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Manuscript No 8216-V
Date of Revision: September 24, 2007
A Metalloproteinase-13 Polymorphism Affecting its Gene Expression
is Associated with Advanced Stages of Oral Cancer
ELEFTHERIOS VAIRAKTARIS1, EMEKA NKENKE2, CHRISTOS YAPIJAKIS1,
ZOE CHARALAMBOUS SEREFOGLOU1, AGAPI CHATZITHEOFYLAKTOU1,
STAVROS VASSILIOU1, SPYRIDOULA DERKA1, ANTONIS VYLLIOTIS1,
FRIEDRICH WILHELM NEUKAM2 and EFSTRATIOS PATSOURIS3
1Department
of Oral 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 D91054, 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: +30210-6443803; e-mail: [email protected]
Key Words: Oral cancer, metalloproteinase-13, polymorphism, tumor progression,
invasion, metastasis.
Vairaktaris et al: Association of MMP-13 with Oral Cancer
1
Abstract. Background: In the light of the known 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 A allele 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 or
subgroups 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.
2
Common DNA 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-13 has
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 identified in the promoter
region of the MMP-13 gene, affecting its transcriptional activity (19). The A allele
confers double the transcriptional activity of 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
3
(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 HardyWeinberg 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 (-77 A/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.
4
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 nonsignificant 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), a significantly increased A allele frequency (76.5%) was
detected in 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 difference in
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 in the 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).
5
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, MMP12 (24), this finding may indeed disclose an 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, 1718, 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 oncogenesis particularly (6, 7, 25). On the
other hand, MMP-13 may play a more important role 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-13 activates 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.
6
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.
7
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Received July 12, 2007
Accepted
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Table I.
Prevalence of MMP-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 (%)
N (%)
Patients
P
AA
40
(41.2%)
75
(46.6%)
GG
10
(10.3%)
8
(4.9%)
A/G
47
(48.5%)
78
(48.5%)
Total
97
(100%)
161
(100%)
A allele
frequency
65.5%
70.8%
p =0.24
(0.85-1.84)
1.25
66.25%
p =0.91
1.02
(0.64-1.61)
76.5%
p=0.0379
1.69
(1.01-2.82)
Carrier
frequency
of A allele
89.7%
95%
p =1.30
(0.76-5.65)
2.07
92.5%
p =0.604
1.20
(0.42-3.5)
97.1%
p=0.125
1.83
(0.5-7.16)
p=0.117
p =0.198
OR (CI)
Patients with cancer stages I/II
N (%)
P
OR (CI)
2.31
(0.81-6.6)
32
(40%)
1(referent)
6
(7.5%)
1.9
(0.67-5.37)
42
(52.5%)
p =0.78
p =0.59
Patients with cancer stages III/IV
N (%)
P
OR (CI)
1.25
(0.41-3.83)
38
(55.9%)
1(referent)
2
(2.9%)
1.23
(0.40-3.8)
28
(41.2%)
80
(100%)
p=0.059
3.26
(0.70-15.1)
1(referent)
p = 0.205
1.53
(0.37-6.3)
68
(100%)
Prevalence
of A allele
Fischer’s p-value corresponds to genotype comparisons and allele frequency comparisons; significant p-value is given in bold; odds ratios (OR) are ageadjusted; CI: 95% confidence interval.
12