Matrix Metalloproteinase-2 Promoter Polymorphism Is ...

Original Article Gynecol Obstet Invest 2008;65:68–72 DOI: 10.1159/000108282

Received: October 11, 2006 Accepted after revision: May 29, 2007 Published online: September 13, 2007

Matrix Metalloproteinase-2 Promoter Polymorphism Is Associated with Breast Cancer in a Mexican Population Iván Delgado-Enciso a Friedman R. Cepeda-Lopez b Elisa A. Monrroy-Guizar a Jose R. Bautista-Lam a Maricela Andrade-Soto a Gregorio Jonguitud-Olguin b Alejandrina Rodriguez-Hernandez a Aristoteles Anaya-Ventura a Luz M. Baltazar-Rodriguez a Martha Orozco-Ruiz b Alejandro D. Soriano-Hernandez a Iram P. Rodriguez-Sanchez a Angel Lugo-Trampe a Francisco Espinoza-Gomez a Martha L. Michel-Peregrina a a

School of Medicine, Universidad de Colima, and b Centro Estatal de Cancerologia, Secretaria de Salud del Estado de Colima, Colima, Col., Mexico

Key Words Breast tumor Gelatinase A Matrix metalloproteinase Mexico Polymorphism Promoter

Abstract Background: Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity on matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C1T transition at –1306 displayed a strong association with several cancers. Our study investigated whether or not the MMP-2 –1306C1T polymorphism contributed to the development of breast cancer (BC) in a Mexican population. Methods: 90 patients with BC and 96 control subjects were analyzed to detect MMP-2 –1306C1T polymorphism. Results: The frequency of MMP-2 CC genotype was significantly higher in BC patients when compared with the control group (OR 2.15; 95% CI 1.1–4.1). MMP-2 CC genotype frequency was more pronounced in younger subjects

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(^50 years) at diagnosis (OR 2.66; 95% CI 1.04–6.96). Conclusion: The data suggest that MMP-2 –1306C1T polymorphism strongly contributes to the development of BC in the population studied, especially among women 50 years old and younger. Copyright © 2008 S. Karger AG, Basel

Introduction

Matrix metalloproteinases (MMPs) are a superfamily of proteolytic enzymes capable of degrading extracellular matrix and basement membrane [1, 2]. Overexpression of MMPs in multiple carcinomas was found to be important for tumor invasion and metastasis. Studies have also indicated that MMPs are involved in early tumorigenesis, modulating proliferation, apoptosis and angiogenesis [3]. Of all the MMPs described to date, MMP-2 (gelatinase A or type IV collagenase) has been the most closely linked Iván Delgado-Enciso School of Medicine, Universidad de Colima Av. Universidad 333, Colonia las viboras CP 28040, Colima, Col. (Mexico) Tel./Fax +52 312 3161 099, E-Mail [email protected]

to breast cancer. The principle activity of MMP-2 is gelatin and type IV collagen hydrolysis, these being the main structural components of basement membranes. A number of studies have shown increased expression of MMP2 protein or mRNA [4–9] in primary breast tumors compared with normal tissues. Most reports also suggest that high serum levels of MMP-2 in breast cancer patients have been shown to be correlated with adverse prognosis [10] and/or with primary tumor conventional prognostic markers that are associated with poor prognosis [11]. Genetic variants that influence the level of MMP-2 gene expression could therefore be expected to influence the role this enzyme plays in tumor invasion and metastasis. Price et al. [12] identified a –1306C1T polymorphism in the MMP-2 promoter [12]. This base transition is located in the CCACC box of the Sp1-binding site and eliminates promoter activity. It is likely that CC genotype may be associated with the high transcription level and enzyme activity of MMP-2, and eventually affects individual susceptibility to neoplasms and/or patient outcome. Grieu et al. [13] found that breast cancer phenotype and outcome can be influenced by MMP-2 –1306C1T polymorphism in their study of an Australian population. Zhou et al. [14] carried out a study on a Chinese population which showed that the –1306CC genotype doubled breast cancer probability. The two studies suggest that this polymorphism could be implicated in tumorigenesis as well as breast cancer evolution and clinical behavior. However, another study carried out in Brazil, showed no association between –1306C1T polymorphism and BC, thus making the topic controversial [15]. As these are the only three studies on the subject, more research needs to be carried out. The objective of the present case-control study is to investigate the contribution of the –1306CC genotype in the MMP-2 promoter towards breast cancer probability in a Mexican population.

