Background
The matrix metalloproteinases (MMPs) are enzymes that cleave various components of the extracellular matrix (ECM) and basement membranes. Upon degradation, the ECM releases and activates ECM-bound cytokines and ECM fragments which modulate cell growth and migration as well as angiogenesis
MMPs are expressed in melanocytes and their overexpression has been linked to tumor development, progression and metastasis
At the genetic level, two functional promoter single nucleotide polymorphisms (SNPs) have been described in the MMP2 gene [rs243865: -1306 C/T; and rs2285053: -735 C/T], and one functional insertion/deletion in the promoter region of the MMP3 gene [rs3025058: -1171 5A/6A]. All changes produce either a disruption or creation of binding sites for transcriptional regulators which modify the gene transcription and, in turn, the enzymatic levels
Functional SNPs in MMP genes' promoter regions may modify the production of proteolytic enzymes, and in turn modify the risk for melanoma progression. Therefore, in this study, we sought to determine whether an association between MMP2 and MMP3 SNPs and disease progression exists. Functional promoter polymorphisms in MMP2 and MMP3 genes were examined in a cohort of 1002 melanoma patients.
Results
This study included 1002 melanoma patients with stages 0 (
MMP2 -1306 C/T and -735 C/T
One sample showed an unexpected profile in the melting temperature analysis of -735 C/T that did not match any of the three possible genotypes (Figure
Genotyping of MMP2 -735 C/T SNP by melting temperature analysis
Genotyping of MMP2 -735 C/T SNP by melting temperature analysis. (A) The top panels depict the derivative melting curve plots obtained for (from left to right): MMP2 -735CC, MMP2 -735CT, MMP2 -735TT, and an unexpected profile (UKN) showing peaks between 62 and 66°C (B) The bottom panel depicts the wild type (left) and a new variant (right) at position -729 near the target SNP (white arrow). Reverse sequencing revealed a novel mutation corresponding to a G to A change at position -729 in the sense strand (black arrow).
The allele frequencies for MMP2 -1306 C/T were similar to those reported in the dbSNP for the Caucasian population CAUC1
MMP2 genotypes
Variables
MMP2
-1306 C/T
p-value*
-735 C/T
p-value*
CC
CT
TT
CC
CT
TT
0
34 (59%)
19 (33%)
5 (9%)
48 (83%)
10 (17%)
0 (0%)
I
320 (64%)
159 (32%)
23 (5%)
365 (73%)
123 (25%)
13 (3%)
II
152 (65%)
72 (31%)
9 (4%)
184 (78%)
49 (21%)
2 (1%)
III
105 (63%)
54 (32%)
9 (5%)
131 (79%)
32 (19%)
3 (2%)
IV
6 (67%)
3 (33%)
0 (0%)
0.92
4 (44%)
5 (56%)
0 (0%)
0.08
I
34 (59%)
19 (33%)
5 (9%)
48 (83%)
10 (17%)
0 (0%)
II
70 (65%)
36 (33%)
2 (2%)
79 (74%)
23 (22%)
5 (5%)
III
89 (60%)
48 (32%)
12 (8%)
115 (77%)
32 (21%)
3 (2%)
IV
308 (64%)
155 (32%)
22 (5%)
358 (74%)
116 (24%)
10 (2%)
V
51 (74%)
14 (20%)
4 (6%)
0.20
54 (78%)
15 (22%)
0 (0%)
0.44
34 (59%)
19 (33%)
5 (9%)
48 (83%)
10 (17%)
0 (0%)
< 1.01
201 (63%)
107 (33%)
12 (4%)
237 (74%)
72 (23%)
10 (3%)
1.01 – 2.00
174 (64%)
81 (30%)
15 (6%)
199 (73%)
68 (25%)
4 (2%)
2.01 – 4.00
100 (63%)
50 (31%)
10 (6%)
124 (78%)
33 (21%)
2 (1%)
> 4.00
81 (68%)
35 (29%)
4 (3%)
0.72
94 (78%)
25 (21%)
1 (1%)
0.44
Absent
129 (63%)
67 (33%)
9 (4%)
