A Metalloproteinase-13Polymorphism Affecting Its Gene Expression Is Associated with Advanced

Manuscript No 8216-V

Date of Revision: September 24, 2007

A Metalloproteinase-13Polymorphism Affecting its Gene Expression is Associated with Advanced Stages of Oral Cancer

ELEFTHERIOS VAIRAKTARIS1, EMEKA NKENKE2, CHRISTOS YAPIJAKIS1, ZOE CHARALAMBOUSSEREFOGLOU1, AGAPICHATZITHEOFYLAKTOU1, STAVROS VASSILIOU1, SPYRIDOULA DERKA1, ANTONIS VYLLIOTIS1, FrIedrich Wilhelm NEUKAM2 andEfstratios PATSOURIS3

1Department ofOral and Maxillofacial Surgery, University of Athens Medical School, “Attikon” Hospital, Rimini 1, GR-12462, Greece and 3Department of Pathology, University of Athens Medical School, Mikras Asias 75, Athens GR-11527, Greece;

2Department of Oral and Maxillofacial Surgery, Universität Erlangen, Klinik und Poliklinik fűr Mund-, Kiefer-, Gesischtschirurgie, Glueckstrasse 11, Erlangen D-91054, Nűrnberg, Germany

Correspondence to:Prof. Eleftherios Vairaktaris, M.D.,D.D.S.,Ph.D,Ph.D., Department of Oral and Maxillofacial Surgery, University of Athens Medical School, “Attikon” Hospital, Rimini 1, GR-12462, Greece. Tel: +30-210-6443035; Fax: +30-210-6443803; e-mail:

Key Words: Oral cancer, metalloproteinase-13, polymorphism, tumor progression, invasion, metastasis.

Vairaktaris et al:Association of MMP-13 with Oral Cancer

Abstract. Background: In the light of theknown contribution of other metalloproteinases to the development of oral cancer, this study investigated the possible association of the -77A/G polymorphism in the matrix metalloproteinase-13 (MMP-13) gene with the risk of oral cancer. Materials and Methods: The polymorphism -77A/G, which affects gene transcription, was examined in DNA samples of 161 patient with oral squamous cell carcinoma and 97 healthy controls of comparable ethnicity, age and sex. Results: The detected allele and carrier frequency for the high expression Aallele in the patient group were not significantly increased in comparison to that of the control group (70.8 % versus 65.5%, and 95% versus 89.7%, respectively). The same pattern was observed between controls and patients orsubgroups of patients in regard to family history of cancer, smoking and heavy alcohol consumption. Only in the subgroup of patients with advanced stages of cancer was the allele frequency for the high expression A allele significantly increased compared to controls (p=0.038). In the same subgroup AA genotypes had a borderline significant difference from controls (p=0.059). Conclusion:MMP-13 gene expression-related polymorphism is associated with risk for the highly aggressive form of oral cancer. The high expression A allele of the -77A/G polymorphism seems to be a prognostic factor for tumor progression.

CommonDNA polymorphisms in genes involved in angiogenesis, inflammation and thrombosis have recently been associated with an increased risk of oral cancer(1-7).These include polymorphisms affecting gene expression of matrix metalloproteinases (MMPs), the enzymes that degrade the extracellular matrix and basement membrane components (6,7).

One such enzyme is MMP-13, also known as collagenase 3 (8).MMP-13has the ability to degrade fibrillar collagens (such as type II collagen), but it may also act as a potent gelatinase by degrading a wide variety of extracellular matrix components (9,10).MMP-13 is overexpressed in a variety of tumors from diverse sources such as head and neck, laryngeal, breast, chondrosarcoma, gastric, colorectal, vulvar carcinomas and cutaneous malignant melanoma (11-18). In most malignancies MMP-13 has been correlated with tumor invasion, metastasis and poor prognosis in patients with head and neck and gastric cancer(12-15, 17, 18).

Only one DNA polymorphism (-77A/G) has been identifiedin the promoter region of the MMP-13 gene, affecting its transcriptional activity (19). The A allele confers double thetranscriptional activityof the G allele (19).The frequency of the high expression A allele ranges between 65 and 73% in populations of European or African origin, but it is more rare (49%) in Oriental Asians(19, 20).In order to investigate if there is any association of the (-77A/G) polymorphism with increased susceptibility for oral cancer, we studied this polymorphism was studied in Greek and German patients and healthy controls.

Materials and Methods

The population under study included 258 Greek and German individuals, consisting of 161 patients with oral squamous cell carcinoma and 97 healthy blood donors (controls) of similar age, ethnicity and sex. In addition to clinical presentation, a biopsy with pathological diagnosis of tumor stages I-IV and a family history regarding cancer and thrombophilia were available.

Patients and controls were informed about the possible results of the study and willingly donated blood samples. DNA was isolated from blood with the use of a NucleoSpin TM kit (Macherey-Nagel GmbH & Co, Düren, Germany). Molecular detection of the -77 A/G polymorphism in the MMP-13 gene was performed using restriction fragment length polymorphism typing with BsrI, as previously described(19).

Statistical analysis was performed using SAS® software (version 9.0; SAS Worldwide Headquarters SAS Institute Inc., Cary, NC, USA). The frequencies of alleles and genotypes of the whole group or subgroups of patients were compared to the respective frequencies of the control group using the Fisher’s exact test and odds ratios, while all genotype distributions were checked for compliance with Hardy-Weinberg estimates. The significance level was set at p<0.05 and the results are presented as hazard ratios using the Mantel - Haenszel method with 95% confidence intervals (CI).