Materials and Methods 90 breast cancer patients and 96 nonhospitalized women presenting with normal mammography (American College of Radiology Breast Imaging Reporting and Data System –BIRADS- 1 or 2) agreed to participate in the study. Participants were recruited from a single public hospital center where patients received cancer treatment or follow-up and the control group received routine mammography. A case history was elaborated for each of the participants (including histopathologic and tumoral immunohistochemical reports) and desquamation cells from the buccal inside cheek were taken to extract DNA. All patients included in the study signed a letter of informed consent. Breast cancer patients

MMP-2 Polymorphism and Breast Cancer

were categorized into stages according to TNM classification [16]. The presence of metastasis was evaluated with a liver function test, chest X-ray, abdominal ultrasound scan and radionucleotide bone scan. Selection criteria for the control group individuals were: (1) that their routine mammographs were carried out in the same hospital in which the breast cancer patients were treated; (2) that they had a normal mammograph (BIRADS 1 or 2); (3) that they belonged to the same age group (less than a 5-year difference) as a patient of the BC group (individuals were paired by age group), and (4) that they had had no personal history of cancer. Additionally, all study participants were non-blood-related Mexican mestizo subjects from the State of Colima, Mexico. The study was approved by the institutional review board of the University of Colima School of Medicine and the Colima State Health Department. Desquamation cells were directly placed in 400 l of lysis buffer (Tris-HCl at 100 mM pH 8.8, EDTA at 5 mM, SDS at 0.5%, NaCl at 100 mM). 10 l of SDS at 10% and 45 l of K proteinase at 10 mg/ml were then added, and the mixture underwent incubation for 3–5 h at 55 ° C. Phenol-chloroform extraction and DNA precipitation with absolute ethanol were immediately carried out. The DNA was resuspended in 100 l water and stored at –20 ° C until analyzed. Polymerase chain reactions (PCRs) for the MMP2 gene polymorphic region were carried out in a total volume of 15 l. The solution contained 50 ng genomic DNA, 0.1 M of each primer, 1! Taq polymerase buffer (1.4 mM MgCI2), 0.2 mM dNTP and 0.15 U of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif., USA). The primers for MMP-2 were forward 5-ccctgtgccccacctttttcagat-3 and backward 5-agctgagacctgaagagctaaacagct-3. The backward primer incorporates the Pvu II cut sequence when the –1306C allele is amplified. PCR was performed in a programmable thermal cycler (Mastercycler personal, Eppendorf AG, Germany). The cycling conditions were set as follows: one cycle at 94 ° C for 3 min, 35 cycles at 94 ° C for 30 s, 63.5 ° C for 40 s, and 72 ° C for 40 s, and one final extension cycle at 72 ° C for 5 min. The 150-bp PCR product was mixed with 2U of Pvu II (Invitrogen, San Diego, Calif., USA) and the reaction buffer, according to the manufacturer’s instructions. Two fragments measuring 122 and 28 bp were present if the product was digestible (–1306C allele). The digestion product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as a ‘CC’ homozygote (cuttable), ‘TT’ homozygote (uncuttable), or ‘TC’ heterozygote. Genotyping of 10% of random individuals was confirmed by sequencing. Evaluation of the Hardy-Weinberg equilibrium was performed by comparing observed and expected genotypes, using the 2 analysis. The Fisher exact test was used to examine differences between cases and controls. The associations between environmental factors/MMP-2 genotype and disease genesis were estimated by odds ratio (OR) and associated 95% confidence interval (CI) using the Epi Info, version 6 (Centers for Disease Control and Prevention, Atlanta, Ga., USA) computer-assisted program. A meta-analysis was carried out using data from previous case-control studies, as well as the results of the present study using the MedCalc (version 8.1.0.0 for Windows; Mariakerke, Belgium) software package. The fixed effects statistical model was adjusted according to the MedCalc software package recommendations. Statistical significance was reached when p ! 0.05.

