A Functional Polymorphism in the Matrix Metalloproteinase-2 Gene Promoter (−1306C/T) Is Associated with Risk of Development but not Metastasis of Gastric Cardia Adenocarcinoma¹ Free

The MMPs⁴ are a large family of proteolytic enzymes that consist of >20 members. These proteolytic enzymes are present in normal healthy individuals and up-regulated in almost every type of human cancers (reviewed in Ref. 1). Historically, MMPs were considered to play an important role in cancer invasion and metastasis because of their features capable of degrading extracellular matrix and basement membrane barriers (2, 3). However, recent studies have demonstrated that MMPs are also involved in several steps of cancer development by modulating cell proliferation, apoptosis, and host immune surveillance (reviewed in Ref. 1). MMP-2, among other MMPs, primarily hydrolyzes type IV collagen, the major structural component of basement membrane (3, 4). This proteinase also has high activity toward many other bioactive molecules, such as growth factor-binding proteins and growth factor receptors (reviewed in Ref. 1), e.g., MMP-2 can cleave insulin-like growth factor-binding proteins and release insulin-like growth factors, which are well known to have a strong effect on stimulating cell proliferation and inhibiting apoptosis. These activities of MMP-2 are believed to be linked to both cancer development and progression.

In contrast to the majority of MMPs, MMP-2 is constitutively expressed by a large number of cell types and overexpressed in a wide variety of human cancer, including gastroesophageal cancer (5, 6, 7, 8). Although the activity of MMP-2 is known to be regulated by post-transcriptional mechanisms, including the activation of proenzyme and inhibition of enzyme activity (1, 4), transcriptional regulation is also believed to be pivotally important because the human MMP2 promoter contains a number of cis-acting regulatory elements, and several transcription factors, including p53, Sp1, Sp3, and AP-2, participate in the control of its constitutive expression (9, 10). Recently, Price et al. (11) reported a single nucleotide polymorphism in the promoter of the MMP2 gene (−1306C/T). The −1306C→T transition is located in a core recognition sequence of Sp1 (CCACC box), which abolishes the Sp1-binding site and consequently diminishes promoter activity. Transient transfection experiments showed that reporter gene expression driven by the C allelic MMP2 promoter was significantly greater than reporter gene expression driven by the T allelic counterpart both in epithelial cells and macrophages, indicating the functional significance of this polymorphism (11).

The cancers in which an effect for MMP-2 has been established are characterized by varying individual susceptibility, implying the role of genetic factors. However, like many MMPs, MMP-2 is not up-regulated by gene amplification or activating mutations, and genetic alternations in the gene of the cancer cells are generally lacking. Therefore, germ-line polymorphisms that alter constitutive and/or induced expression and enzyme activity of MMP-2 may affect individual susceptibility to certain cancers. It has been hypothesized that the MMP2 −1306C alleles might render increased risk for carcinogenesis because of an elevated level of MMP-2 expression over a lifetime. We have recently shown in a molecular epidemiological study that the frequency of the MMP2 −1306CC genotype was significantly higher in patients with lung cancer than in the healthy population, and this genotype was associated with several folds of increased risk of lung cancer solely or in a manner of interaction with smoking exposure (12). The present report described a case control study that aimed to test the hypothesis in GCA. In addition, we also assessed the contribution of the MMP2 polymorphism to risk of metastasis of the cancer.

This study consisted of 356 patients with GCA and 789 healthy controls, and all subjects were ethnic Chinese. Patients were consecutively recruited from January 1997 to July 2002, at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing). In this study, GCA was defined as tumors arising at the gastric cardia and/or gastroesophageal junction with or without involvement of other esophageal and/or gastric subsites. Tumors confined entirely to the esophageal or gastric subsites were excluded. All patients with histopathologically confirmed GCA were enrolled, yielding a 94% response rate. Because this was a study of genotype and the marker was a constitutional one, both incident and prevalent cases were included. A portion of cases (217) was participants in a molecular epidemiological study of MTHFR and GCA as reported previously (13). Of the 356 patients, 275 (77.2%) underwent surgical resection and had detailed metastatic data. The presence (M+) or absence of detectable metastases (M−) were evaluated according to the Tumor-Node-Metastasis classification (14) on the basis of the postoperative histophathological examination. Population controls were cancer-free individuals living in Beijing region; they were selected from a community cancer screening program for early detection of cancer conducted during the same period as the cases were collected (13). Briefly, these controls were randomly selected from a pool of 2800 individuals based on a physical examination, and the response rate was 96%. The selection criteria included no individual history of cancer, and frequency matched to GCA cases on sex and age (±5 years). At recruitment, informed consent was obtained from each subject, and each participant was then interviewed to solicit detailed information on demographic characteristics and lifetime history of tobacco use. This study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.

