Major factors in the morbidity and mortality of malignant melanoma are tumor invasion and metastasis, which are mediated by Matrix Metalloproteinases (MMPs). MMPs degrade the extracellular matrix, and are associated with invasive cancers. Degradation of interstitial collagens (types I and III) is needed for invasion, and is accomplished by the ubiquitously expressed collagenase-1 (MMP-1). A single nucleotide polymorphism (SNP; 2G allele) at -1607 bp (5'-GGAA-3') in the MMP-1 promoter creates a binding site for the Ets family of transcription factors and, compared to the 1G allele (5'-GAA-3'), enhances MMP-1 transcription. This 2G SNP is associated with the incidence/progression of seven malignancies, including melanoma. Levels of Basic Fibroblast Growth Factor (bFGF) correlate with invasive melanoma, and bFGF induces MMP-1 expression. In addition, an activating mutation in BRAF found in 80% of melanomas in the signaling cascade of Ras/Raf/Mek/Erk also induces MMP-1. We hypothesize that: (a) the 2G allele increases MMP-1 expression and melanoma invasion, (b) bFGF and the activating mutation in BRAF orchestrate an invasive phenotype in the tumor cells by preferentially increasing MMP-1 expression through the 2G allele, and (c) host/tumor/matrix interactions target the 2G allele in stromal cells adjacent to the tumor to further enhance MMP-1 expression and tumor invasion. We will, therefore, (1) Generate transgenic mice harboring 4 kb of the human MMP-1 promoter, containing either 1G or 2Gs at -1607 bp, linked to the beta-galactosidase reporter. Transgenes will be inserted as a single copy at the hprt locus to provide information on the transcriptional activity of MMP-1 in stromal cells. Mice will be challenged with B16 melanoma and expression of the transgenes, which reflects collagenolytic potential, will be examined in stromal cells adjacent to the tumor; (2) Convert a diploid 1G homozygous melanoma cell line to 2G homozygous, and conversely, a diploid 2G homozygous melanoma cell line to 1G homozygous, to compare effects of the 1G and 2G alleles on MMP-1 promoter and enzyme activities, and on tumor invasion in vitro and in vivo in nude mice. We will determine how bFGF and mutant BRAF target the 2G allele to enhance the invasive phenotype of melanoma cells. These studies will provide novel mechanisms for studying MMP-1 gene expression in mouse models, and contribute to our understanding of how this human interstitial collagenase contributes to the pathology of invasive melanoma.
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