Matrix Metalloproteinase-1 Promoter Polymorphism and Lung Cancer Risk

The matrix metalloproteases (MMP) are a family of secreted zinc metalloproteases that degrade the extracellular matrix collagens. MMP-1 (collagenase) may degrade the interstitial types I, II, and III collagens (1) and contribute to tumor initiation and development by altering the cellular microenvironment that facilitates tumor formation (2-4). There is a single nucleotide polymorphism at −1,607 bp in the MMP-1 promoter, with the 2G allele associated with higher expression levels (5, 6). Cells expressing the 2G allele may provide a mechanism for more aggressive matrix degradation, thereby facilitating cancer progression. The MMP-1 2G/2G genotype has been associated with significantly higher risk of lung cancer in one previous study, specifically for males, current smokers, and heavy smokers (3). We hypothesized that the 2G allele of the MMP-1 1G/2G polymorphism is associated with higher risk of lung cancer in our large case-control population.

The study was approved by the Human Subjects Committees of Massachusetts General Hospital and the Harvard School of Public Health, Boston, MA. Details of this case-control population have been described previously (7, 8). In brief, all eligible histologically confirmed lung cancers were recruited at Massachusetts General Hospital between December 1992 and April 2003. Unmatched controls were recruited among healthy friends and non–blood-related family members (usually spouses) of cancer patients or patients with a cardiothoracic condition undergoing surgery. Over 85% of eligible cases and >90% of controls participated in this study and provided blood samples. Interviewer-administered questionnaires collected information on demographic and detailed smoking histories from each subject. The MMP1 −1607 1G/2G polymorphism was genotyped by the 5′ nuclease assay (TaqMan) using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).

Although individuals of all races were recruited for this study, we restricted our analyses to Caucasians (97%) in order to minimize confounding due to allele frequency variation by ethnicity. Logistic regression models were fit to examine the relationship between the log odds of lung cancer and each covariate, after adjusting for possible confounding factors such as age, gender, smoking status, pack-years of smoking, and years since smoking cessation (if ex-smoker, defined as those who quit smoking more than a year before recruitment). As suggested before, square root transformed pack-years of smoking were used in the analyses instead of the original untransformed variable (7). In addition to the overall association analysis, we did a stratified analysis by gender, age, smoking status, histology, and clinical stages, to further explore the association between MMP-1 polymorphism and the risk of lung cancer in each stratum. Case-only analyses were done to investigate the effect of MMP-1 polymorphism on different histologic subtypes or clinical stages among lung cancer patients. Statistical analyses were undertaken using SAS statistical packages (SAS Institute, Cary, NC).

A total of 3,413 (99.6%) of 3,426 enrolled subjects were genotyped successfully for the MMP-1 polymorphism. Complete information on race, age, gender, and detailed smoking variables was available for 3,219 subjects (94%). Of these, there were 1,752 Caucasian lung cancer cases and 1,363 Caucasian controls. The distributions of demographic characteristics for cases and controls are summarized in Table 1. Compared to the controls, cases were older, had a higher proportion of males, were more likely to be current smokers or heavy smokers, and had a shorter time since smoking cessation (if an ex-smoker) and larger pack-years of cigarette smoking.

The MMP-1 polymorphism in this control population was consistent with Hardy-Weinberg Equilibrium (P = 0.46 by χ² goodness of fit). MMP-1 genotype frequencies were very similar between cases and controls, with the frequencies of 1G/1G, 1G/2G, and 2G/2G genotypes of 26%, 51%, and 23% in cases, and 27%, 49% and 24% in controls, respectively. There is no statistical difference in genotype distributions between cases and controls for different strata of age, gender, smoking status, pack-years of smoking, and clinical stage subgroups (P > 0.10; Table 2).

There was no overall association between MMP-1 polymorphism and lung cancer risk, with the crude and adjusted odds ratios (OR) of 1.08 [95% confidence interval (CI), 0.91-1.28] and 1.14 (95% CI, 0.94-1.39) for the 1G/2G genotype, and 0.99 (95% CI, 0.81-1.21) and 1.13 (95% CI, 0.89-1.42) for the 2G/2G genotypes, respectively, when compared with the 1G/1G genotype.