Genomic DNA was isolated from peripheral blood of the controls and most of the cases. Approximately 30% of DNA samples from cases were isolated from surgically resected “normal” tissues adjacent to the tumor of GCA patients. MMP2 genotyping was accomplished by PCR-based DHPLC analysis and direct DNA sequencing as reported previously (12). The primers used to amplify a 295-bp fragment of MMP2 promoter containing –1306 C/T site were: MMP-2F, 5′-CTG ACC CCC AGT CCT ATC TGC C and MMP-2R, 5′-TGT TGG GAA CGC CTG ACT TCA G. PCR was accomplished with a 25-μl reaction mixture containing ∼100 ng of DNA, 1 μm each primer, 0.2 mm deoxynucleotide triphosphate, 2 mm MgCl₂, 1 unit of Taq DNA polymerase with 1 × reaction buffer (Promega, Madison, WI), and 2% DMSO. The PCR profile consisted of an initial melting step of 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 64°C, and 45 s at 72°C, and a final elongation of 7 min at 72°C. DHPLC analysis was performed on a Transgenomic WAVE System (Transgenomic, Inc., Omaha, NE). The detailed genotyping process was described in our recent publication (12). Briefly, each PCR product was denatured for 1 min at 94°C and then gradually reannealed by decreasing sample temperature from 94°C to 45°C over a period of 30 min to form homo- and/or hetero-duplexes. The PCR products were then applied to the DHPLC column and eluted with a linear acetonitrile gradient at a flow rate of 0.9 ml/min. The genotypes of MMP2 –1306 C/T revealed by DHPLC analysis were further confirmed by DNA sequencing. To ensure quality control, genotyping was performed with blinding to case control status, and a 10% random sample of cases and controls was genotyped twice by different persons; the reproducibility was 100%.

Pearson’s χ² test was used to examine differences in demographic variables, smoking, and distributions of genotypes between cases and controls and metastatic and nonmetastatic cases. The associations between the MMP2 polymorphism and risk of development and metastasis of GCA were estimated by ORs and their 95% CIs, which were calculated by unconditional logistic regression models. Smokers were considered current smokers if they smoked ≤1 year before the date of diagnosis for cases or up to the date of the interview for controls. Information was collected on the amount of cigarettes smoked per day, the age at which the subjects started smoking, and the age at which ex-smokers stopped smoking. Light or heavy smokers were categorized by the ∼50th percentile pack-year value among controls, i.e., <26 or ≥26 pack-years [(cigarettes per day ÷ 20) × (years smoked)]. Because only 15 cases and 27 controls were ex-smokers, they were combined with current smokers for analysis. The ORs were adjusted for age, sex, and pack-years smoked. All analysis was carried out with Statistical Analysis System software (Version 6.12; SAS Institute, Cary, NC).

The relevant characteristics of the study subjects are shown in Table 1. The age and gender distributions among cases and controls were not statistically different. The median age was 58.4 years (range 41–72) for the case group and 57.6 years (range 45–76) for the control group. More smokers were presented in cases compared with controls (55.9 versus 48.8%; P < 0.05), and the mean duration of smoking among cases was significantly longer than among controls (15.5 versus 13.2 years; P < 0.05). However, the distribution in lighter (<26 pack-years) and heavier smokers (≥26 pack-years) between cases and controls was not significantly different (P = 0.97).

