Loss of heterozygosity on chromosome 11q22-23 in melanoma is associated with retention of the insertion polymorphism in the matrix metalloproteinase-1 promoter

Abstract

Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at -1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma.

Figure 1.

PCR primers for MMP-1 SNP analysis. The 1G/2G polymorphism in the promoter is indicated in bold upper case. A: Primers for RFLP analysis of the SNP. Note the base change (t->g) in the 3′ primer that creates a BglII site in the 1G allele and an Alw I site in the 2G allele. B: Primers used for quantitative autoradiographic analysis of LOH at the MMP-1 SNP locus. The 3′ primer for the 82-bp amplicon has an added 5′ terminal “g”. This additional base eliminated shadow bands that interfered with accurate quantification of the alleles.

Figure 2.

RFLP analysis of MMP-1 SNP. Alw I digests the 103-bp 2G amplicon (88 bp + 15 bp), leaving intact the 1G amplicon (102 bp); BglII digests the 102-bp 1G amplicon (79 bp + 23 bp), leaving intact the 2G amplicon (103 bp). Samples 3 and 4 are 1G/1G homozygotes. Samples 1, 5, and 7 are 1G/2G heterozygotes. Samples 2 and 6 are 2G/2G homozygotes.

Figure 3.

Autoradiographic detection of the MMP-1 SNP and quantification of LOH in tumor tissue. Data from the 72-bp amplicon assay (A) and the 82-bp amplicon assay (B) are shown. Normal tissue/tumor pairs from three patients are at the left; standards, composed of known mixtures of DNA from 1G and 2G homozygous controls, are at the right. The “%2G” shown for the tumor samples was determined by quantitative phosphorimager analysis and calculated from a standard curve (see Figure 4 ▶ ) constructed from standards run on the same gel. Tumors from patients 65 and 34 have lost the 1G allele. The tumor from patient 22 has lost the 2G allele.

Figure 4.

Standard curves for the 72-bp amplicon assay (A) and the 82-bp amplicon assay (B). Standards were composed of mixtures of DNA from 1G and 2G homozygous controls that were analyzed concurrently with patient samples. The actual composition of the standards is plotted on the x axis as “Actual %2G.” Data from quantitative phosphorimager analysis of the standards (see text) is plotted on the y axis as “Observed %2G.” Data from multiple experiments are shown, plotted as mean ± SD. Standard curves were used to convert “Observed %2G” of tumor samples to “Actual %2G.”

Figure 5.

Summary of MMP-1 LOH data from all tumors where LOH was detected, using the 72-bp amplicon assay (A) and the 82-bp amplicon assay (B). The “%2G” for each tumor was determined as described in Figures 4 and 5 ▶ ▶ , and is represented here as a bar. Replicate assays are indicated by individual bars. Fifty percent 2G represents no allelic loss, characteristic of normal tissues. The hatched lines represent the imprecision (2 SD) of the estimate of “%2G” in normal heterozygous tissue samples and control blood samples. Tumor samples whose “%2G” was at or exceeded the imprecision limits in both assays were classified as having undergone LOH. Ten tumors lost the 1G allele; two tumors lost the 2G allele.