Cancer Research Cell Death Mechanisms and Cancer Therapy Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 68, 6453, August 1, 2008. Published Online First July 22, 2008;
doi: 10.1158/0008-5472.CAN-08-0636
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
0008-5472.CAN-08-0636v1
68/15/6453    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beeghly-Fadiel, A.
Right arrow Articles by Zheng, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beeghly-Fadiel, A.
Right arrow Articles by Zheng, W.

Epidemiology

Common MMP-7 Polymorphisms and Breast Cancer Susceptibility: A Multistage Study of Association and Functionality

Alicia Beeghly-Fadiel1, Ji-Rong Long1, Yu-Tang Gao3, Chun Li1, Shimian Qu1, Qiuyin Cai1, Ying Zheng4, Zhi-Xian Ruan3, Shawn E. Levy2, Sandra L. Deming1, Jay R. Snoddy2, Xiao-ou Shu1, Wei Lu4 and Wei Zheng1

1 Department of Medicine, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine and 2 Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, Tennessee; and 3 Department of Epidemiology, Shanghai Cancer Institute and 4 Shanghai Center for Disease Prevention and Control, Shanghai, China

Requests for reprints: Wei Zheng, Vanderbilt Epidemiology Center, Institute of Medicine & Public Health, Vanderbilt University Medical Center, 2525 West End Avenue, 8th Floor, Nashville, TN 37203-1738. Phone: 615-936-0682; Fax: 615-936-8241; E-mail: wei.zheng{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Matrix metalloproteinase-7 (MMP-7) is a small secreted proteolytic enzyme with broad substrate specificity against ECM and non-ECM components. Known to be vital for tumor invasion and metastasis, accumulating evidence also implicates MMP-7 in cancer development. Using data from the Shanghai Breast Cancer Study, we conducted a two-stage study to evaluate the association of MMP-7 single nucleotide polymorphisms (SNPs) with breast cancer risk. Additionally, associated SNPs were characterized by laboratory assays. In stage 1, 11 SNPs were genotyped among 1,079 incident cases and 1,082 community controls using an Affymetrix Genotyping System. Promising SNPs were selected for stage 2 evaluation and genotyped by TaqMan allelic discrimination assays in an independent set of 1,911 cases and 1,811 controls. Three SNPs were selected for stage 2 validation (rs880197, rs10895304, and rs12184413); one had highly consistent results between the two stages of the study. In combined analysis, homozygosity for the variant T allele for rs12184413 was associated with an odds ratio (OR) of 0.7 [95% confidence interval (95% CI), 0.6–0.9] compared with the common C allele. This effect was slightly more pronounced in postmenopausal women (OR, 0.6; 95% CI, 0.4–0.8) than in premenopausal women (OR, 0.8; 95% CI, 0.6–1.1). This SNP is located 3' of the MMP-7 gene, in an area enriched with CTCF binding sites. In silico analysis suggested a regulatory role for this region, and our in vitro assays showed an allelic difference in nuclear protein binding capacity. Results from our study suggest that common MMP-7 genetic polymorphisms may contribute to breast cancer susceptibility. [Cancer Res 2008;68(15):6453–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Matrix metalloproteinase-7 (MMP-7, matrilysin, PUMP-1) is an important member of the MMP family that has broad substrate specificity against both extracellular matrix (ECM) and non-ECM components (1). Best known as a contributor to tumor invasion and metastasis, a growing body of evidence also implicates MMP-7 in earlier stages of tumorigenesis, including cellular transformation, cell survival, tumor growth, angiogenesis, and evasion of immune surveillance (2–8). MMP-7 is normally expressed in both the ductal and glandular epithelium of the breast (9), and significantly higher levels of expression have been found in breast carcinomas compared with adjacent nontumor tissues (10). MMP-7 overexpression has also been reported for a variety of other cancers, including those of the esophagus, stomach, pancreas, lung, and colon/rectum (11–17).