The allelic frequencies for MMP2 −1306C and -1306Twere 0.83 and 0.17, respectively, among the 789 controls and 0.94 and 0.06, respectively, among the 356 cases (Table 2). The frequencies of three MMP2 genotypes among controls were CC, 68.7%; CT, 29%; and TT, 2.3%, which fit in with the Hardy-Weinberg equilibrium law (P = 0.74). The distribution of MMP2 genotypes was then compared among cases and controls. It was found that 12.4% of cases were heterozygous carriers of −1306T; this was significantly lower than that of controls (29%; χ² = 37.5, P = 0). None of the case subjects carried the TT genotype, whereas 2.3% of control subjects carried this genotype. Because the TT homozygotes were rare in our study, this genotype was combined with the CT genotype as the reference group for subsequent estimation of risk by using logistic regression analysis. It was found that the adjusted OR of developing GCA for the CC genotype compared with the CT and TT genotypes was 3.36 (95% CI 2.34–4.97).

Associations between the MMP2 genotype and GCA risk stratified on age, sex, and smoking are shown in Table 3. Although the risk of GCA associated with the CC genotype was similar in males and females, it appeared to be more pronounced in subjects who were younger (<60 years) at diagnosis (OR 3.72, 95% CI 2.52–5.14), in subjects who were smokers (OR 4.41, 95% CI 2.62–7.74), and in smokers who smoked ≥26 pack-years (OR 3.91, 95% CI 1.87–8.11).

The correlation between MMP2 genotype and metastasis of GCA was also analyzed. Of the 275 patients with GCA who underwent surgical resection, 91 (33.1%) had no detectable metastasis (M− and N−), and 184 (66.9%) had detectable metastasis (M+ and/or N+) at the time of diagnosis. The distribution of the CC genotype showed no significant difference between subgroup M+ and subgroup M− (90.1 versus 89.1%; P = 0.804), with the OR of 0.9 (95% CI 0.36–2.2) for the CC genotype compared with the CT and TT genotypes (data not shown).

In the previous study, we have reported that the −1306C/T polymorphism in the promoter of MMP2 is a strong genetic susceptibility factor for lung cancer, and the CC genotype confers several-fold increased risk for developing the cancer (12). Because MMP-2 has been shown to be overexpressed in gastroesophageal cancer and appears to be a feature of the malignant phenotype (7, 8, 15), we therefore examined whether the functional polymorphism in the MMP2 promoter could have some impact on the risk of GCA development and metastasis. On the basis of analysis with 356 cases with GCA and 789 controls, we found that subjects carrying the CC genotype had a >3-fold increased risk for developing GCA, whereas no effect of this polymorphism on metastasis of the cancer was observed. These results are consistent with our previous findings from the lung cancer study (12) and further support the hypothesis that the MMP2 −1306CC genotype may constitute a common susceptibility factor for certain cancers.

Our molecular epidemiological results are parallel to the laboratory findings showing that the –1306C→T transition in the promoter region of MMP2, which disrupts an Sp1-binding site (CCACC box), leads to a strikingly lower promoter activity with the T allele (11). Deletion or site-directed mutagenesis analysis of MMP2 promoter has also shown that the Sp1 site, among other promoter elements such as AP-2, is critical for constitutive activity of this gene (10). On the other hand, recent study also demonstrated that reduction of Sp1 DNA-binding activity or phosphorylation by nonsteroidal anti-inflammatory drugs suppresses MMP2 expression (16). Taken together, these data clearly suggest that the presence of Sp1 consensus sequence in the MMP2 –1306C allele may enhance transcription, which in turn would produce higher levels of MMP-2 protein in individuals carrying the CC genotype than those carrying the TT or CT genotype. Because MMP-2 plays an important role in multiple ways to all stages of cancer initiation and development (see above), one would expect that individuals who carry the CC genotype and therefore have increased expression of this enzyme over a lifetime may be more susceptible to cancer. The association between high levels of constitutive expression of MMP-2 and susceptibility to tumor formation has been tested in several studies with genetically modified animals. It was found that when induced by carcinogenic stimulus, mice that lack the Mmp2 or Mmp9 gene developed fewer tumors than wild-type mice (17). Cancer cells injected via vein were found to be less capable of colonizing the lungs of Mmp2-knockout mice than lungs of wild-type mice (18). Conversely, transgenic mice that overexpress membrane type-MMP-1, a known activator of pro-MMP-2, were at increased susceptibility to mammary tumor formation and metastasis (19). These data strongly support our epidemiological observation that the genetic polymorphism resulting in high expression of MMP-2 over a lifetime may increase cancer susceptibility. In addition, functional polymorphisms in some other MMP genes have also been reported to be associated with risk of certain cancers, e.g., a single guanine insertion polymorphism in the MMP1 promoter region, which creates an Ets-binding site and enhances transcriptional activity, has been associated with increased risk for lung (20), colorectal (21, 22), endometrial (23), and ovarian cancer (24). A single adenosine insertion polymorphism in the MMP3 promoter (6A allele), which has half the transcriptional activity of the 5A allele, has been linked to reduced susceptibility to breast cancer (25).