Results

The prevalence of detected MMP-13 genotypes,allele and carrier frequencies are shown in Table I. The data for the two populations under study (Greek and German healthy controls and patients) were analyzed together, since there were no significant differences in genotype and allele frequencies of the (-77A/G) polymorphism among the two populations. The genotype distributions were as expected in Hardy-Weinberg equilibrium in the control group, as well as in the whole group of patients.

The detected allele and carrier frequencies for the high expression A allele in the patient group (70.8% and 95%, respectively) were not significantly different from the respective ones (65.5% and 89.7%) in the controls (Table I). The same non-significant pattern was observed in all subgroups of patients in regard to categorization of family history of cancer or thrombophilia, initial stages of cancer, smoking or heavy drinking habits, gender, age and age at onset of oral cancer, with one exception (Table I),asignificantly increasedA allele frequency (76.5%) was detectedin the subgroup of patients with advanced stages of cancer in comparison to controls (p=0.038, Table I).

In regard to genotypes, there was no statistical difference between patients with oral cancer in comparison to controls (Table I). The same pattern was observed in all subgroups of patients compared to normal subjects, but in the patients with advanced stages of cancer only, there was a trend towards a statistical differencein the AA genotype (p=0.059, Table I).

Discussion

High levels of MMP-13 have been associated with tumor invasion, metastasis and poor prognosis in several types of cancer (11-15, 17, 18, 21-23).

Despite the modest size of the studied population, MMP-13 clearly did not seem to be a major contributing factor in the development of oral cancer. No significant difference,in comparison to controls,was observed in the total group of patients or inthe subgroups of patients with or without family history of cancer or thrombophilia, initial stages of cancer, smoking or drinking habits.In accordance with these findings no significant difference was found in patients with esophageal cancer, between (-77A/G) MMP-13 polymorphism and esophageal cancer (20).

Nevertheless, a significantly increased allele frequency of the high expression A allele, and a trend for a statistical difference in the AA genotypes was detected in the subgroup of patients with advanced stages of cancer. Although it is possible that the MMP-13 (-77A/G) polymorphism might be in linkage disequilibrium with other loci found on chromosome 11q22, such as those in genes of MMP-1, MMP-7, MMP-12 (24), this finding may indeed disclosean important role of MMP-13 in tumor progression and invasion of oral cancer.Interestingly, the findings of this study are in accordance with the results of a previous study regarding malignant melanoma in which MMP-13 was highly expressed only in grades III and IV, and not in premalignant and grade I tumors (22). Furthermore, MMP-13 has been reportedly found to be significantly increased in aggressive and invasive carcinomas(12-15, 17-18, 21-23).In squamous cell carcinomas of the head and neck in particular, MMP-13 was expressed mostly in tumor cells at the invading front, and only in a subset of intermingled stromal cells,while in laryngeal carcinomas MMP-13 was detected only in well and moderately differentiated carcinomas(14, 23).

According to the present study and others, MMP-13 does not seem to affect the initial stages of oncogenesis. It seems that many other MMPs are involved during these stages, as previously found in oral oncogenesisparticularly (6,7,25). On the other hand, MMP-13 may playa more importantrole in the cascade of MMP activation during advanced stages. MMP-13 is activated by MMP-2, which plays a significant role in advanced oral cancer stages and in non-lymph node metastasis and subsequently MMP-13activates MMP-9, previously associated with oral cancer (7, 16,25).The activation of MMP-13 by MMP-2 in advanced tumors may possibly explain the significant increase of high expression A allele frequency only in the advanced stages of oral cancer.

In conclusion, the present study indicates that MMP-13 is not a major contributing factor for the initiation of oral cancer. Nevertheless, once oral oncogenesis starts, it seems that the presence of high expression A allele may serve as a prognostic factor of tumor progression (especially in the homozygous state). A similar association with the high expression A allele may exist in other types of cancer, since increased levels of MMP-13 have been detected in advanced stages of other malignancies as well.

Acknowledgements

This work was co-funded by the European Social Fund and National Resources (EPEAEK II “Pythagoras” 70/3/7391) grant to E.V.

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Received July 12, 2007

Accepted

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Table I.

Prevalence ofMMP-13 (-77 A/G) polymorphism in healthy controls as well as patients with oral cancer and their subgroups in regard to cancer stage.

Genotype / Controls
N (%) / Patients
N (%) P OR (CI) / Patients with cancer stages I/II
N (%) P OR (CI) / Patients with cancer stages III/IV
N (%) P OR (CI)
AA
GG
A/G
Total
Prevalence
of A allele
A allele
frequency
Carrier
frequency
of A allele / 40
(41.2%)
10
(10.3%)
47
(48.5%)
97
(100%)
65.5%
89.7% / 75 p=0.117 2.31
(46.6%) (0.81-6.6)
8 1(referent)
(4.9%)
78 p=0.198 1.9
(48.5%) (0.67-5.37)
161
(100%)
70.8% p=0.24 1.25
(0.85-1.84)
95% p=1.30 2.07
(0.76-5.65) / 32 p=0.78 1.25
(40%) (0.41-3.83)
6 1(referent)
(7.5%)
42 p=0.59 1.23
(52.5%) (0.40-3.8)
80
(100%)
66.25% p=0.91 1.02
(0.64-1.61)
92.5% p=0.604 1.20
(0.42-3.5) / 38 p=0.059 3.26
(55.9%) (0.70-15.1)
2 1(referent)
(2.9%)
28 p= 0.205 1.53
(41.2%) (0.37-6.3)
68
(100%)
76.5% p=0.0379 1.69
(1.01-2.82)
97.1% p=0.125 1.83
(0.5-7.16)

Fischer’s p-value corresponds to genotype comparisons and allele frequency comparisons; significant p-value is given in bold; odds ratios (OR) are age-adjusted; CI: 95% confidence interval.

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