No statistically significant association was found between MMP-1 polymorphism and lung cancer risk in younger or older subjects (Table 2). Statistically significant higher lung cancer risk (only for the 1G/2G genotype) was found in males but not in females, with the adjusted ORs for 1G/2G and 2G/2G genotypes (when using the 1G/1G genotype as reference) of 1.30 (95% CI, 1.00-1.75) and 1.23 (95% CI, 0.88-1.73) in males, and 0.99 (95% CI, 0.74-1.32) and 1.05 (95% CI, 0.75-1.46) in females, respectively. For different smoking categories, statistically higher lung cancer risk (only for the 1G/2G genotype) was found in never-smokers, with the adjusted ORs for 1G/2G and 2G/2G genotypes (when versus the 1G/1G genotype) of 1.67 (95% CI, 1.02-2.76) and 1.50 (95% CI, 0.86-2.62), respectively. No associations were found in ex-smokers or current smokers, or in different pack-years of smoking (Table 2).

For different histologic subtypes of lung cancer, higher lung cancer risk of MMP-1 polymorphism was found for patients with adenocarcinoma than for patients with squamous cell carcinoma (when each was compared with all controls), with the adjusted ORs for 1G/2G and 2G/2G genotypes (when versus the 1G/1G genotype) of 1.20 (95% CI, 0.95-1.52) and 1.21 (95% CI, 0.92-1.59), respectively, for patients with adenocarcinoma; and 0.93 (95% CI, 0.66-1.31), and 0.72 (95% CI, 0.47-1.11), respectively, for squamous cell carcinoma patients (Table 3). In case-only analysis, the adjusted ORs of adenocarcinoma versus squamous cell carcinoma for 1G/2G and 2G/2G genotypes were 1.12 (95% CI, 0.83-1.51) and 1.51 (95% CI, 1.04-2.20), respectively. By gender, the histologic difference of MMP-1 polymorphism was found in males only, and not in females, with the case-only adjusted ORs (adenocarcinoma versus squamous cell carcinoma) for the 1G/2G and 2G/2G genotypes were 1.25 (95% CI, 0.85-1.85) and 1.88 (95% CI, 1.16-3.07) in males; and 0.90 (95% CI, 0.55-1.48) and 1.05 (95% CI, 0.58-1.91) in females.

The associations between MMP-1 polymorphism and lung cancer risk for different disease stages (early or late) are also shown in Table 3. Although we observed a slightly higher lung cancer risk associated with MMP-1 genotypes in early stage patients [adjusted ORs, 1.30 (95% CI, 1.01-1.66) for the 1G/2G genotype and 1.13 (95% CI, 0.84-1.52) for the 2G/2G genotype, respectively] than for late stage patients [adjusted ORs, 0.99 (95% CI, 0.78-1.27) for the 1G/2G genotype and 1.08 (95% CI, 0.81-1.44) for the 2G/2G genotype, respectively], these differences were not statistically significant in the case-only analysis, overall or stratified by gender (P > 0.30, early stage versus late stage).

Carcinogenesis is a multicellular and multistage process in which the destruction of the microenvironment is required for the conversion of normal tissue to tumor. MMPs may alter cell cycle checkpoint controls, conceivably promote genomic instability by affecting cell adhesion (9), and alter the microenvironment that can influence tumor formation and play an important role in several steps of cancer development (3). MMP-1 is the most highly expressed interstitial collagenase degrading fibrillar collagens. Overexpression of MMP-1 has been associated with higher risks of lung cancer (10, 11).

In this study, the MMP-1 2G allele was associated with higher lung cancer risk in never-smokers, but not in ex- or current smokers, nor in any of the smoking subgroups. One possible explanation is that although cigarette smoking may increase MMP-1 mRNA levels and MMP-1 activity (12, 13), smoking may also increase the mRNA levels of tissue inhibitors of metalloprotease (14, 15). Therefore, the effect of MMP-1 polymorphism in smokers may depend on the balances between MMPs and tissue inhibitors of metalloprotease. The results are inconsistent with a previous study where the 2G/2G genotype was found to be associated with higher lung cancer risk in current smokers and heavy smokers, and not in never-smokers (3). However, the genotype distribution of this polymorphism was out of Hardy-Weinberg equilibrium in the control population for the previous study, and the 1G/2G genotype was combined with the 1G/1G genotype in all analyses. Further studies are needed to investigate the effect of cigarette smoking on the expression levels of different genotypes of MMP-1.