Gynecol Obstet Invest 2008;65:68–72

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Table 1. Distribution of MMP-2 –1306 genotypes by control-case

status

T/T C/T C/C

Control (%)

Case (%)

4 (4.2) 42 (43.7) 50 (52.1)

2 (2.2) 25 (27.8) 63 (70.0)

Results

The mean age was 47.9 (SD 9.8) and 50.2 (SD 11.3) years in the control and case groups, respectively. No significant differences were found in this variable (p = 0.19). The gestation average in both groups was close to 4, so the groups were divided into one of 0–3 gestations and another of 4-or-more gestations. This division was taken into account in later analyses. Forty-five percent of the case group and 54% of the control group had 4 or more pregnancies and this did not represent a significant difference (p = 0.2). Twenty-two percent of the case group and 15% of the control group were smokers, not representing a significant difference between groups (p = 0.2). Sixteen percent of breast cancer patients presented with systemic metastasis when diagnosed. Ten percent of the tumors were well-differentiated, 71% moderately differentiated and 19% poorly differentiated. Upon carrying out histochemistry on the tumoral tissues, 63% were shown to be positive estrogen receptors, 47% progesterone receptors and 27% positive HER2 receptors. The allele frequencies for the MMP2 –1306C and –1306T were 74 and 26%, respectively, in the control group, compared with 84 and 16%, respectively, in breast cancer patients (p = 0.02). The distribution of MMP-2 genotypes in the case and control groups (table 1) were in Hardy-Weinberg equilibrium. Since TT genotype was rare (2.2% of the case group and 4.2% of the control group), it was combined with the CT genotype as the variant group for subsequent analysis. The OR for subjects carrying the CC genotype was 2.15 (95% CI 1.13–4.11, p = 0.01) for breast cancer, suggesting that the CC genotype could be a genotype that increases the probability of breast cancer. Effects of the MMP2 polymorphism were additionally examined with stratification by age at diagnosis, pregnancies, smoking, presence of metastasis, histological grade and immunohistochemical parameters. It was found that increased probability of breast cancer associated with the MMP-2 CC genotype was more pronounced 70


in subjects who were younger (^50 years old) at diagnosis (OR 2.66; 95% CI 1.04–6.96, p = 0.02), compared with subjects who were 651 years old (OR 1.37; 95% CI 0.40– 4.73, p = 0.57). However, no significant associations were observed between the polymorphisms and the presence of systemic metastasis at the time of diagnosis, histological differentiation or detection of estrogen, progesterone or HER2 receptors by immunohistochemistry. No association between the polymorphism and BC was detected when the women were divided into groups according to the number of gestations (0–3 pregnancies; 4 or more pregnancies) or smoking status (data not shown).

Discussion

A number of studies have shown that MMP-2 is overexpressed in various cancer tissues and its involvement in tumor initiation, invasion, angiogenesis and metastasis was critical [3]. One of the causes of MMP-2 overexpression could be the polymorphisms within its gene promoter. The –1306C1T stands out among them for its demonstrated capacity to alter MMP-2 expression levels [12] along with its being associated with the development of lung, stomach, colorectal, oral cavity and breast cancers [14, 17–20]. However, studies on this topic are scarce and have mainly been carried out on Chinese populations. Studies in other populations have produced conflicting results, such as the Elaner et al. [15] report, in which no association was found between the MMP2 promoter polymorphisms and colorectal cancer in a Swedish population [21]. Another recent study carried out on a population in Brazil showed no association between the polymorphism and BC. The MMP-2 –1306C1T polymorphism has been studied very little throughout the world. Only taking into account the control group results of the present study, the –1306C allele frequency observed (0.74) is one of the lowest reported world-wide, compared with frequencies reported in Chinese (0.84), French (0.83), Brazilian (0.82) and Australian (0.76) control group populations [14, 15, 22, 23]. The CC genotype also had a low frequency (52%), as the reported frequencies vary from 58% in an Australian population to 73% in a Chinese population. On the other hand, the TT genotype showed an intermediate frequency (4%) with respect to that previously reported (1–6%). The present study demonstrates that the –1306C allele, previously associated with MMP-2 overexpression, is associated with breast cancer in the Mexican population Delgado-Enciso et al.

