METASTASIS INHIBITION BY FIBRONECTIN-TUMOR INTERACTIONS

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Our results indicate an important role for melanoma cell surface heparin sulfate proteoglycans (HSPG) in mediating arginyl-glycyl-aspartyl (RGD)- independent cell adhesion to the 33kD carboxyl-terminal heparin binding fragment of plasma fibronectin (FN) A-chains. This is supported by several observations made during our initial funding period. First, HSPGs, isolated from metabolically-labelled tumor cells in vitro, bind to substrate coated with the 33kD fragment in a concentration dependent, saturable fashion. Secondly, HSPG binding to this fragment is specifically abolished by a cell adhesion-inhibiting monoclonal antibody (MAB) generated against the 33 kD fragment. Thirdly, we have identified novel cell adhesion-promoting synthetic peptides from the 33 kD fragment which also bind 3H-heparin and melanoma cell surface HSPG. Furthermore, the cell adhesion promoting activity of these synthetic peptides can be specifically inhibited by exogenous heparin and heparin sulfate glycosaminoglycans (GAGs). These peptides, termed I and II, have the primary sequences YEKPGSPPREVVPRPRPGV and KNNQKSEP-LIGRKKT, respectively (McCarthy, 1988b). We also have evidence for the involvement of an alpha 4 beta 1 integrin in cell recognition of this region of FN A-chains. This is based, in part, on recently published results (Wayner, et al, 1989) demonstrating a role for the interaction of alpha 4 beta 1 integrin with another cell adhesion promoting sequence within this fragment, termed CS1 (DELPQLVTLPHPNLHPGPEILDVPSKT; Humphries, 1987). Unlike peptides I and II, peptide CS1 fails to bind 3H-heparin and is only expressed in plasma FN A- chains. Although cross competition experiments suggest that peptides I, II and CS1 promote murine melanoma cell adhesion by distinct molecular mechanisms, our preliminary results demonstrate that specific alpha 4 beta 1 MABs also inhibit cell adhesion to heparin binding peptides I and II, suggesting that peptides I, II, and CS1 represent portions of a larger active site on intact FN A-chains which can bind both alpha 4 beta 1 integrins and cell surface HSPG. The underlying, and central, hypothesis to be tested in these studies is that these three sites, within the larger domain, drive the association of HSPG and alpha 4 beta 1 integrin on the cell surface as a consequence of melanoma cell adhesion to this region of FN A-chains. We will use specific anti-integrin and anti-HSPG MABs, in conjunction with other reagents such as synthetic peptides and restricted recombinant FN fragments containing this domain, to confirm or refute predictions stemming from this hypothesis. Specific comparisons will be made between highly metastatic melanoma cells and poorly metastatic counterparts, to attempt to identify specific changes in cell adhesion phenotype which correlate with invasive or metastatic behavior.