disease location, and need for surgery.
Genomic DNA isolation and purification.Genomic DNA was isolated from whole blood samples using QIAamp R DNA Blood Min Kit Cat.No. 51106 (Qiagen) according to manufacturer's instructions.Concentrations and purity of DNA samples were assessed using Nan-oDrop 8000 (Thermo Scientific).
Genotyping for MMP-2.Genotyping for the C -1306 T (rs 243865) in the 5'-flanking region of MMP-2 gene was performed by real time polymerase chain reaction (RT-PCR) using TaqMan Allele Discrimination assay (Applied Biosystems, Carlsbad, CA, USA).Probes and primers used for the C -1306 T genotyping assay were customized as follows: forward 5'-GCCATTGTCAATGTTC-CCTAAAACA-3'; reverse 5'-TGACTTCTGAGCT-GAGACCTGAA-3' and probes 5'-CAGCACTC[T/C] ACCTCT-3'.TaqMan PCR was performed in a total volume of 20 µl containing 20 ng of genomic DNA, 1x TaqMan master mix and 1x assay mix placed in 96-well PCR plate.Fluorescence from PCR amplification was detected using Chromo 4 detector (Applied Biosystems 7500 Fat Real Time PCR System) and analyzed with the manufacturer's software.
PCR and sequencing.PCR was carried out in a final volume of 50 µl containing 25 µl high fidelity PCR master mix (GE Healthcare, USA), 3 µl of genomic DNA (50 ng) and 3 µl (30 pmole) of each primer (MMP-2 forward 5'-CTGACCCCCAGTCCTATCTGCC-3'; reverse 5'-TGTTGGGAACGCCTGACTTCA-3').The PCR conditions were 1 cycle at 95°C for 5 min followed by 30 cycles at 95°C for 40 seconds, 54°C for 40 seconds, and 68°C for 1 min.The final extension step was carried out at 72°C for 5 min.The PCR products were analyzed using 2.0% agarose gel stained with 0.5 µg/mL ethidium bromide and visualized using an ultraviolet transilluminator.Sequencing of the PCR products was carried out according to Sanger et al., (1977) using the MegaBACE 1000 DNA Sequencing System (Pharmacia/Amersham Co.).The chain termination sequencing reaction was conducted utilizing the DYEnamic ET terminator kit as an integral part of the MegaBACE 1000 DNA sequencing system.The sequencing reaction products were purified using DyeEx 2.0 Spin Kit (63206 Qiagen) and applied to MegaBace 1000 Sequencing machine.
Statistical analysis.Fisher's exact test and the odds ratio (OD) with 95% confidence interval (CI) were used to test the association between cancer and the studied genetic polymorphisms and to describe the strength of the association.The associations were considered to be statistically significant if the Fisher's exact p-value was less than 0.05 and if the 95% CI excluded the value 1.0.All statistical calculations were done using MedCalc Software (Version 11.3.1.0© 2010 MedCalc Software bvba).

Characteristic of breast cancer patients
The clinical characteristics of the studied breast cancer patients are summarized in Table 1.The median age of the 90 patients was 49 years.Among a total of 90 breast cancer samples, 54 were staged as early disease (10 patients at Stage 0, 1 patient at Stage I and 43 patients at Stage II), 34 patients (37.7%) as locally advanced disease (Stage III) and 2 patients (2.2%) had metastatic disease (Stage IV) at the time of diagnosis.

Genotyping for MMP-2 C -1306 T SNP
The identification of MMP-2 C -1306 T polymorphism was carried out by two different PCR based methods.
The first method was the amplification of 296 bp DNA fragment of the MMP-2 promoter followed by sequencing (Figs. 1 and 2).The second method was the TaqMan Allele Discrimination Assay utilizing primers and probes that identify both C and T alleles.
The MMP-2 genotype distribution for cases and controls are shown in Table 2.In the present study there was a significant difference in the distribution of MMP-2 C/C genotype between breast can- U n c o r r e c t e d P a p e r i n P r e s s cer cases and the matched healthy control samples (Table 2).The frequencies of CC, CT and TT genotypes in breast cancer cases were 58 (0.65), 30 (0.33) and 2 (0.02), respectively, whereas, for the healthy controls the frequencies were 73 (0.79), 19 (0.21) and 0 respectively.The homozygous CC (OR=2.022,χ 2 =5.36, p= 0.02) and heterozygous CT (OR=1.98,χ 2 =4.1, p=0.04288) in breast cancer patients show higher risk when compared to healthy individuals (Table 2).Moreover, a significantly increased risk was also observed in women when C/T + T/T were combined together as compared with healthy women (OR=2.1,χ 2 =5.01, p=0.02).The correlation of MMP-2 polymorphic status with the clinicopathological characteristics was analyzed.Stratification of patients by age groups indicated that MMP-2 C 1306 T SNP had a stronger association in above 48 years old patients than in patients younger than 48 years.Our results indicated that patients older than 48 years age with the homozygous variant (CC) genotype had an increased risk of breast cancer (OR=2.35,χ 2 =4.26, p=0.038) (Table 3).No significant association was observed between the HER, ER, and PR status and the genotypes under investigation in breast cancer patients and healthy individuals.