MMP-7 expression is primarily regulated at the transcriptional level (18). Two functional single nucleotide polymorphisms (SNPs) in the promoter, rs11568818 (–181 A/G) and rs11568819 (–153 C/T), were shown to modulate transcription by affecting the interaction of nuclear binding proteins (19). In transient transfection assays, the presence of both minor alleles conferred an approximate 2- to 3-fold elevated level of protein expression (19). In population studies, the MMP-7 –181 (rs11568818) G allele has been shown to be associated with susceptibility for several cancers, including esophageal, gastric, lung, colorectal, and ovarian carcinomas (20–23). To our knowledge, MMP-7 polymorphisms have not been evaluated in relation to breast cancer risk. In the present study, we systematically searched for and evaluated common genetic polymorphisms in the MMP-7 gene in relation to breast cancer risk using data from a population-based case-control study involving 2,990 breast cancer cases and 2,893 controls. Furthermore, SNPs found to be associated with breast cancer were functionally characterized by in vitro experiments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Study Design and Population
A two-stage (stage 1 and stage 2) case-control study design was used to first comprehensively evaluate MMP-7 polymorphisms in relation to breast cancer risk and then to validate promising associations in a second independent patient population. Study subjects were participants of the Shanghai Breast Cancer Study (SBCS), a population-based case-control study among women in urban Shanghai; detailed information on the study design and data collection procedures has previously described (24). Briefly, stage 1 cases were women diagnosed with breast cancer between August 1996 and March 1998, ages 25 to 64, without a previous cancer diagnosis, and alive at the time of interview. Recruitment for stage 2 occurred between April 2002 and February 2005, and eligibility criteria were expanded to include women up to age 70 (25). Cases were identified via the population-based Shanghai Cancer Registry; diagnoses were confirmed by two senior pathologists. Controls were randomly selected from the general population using the Shanghai Resident Registry, a population registry of adult residents in urban Shanghai; women with previous cancer diagnoses were excluded. Structured questionnaires were used to obtain detailed information on demographic, reproductive, and behavioral factors. Of eligible participants, 1,459 (91.1%) cases and 1,556 (90.3%) controls, and 1,989 cases (83.7%) and 1,989 controls (70.4%) completed in-person interviews for stage 1 and stage 2, respectively. In stage 1, 1,193 cases (81.8%) and 1,310 controls (84.2%) donated blood samples. In stage 2, 1,932 (97.1%) cases and 1,857 (93.4%) controls donated either blood or buccal cell samples.

SNP Selection
Polymorphisms were selected by searching Han Chinese data from the HapMap Project (26) using the Tagger program (27). Haplotype-tagging SNPs (htSNP) were selected to cover SNPs with an r2 of 0.90 or greater in the MMP-7 gene ± 5 kb with a minor allele frequency (MAF) of at least 0.05. Known or potentially functional SNPs were forced into the htSNP selection process. Twelve MMP-7 SNPs were selected, including two previously reported to affect promoter activity (rs11568818 and rs11568819) (19). Of the selected htSNPS, design of the assay for one (rs10502001) failed, leaving 11 SNPs that were genotyped in stage 1 (Table 1 ).


View this table:
[in this window]
[in a new window]

  		Table 1. Description of MMP-7 SNPs and genotype distributions among 2,161 stage 1 SBCS participants

 
DNA Extraction and SNP Genotyping
Genomic DNA was extracted from buffy coats or buccal cell samples using Puregene's DNA Purification kits (Gentra Systems) or Qiagen's DNA Purification kits according to manufacturers' instructions. Of the 2,503 stage 1 participants, 2,219 (88.7%) were genotyped for the 11 MMP-7 SNPs using the Targeted Genotyping System (Affymetrix) using an advanced Molecular Inversion Probe method (28) as part of a large-scale genotyping effort. Successful genotyping data were obtained from 2,161 (97.4%) stage 1 samples. Blinded (n = 39) and HapMap samples (n = 12) were also included with the stage 1 genotyping, consistency rates averaged 99.6%. In stage 2, three promising SNPs (rs880197, rs10895304, and rs12184413) were genotyped using the TaqMan allelic discrimination assay and the ABI PRISM 7900 Sequence Detection System (Applied Biosystems). TaqMan assays C_7492868_10, C_32018622_10, and C_32018621_10 were used for the three SNPs, respectively. The final volume for each reaction was 5 µL, and included 2.5 µL TaqMan Universal PCR Master Mix, 0.25 µL primers/TaqMan probe mix and 5.0 ng genomic DNA. The PCR profile consisted of an initial denaturation step at 95°C for 10 min followed by 40 amplification cycles of 92°C for 15 s each and a final elongation step of 60°C for 1 min. Results were measured using the ABI PRISM 7900HT sequence detector (Applied Biosystem); allele frequencies were determined by ABI SDS software. Call rates for the three MMP-7 stage 2 SNPs ranged from 92.4% to 97.5%. Laboratory personnel were blinded to the case-control status of all samples.

