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Matrix Metalloproteinase Polymorphisms and Bladder Cancer Risk

Departments of ¹ Epidemiology and ² Urology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Xifeng Wu, Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, Unit 1340, 1155 Pressler Boulevard, Houston, TX 77030. E-mail: xwu{at}mdanderson.org.

	Abstract

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Matrix metalloproteinases (MMP) contribute to tumor microenvironment and are associated with bladder cancer. A study examining the association between MMP polymorphisms and bladder cancer risk has never been published. We analyzed the association of 11 single nucleotide polymorphisms (SNPs) and one microsatellite polymorphism in MMP genes MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-12 with bladder cancer risk in 560 Caucasian patients and 560 controls matched on age, gender, and ethnicity. Individual, combination, haplotype, and diplotype analyses were done. No associations between individual MMP polymorphisms and overall bladder cancer risk were seen. The MMP-9 microsatellite 24 CA repeat allele and the MMP-12-82 GG polymorphisms were associated with invasive bladder cancer risk [odds ratio (OR), 2.60; 95% confidence interval (95% CI), 1.07–6.26; and OR, 4.59; 95% CI, 1.21–17.32, respectively]. Smoke-stratified analyses revealed several associations between MMP polymorphisms, alone and in combination, with bladder cancer risk, particularly in light smokers. Linkage disequilibrium was seen in all of the MMP-1, MMP-3, MMP-8, and MMP-12 SNPs and in four of five MMP-9 polymorphisms tested. Several MMP-9 haplotype and diplotypes were associated with overall and invasive bladder cancer risk. Our study suggests that genetic variations in the MMP family are associated with bladder cancer risk. Heavy carcinogen exposure may overwhelm some of the genetic effects of MMP polymorphisms. Our study confirms the importance of taking a multigenic pathway–based approach to risk assessment. (Cancer Res 2006; 66(24): 11644-8)

	Introduction

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Urinary bladder cancer is a complex disease influenced by genetic factors and environmental exposures (1–3). Cigarette smoking, the predominant risk factor for bladder cancer, increases the overall risk for bladder cancer up to 4-fold (2). However, it is increasingly clear that genetic factors also play an important role in determining bladder cancer risk (3). Although many genetic factors are involved in the development of bladder cancer, interactions between neoplastic cells and the surrounding microenvironment are crucial for tumorigenesis.

The matrix metalloproteinase (MMP) family of enzymes plays an essential role in maintaining the cellular microenvironment. They have a range of biological functions, including the liberation of cytokines and membrane-bound receptors (4), and the promotion of tumor invasion and angiogenesis (5). MMPs have been implicated in the development of cancer (4). Previous studies have shown that MMP-1, MMP-2, MMP-3, and MMP-9 are associated with bladder cancer and are able to predict stage, grade, and disease outcome (6, 7).

Several promoter single nucleotide polymorphisms (SNPs) known to alter MMP expression were selected for analysis in this study. Four of these have been linked with a variety of cancers, including MMP-1-1607 and MMP-3-1612 SNPs in colon cancer (8), and MMP-2-1306 and MMP-9-1562 in breast cancer (9). Despite causing altered expression, the MMP-9 promoter CA repeat microsatellite polymorphism and the MMP-12-82 SNP have not yet been associated with cancer risk. In addition, six nonsynonymous coding SNPs involving MMP-3 (E45K), MMP-8 (K87E), and MMP-9 (A2V, R279Q, and P574R) were selected for study. All of these SNPs result in an amino acid change of the protein product, potentially resulting in altered enzymatic function.

Given the genetic complexity of bladder cancer, individual polymorphisms are likely to have a modest effect on risk. However, examining several polymorphisms within biologically relevant pathways may reveal subgroups of individuals who are at significantly elevated risk for this disease. In the present analysis, we examined six MMP genes that included 11 SNPs and 1 microsatellite repeat to determine their association with the overall risk of bladder cancer and the risks for superficial and invasive disease. To our knowledge, there have been no other studies examining the association of MMP polymorphisms on bladder cancer risk.

