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Technical Briefs |
1 Dipartimento MCO, Clinica Chirurgica Generale, Università degli Studi di Milano, Polo S. Paolo, Via A. Di Rudinì 8, I-20142 Milan, Italy;
2
Laboratorio di Chimica Clinica e Microbiologia, Ospedale S. Paolo, Polo Universitario, Via A. Di Rudinì 8, I-20142 Milan, Italy
aauthor for correspondence: fax 39-02-8137613, e-mail giorgio.ghilardi{at}unimi.it
Tumor invasion and metastasis are important aspects of tumor progression, and the formation of tumor metastasis is a principal contributing factor to cancer morbidity and mortality (1). Basal membrane and extracellular matrix represent two physical barriers to malignant invasion: their degradation by matrix metalloproteinases (MMPs) plays a key role in tumor progression and metastatic spread (2)(3).
MMP expression in tumors is regulated in a paracrine manner by growth factors and cytokines secreted by tumor-infiltrating inflammatory cells as well as by tumor or stromal cells. Recent studies have suggested continuous cross-talk between tumor cells, stromal cells, and inflammatory cells during the invasion process (1)(2)(4)(5). Expression of most MMPs is usually low in tissues and is induced when remodeling of extracellular matrix is required. MMP gene expression is primarily regulated at the transcriptional level, but there is also evidence of modulation of mRNA stability in response to growth factor and cytokines (2)(6).
Several lines of evidence indicate a significant association between variations in MMP genes and susceptibility to cancer. The promoter region of inducible MMP genes (i.e., MMP1 and MMP3) shows remarkable conservation of regulatory elements, and their expression is induced by growth factors, cytokines, and other environmental factors, such as contact with extracellular matrix (7)(8)(9).
Recently, a naturally occurring sequence variation in the human MMP1 gene promoter was reported (10). Many studies have demonstrated the correlation between the 2G allele and several malignant tumors with different histogenetic origins (11)(12)(13)(14)(15). Recently, our group demonstrated a correlation between the MMP3 polymorphism and breast cancer (16).
Matrilysin (MMP7) is a protease with broad substrate specificity, being able to degrade elastin, proteoglycans, fibronectin, and type IV collagen. MMP7 is among the smallest members of the MMP family (17).
MMP7 was first characterized from a human rectal carcinoma cell line, and overexpression of MMP7 has been shown to correlate with Duke’s stage and increased metastatic potential in colorectal carcinoma (CRC) (18)(19).
Jormsjö et al. (20) recently described two common polymorphisms in the promoter region of the MMP7 gene that are functional in vitro and seem to influence coronary artery dimensions. Both polymorphisms influenced the binding of nuclear proteins. Furthermore, in transient transfection studies, the combination of the two rare alleles conferred an increased promoter activity.
The aim of this prospective study was to investigate a possible correlation between MMP7 promoter polymorphisms and CRC clinical phenotypes, specifically the ability of genetic analysis to identify a subgroup of CRC patients with a disease that appears more aggressive or prone to metastasis.
The study started during June 2000, and recruitment was stopped during June 2002. Our Institutional Ethical Committee approved this study, and informed consent was obtained from patients and controls.
MMP7 gene promoter sequences were obtained from peripheral blood samples from 58 consecutive patients with CRC of different stages who underwent surgery and from 111 sex- and age-matched healthy individuals (control group). CRC patients were 38 males and 20 females (median age, 68 years; range, 32–88 years). Patients and controls were all Italian. CRC patients were grouped according to Duke’s classification, as modified by Astler and Coller (21), on the basis of the postoperative histopathologic evaluation. The group assignment was then reevaluated at the end of the follow-up period, which ranged from 6 to 30 months (median, 21 months). The 58 patients with CRC were assigned to two subgroups according to the presence (M+) or absence (M-) of detectable metastasis at the time of diagnosis and at the end of the follow-up.
Genetic polymorphisms were detected with PCR followed by direct sequencing. The Insta Gene (Bio-Rad) commercial reagent set was used for DNA extraction from whole blood. The PCR reaction for MMP7 was carried out in a total volume of 25 µL with 5 µL of extracted genomic DNA; 100 µM dATP, dGTP, dTTP and dCTP; 1.5 mM MgCl2; 1 U of Taq Gold polymerase; and the two primers, forward and reverse, each at a concentration of 80 nM. The primers were designed with the Primer Express software. The MMP7 primer sequences were as follows: forward primer, 5'-CCTGAATGATACCTATGAGAGCAGTC-3'; reverse primer, 5'-AGAGTCTACAGAACTTTGAAAGTATGTGTTATT-3'. The PCR began with a 5-min incubation at 94 °C to activate the enzyme, followed by 35 cycles of 20 s at 94 °C, 20 s at 55 °C, and 30 s at 72 °C.
The amplification was verified on an agarose gel (2%) followed directly by sequencing with an automatic sequencer using fluorescent DNA capillary electrophoresis (ABI Prism 310; Applied Biosystems). The forward primer was used for the sequencing primer.
Differences between groups were examined by the 
2 test or Fisher exact test when appropriate. Odds ratios (ORs; approximate relative risk) were calculated as an index of the association of the MMP genotypes with each phenotype. For each OR, two-tailed probability values and 95% confidence intervals (CIs) were calculated. P <0.05 was assumed as the cutoff for statistical significance.
All statistical analyses were two-sided and were performed with Stata Statistical Software (Stata Corporation). The polymorphism distributions in patients and controls were, as expected, according to Hardy–Weinberg equilibrium.