In vitro Functional Experiments
MMP-7 reporter constructs and luciferase assays. To evaluate the function of SNPs located in the 3' flanking region of the MMP-7 gene, a series of experiments were conducted. First, a 3.5 kb fragment that included rs12184413 and rs11225297 was generated by PCR; the forward primer 5'-CACCACACTATTTTGAGGTCTTCCGCAG-3' and backward primer 5'-CATCCTAGGCTGAGGCTGGTATGTTTTG-3' were used. Template DNA from individuals known to have either the major or minor alleles for rs12184413 and rs11225297 was used; resulting fragments were cloned into a pGL3-Promoter vector (Promega). All DNA constructs were verified by sequencing. Transfection was performed with the use of FuGene 6 Transfection Reagent (Roche Diagnostics) in triplicate for each construct. MDA231 and MCF7cells (2 x 105 each) were seeded in 24-well plates and cotransfected with pGL4.73, a Renilla expressing vector to serve as a reference for transfection efficiency, and the different allelic combination containing luciferase reporter constructs. Thirty-six to 48 h later, cells were lysed with Passive Lysis Buffer and luminescence (relative light units) was measured using the Dual-Luciferase Assay System (Promega). Reporter activity was measured as the ratio of firefly to Renilla luciferase activity, and results from five independent experiments were averaged.

Electrophoretic mobility shift assay. Biotin-labeled, double-stranded oligonucleotide probes containing sequences surrounding rs12184413 and rs11225297 were synthesized. Probes for the major and minor alleles for the two SNPs were 5'-GTAGGGTGATGGGATGCTGGGCAGGTAAGAAC-3' and 5'-GTAGGGTGATGGGATGTTGGGCAGGTAAGAAC-3', and 5'-GACTTCTATAAATACATAGTACCCTGGCTTTG-3' and 5'-GACTTCTATAAATACTTAGTACCCTGGCTTTG-3', respectively. Probes were incubated with nuclear protein extracts from MDA-MB-231 and MCF-7 breast cancer cells, in the presence or absence of competitors, i.e., unlabeled double-stranded probe. Protein-DNA complexes were resolved by PAGE and then detected using a LightShift Chemiluminescent EMSA kit (Pierce Biotechnology).

Statistical Analysis
Hardy-Weinberg equilibrium (HWE) was tested by comparing the observed and expected genotype frequencies ({chi}2 test) for the cases and controls separately. Characteristics between cases and controls were compared with the {chi}2 test or Student's t test for categorical or continuous variables, respectively. Odds ratios (OR) and corresponding 95% confidence intervals (95% CI) were determined by logistic regression analyses; additive, dominant, and recessive models were applied. Covariates considered included age at diagnosis, age at menarche, age at first live birth among parous women, age at menopause among postmenopausal women, history of breast fibroadenomas, body mass index (BMI), and leisure physical activity in the decade preceding diagnosis. Tests for trend were conducted by coding for the number of variant alleles (0,1,2) and reporting the P value for the β coefficient from the logistic regression models. Linkage disequilibrium between polymorphisms was assessed by Haploview (29); haplotype blocks were defined using the methods of Gabriel and colleagues (30). Associations between haplotypes and breast cancer risk were analyzed with HAPSTAT software (31). Haplotype analysis was based on additive models of effect; P values for trends are shown. All statistical tests were two tailed, and P values of ≤0.05 were interpreted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Table 2 presents demographic, reproductive, and other known breast cancer risk factors by case-control status for the SBCS participants included in the current study. As with studies conducted elsewhere (32, 33), early age at menarche, late age at menopause, late age at first live birth, use of estrogen replacement therapy, having a first-degree relative with breast cancer, prior history of fibroadenomas, high BMI or waist-to-hip ratio, and less regular physical activity were found to be associated with the risk of breast cancer in this study. Women participating in stage 1 and stage 2 were comparable in all listed characteristics, with the exceptions of age and leisure activity; these differences resulted from the expansion of the inclusion criteria between the two study stages.


View this table:
[in this window]
[in a new window]

  		Table 2. Description of demographic characteristics and known breast cancer risk factors in the SBCS

 
Descriptive information on the 11 SNPs evaluated in stage 1 is shown in Table 1. One SNP (rs11568819) was not found to be polymorphic in our study population. None of the genotype distributions of the remaining 10 SNPs deviated from HWE.

Additive models assessing the association between MMP-7 polymorphisms and breast cancer for stage 1 are shown in Table 3 . Estimates included adjustment for age and education; additional adjustment for age at menarche, age at menopause if postmenopausal, age at first live birth if parous, history of breast fibroadenomas, BMI, and leisure physical activity in the past 10 years did not appreciably alter the estimates of effect and were therefore not included. The rare allele of rs880197 was less common among breast cancer cases, and the gene-dose association was of borderline significance (P = 0.07). Homozygotes for the rs12184413 variant allele had a decreased risk of breast cancer, particularly among postmenopausal women (OR, 0.4; 95% CI, 0.2–0.9). On the other hand, having a rare allele for either rs10895304 or rs7935378 was associated with increased breast cancer risk among premenopausal women, and the trend tests for both SNPs was statistically significant (P < 0.02). Premenopausal homozygotes for either rs10895304 or rs7935378 were at a significantly increased risk of breast cancer (OR, 1.9; 95% CI, 1.2–3.0, for either SNP), whereas postmenopausal homozygotes had nonsignificant reductions in risk (rs10895304: OR, 0.7; 95% CI, 0.4–1.2; and rs7935378: OR, 0.6; 95% CI, 0.3–1.2). In stage 1, only 2.7% (n = 29) of patients were diagnosed with in situ carcinomas; excluding these patients did not appreciably alter the results (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 3. Risk of breast cancer associated with MMP-7 SNPs in the SBCS (stage 1)