	Materials and Methods

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Study population. Between 1999 and December 2004, 560 patients with newly diagnosed and histologically confirmed bladder cancer and 560 healthy controls were accrued from an ongoing case-control study of bladder cancer (10). Patients who had not received any previous chemotherapy or radiotherapy before enrollment were recruited from the Departments of Urology and Genitourinary Oncology at The University of Texas M. D. Anderson Cancer Center and the Scott Department of Urology at Baylor College of Medicine. There were no recruitment restrictions on age, gender, ethnicity, or cancer stage. In this analysis, we subsequently restricted our study population to Caucasians, because 90% of patients were self-reported Caucasian.

Healthy control subjects without a prior history of cancer other than nonmelanoma skin cancer were recruited from a multidisciplinary managed care organization in Houston, Texas (Kelsey-Seybold Clinic). The overall study had 737 Caucasian patients and 656 controls. MMP genotyping data was available for 625 cases and 611 controls. Controls were one-to-one matched with cases on age (±5 years), gender, and ethnicity, leaving 560 eligible pairs. The M. D. Anderson Cancer Center, Baylor College of Medicine, and Kelsey-Seybold Institutional Review Boards approved this research. The response rates were 92% for patients and 75% for control subjects.

Epidemiologic and clinical data. All study participants signed an informed consent, completed a personal interview to collect demographic information and smoking history, and provided a 40-mL blood sample. Ever smokers were defined as individuals who had smoked at least 100 cigarettes in their lifetime; of those, a former smoker had quit smoking at least 1 year before diagnosis (cases) or before interview (controls). Pack-years smoked was defined as the number of cigarettes per day divided by 20 and then multiplied by the number of years smoked.

An M. D. Anderson Cancer Center pathologist confirmed the pathologic diagnosis and staging of all tumors. Patients with bladder cancer were classified as superficial if their tumor was confined to the urothelium or lamina propria. Those with tumors involving muscularis propria or beyond were classified as muscle invasive.

Genotyping. Genomic DNA was isolated from peripheral blood lymphocytes by proteinase K digestion followed by isopropanol extraction and ethanol precipitation. All of the polymorphisms except for MMP-9-1571, MMP-9 P574R, and the MMP-9 microsatellite were determined using TaqMan real-time PCR. The primer and probe sequences were either obtained from the National Cancer Institute SNP500 database or designed using Primer Express Software (Applied Biosystems, Foster City, CA). The probes were fluorescently labeled with either FAM or VIC on the 5' end and a nonfluorescent minor groove binder quencher on the 3' end (Applied Biosystems). Typical amplification mixes (5 µL) contained sample DNA (5 ng), 1x TaqMan buffer A, 200 µmol/L deoxynucleotide triphosphates, 5 mmol/L MgCl₂, 0.65 units of AmpliTaq Gold, 900 nmol/L of each primer, and 200 nmol/L of each probe. The thermal cycling conditions consisted of one cycle for 10 minutes at 95°C, 40 cycles for 15 seconds at 95°C, and 1 minute at 60°C. SDS version 2.1 software (Applied Biosystems) was used to analyze end-point fluorescence. Water control, internal controls, and previously genotyped samples were included in each plate to ensure accuracy of genotyping. Genotyping for MMP-9-1571 and MMP-9 P574R were done as described previously using PCR-RFLP analysis (11). The MMP-9 microsatellite polymorphism was genotyped by capillary electrophoresis of PCR products as described by Fiotti et al. (12).

Statistical analyses. Pearson's ² test was used to test for differences between the cases and controls in terms of gender, ethnicity, smoking status, and the distribution of the MMP genotypes. Wilcoxon rank-sum test was used to test for differences between the cases and controls in median age. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated as an estimate of the relative risk by using unconditional multivariable logistic regression analyses controlling for confounding by age, gender, and smoking, where appropriate. For the MMP-9 microsatellite, the data were dichotomized at 24 CA repeats based on the literature, suggesting that this level was associated with the greatest MMP-9 expression (13). Combined analysis involved investigation of those polymorphisms (i.e., MMP-1-1607, MMP-2-1585, MMP-3-1612, MMP-8 K87E, MMP-9-1562, microsatellite, and A2V), which were deemed not to be highly linked (D' < 0.6). ORs were inverted for the promoter SNPs MMP-1-1607, MMP-2-1306, and MMP-3-1612 so that all polymorphisms used the lower expressing genotype, as per the literature (14), as the reference.