161 (79%)
40 (20%)
3 (2%)
Non-brisk
262 (67%)
111 (28%)
21 (5%)
289 (73%)
95 (24%)
10 (3%)
Brisk
19 (63%)
9 (30%)
2 (7%)
0.80
21 (68%)
10 (32%)
0 (0%)
0.32
Yes
86 (67%)
39 (30%)
4 (3%)
97 (76%)
29 (23%)
2 (2%)
No
543 (63%)
281 (32%)
42 (5%)
0.28
653 (75%)
196 (23%)
17 (2%)
0.88
No
590 (63%)
302 (32%)
46 (5%)
705 (75%)
216 (23%)
16 (2%)
Yes
18 (67%)
9 (33%)
0 (0%)
0.44
22 (82%)
3 (11%)
2 (7%)
0.96
None
161 (62%)
84 (33%)
13 (5%)
201 (77%)
54 (21%)
5 (2%)
Few
311 (63%)
166 (33%)
20 (4%)
380 (77%)
105 (21%)
11 (2%)
Moderate
112 (67%)
51 (30%)
5 (3%)
115 (69%)
49 (30%)
2 (1%)
Many
22 (55%)
11 (28%)
7 (18%)
< 0.01
29 (73%)
10 (25%)
1 (3%)
0.40
1 (low risk)
26 (68%)
11 (29%)
1 (3%)
31 (82%)
6 (16%)
1 (3%)
2
132 (63%)
66 (32%)
11 (5%)
156 (75%)
47 (23%)
4 (2%)
3
206 (65%)
93 (29%)
18 (6%)
231 (73%)
76 (24%)
10 (3%)
4
191 (59%)
122 (38%)
11 (3%)
254 (78%)
68 (21%)
4 (1%)
5 (high risk)
73 (70%)
27 (26%)
5 (5%)
0.28
76 (73%)
28 (27%)
0 (0%)
0.36
* The associations were examined in three different ways (see statistical methods for a detailed explanation). The p-values shown refer to the analysis of the three individual genotypes, and appear in bold font if ≤ 0.05. The significance was lost after adjustment for age, sex, phenotypic index, moles, freckles, and race.
MMP3 -1171 5A/6A
The MMP3 genotypes were in Hardy-Weinberg equilibrium. In our population, this polymorphism showed a similar distribution to the one reported in the dbSNP for the Italian panel, but the inverse distribution was seen when compared to the PGA-European panel. Although this difference was not significant (p = 0.73). The 5A allele was more common in patients with low Clark level melanomas (p = 0.04) and with tumors with infiltrating lymphocytes (p = 0.04) (Table
MMP3 genotypes
Variables
MMP3 -1171 5A/6A
5A5A
5A6A
6A6A
p-value*
0
9 (16%)
36 (63%)
12 (21%)
I
96 (19%)
238 (48%)
162 (33%)
II
32 (14%)
124 (54%)
75 (33%)
III
27 (16%)
81 (49%)
57 (35%)
IV
2 (22%)
5 (56%)
2 (22%)
0.36
I
9 (16%)
36 (63%)
12 (21%)
II
22 (21%)
54 (51%)
29 (28%)
III
28 (19%)
73 (49%)
47 (32%)
IV
72 (15%)
241 (50%)
165 (35%)
0.36
V
9 (13%)
39 (57%)
21 (30%)
0.04£
9 (16%)
36 (63%)
12 (21%)
< 1.01
60 (19%)
150 (48%)
105 (33%)
1.01 – 2.00
45 (17%)
132 (49%)
90 (34%)
2.01 – 4.00
28 (18%)
78 (49%)
53 (33%)
> 4.00
12 (10%)
71 (60%)
35 (30%)
0.16
Absent
28 (14%)
101 (50%)
73 (36%)
Non-brisk
65 (17%)
208 (54%)
114 (30%)
0.32
Brisk
4 (13%)
20 (65%)
7 (23%)
0.04£
Yes
21 (16%)
69 (54%)
39 (30%)
No
148 (17%)
428 (50%)
277 (33%)
0.84
No
160 (17%)
468 (51%)
296 (32%)
Yes
6 (22%)
12 (44%)
9 (33%)
0.80
None
41 (16%)
136 (53%)
79 (31%)
Few
87 (18%)
240 (49%)
165 (34%)
Moderate
27 (17%)
87 (54%)
48 (30%)
Many
9 (23%)
21 (53%)
10 (25%)
0.76
1 (low risk)
6 (16%)
15 (41%)
16 (43%)
2
28 (14%)
105 (51%)
73 (35%)
3
59 (19%)
157 (50%)
98 (31%)
4
59 (18%)
172 (54%)
89 (28%)
5 (high risk)
18 (18%)
47 (46%)
38 (37%)
0.36
*The associations were examined in three different ways (see statistical methods for a detailed explanation). The p-values shown refer to the analysis of the three individual genotypes, and appear in bold font if ≤ 0.05. The significance was lost after adjustment for age, sex, phenotypic index, moles, freckles, and race.