 
Based on the LD structure of the MMP-7 gene among the SBCS stage 1 controls (Supplementary Fig. S1), there are two haplotype blocks for this gene, one consisting of three promoter and two intronic SNPs, the other of two 3' FR SNPs. Haplotype analysis, based on additive models of effect, is shown in Table 4 . The 5 SNPs in block 1 generated 5 common haplotypes, which accounted for 99.8% of all block 1 haplotypes. The second (H2: AGAAA) was associated with a significant increase in breast cancer risk (OR, 1.2; 95% CI, 1.0–1.4) compared with the reference haplotype (H1: TGAAA) that contained the variant allele of rs880197. This is in agreement with single SNP analysis, as the rare allele for rs880197 (T) tended to have a decreased risk of breast cancer compared with the common allele. Analysis of the 3 SNPs in between blocks 1 and 2 yielded 3 common haplotypes, accounting for 93.6% of all haplotypes in this region. The third (H3: TGC) had the rare alleles for rs495041, rs10895304, and rs7935378 and was associated with an increased breast cancer risk (OR, 1.2; 95% CI, 1.0–1.4), which is also consistent with the single marker analyses shown in Table 3. No significant associations were seen for block 2, although both haplotypes with the variant allele of rs12184413 tended to have reduced risk compared with the reference haplotype. This is not surprising, as the single marker association for this SNP was not additive, but only evident among homozygotes. When all 10 MMP-7 SNPs were included, the significant findings above were better explained. The two haplotypes with increased risk (block 1, H2: AGAAA, and in between SNPs, H3: TGC), were found to occur together (H2: AGAAATGCCA). Haplotypes that differed from the reference haplotype by three polymorphisms (rs880197, rs10895304, and rs7935378) had an ~30% greater risk of breast cancer (OR, 1.3; 95% CI, 1.1–1.5). Recessive models for haplotype effects were not permitted, as estimates of effect were unstable. Similarly, analyses stratified by menopausal status for less common haplotypes were not permitted. Interactions with menopausal status were evaluated for common haplotypes, and no significant interactions were observed (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 4. Haplotype analysis of MMP-7 SNPs and breast cancer risk among the stage 1 SBCS participants

 
Of the 4 promising SNPs revealed from the single marker and haplotype analysis presented above, 2 (rs10895304 and rs7935378) were in high LD (D = 92%; r2 = 0.81); rs10895304 was selected for stage 2 validation because it had a slightly greater MAF. Also included in stage 2 was rs880197 in the promoter and rs12184413 in the 3' flanking region. After genotyping ~1,000 samples, preliminary analysis for stage 2 was conducted. Results from rs880197 for an association with breast cancer were in the opposite direction of those seen in stage 1 (data not shown), indicating that the findings for this SNP in stage 1 were unlikely to be replicated in stage 2. Therefore, this SNP was not further evaluated. Stage 2 genotyping was completed for rs10895304 and rs12184413. The results for rs10895304 were unsupportive of the stage 1 hypothesis; no association was seen (OR, 1.0; 95% CI, 0.8–1.1 for heterozygotes; OR, 1.0; 95% CI, 0.7–1.3 for homozygotes). Results for rs12184413 showed an association in agreement with stage 1, as shown in Table 5 . A reduced risk of breast cancer was found to be associated with the TT genotype (OR, 0.7) in both stage 1 and stage 2. Only 2.1% (n = 39) of stage 2 patients were diagnosed with in situ carcinomas; their exclusion from the analyses did not materially alter the results (data not shown). In combined analysis of the two stages of the study, women homozygous for the rare allele had a significantly decreased risk of breast cancer (OR, 0.7; 95% CI, 0.6–0.9). The reduction in risk associated with this SNP was consistent with a recessive effect and was more pronounced in postmenopausal women (OR, 0.6; 95% CI, 0.4–0.8).


