) Recent evidence has established the importance of the alpha v Beta 3 and alpha v Beta 5 integrins and their interaction with matrix in tumor-induced angiogenesis. High-risk neuroblastoma is associated with active angiogenesis, suggesting that integrins and matrix degrading proteases play a role in this disease. We recently showed that ceramide, a lipid second messenger important in apoptosis, increases in endothelial cells following inhibition of integrin alpha v Beta 3- and alpha v Beta 5-mediated anchorage. The goals of this project are to define the potential of alpha v Beta 3 and/or alpha v Beta 5 and their interaction with proteolyzed collagen as targets for anti-angiogenic treatment in neuroblastoma and to improve the efficacy of this anti-angiogenic approach by combining it with modifiers of intracellular ceramide metabolism. Our preliminary data supports the following hypothesis: (1) antagonism of integrins alpha v Beta 3 and alpha v Beta 5, which are potentially important to neuroblastoma angiogenesis, will provide a mechanism for attack of tumor endothelium and improve treatment of high-risk neuroblastoma; (2) manipulation of signaling pathways to increase endogenous endothelial ceramide and/or inactivate survival kinases will enhance the anti-angiogenic response of neuroblastoma. Our work will consist of three Specific Aims: (1) to define the angiogenic phenotype of primary untreated neuroblastomas with regard to expression of alpha v Beta 3, alpha v Beta 5, proteolyzed collagen, MMP-9 and PAI-1 using immunohistochemistry; (2) to analyze endothelial signaling pathways regulated by ceramide under conditions of anoikis induced by antagonism of alpha v Beta 3 or alpha v Beta 5 integrins and/or ceramide challenge; (3) to use alpha v Beta 3 and alpha v Beta 5 antagonists and agents which interfere with ceramide-regulated pathways in in vivo models for local and metastatic neuroblastoma to determine which individual or synergistic antitumor effects. Research Design: The feasibility of treating neuroblastoma with anti-angiogenic treatments directed against integrin alpha v Beta 3 and alpha v Beta 5 or direct antagonism of proteolyzed type V collagen will be examined using in vivo and in vitro models. The signal transduction pathways regulated by ceramide that function in endothelial anoikis will be identified using experiments in cell culture. This integrated approach will likely yield novel therapeutic agents for the treatment of neuroblastoma and other tumors.
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