The long-term objective of this grant proposal is to gain in-depth understanding of host tumor cell interactions in the multi-step process of metastasis. Tumor cells dissolve extra-cellular matrix, activate ubiquitous pro-enzymes and interact with host cells. In this regard, recent evidence suggests a functional role in the metastatic process for both plasminogen activators (PA), in particular urokinase (UK), and procoagulant activity, mediated by tissue factor (TF), a cell surface receptor on tumor cells. In these studies, human melanoma cell lines will be used as a model system, since tumor progression is well defined for cutaneous melanoma and because human melanoma metastasis can be readily studied in animal models. The goals of this proposal are to demonstrate that UK and TF catalyze rate limiting steps in melanoma metastasis and to show that a balance between fibrinolytic and procoagulant activity is-necessary for the metastatic phenotype. To this end, poorly (WM35, M2l) and aggressively (M24met, C8l6l) metastatic human melanoma cell lines will be analyzed in a number of in vitro assays, including those that measure matrix degradation, invasion, and coagulation, and tested for their ability to produce experimental and spontaneous metastasis in severe combined immune deficient (SCID) mice. Specific molecular probes will be used to modulate the behavior of metastatic cells in vitro and in vivo. These include neutralizing monoclonal antibodies directed against UK and TF, as well as highly specific inhibitors, such as plasminogen activator inhibitor (PAI), and hirudin. The phenotype of melanoma cell lines with regard to PA and procoagulant activity will be altered using transfection approaches. Highly metastatic melanoma cell lines will be transfected stably with cDNA encoding for PAI-2 to study the effect of low or absent net PA activity on melanoma metastasis in SCID mice. Melanoma cells with high metastatic potential will be transfected with TF antisense cDNA, melanoma cells with low metastatic potential with TF sense cDNA to determine the contribution of procoagulant activity to melanoma metastasis. It is anticipated that the results from these proposed studies will ultimately lead to the identification of specific targets for the therapy of metastatic tumors.
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