View this table:
[in this window]
[in a new window]

  		Table 5. Association of MMP-7 rs12184413 with breast cancer risk, the SBCS

 
The rs12184413 SNP is located ~1.7 kb downstream of the MMP-7 gene and is within a predicted potential regulatory region spanning 1.3 to 2.1 kb downstream of the gene (34). This purported CCCTC-binding factor (CTCF)-enriched binding site has been implicated in several forms of transcriptional regulation, including activation, repression, and genomic insulation (35, 36). To evaluate if this downstream region influences MMP-7 gene expression, in vitro luciferase assays were conducted in nonmetastatic MCF-7 and metastatic MDA-MB-231 breast cancer cells with reporter constructs containing the major and minor allele combinations of two SNPs, rs12184413 and rs11225297. Although a significant difference between constructs with the different allele combinations was not detected (data not shown), there was an average 10% decrease in luciferase activity from all constructs that included the 3.5 kb region from downstream of the MMP-7 gene (data not shown). To further investigate whether rs12184413 or rs11225297 interacts with nuclear proteins in breast cancer cells, and if so, whether a single nucleotide change in these sites can alter the protein-DNA interactions, we performed electrophoretic mobility shift assays. Results clearly show a difference in binding between the rs12184413 major and minor alleles (Fig. 1 ). The variant rs12184413 (T) resulted in weaker DNA-protein complex intensity compared with that of the major allele (C) in both MCF-7 and MDA-MB-231 cells, whereas no differential binding resulted from the different alleles of rs11225297 (data not shown).


Figure 1
View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Electrophoretic mobility shift assay for rs12184413 alleles. Nuclear protein extracts from MCF7 (top) and MDA-MB-231 (bottom) breast cancer cells were incubated with biotin-labeled probes corresponding to the major allele (lanes 1–5) or the minor allele (lanes 6–10) of rs12184413 in the absence or presence of competitors. Lanes 1 and 6, no nuclear protein extract; lanes 2 and 7, competitor in 200-fold molar excess; lanes 3 and 8, 5 mmol/L MgCl2; lanes 4 and 9, 2.5 mmol/L MgCl2; and lanes 5 and 10, 1.25 mmol/L MgCl2, without competitor. Arrowhead, DNA-protein complexes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
We evaluated common genetic variation in the MMP-7 gene in association with breast cancer risk in a large, population-based, two-stage case-control study of Chinese women, including 2,990 breast cancer cases and 2,893 controls. Women with rs12184413 TT were 30% less likely to be breast cancer cases than women with the CT and CC genotypes. Furthermore, this SNP was found to affect DNA-protein interactions such that the T allele had decreased protein binding. These findings are novel and suggest that common MMP-7 polymorphisms may contribute to the susceptibility of breast cancer.

Previous studies on MMP-7 genetic variation and cancer susceptibility have focused on two promoter polymorphisms that have been shown to affect expression (19). These studies have generally found positive associations with the risk of cancer (20, 21, 23, 37). A small Italian study reported that the rare alleles for both rs11568818 (–181 A/G) and rs11568819 (–153 C/T) occurred more frequently among colorectal cancer cases than controls (20). A small study among Han Chinese found that rs11568818 was associated with esophageal squamous cell carcinoma, gastric cardiac carcinoma, and non–small cell lung carcinoma (21). A hospital-based study reported a positive association with astrocytoma among Han Chinese (37), and a similar positive association with epithelial ovarian cancer was found among almost 300 women from Northern China (23). On the contrary, one study found that the rs11568818 G allele was associated with a reduced risk of gastric cancer among 248 Caucasians (22). In our study, we did not find a significant association for this SNP, although an increase in risk was suggested among premenopausal women. The MAF of rs1156818 in our study was low (8.6%), which may have limited our power to detect an association. In addition, rs11568819 was not found to be polymorphic in our study population. Our genotype frequencies for these two SNPs agrees with other reports (20–23) and indicates they have lower MAFs among Asian populations compared with Caucasians.

Our finding of a significant association between rs12184413 and breast cancer risk is supported by several lines of evidence. First, it is unlikely that this association occurred by chance, as we analyzed two separate and large study populations. Additionally, in silico analyses indicated that the MMP-7 3' flanking region is conserved across eutherian mammals and, therefore, may have some important functional role. The region surrounding rs12184413 was found to be enriched with CTCF binding sites, a factor implicated in several forms of regulation, including gene activation, repression, silencing, and genomic insulation (35, 36). Furthermore, in vitro analyses showed that the major and minor alleles of rs12184413 have different capacities to bind nuclear proteins, whereas no difference was observed for rs11225297. Thus, it is possible that the potential CTCF binding region is a transcriptional repressor, and that binding of a nuclear factor relinquishes this repression, thereby allowing expression. Alternately, this region may be a genomic insulator, separating the chromosome 11 MMP gene cluster from other genes further away. In this case, CTCF binding would protect against the spreading of methylation (38), such that when unbound, MMP-7 expression would be reduced. Finally, we cannot exclude the possibility that rs12184413 may be in high LD with another region that is actually responsible for the alteration in cancer risk. In our data, this SNP was not in high LD with either of the known functional SNPs, and the two polymorphisms that were most linked were also both in the 3 flanking region: rs11225297 (D = 95%), and rs10895304 (D = 92%). Although the true sequence of interest may still be elsewhere in the MMP-7 gene, this seems unlikely as no tagging SNPs were indicative of such a relationship.