China (2004)

Brazil (2007)

Mexico (present study)

Combined

0.3

1.0 Odds ratio

6.0

Fig. 1. Meta-analysis of 3 studies on the MMP-2 –1306CC genotype and breast cancer. The center of the box and the horizontal line indicate the odds ratio and a 95% confidence interval in each study [14, 15]. The summary of combined odds ratio (fixed effect model) is also shown.

studied. The –1306CC genotype was found to produce a 2.15 increase in the probability of developing this neoplasia. This result is in concordance with the study carried out by Zhou et al. [14] on a Chinese population, but differs from that found by Roehe et al. [15] in Brazil. Zhou demonstrated that the CC genotype produced a 2.16 increase in breast cancer probability (OR 2.16, 95% CI 1.57–2.96), whereas Roehe found no association between the polymorphism and BC. A meta-analysis (fig. 1) which brings together the data of case and control studies carried out up to the present (two previous reports and the present one) show that the MMP-2 –1306CC genotype produced a 1.9 increase in breast cancer probability (OR 1.97, 95% CI 1.54–2.52). This last result is congruent with those found in the present study, although there are still very few reports with respect to this subject. Grieu et al. [13] did not carry out a study to examine the association between breast cancer and MMP-2 polymorphism, given that they did not analyze a control group. They suggest that this genetic variant may not represent a breast cancer risk in the Australian Caucasian population. The objective of their study was to look for associations in the MMP2 –1306 polymorphism with phenotypical characteristics of breast cancer. Standing out among their results was the fact that the TT genotype carriers presented significantly smaller tumors than the CT or CC genotype carriers. MMP-2 Polymorphism and Breast Cancer

Additionally, it was observed that the probability of developing breast cancer conferred on CC genotype was accentuated in women 50 years old and younger (OR, 2.66). The CC genotype represented no association in older women (OR, 1.37). This is in accordance with the study by Zhou et al. [14] which shows an increase in OR from CC genotype among women 55 years old and younger and a significant loss of association in older women. Grieu et al. [13] found that the CC or CT genotypes were significantly more frequent among younger breast cancer patients (^57 years old), lending support to the idea that the C allele plays a more accentuated role in breast cancer in women of that age range (^50–57 years old). This is all in line with the concept that genetic susceptibility is often associated with an early age of disease onset [14]. No association was found between polymorphism and the presence of systemic metastasis, histological differentiation or estrogen, progesterone or HER2 receptor detection, which is similar to previously found results (Chinese and Australian phenotypes with breast cancer). Multigestation and tabacism, processes which are associated with increases in MMP expression [24, 25], did not modify genotype CC breast cancer probability, which had not been previously studied. Unfortunately, other variables associated with the MMP-2 –1306 polymorphism in breast cancer, such as tumor size and survival, were not analyzed in this study. In conclusion, the results indicate that on a genetic level, MMP-2 may be implicated in breast cancer genesis and that the –1306C1T polymorphism is of great importance since the CC genotype significantly increases the probability of neoplasia, especially in women 50 years old and younger. More such studies on different populations need to be carried out.

Acknowledgments The author acknowledges support of the ‘Fondo Ramon Alvarez Buylla’ of the University of Colima. We wish to thank Gusti Gould de Pineda of CUMBRE Centro de Inglés for English language editorial assistance.

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