In CRC patients, the number of -181G homozygotes [patients vs controls, 15 (26%) vs 14 (13%); OR, 2.41; 95% CI, 0.98–5.89; P = 0.03] and the T allele frequency in position -153 (0.11 vs 0.05; OR, 2.20; 95% CI, 0.89–5.48; P = 0.05) were significantly higher than in controls. The -181G/-153T haplotype was more represented, although not significantly, in CRC patients than in controls (P = 0.08; Table 1
).
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Subgroups were compared with controls and among allelic variants. The -153C/T promoter allelic variant distribution was not statistically significantly different among the subgroups. For the -181A/G promoter polymorphism, however, we found a strong correlation between -181G homozygosity and the M+ subgroup at the time of diagnosis [M+ vs M-, 9 (56%) vs 6 (14%); OR, 7.5; 95% CI, 2.07–27.19; P = 0.0013].
The frequency of the -181G allele was not statistically different between controls and metastasis-free (M-) patients (P = 0.95). Moreover, the -181A/G polymorphism was correlated with lymph node metastasis at the beginning of follow-up. In the N1+N2 patients, the number of -181G homozygotes and G allele frequencies were greater than in the N0 patients [number of homozygotes, 13 (35%) vs 2 (10%; P = 0.03); G allele frequency, 0.53 vs 0.36 (P = 0.02), respectively].
Our data demonstrate, for the first time, that the two common polymorphisms on MMP7 promoter region are correlated to CRC susceptibility. In addition, our results may suggest that -181G homozygosity is associated with the presence of distant metastasis as well as lymph node involvement. From this point of view, G homozygosity might be considered as a factor of worse prognosis.
MMP7 has unique characteristics, such as a minimum MMP structure, wide spectrum of substrate specificity, and potency to start an activation cascade of MMPs (17). In addition to its overexpression in a variety of cancer tissues, in vitro and animal data suggest that MMP7 may play a key role in tumor invasion and metastasis as well as tumor initiation and growth. Many studies have reported that MMP7 mRNA is overexpressed in human CRC and is correlated with increasing Duke’s stage (22)(23)(24).
Recently Jormsjö et al. (20) identified two novel polymorphisms in the MMP7 promoter region. Electrophoretic mobility shift assay results demonstrated that the -181A/G and the -153C/T polymorphisms influence the binding of nuclear protein(s). Furthermore, basal promoter activity was higher in promoter constructs harboring the combination of the two rare alleles in transient transfection studies. In our study the -181G/-153T haplotype was more represented in CRC patients than in controls, although statistical significance was not reached (19% vs 10%; P = 0.08). We did observe a statistically significant correlation between the -181A/G substitution and susceptibility, invasiveness, and prognosis in CRC. In CRC patients, the frequency of -181G homozygotes was 2-fold higher than in controls, and 4.2-fold higher in patients who developed metastasis at the end of the follow-up (OR, 4.2; 95% CI, 1.19–14.59; P = 0.024). Lymph node metastasis is the most important prognostic factor in colon cancer, and the -181A/G substitution correlated well with lymph node involvement at the time of diagnosis. The G allele frequency was 2.5-fold higher in lymph node-positive patients than in lymph node-negative ones (OR, 2.49; 95% CI, 1.14–5.42; P = 0.02). The -153C/T polymorphism seems in our series less involved in CRC susceptibility, although the mutant T allele was more represented in CRC patients (P = 0.05).
In conclusion, our study suggests that the presence of G allele for the MMP7 gene promoter sequence may be a facilitating factor for cancer growth, lymph node invasion, and metastasis in CRC patients. The good correlation of lymph node involvement and distant metastasis with -181G homozygosity might suggest the use of this common polymorphism as a prognostic factor at the beginning of follow-up for influencing the decision for adjuvant therapy in N0M0 patients. Our data suggest a role of MMP7 in the matrix remodeling associated with CRC. However, no firm conclusions regarding the clinical significance of MMP7 promoter polymorphisms can be drawn until much larger cohorts have been analyzed.
References
The following articles in journals at HighWire Press have cited this article:
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  W. Qiu, G. Zhou, Y. Zhai, X. Zhang, W. Xie, H. Zhang, H. Yang, L. Zhi, X. Yuan, X. Zhang, et al. No Association of MMP-7, MMP-8, and MMP-21 Polymorphisms with the Risk of Hepatocellular Carcinoma in a Chinese Population Cancer Epidemiol. Biomarkers Prev., September 1, 2008; 17(9): 2514 - 2518. [Abstract] [Full Text] [PDF]
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 A. Beeghly-Fadiel, J.-R. Long, Y.-T. Gao, C. Li, S. Qu, Q. Cai, Y. Zheng, Z.-X. Ruan, S. E. Levy, S. L. Deming, et al. Common MMP-7 Polymorphisms and Breast Cancer Susceptibility: A Multistage Study of Association and Functionality Cancer Res., August 1, 2008; 68(15): 6453 - 6459. [Abstract] [Full Text] [PDF]
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 K. Shan, Z. Lian-Fu, D. Hui, G. Wei, W. Na, J. Xia, and L. Yan Polymorphisms in the promoter regions of the matrix metalloproteinases-7, -9 and the risk of endometriosis and adenomyosis in China Mol. Hum. Reprod., January 1, 2006; 12(1): 35 - 39. [Abstract] [Full Text] [PDF]
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 J. Zhang, X. Jin, S. Fang, R. Wang, Y. Li, N. Wang, W. Guo, Y. Wang, D. Wen, L. Wei, et al. The functional polymorphism in the matrix metalloproteinase-7 promoter increases susceptibility to esophageal squamous cell carcinoma, gastric cardiac adenocarcinoma and non-small cell lung carcinoma Carcinogenesis, October 1, 2005; 26(10): 1748 - 1753. [Abstract] [Full Text] [PDF]
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