MMP-7 is a small secreted metalloproteinase that differs from most other MMPs in that it is expressed in epithelial rather than only stromal cells (1). MMP-7 is normally expressed in the breast and has been shown to contribute to the development and proper branching of the mammary system in mice (9, 39). In animals lacking MMP-7, reduced mammary lesions after chemical induction were found (7), while overexpression resulted in higher occurrences of mammary hyperplasia and accelerated tumor development (40). There are several mechanisms by which MMP-7 may contribute to cancer initiation and promotion. The MMPs provide regulatory signals for cellular adhesion, differentiation, division, and death; alterations in these pathways can cause loss of contact inhibition, aberrant cell growth, and evasion of apoptosis (4, 8). MMP-7 contributes to these events via the activation, degradation, and shedding of target molecules (4, 6). In addition to ECM substrates such as elastin, proteoglycans, fibronectin, type IV collagen, and E-cadherin (1), MMP-7 can degrade non-ECM components including Fas ligand, protumor necrosis factor-{alpha}, insulin-like growth factor binding proteins, and heparin-binding epidermal growth factor (1, 4, 6, 8). Resulting effects include potent mitogenic stimulation for cellular transformation and growth, as well as the dissociation of normal cellular organization, which may contribute to the epithelial-to-mesenchyme transition that occurs frequently during cancer progression (1, 6, 8). In addition, downstream effects of MMP-7 may also include either antiapoptotic signaling for increased cell survival, or stimulation for cell death, which is thought to select against susceptible cells in the early tumor microenvironment and leave more robust cells to continue to grow and acquire malignant characteristics (4, 6, 8).

In summary, MMP-7 has a myriad of functions that may contribute to the development and progression of cancer. Naturally occurring genetic variation has been previously shown to modify the expression of the MMP-7 gene and may thereby influence individual cancer risk. In a large two-stage case-control study of Chinese women, we found a significant association between a polymorphism 3' downstream of the MMP-7 gene and a reduced risk of breast cancer. This association was detected in two independent and large population-based sets of study participants, and further, a functional difference for the alleles of this SNP was confirmed by in vitro experiments. Given the oncogenic effects of MMP-7 proteolysis, the decreased risk of breast cancer found, and the proposed 3' regulatory region, these results not only show how small genetic differences may influence individual cancer susceptibility, but are also supportive of a role for MMP-7 in breast cancer etiology.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: U.S. Public Health Service grants R01CA64277 and R01CA90899.

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.

We thank the participants and research staff of the SBCS for their contributions and commitment to this project, Regina Courtney for genotyping, and Brandy Venuti for clerical support in the preparation of this manuscript. Stage 1 genotyping was conducted by Melanie Robinson at the Vanderbilt Microarray Shared Resource, which is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485), the Vanderbilt Diabetes Research and Training Center (P60 DK20593), the Vanderbilt Digestive Disease Center (P30 DK58404), and the Vanderbilt Vision Center (P30 EY08126).