In this study, we found that the association between the MMP2 –1306CC genotype and GCA risk appeared to be more pronounced in younger subjects (<60 years old). This finding is in line with the conception that genetic susceptibility is often associated with an early age of disease onset. We also found a significantly higher risk for GCA related to the MMP2 –1306CC genotype among smokers and smokers who smoked ≥26 pack-years. Because the expression of MMPs can be induced by smoking (26, 27), one suggestive explanation of these findings is that, in addition to higher constitutive expression attributable to gain of an Sp1 promoter site, the inducibility by smoking of the C allele of MMP2 may also be higher than that of the T allele, which loses an Sp1 site. Given these conditions, it would be expected that smokers, especially heavy smokers, who carried the CC genotype were at the highest risk. Numerous studies have consistently shown that tobacco smoke may play an important role as an environmental etiological factor in the development of GCA (28, 29, 30, 31). Therefore, another explanation for a higher risk of GCA among smokers and heavy smokers with the CC genotype could be that these subjects had larger numbers of transformed cells caused by tobacco carcinogens in the target tissue, which consequently increases the possibility that one of these cells will form malignancy under the condition of higher MMP2 expression.

Local overexpression of MMP-2 has been shown to be correlated to invasion and metastasis of certain cancers, including gastroesophageal cancers (15, 32, 33). Several studies have suggested that genetic polymorphism in the promoter of MMP1 (1G/2G) or MMP3 (5A/6A), which alters the transcription activity of the genes, may influence invasiveness or metastasis of some types of cancer, such as melanoma (34), colorectal cancer (21), and breast cancer (25). In the present study, we did not find any significant association between MMP2 genotype and status of lymph node metastasis of GCA, questioning the major role of the CC genotype as a relevant genetic factor inducing the local overexpression of MMP-2 and as a risk marker of metastatic disease. However, these data on metastasis should be considered as preliminary data because of limited sample size and metastatic parameter. Additional examinations of larger patient series with more detailed clinicopathological features and clinic outcome, especially the survival rate, may be required.

Our study may have certain limitations because of the study design. Because it was a hospital-based, case control study and the cases were from the hospital and the controls were from the community, selection bias and/or systematic error may occur. It would therefore be important to confirm these findings in a population-based prospective study. In addition, excepting for tobacco smoking, other factors, such as gastroesophageal reflux disease, obesity, and certain dietary components, are known to be associated with risk of GCA (35, 36). These factors might interact with MMP2 genotype or act as potential confounders in the analysis. Unfortunately, information on these factors in our case control study is not available. It would be interesting to investigate the interaction between MMP2 genotype and these risk factors, especially gastroesophageal reflux disease on risk of GCA in additional studies.

In conclusion, our study provides the first evidence that the MMP2 −1306C/T polymorphism is a genetic susceptibility factor for the development of GCA, with the CC genotype being associated with increased risk of the cancer in Chinese population. This association is especially noteworthy in younger individuals and smokers. These results are consistent with our previous findings in the lung cancer study, further supporting the hypothesis that MMP-2 may play an important role in carcinogenesis. The MMP2 genotype seems not associated with the risk for metastatic of GCA, although additional studies are needed to clarify this important issue.