£Genotypes 5A6A and 6A6A combined.
We did not find any significant cumulative effect between the number of high-activity alleles and the clinical and epidemiological variables, except for ulceration. Presence of ulceration occurred less frequently in individuals with higher numbers of alleles (p = 0.03). None of the polymorphisms showed associations with progression, survival and recurrence when we computed the Kaplan-Meier estimates and compared differences in survival based on genotypes using a log rank test (data not shown).
Discussion
After adjustment for age, sex, race, phenotypic index, and freckles, we did not observe any significant associations between the matrix metalloproteinase 2 or matrix metalloproteinase 3 polymorphisms and the clinicopathological and epidemiological variables studied.
Associations between polymorphisms in MMPs and risk of development or progression of the disease have been previously reported in various types of malignancies, including esophageal, breast, and lung cancers
Our genotyping analysis identified a new G to A variation in the MMP2 promoter at position -729. This new variation is not situated within a conserved sequence; therefore, it might not have a functional consequence on the regulation of transcription of MMP2. Even though the functional potential of this nucleotide substitution remains to be determined by
The MMP3 allele with high transcriptional activity 5A was found more frequently in patients with melanomas containing infiltrating lymphocytes and showing low Clark level lesions. It is of note that we are unable to verify whether the missingness of data on TILs in this study is informative therefore these results should be considered preliminary. Although it seems to be a trend between this SNP and TILs and Clark level, the associations lose significance after adjusting for age, sex, phenotypic index, moles, freckles, and race.
Conclusion
The MMP2 and MMP3 transcriptionally more active polymorphisms appear more frequently among individuals with many moles, tumor infiltrating lymphocytes, and low Clark level, and the number of alleles seems associated with absence of ulceration. However, after controlling for known melanoma confounders the associations are non-significant. This study does not provide strong evidence for further investigation of MMP2 and MMP3 genetic variants in melanoma progression.
Methods
Study population
Melanoma patients with stages 0 to IV were recruited at Memorial Hospital (New York, USA) between March 1974 and August 2005. Of these, 763 were newly diagnosed at Memorial Hospital and 239 were prevalent cases. The study protocol was approved by the Memorial Sloan-Kettering Cancer Center (MSKCC) Institutional Review Board (IRB). Ninety-six percent of the patients approached agreed to participate in the study and signed an informed consent. Patients filled out a short self-administered questionnaire that included information on gender, race, age, family history, moles and freckling pattern, nevus density, hair and eye color, propensity to sunburn and ability to tan after sun exposure. The information on hair color, eye color and propensity to tan or sunburn were combined into a single variable, the 'phenotypic index'
Clinico-pathological characteristics of the study group
Variable*
Patients
%
Males
573
57.2
Females
429
42.8
Yes
167
16.7
No
826
82.4
Unknown
9
0.9
Yes
152
15.2
No
849
84.7
Unknown
1
0.1
0
58
5.8
I
504
50.3
II
235
23.4
III
169
16.9
IV
9
0.9
Unstagable¥
27
2.7
0
56
5.6
I
435
43.4
II
159
15.9
III
217
21.7
IV
129
12.9
Unstagable¥
6
0.5
I
58
5.8
II
108
10.8
III
150
14.9
IV
489
48.8
V
69
6.9
Unknown
128
12.8
58
5.8
< 1.01
321
32.0
1.01 – 2.00
272
27.1
2.01 – 4.00
161
16.1
> 4.00
121
12.1
Unknown
69
6.9
Absent
206
20.6
Non-brisk
397
39.6
Brisk
31
3.1
unknown
368
36.7
Yes
130
13.0
No
870
86.8
N/Aϕ
2
0.2
No
942
94.0
Yes
27
2.7
N/Aϕ
33
3.3
None
260
25.9
Few
498
49.7
Moderate
169
16.9
Many
40
4.0
N/Aϕ
35
3.5
1 (low risk)
38
3.8
2
210
21.0
3
317
31.6
4
328
32.7
5 (high risk)
105
10.5
N/Aϕ
4
0.4
Extremities
535
53.4
Trunk
342
34.1
Head and Neck
71
7.1
Non-cutaneousω
12
1.2
Unknown
42
4.2
* With the exception of 'current stage', the variables were recorded at the initial diagnosis.