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 2/20/08. Revised 5/ 8/08. Accepted 6/ 4/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Wilson CL, Matrisian LM. Matrilysin: an epithelial matrix metalloproteinase with potentially novel functions. Int J Biochem Cell Biol 1996;28:123–36.[CrossRef][Medline]
  2. Fingleton B, Vargo-Gogola T, Crawford HC, Matrisian LM. Matrilysin [MMP-7] expression selects for cells with reduced sensitivity to apoptosis. Neoplasia 2001;3:459–68.[CrossRef][Medline]
  3. Nishizuka I, Ichikawa Y, Ishikawa T, et al. Matrilysin stimulates DNA synthesis of cultured vascular endothelial cells and induces angiogenesis in vivo. Cancer Lett 2001;173:175–82.[CrossRef][Medline]
  4. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161–74.[Medline]
  5. Huo N, Ichikawa Y, Kamiyama M, et al. MMP-7 (matrilysin) accelerated growth of human umbilical vein endothelial cells. Cancer Lett 2002;177:95–100.[CrossRef][Medline]
  6. Hojilla CV, Mohammed FF, Khokha R. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Br J Cancer 2003;89:1817–21.[CrossRef][Medline]
  7. Hulboy DL, Gautam S, Fingleton B, Matrisian LM. The influence of matrix metalloproteinase-7 on early mammary tumorigenesis in the multiple intestinal neoplasia mouse. Oncol Rep 2004;12:13–7.[Medline]
  8. Ii M, Yamamoto H, Adachi Y, Maruyama Y, Shinomura Y. Role of matrix metalloproteinase-7 (matrilysin) in human cancer invasion, apoptosis, growth, and angiogenesis. Exp Biol Med (Maywood) 2006;231:20–7.[Abstract/Free Full Text]
  9. Saarialho-Kere UK, Crouch EC, Parks WC. Matrix metalloproteinase matrilysin is constitutively expressed in adult human exocrine epithelium. J Invest Dermatol 1995;105:190–6.[CrossRef][Medline]
  10. Pacheco MM, Mourao M, Mantovani EB, Nishimoto IN, Brentani MM. Expression of gelatinases A and B, stromelysin-3 and matrilysin genes in breast carcinomas: clinico-pathological correlations. Clin Exp Metastasis 1998;16:577–85.[CrossRef][Medline]
  11. Mori M, Barnard GF, Mimori K, Ueo H, Akiyoshi T, Sugimachi K. Overexpression of matrix metalloproteinase-7 mRNA in human colon carcinomas. Cancer 1995;75:1516–9.[CrossRef][Medline]
  12. Ohashi K, Nemoto T, Nakamura K, Nemori R. Increased expression of matrix metalloproteinase 7 and 9 and membrane type 1-matrix metalloproteinase in esophageal squamous cell carcinomas. Cancer 2000;88:2201–9.[CrossRef][Medline]
  13. Crawford HC, Scoggins CR, Washington MK, Matrisian LM, Leach SD. Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-to-ductal metaplasia in exocrine pancreas. J Clin Invest 2002;109:1437–44.[CrossRef][Medline]
  14. Zheng HC, Sun JM, Li XH, Yang XF, Zhang YC, Xin Y. Role of PTEN and MMP-7 expression in growth, invasion, metastasis and angiogenesis of gastric carcinoma. Pathol Int 2003;53:659–66.[CrossRef][Medline]
  15. Kitoh T, Yanai H, Saitoh Y, et al. Increased expression of matrix metalloproteinase-7 in invasive early gastric cancer. J Gastroenterol 2004;39:434–40.[CrossRef][Medline]
  16. Lin TS, Chiou SH, Wang LS, et al. Expression spectra of matrix metalloproteinases in metastatic non-small cell lung cancer. Oncol Rep 2004;12:717–23.[Medline]
  17. Nosho K, Yoshida M, Yamamoto H, et al. Association of Ets-related transcriptional factor E1AF expression with overexpression of matrix metalloproteinases, COX-2 and iNOS in the early stage of colorectal carcinogenesis. Carcinogenesis 2005;26:892–9.[Abstract/Free Full Text]
  18. Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem 2003;253:269–85.[CrossRef][Medline]
  19. Jormsjo S, Whatling C, Walter DH, Zeiher AM, Hamsten A, Eriksson P. Allele-specific regulation of matrix metalloproteinase-7 promoter activity is associated with coronary artery luminal dimensions among hypercholesterolemic patients. Arterioscler Thromb Vasc Biol 2001;21:1834–9.[Abstract/Free Full Text]
  20. Ghilardi G, Biondi ML, Erario M, Guagnellini E, Scorza R. Colorectal carcinoma susceptibility and metastases are associated with matrix metalloproteinase-7 promoter polymorphisms. Clin Chem 2003;49:1940–2.[Free Full Text]
  21. Zhang J, Jin X, Fang S, 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 2005;26:1748–53.[Abstract/Free Full Text]
  22. Kubben FJ, Sier CF, Meijer MJ, et al. Clinical impact of MMP and TIMP gene polymorphisms in gastric cancer. Br J Cancer 2006;95:744–51.[CrossRef][Medline]
  23. Li Y, Jin X, Kang S, et al. Polymorphisms in the promoter regions of the matrix metalloproteinases-1, -3, -7, and -9 and the risk of epithelial ovarian cancer in China. Gynecol Oncol 2006;101:92–6.[CrossRef][Medline]
  24. Gao YT, Shu XO, Dai Q, et al. Association of menstrual and reproductive factors with breast cancer risk: results from the Shanghai Breast Cancer Study. Int J Cancer 2000;87:295–300.[CrossRef][Medline]
