Metastasis (i.e. tumor spread) is the major obstacle to cancer cure. The metastatic process consists of many steps. If this cascade of events is interrupted at any step, metastasis will not occur. Malignant melanoma cells interacted with their environment using the same repertoire of molecules (e.g. receptors) found on normal cells surfaces. Since cell-to- cell interactions occur at many pints of the cascade, blockade of receptor(s) on melanoma cells, through which they interact with other cells, represents a rational target for interruption of tumor spread. Eristostatin, am member of the disintegrin family of viper venom proteins, has been found to inhibit melanoma metastasis in vivo using immunodeficient mice. To date, the basis for eristostatin's anti-metastatic effect remains unknown. The long term objective of this research is to identify molecular mechanisms by which melanoma metastasis may be inhibited. The specific goal proposed here is to investigate hoe eristostatin inhibits experimental melanoma metastasis by pursuing three specific aims: i) Create a panel of eristostatin mutations designed to alter single amino acid residues along its entire length. ii) Characterize the interactions of eristostatin and its mutants with four melanoma cell lines possessing distinct combinations of well-defined surface receptors. iii) Identify the structural sequence within eristostatin which is most critical for its anti-metastatic ability. Recombinant eristostatin mutations will be created by alanine scanning mutagenesis, and made as bacterial fusion proteins with glutathione-S transferase. These purified mutants will be used in a series of functional assays with four metastatic, human melanoma cell lines. We will identify the specific molecule with which eristostatin interacts on each type pf melanoma cell through crosslinking and immunological techniques. To determine which residue(s) of eristostatin are responsible for its cellular interactions, each mutant will be compared to wild type eristostatin's activity in cellular assays. Those mutants which show the greatest difference in cellular interactions will be used in experimental metastasis assays. After I.V. injection of immonodeficient mice with melanoma cells and mutated eristostatin, mice will be observed for 4 weeks, and then necropsied. Metastatic potential will be assessed by counting lung metastases. Cryosections of how lungs will be evaluated using immunohistochemical stains. Taken together, these studies will provide insights into how one naturally occurring protein possesses the """"""""right fit"""""""" to bind melanoma cells and block their metastatic ability. This information will, in turn, lead to a rational design of therapeutic agents which would target these cells and triumph over tumor spread the major obstacle to cancer cure.
