The overall objective of this proposal is to understand the contribution of over-expression of one of the matrix metalloproteinases (MMPs), MMP- 9, to the invasive and/or metastatic properties of carcinomas and the molecular mechanisms which regulate its expression using the mouse skin model. The MMPs degrade extracellular matrix components and aberrant expression of several MMPs, MMP-9 in particular, in malignant invade into the underlying stromal tissue and into and out of blood vessels during metastasis Thus, understanding the mechanisms that allow up-regulated expression of MMP-9 in tumor cells could lead to strategies to block expression and so inhibit invasion and metastasis, the primary cause of cancer deaths. The first specific aim is to determine the functional consequences of MMP-9 expression in tumor cells in terms of invasiveness and metastatic potential MMP-9 cDNA will be stably transfected into non-malignant mouse skin cells will then be used to examine tumor growth, invasive, and metastatic properties in vivo. The second specific aim is to examine what are the possible molecular mechanisms for the lack of MMP-9 expression in non-malignant cells compared to constitutive and strongly inducible expression in malignant carcinoma cells. MMP-9 promoter activity will be determined with a luciferase reporter gene in transient transfections of non-malignant versus carcinoma cells. MMP-9 promoter deletions will also be used to determine which regions of the mouse MMP-9 gene promoter are responsible for its repressed expression in non-malignant cells. We will also do gel shift assays to identify the nuclear factors which bind to these promoter regions and so modulate the expression of MMP-9. Co- transfections of specific nuclear factors with the MMP-9 promoter/luciferase construct will also be done in the non-malignant ells, if necessary, to determine if the lack of MMP-9 expression in these cells is due to a lack of positive transactivating factors rather than the presence of repressor proteins.