Haplotypes and diplotypes were estimated using HelixTree program (Golden Helix, Inc., Bozeman, MT); the other analyses were done using Stata v8.0 (Stata Corporation, College Station, TX). Haplotypes with a probability <95% were excluded from the final data to ensure analytic reliability. All statistical analyses were two-sided.

	Results

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Host characteristics. Data were available on 560 Caucasian patients and 560 healthy control subjects (Table 1 ). There were no differences between the cases and the controls in terms of gender (P = 0.89). Median patient age was 65 years, which was comparable with the 64 years of control subjects despite statistical significance (P = 0.03). Approximately 43% of the cases were heavy smokers (>31 pack-years smoked) compared with 21% in the controls (P < 0.01), whereas 45% of the controls were never smokers compared with 26% in the cases (P < 0.01). Among the cases, 243 patients (44%) had muscle-invasive disease and 315 (56%) had superficial disease; stage data was not available for two patients.

MMP genotypes and smoking status. We also analyzed the data by smoking status (i.e., never smokers, light smokers, and heavy smokers, data not shown). Overall, significant protective effects were noted in light smokers (31 pack-years) with the CT genotype of the MMP-9-1562 SNP and the AG genotype of the R668Q SNP (OR, 0.53; 95% CI, 0.32-0.88; and OR, 0.57; 95% CI, 0.34-0.94, respectively, P_interaction = 0.04 and 0.07, respectively).

Light smokers homozygous for the 1G allele of MMP-1-1607 polymorphism had an elevated risk for invasive bladder cancer (OR, 2.42; 95% CI, 1.11-5.28; P_interaction = 0.09). In never smokers, the A-allele genotypes of the MMP-8 K87E were associated with a 2.58-fold increased risk of invasive bladder cancer (95% CI, 1.12-5.92; P interaction = 0.11). The only significant effect associated with superficial bladder cancer risk was a 50% protective effect for the T-allele genotypes of MMP-9-1562 (OR, 0.51; 95% CI, 0.27–0.97; P_interaction = 0.05). Additional analysis revealed no significant associations with the remaining polymorphisms.

Linkage analysis. MMP-1, MMP-3, MMP-8, and MMP-12, which are located on chromosome 11q22.3, were found to be in linkage disequilibrium (P < 0.05, data not shown). The A2V polymorphism of MMP-9 was not in linkage disequilibrium with the other MMP-9 polymorphisms tested (P = 0.11–0.88). The remaining MMP-9 SNPs were in linkage disequilibrium (P < 0.05, data not shown).

Number of MMP variant alleles and bladder cancer risk. The MMP variant alleles were categorized (<3 alleles, 4–6 alleles, and 6 alleles) and analyzed for gene-dosage effects (data now shown). The only statistically significant effect was seen in light smokers where there was a 55% protective effect (OR, 0.45; 95% CI, 0.22–0.92) in those with four to five MMP alleles and a 57% protective effect (OR, 0.43; 95% CI, 0.20–0.92) in those with 6 alleles on invasive bladder cancer risk.

MMP-9 haplotype and diplotype analyses. The MMP-9 haplotype analyses consisted of six alleles, five of which were in linkage disequilibrium (Table 3 ). The haplotypes were labeled and categorized H1 to H6 based on their computer-estimated frequency, with H1 being the most frequent. Individuals with the H6 haplotype had an overall significantly increased risk of bladder cancer (OR, 2.76; 95% CI, 1.14–6.68). Diplotype analysis was also done using computer-generated estimates of diplotype frequency. Those with the highest estimated frequencies were selected for analysis, with the most frequent diplotype (H1-H1) used as the reference. The H1-H6 diplotype conferred an elevated risk of overall bladder cancer (OR, 6.99; 95% CI, 1.51–32.23) and for invasive bladder cancer (OR, 9.56; 95% CI, 1.89–48.29) but not superficial disease (OR, 3.65; 95% CI, 0.61–21.70). The MMP-1, MMP-3, MMP-8, and MMP-12 haplotypes and diplotypes did not yield any statistically significant associations.

