We evaluated the effect of three polymorphisms in the promoter regions of two human gelatinases, MMP2 and MMP9, and the human stromelysin MMP3 on the risk and survival time of lung cancer.
In this study, the distribution of the MMP2 genotypes in controls is not in the Hardy-Weinberg equilibrium as reported in Caucasian [19
] and Asian [26
] populations. Although the explanation is not known, the random recruitment of the healthy individuals, the reproducible genotyping method and the consistence with the Hardy-Weinberg equilibrium in several other polymorphic loci [8
], suggests that the controls in the present study may reasonable be used in case-control investigations.
Our results suggest that the studied polymorphism in the promoter region of the MMP9 gene is associated with the risk of the development of lung cancer. Thus, individuals with the MMP9 -1562 T/T genotype have shown a protective effect against the development of lung cancer compared to the reference genotype (-1562 C/C). In relation to survival analysis, the MMP2 -735 T/T genotype was significantly associated with an unfavourable survival prognosis in patients with NSCLC.
The MMP family comprises 23 human enzymes that traditionally have long been associated with cancer invasion and metastasis because of their ability to degrade the extracellular matrix. However, recent studies have showed that the roles of MMPs in tumour development and metastasis are much more complex than was originally envisioned. In vitro and animal studies have demonstrate that MMPs are also the key mediators of growth factor activation, bioavailability and receptor signalling, cell adhesion and motility, apoptosis and survival mechanisms, angiogenesis, and inflammatory responses and immune surveillance [10
]. In this sense, high levels of MMP2, MMP3 and MMP9 proteins have been implicated in several malignancies including oesophageal, renal, head and neck, oral, colorectal, NSCLC, breast carcinomas and melanomas [27
]. However, recent studies have shown that several members of this family, including MMP9, which were originally recognised as pro-tumourigenic proteases [35
], provide a protective effect in different stages of cancer progression [36
]. These experimental analyses support the results obtained in our study where individuals with the MMP9
-1562 T/T genotype showed a decreased risk of developing lung cancer. Only two studies have evaluated the association between the MMP9
-1562 C/T polymorphism and lung cancer risk, both finding a non-statistically significant association [19
]. Several hypotheses can explain this apparent discrepancy. First, Zhou et al. carried out their study among the Chinese population, whereas all individuals included in our study were Caucasians. In this sense, numerous differences have already been reported concerning genotype frequencies and cancer susceptibility between Asian and Caucasian populations. For example, a recent meta-analysis for the MMP1
1 G/2 G polymorphism found a statistically significant association with cancer risk in European populations, whereas no association was found in Asian populations [18
]. Second, Rollin et al. analysed the risk in 90 cases and 90 controls, though this sample size was too small to yield a real association [19
With regard to the MMP3
-1171 5A/6A polymorphism, two studies have investigated the association between this polymorphism and lung cancer risk, showing a non-statistically significant association [17
]. Thus, our findings are consistent with previous studies.
Finally, two studies have evaluated the lung cancer risk for individuals with the -735C/T polymorphism in the MMP2
gene, showing an 1.6-fold increased risk associated with the -735C/C genotype in Asian populations [26
] and no significant association in Caucasian populations [19
], which is in line with our results (although it should be noted that Rollin's study includes only 90 cases).
Alternatively, in this study, we investigated the effects of these three polymorphisms on the survival time of 816 lung cancer cases, subdivided on the basis of their different histopathological presentation and clinical stages (NSCLC: I, II, III, IV and SCLC: extended or limited). To date, a large number of studies have investigated the relationship between variants in the MMP2, 3
genes and cancer susceptibility or metastasis, including lung cancer [26
]. However, only three studies have explored the relationship between the polymorphisms in these MMPs
and survival rates among patients with NSCLC [19
]. Rollin et al. explored the effect of the MMP9
-1562 C/T and the MMP2
-735C/T polymorphisms in NSCLC survival among Caucasian patients and found that the MMP9
-1562 C/T polymorphism did not present a significant increase in survival rate, in accord with our results (although it should be noted that Rollin's study includes only 90 patients). However, the homozygous individuals for the MMP2
-735C allele had a shorter survival time than those carrying the T allele (P
= 0.02), and Cox's proportional hazard regression analysis demonstrated that this polymorphism was an independent risk factor for a shortened survival time (P
= 0.045) [19
]. Similarly to the risk analysis, one possible reason for this discrepancy is the relatively small sample size (90 patients). In another study, Heist et al. investigated the association of five polymorphisms, including the MMP3
-1171 5A/6A polymorphism, in 382 patients with stage I lung cancer, finding that individual carriers of a variant genotype did not present a significant increasebetter in survival rate. To verify these results in our study, we analysed the effects of the MMP3
-1171 5A/6A polymorphism in the group of patients with stage I NSCLC and obtained similar results (adjusted HR = 1.20; 95% CI: 0.72-2.00) [data not shown]. Finally, a recent study of 561 NSCLC patients in a Chinese population analysed the effects of 14 SNPs in MMPs
genes in the overall survival of patients with NSCLC and found that the MMP2
-735 T/T and MMP3
-1171 5A/5A genotypes did not decrease survival time. However, large sample size studies in Caucasian and Asian populations are needed to corroborate and validate these findings.
Our study is the first to analyse the effects of three polymorphisms in the MMPs
genes on the survival time in SCLC patients. Our results indicated that the MMP2
-735 T/T genotype did not show significant differences in survival time in patients with SCLC, whereas it was associated with a significant decrease in survival among patients with NSCLC. Studied polymorphisms in the MMP3 and MMP9
genes showed no effect on the survival time in both NSCLC and SCLC. The discrepancy of these findings may be explained by different expressions of MMPs between SCLC and NSCLC tissues. Thus, one study among 45 patients with SCLC, where expression was evaluated by Immunohistochemistry (IHC) for several MMPs, showed a wide expression for all MMPs, except for MMP2, whose expression was not detected [39
]. However, a recent meta-analysis showed that MMP2 is highly expressed in NSCLC patients and that decreased the survival time [40
]. These results seem to show that some MMPs may be more specific to NSCLC tissues than to SCLC tissues.
Our study has several strengths, including high participation of eligible cases (rate of 91.4%) and quite a large sample size from a homogeneous population of similar ancestry (879 cases and 803 controls). Furthermore, all of our cases were pathologically confirmed, and we finally applied a strong quality control for genotyping. Inevitably, the use of hospital-based controls is a potential limitation. Although there is always a chance of recall bias because information on confounding variables was obtained retrospectively, the estimations obtained for the most important confounding variable (tobacco) was nevertheless in line with the literature. Even though our sample size is quite large because of the low allelic frequency in the studied polymorphisms, our patient and control groups with variant genotypes were probably not large enough to study the impact of these gene polymorphisms on lung cancer risk. Therefore, further studies with larger populations are necessary to reach conclusions. Along the same line, the approach taken in this manuscript (over-simplified with only 1 genetic variant in each MMP gene) is a limitation. To properly assess the association between the MMP genes and lung cancer risk and survival, it would be preferable to take a tagging variant approach.