Consistent with the previous study (3), we found that lung cancer risk conferred by the 2G allele was higher in males than females. Similar gender differences in risk for the MMP-1 polymorphism were also found for renal cell and gastric cancer patients (16, 17). Sex hormones including progesterone, estradiol, and ovarian steroids levels may decrease the MMP-1 expression levels and increase the tissue inhibitor levels of MMP-1 proteins (18-20). Therefore, females may have lower MMP-1 levels than males, and estrogen levels may be more important in the influence on the expression levels of MMP-1 than the polymorphism (16).

For different histologic subtypes of patients, we observed a higher risk of the 2G allele for patients with adenocarcinoma when compared with patients with squamous cell carcinoma, specifically for males. This finding is echoed in a study that reported adenocarcinoma overexpressed MMP-1 more frequently than squamous cell carcinoma in non–small cell lung cancer tissues (21). The mechanism for this histologic difference is still under investigation, but there are two distinct theories. The MMP-1 −1607 1G/2G polymorphism is adjacent to an activating protein-1 site at −1,602 bp, which may cooperate with the 2G allele (Ets site) to induce higher levels of transcription (5), and the activator protein-1 site is inhibitory in the context of the 1G allele, but activating the 2G allele (6). If this activator protein-1 site is mutated, it could lead to a substantial increase in expression of the 1G allele where the difference in transcription between 1G and 2G alleles is abolished and the MMP-1 expression is similar between the two alleles (6). It is possible that activator protein-1 mutant frequencies are different between different histologic subtypes of lung cancer, which may induce the differential association between MMP-1 polymorphism and lung cancer risk by histologic subtypes. An alternative explanation is that squamous cell carcinoma tumor tissues have been found to have higher rates of p53 mutations than adenocarcinoma tissues (22). MMP-1 is a target of the p53 protein, and wild-type p53 can exert a strong inhibitory effect on the human MMP-1 promoter by disrupting communications between the transactivator −72AP-1 and the basal transcriptional complex, whereas p53 mutants lose such repressive activity (23, 24). The down-regulation of the human MMP1 promoter by p53 was abolished after the proximal −72AP-1 site was deleted or mutated (24). Therefore, mutated p53 may lose the ability of regulating MMP-1 transcription.

There are several limitations to this study. Firstly, this is a hospital-based case-control study, where a subset of the controls included healthy spouses and friends of patients with lung cancer. Secondly, we only evaluated the MMP-1 −1607 1G/2G polymorphism that has been suggested to be associated with MMP-1 expression levels, which may result in some misclassification in MMP-1 action. It is possible that other polymorphisms might also be associated with MMP-1 expression levels, which may be one reason why we observed a stronger association for the 1G/2G genotype than the 2G/2G genotype in most of the comparisons. Thirdly, although we adjusted for various smoking variables in all of the analyses, secondhand smoke exposure, alcohol consumption, diet, and environmental and occupational exposure data were not adjusted in our logistic regression models because of missing or incomplete information. Lastly, we did multiple comparisons through subgroup analyses. It is possible that the borderline significant results could be due to multiple comparisons. Given our sample size, we have tried as much as possible to avoid the bias by careful a priori specification of our primary hypotheses.

In conclusion, the 2G allele of the MMP-1 −1607 1G/2G polymorphism is associated with higher lung cancer risk in never-smokers and males, where the risk is mostly attributable to patients with adenocarcinoma.

We thank the following staff members of the Lung Cancer Susceptibility Group: Barbara Bean, Jessica Shin, Andrea Solomon, Andrea Shafer, Thomas Van Geel, Lucy Ann Principe, Salvatore Mucci, Richard Rivera-Massa, David P. Miller, Steven Su, and the generous support of Dr. Panos Fidias and the physicians and surgeons of the Massachusetts General Hospital Cancer Center.