¥ patients with missing data on the 'T' classification.
ω 90% mucosal melanomas and 10% other sites.
ϕ N/A: data not available.
Biospecimens
Buccal cells were collected from mouthwash or buccal swabs (n = 985). Blood was also obtained from some individuals (n = 17). DNA from buccal cells was extracted using Puregene® kits (Gentra Systems Inc., Minneapolis, USA), and blood was extracted with the QIAamp DNA Blood kit (QIAGEN Inc. Valencia, USA) using manufacturer's recommendations. DNA concentration was measured by spectrophotometry at 260 nm in a Spectramax Plus 384 (Molecular Devices, Sunnyvale, USA). The DNA quality was determined by the ratio A260/A280.
Genotyping
The polymorphism MMP3 -1171 5A/6A were studied by fragment size analysis as previously described by Zinzindohoue
Sequencing
Direct sequencing was done in samples that showed unclear genotyping profiles. Briefly, samples were PCR amplified and then purified with a Qiagen purification kit following the manufacturer's recommendations (QIAGEN Inc., Valencia, USA). One to 10 ng of each purified sample were sequenced in the DNA Sequencing Core Facility at Memorial Sloan-Kettering Cancer Center. Samples were run in an ABI 3730-XLDNA Analyzer (Applied Biosystems, Foster City, USA). Sequencing electropherograms were read at least twice, reviewed manually and with the Mutation Surveyor software, version 2.41 (SoftGenetics LLC, State College, USA).
Bioinformatics
The University of California Santa Cruz (UCSC) Genome Browser Database
Statistical analysis
Two-sided Chi-Square tests, Cochran-Armitage tests for trend, and Fisher's exact tests were performed to test for association between genotype and various clinical and epidemiologic factors. Associations were examined in three different ways: comparing the homozygote high-transcriptional-activity allele group versus those having at least one copy of the low-transcriptional-activity allele, comparing the homozygote low-transcriptional-activity allele group versus all others, and looking at all three genotypes separately. Multivariate analyses were conducted using logistic regression, adjusting for age, sex, phenotypic index, moles, freckles, and race. Associations between number of high-transcriptional-activity alleles and the clinical and epidemiological variables were examined using the Chi-square and Kendall's Tau tests. Associations were considered significant when p < 0.050. To investigate associations between SNP and overall survival, time was measured from initial date of diagnosis with melanoma to date of death or last follow-up. Potential associations between genotypes and time to recurrence, defined as a patient's first recurrence of melanoma (local, intransit, nodal and/or systemic), and time to disease progression defined as progression to stage III or IV, were also examined. Survival estimates were computed using the methods of Kaplan and Meier and comparisons between genotypes were made using the log-rank test. All statistical analyses were carried out using SAS version 9.1 (SAS Institute, Cary, NC).
Abbreviations
AJCC, American Joint Committee on Cancer ; CI, confidence interval; CMM, cutaneous malignant melanoma; dbSNP, SNP database from the NCBI; ECM, extracellular matrix; MMP, matrix metalloproteinase; OR, odds ratio; SNP, single nucleotide polymorphism; TILs, tumor infiltrating lymphocytes; UCSC, University of California Santa Cruz.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
JC carried out the genotyping, participated in the selection of SNPs, analysis, and prepared the manuscript; PR and SS participated in the genotyping; AP coordinated the patients' accrual and updated the clinicopathological and epidemiological database; NI performed the statistical analysis and contributed to the materials and methods section; DC, AH and AH contributed with subject accrual and discussions; KB contributed with pathology review; BR and CS participated in the analysis and interpretation of the results obtained with the