Transformation of normal cells to malignant tumors is a multi-step process which results from genetic alterations and environmental insults. Most malignant cells possess the ability to metastasize via the detachment of cells form the primary tumor, intravasation, extravasation and establishment of secondary foci in distal organs. Extracellular matrices (ECM) and basement membranes are composed of a variety of proteins that surround tissues and organs and present impermeable barriers to active cell mobility. The metastatic process involves interactions between wandering malignant cells and macromolecules of basement membranes and extracellular matrices, whereby the cells attach to the matrix and gain egress via the increased synthesis of proteolytic enzymes. Various investigations have shown altered regulation of the synthesis of ECM proteins in various malignant cells. The hypothesis of this proposal is that there is altered transcriptional regulation of ECM protein synthesis as a direct response to the transformed state, and that this alteration enhances metastasis. In this proposal, we propose a systematic approach to exploring this hypothesis. A battery of paired cell lines and tumor specimens (normal cells, polyps, primary tumors and metastatic samples) from breast, colon and skin will be analyzed by Northern, Southern and slot blot techniques for the increased/decreased transcription of genes encoding osteonectin, decorin, type I collagen, fibronectin, and various oncogenes. The housekeeping proteins, beta-actin and glycerol phosphate dehydrogenase, will serve as controls in these experiments. Nuclear extractions, DNA footprinting, gel shift mobility and site directed mutagenesis assays will be performed to determine the molecular mechanism(s) of the altered transcription. Data from these studies may provide a mechanism by which to determine the probability of metastasis at a specific stage of tumor growth, and provide molecular markers to determine if micro-metastasis has already occurred.