	Discussion

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MMPs have been associated with a change in the tumor cell cytokine milieu and may provide a fertile ground for angiogenesis and invasion (4, 5). Previous studies suggest that MMPs play a role in bladder cancer. Elevated urinary MMP levels and immunohistochemical staining have been seen in a grade- and stage-dependent fashion (6, 15). Furthermore, the MMP inhibitor halofuginone repressed bladder tumor growth and angiogenesis in a murine model (16).

MMP-9 likely has an important role in a variety of cancers, including bladder cancer (17). MMP-9 degrades type IV collagen, a major component of the basement membrane, which is breached in invasive bladder cancer. The expression of MMP-9 is regulated by cis-acting elements, including binding sites for the activating protein 1 (AP-1), nuclear factor-B, AP-2, and SP-1 (18). The MMP-9 CA microsatellite polymorphism is located at –90 within the promoter region just proximal to an AP-1 binding site (18). Previous studies have shown that individuals with 24 CA repeats have 20 times greater MMP-9 expression compared with individuals with <20 CA repeats (13, 18). In this study, we noted a significantly elevated risk of invasive bladder cancer for individuals with 24 CA repeats, suggesting that higher MMP-9 expression is a putative risk factor.

The MMP-12-82 A/G SNP may also alter expression by virtue of its location in the AP-1 transcription factor–binding site (19). In contrast to what was seen with the MMP-9 microsatellite, our data suggest that the lower-expressing GG genotype is associated with an elevated risk of invasive bladder cancer. MMP-12 is believed to be angiostatic (20). It may be that loss of the angiostatic effects of MMP-12 promotes the development of muscle invasive disease.

Smoke-stratified analyses showed several protective effects of SNPs, alone and in combination, on overall and stage-stratified bladder cancer risk in light smokers. Given the small sample size in some strata and the multiple comparisons, these data need to be interpreted with caution, as it is possible that these results may have occurred due to chance. Only the protective effect of the MMP-9-1562 SNP withstood interaction testing with P interaction values of 0.04 for overall and 0.05 in superficial disease in light smokers. However, these results are hypothesis generating. Perhaps these enzymes counteract the effects of light carcinogen exposure. Elevated tissue inhibitor of metalloproteinase levels have been associated with invasive disease (21), suggesting a potential protective role of some MMPs given the right balance between enzyme and inhibitor. This protective effect may be overwhelmed by heavy carcinogen exposure.

Because individual polymorphisms likely confer modest effects to the risk of bladder cancer, we examined the effects of multiple MMP polymorphisms by performing haplotype and diplotype analyses. Individuals with the MMP-9-H6 haplotype had an almost 3-fold increased risk of developing bladder cancer, and those with the H1-H6 diplotype had an even greater risk. Additional research is required to investigate the functional effect of these haplotypes and diplotypes. Nevertheless, these results suggest that an appropriate combination of MMP-9 polymorphisms modifies the risk of overall and invasive bladder cancer.

Limitations and sources of bias should be considered. Case-control studies are subject to selection bias and recall bias. The biological role for many of the MMPs in bladder epithelium and the functional effects for many of the SNPs are unknown. Furthermore, the frequency-based diplotype inference done in this analysis may inflate type I error. Therefore, these novel findings require further research in larger studies.

In summary, our results suggest an association between MMP polymorphisms and bladder cancer risk. When the variant alleles were combined, we found significant protective effects in light smokers; however, there was an increased risk for bladder cancer when MMP-9 haplotypes and diplotypes were analyzed. These results, upon validation and confirmatory mechanistic studies, may one day be incorporated into a risk assessment tool as well as help to further our understanding of the biology of bladder cancer.

	Acknowledgments

 
Grant support: National Cancer Institute Public Health Service grants CA 74880 (A.K. Kader, L. Shao, Y. Wu, J. Liu, J. Gu, and X. Wu) and CA 91846 (C.P. Dinney, H.B. Grossman, and X. Wu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 3/06. Revised 9/29/06. Accepted 10/18/06.

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