The metastatic potential of tumor cells is the most important determinant for the prognosis of neoplastic disease. Among the molecular steps required for successful metastasis are several discrete events that involve the invasion of tumor cells through connective tissue barriers. Cell invasiveness concerns adhesion to and proteolysis of extracellular matrices and basement membranes, as well as cell motility. This proposal focuses on plasma membrane-associated proteases and adhesion molecules of human malignant melanoma cells that are important in mediating the invasiveness and metastatic potential of these cells. Recent work from this laboratory identifies several novel membrane proteases from human malignant melanoma cells, chicken embryonic cells, chicken transformed cells, and chicken embryos. Unique cell surface structures called invadopodia are present on cells expressing the malignant phenotype and have been shown to be involved in the invasiveness of cells. The invadopodia are sites of active tyrosine-phosphorylation, localization of beta1 integrins, and active degradation of the extracellular matrix by membrane proteases. The goals of this proposal are (1) to determine the biochemical nature of membrane proteases by the combined approaches of protease activity assays, cellular localization by monoclonal antibody production, sub-cellular fractionation, inhibitor- and antibody-affinity chromatography, protein biochemistry including microsequencing, and molecular cloning, (2) to determine how membrane proteases interact with other proteases to activate degradation, and their role in the expression of cell invasiveness, (3) to investigate the control mechanisms of cell invasion, particularly focusing on the direct or indirect interaction of membrane proteases with putative motility factors including the laminin A chain peptide, autocrine motility factor, acidic fibroblast growth factor, and interleukin-I during the induction of invadopodia and with the human integrins that localize to invadopodia during the stabilization of invasion-associated membranes, and (4) to determine the expression and localization of protease and adhesion molecules in experimental invasion and metastasis models and human tumors by scanning confocal microscopy and immuno-electron microscopy.
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