This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Metastasis (i.e., tumor spread) is the major obstacle to cancer cure. The metastatic process consists of many steps, including those involving motility and adhesion. If the cascade of events is interrupted at any step, metastasis will not occur. Eristostatin, a member of the disintegrin family of viper venom proteins, can inhibit melanoma cell colonization in the lungs of mice injected with either murine or human melanoma cells. We have shown that the presence of eristostatin can cause changes in adhesion and motility in melanoma cells, but does not affect cell growth or survival. To date, the basis for eristostatin's anti-metastatic effect remains unknown, but these functional studies have opened the way to unlocking this natural protein's anti-cancer mechanism. The long term objective of this research is to identify mechanisms by which melanoma metastasis may be inhibited. The specific goals proposed here are to investigate the molecular mechanism used by eristostatin to affect melanoma cell function. We will do this by pursuing three specific aims: (i) Characterize the direct interaction of eristostatin with melanoma cell molecule(s). We will use atomic force microscopy, confocal microscopy and chemical crosslinking to identify the binding partner for eristostatin which is present on 6 different melanoma cell lines with varying degrees of metastatic potential. (ii) Compare the interaction differences between eristostatin and melanoma cells in a 2-dimensional versus 3-dimensional environment. Cellular responses can be very different in tissue culture (2-D) compared with what happens in the body (3-D). We will investigate eristostatin-melanoma cell interactions involving adhesion, proliferation and signaling in a 3-dimensional environment using matrices containing fibronectin and/or collagen. (iii) Identify intracellular signaling events associated with eristostatin's interaction with melanoma cells. The clearest example of melanoma cell activity inhibition by eristostatin is with motility when placed on fibronectin. We will perform immunochemical assessment of the intracellular signaling occurring when melanoma cells are exposed to eristostatin, focusing on known pathways associated with motility. Taken together, these studies will provide insights into how one naturally occurring protein, eristostatin, possesses the """"""""right fit"""""""" to bind melanoma cells and block their metastatic ability. The information will, in turn, lead to a rational design of therapeutic agents which will target these cells.
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