  25. Ye C, Dai Q, Lu W, et al. Two-stage case-control study of common ATM gene variants in relation to breast cancer risk. Breast Cancer ResTreat 2007;106:121–6.[CrossRef]
  26. The International HapMap Project. Nature 2003;426:789–96.[CrossRef][Medline]
  27. de Bakker PIW, McVean G, Sabeti PC, et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat Genet 2006;38:1166–72.[CrossRef][Medline]
  28. Hardenbol P, Yu F, Belmont J, et al. Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res 2005;15:269–75.[Abstract/Free Full Text]
  29. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263–5.[Abstract/Free Full Text]
  30. Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science 2002;296:2225–9.[Abstract/Free Full Text]
  31. Lin DY, Zeng D, Millikan R. Maximum likelihood estimation of haplotype effects and haplotype-environment interactions in association studies. Genet Epidemiol 2005;29:299–312.[CrossRef][Medline]
  32. Abeloff MD, Wolf AC, Wood WC, McCormick B, Weber BL. Cancer of the Breast. In: Abeloff MD, Armitage JO, Lichter AS, et al., editors. Clinical Oncology. 3rd ed. Philadelphia: Elsevier; 2004. p. 2369–470.
  33. Wood WC, Muss HB, Solin LJ, Ologade OI. Malignant tumors of the breast. In: DeVita VT, Hellman S, Rosenberg SA, editors. Principles and Practice of Oncology. 7th ed. Philadelphia: Lippincott-Williams & Wilkins; 2005. p. 1415–77.
  34. Flicek P, Aken BL, Beal K, et al. Ensembl 2008. Nucleic Acids Res 2008;36:D707–14.[Abstract/Free Full Text]
  35. Ohlsson R, Renkawitz R, Lobanenkov V. CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet 2001;17:520–7.[CrossRef][Medline]
  36. Kim TH, Abdullaev ZK, Smith AD, et al. Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 2007;128:1231–45.[CrossRef][Medline]
  37. Lu Z, Wang Y, Zhang Q, et al. Association between the functional polymorphism in the matrix metalloproteinase-7 promoter and susceptibility to adult astrocytoma. Brain Res 2006;1118:6–12.[CrossRef][Medline]
  38. Recillas-Targa F, De La Rosa-Velazquez IA, Soto-Reyes E, Itez-Bribiesca L. Epigenetic boundaries of tumour suppressor gene promoters: the CTCF connection and its role in carcinogenesis. J Cell Mol Med 2006;10:554–68.[Medline]
  39. Steffgen K, Dufraux K, Hathaway H. Enhanced branching morphogenesis in mammary glands of mice lacking cell surface [β]1,4-Galactosyltransferase. Dev Biol 2002;244:11-4–33.[CrossRef][Medline]
  40. Rudolph-Owen LA, Chan R, Muller WJ, Matrisian LM. The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res 1998;58:5500–6.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
T. A. O'Mara, J. A. Clements, and A. B. Spurdle
The Use of Predictive or Prognostic Genetic Biomarkers in Endometrial and Other Hormone-Related Cancers: Justification for Extensive Candidate Gene Single Nucleotide Polymorphism Studies of the Matrix Metalloproteinase Family and their Inhibitors
Cancer Epidemiol. Biomarkers Prev., September 1, 2009; 18(9): 2352 - 2365.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
A. Beeghly-Fadiel, Y.-B. Xiang, S. L. Deming, J.-R. Long, W.-H. Xu, Q. Cai, W. Zheng, and X. O. Shu
No Association between Matrix Metalloproteinase (MMP)-1, MMP-3, and MMP-7 SNPs and Endometrial Cancer Risk
Cancer Epidemiol. Biomarkers Prev., June 1, 2009; 18(6): 1925 - 1928.
[Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
A. Beeghly-Fadiel, W. Lu, J.-R. Long, X.-o. Shu, Y. Zheng, Q. Cai, Y.-T. Gao, and W. Zheng
Matrix Metalloproteinase-2 Polymorphisms and Breast Cancer Susceptibility
Cancer Epidemiol. Biomarkers Prev., June 1, 2009; 18(6): 1770 - 1776.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
A. Beeghly-Fadiel, Q. Cai, W. Lu, J. Long, Y.-T. Gao, X.-o. Shu, and W. Zheng
No Association between Matrix Metalloproteinase-1 or Matrix Metalloproteinase-3 Polymorphisms and Breast Cancer Susceptibility: A Report from the Shanghai Breast Cancer Study
Cancer Epidemiol. Biomarkers Prev., April 1, 2009; 18(4): 1324 - 1327.
[Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
Y.-S. Hong, S. L. Deming, Y.-T. Gao, J.-R. Long, X.-O. Shu, Q. Cai, W. Lu, and W. Zheng
A Two-stage Case-Control Study of EGFR Polymorphisms and Breast Cancer Risk
Cancer Epidemiol. Biomarkers Prev., February 1, 2009; 18(2): 680 - 683.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
0008-5472.CAN-08-0636v1
68/15/6453    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beeghly-Fadiel, A.
Right arrow Articles by Zheng, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beeghly-Fadiel, A.
Right arrow Articles by Zheng, W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online