DISCUSSION
The human matrix metalloproteinase-2 possesses proteolytic activity against type IV collagen, a major constituent of the basement membrane, and is therefore implicated in an extensive array of pathologies including atherogenesis, arthritis and tumor growth and metastasis (Clifford et al., 1984;Itoh et al., 1998;Yrjö et al., 1999;Fang et al., 2000).Thus, any naturally occurring genetic variants that directly affect gene expression, and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling.Recently, a number of function altering polymorphisms have been found in the promoter of MMP-2.Some of these polymorphisms have allele-specific effects on the regulation of MMP-2 gene transcription and are associated with the development and progression of several diseases including breast cancer (Grieu et al., 2004), colorectal cancer (Enping, 2004) and prostate cancer (Priyanka et al., 2012).
In the present study, we investigated the role of MMP-2 C -1306 T polymorphism within the promoter site in breast cancer Saudi patients.Our results demonstrated a significant difference in MMP-2 allelic variant distribution with a 2.0 relative risk of breast cancer for C/C and a 1.987 for C/T heterozygous patients (Table 2) and this polymorphism may influence the invasiveness of breast cancer.These molecular epidemiological results are consistent with previous findings of Grieu et al., showing that, the C>T transition at -1306, disrupts the Sp1-type promoter site and results in a strikingly lower promoter activity with the T allele of the MMP-2 gene (Grieu et al., 2004).A study in Chinese population showed that the -1306 CC genotype doubles breast cancer risk (Zhou et al., 2004).In contrast, Roehe et al., (2007) reported,    no association between -1306 C>T polymorphism and breast cancer.Studies in a Swedish population found, no association between the MMP-2 promoter polymorphisms and colorectal cancer (Elander et al., 2006).Xu et al. (2004) investigated the role of the -1306 C>T polymorphism within the MMP-2 gene promoter in colorectal cancer in the Chinese population and found that C/C homozygotes were significantly more common among cancer patients.Our study showed that, the MMP-2C - 1306 T SNP had a stronger association in patients older than 48 years than those under 48 years of age indicating that patients above 48 years with the homozygous variant CC genotype had an increased risk of breast cancer (OR=2.35,χ 2 =4.26, p=0.038) (Table 3).Breast cancer is an epithelial tumor with high invasive and metastatic potential.Tumor growth, invasion and metastasis is a multistep process that is facilitated by the proteolytic degradation of the extracellular matrix and basement membrane (Zhang et al., 2012).The role of MMPs in this process has been firmly established based on numerous previously published studies (Azzam et al., 1993;Talvensaari-Mattil et al., 1998;Nagase & Woessner Jr, 1999;Duffy et al., 2000).Because of its ability to degrade the basement membrane, MMP-2 has been postulated to be a potential marker of tumor progression and prognosis (Zhang et al., 2012).The MMP-2 promoter a 1.9 kbp in size and contains Sp1 a ubiquitously expressed transcription factor that binds to GC/GT-rich elements and regulates a variety of genes in a constitutive or inducible manner (Rutter et al., 1998;Simon et al., 2001).One such motif, the CCACC box, has been shown to be essential for Sp1 binding and promoter function in several genes (Alory et al., 1999;Maouche et al., 1995).Sp1 is a multifunctional protein that can directly interact with the basal transcriptional complex as shown for the MMP-2 proximal promoter (Qin et al., 1999;Price et al., 2001), or alternatively function as a more general transcription factor and play an important role in directing tissue-specific expression (Block et al., 1996;Margana & Boggaram, 1997).Clearly, any variant that abolishes Sp1 binding, such as the MMP-2 C -1306 T polymorphism, has the potential to affect the level and specificity of gene transcription.
In conclusion this study indicated that the MMP-2 C -1306 T polymorphism will be informative in tests of associations for breast cancer.In addition, this is the first study to demonstrated that MMP-2 C -1306 T polymorphism may be associated with the risk of developing breast cancer in a Saudi population.

Table 3 . Association between MMP2 gene polymorphism and clinicopathologic characteristics
, odds ratio; p, Fisher's exact p; CI, confidence interval.The frequency of the allele is in brackets. OR