REGULATION OF ECM PROTEIN BIOSYNTHESIS IN NORMAL, MALIGNANT AND METASTATIC CELLS

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It is known that metastasis results from increased metalloprotease production/activity by cancerous cells resulting in the breaching of the extracellular matrix (ECM) and basement membranes, which serve as barriers to ingress and egress of metastatic cells through the circulatory and lymphatic systems. Few studies have been done to ascertain the regulatory and synthetic status of the connective tissue proteins (CTP) during tumor progression. The goal of this research is to determine what other regulatory events occur in cancerous cells which further facilitate the process of metastasis. Specifically, this research will explore the possibility that down-regulation of ECM protein synthesis is an integral component in the loss of structural integrity of the basement membrane and ECM, thus facilitating metastasis. Our previous studies demonstrated altered expression of decorin, fibronectin, osteonectin and type I collagen when transcriptional profiles were compared among breast colon and skin (normal, benign, primary cancerous and metastatic) cell lines and clinical samples. Additionally, restriction fragment length polymorphism (RFLP) analyses have revealed mutationally altered genes (decorin, fibronectin and osteonectin) in two metastatic cell lines (melanoma and breast). The specific aims of this proposal are: (1) perform a systematic analysis of normal, benign, primary cancerous and metastatic cell lines and clinical samples for further correlations of decreased ECM synthesis and increased metastatic potential, (2) develop a metastatic profile of decreased ECM production and increased metalloprotease activity in clinical derived tissues, (3) determine the structural and functional domains of decorin and osteonectin which demonstrate RFLP polymorphisms via single strand conformational polymorphism (SSCP) analysis, RT-PCR cloning and sequence analysis and (4) determine regulatory regions of selected ECM genes and their binding proteins which are involved in the observed altered regulation. Completion of the above specific aims should provide a means of determining the metastatic capability of a primary tumor in situ.