The Principal Investigator's laboratory focuses on the study of sphingolipids in regulation of apoptosis. The Principal Investigator's group was the first to identify and clone sphingosine-l-phosphate (S1P) phosphatase. This led to studies on the role and regulation of S1P. S1P has recently been demonstrated to be the ligand for the endothelial differentiation gene (Edg) family of receptors capable of inducing endothelial cell proliferation, migration, and tube formation, thus turning on an angiogenic program. Sphingosine kinase (SK) is the enzyme that generates S1P in cells. The Principal Investigator's lab recently cloned the cDNA for human SK1, raised antibodies to the protein, chemically synthesized several analogues of S1P and begun testing them for biologic activity. We have compelling new data that (i) SK1 is significantly over expressed in several lung cancer tissues; (ii) hypoxia, a key factor in driving angiogenesis in cancer, up regulates SK1; (iii) S1P synergizes with VEGF in stimulating angiogenesis; and (iv) SIP and SK are critical regulators of COX-2. This led to the following hypothesis: SK and SIP regulate tumor angiogenesis, and that inhibiting this pathway could inhibit tumor angiogenesis. This will be demonstrated by: 1) Establishing the role of SK and S1P in regulation of tumor angiogenesis and determining the mechanism of this regulation. A) We will evaluate the effect of over expression of SK and of SIP treatment on in vitro and in vivo models of angiogenesis, and B) determine the mechanism by which SK and S1P synergize with VEGF and regulate COX-2. 2) Establishing that the SK and S1P pathway is regulated in cancer angiogenesis and study the mechanism of this regulation. A) We will study the expression and cell-type distribution of SK in cancer tissues and compare to matched normal tissues, B) determine if up regulation of this pathway in cancer is functional, and C) study the mechanism of regulation of SK and S 1P in cancer (by hypoxia). 3) Determining if SK activity and SIP levels are necessary for tumor angiogenesis. A) We will block SKI activity in cells by using small interfering RNA as well as a dominant negative SK1, B) we will synthesize different compounds that will act either as inhibitors of SK or as antagonists of S1P, C) determine the role of the SK/S1P pathway in vitro angiogenesis using the molecular approaches from Specific Aim 3A and the compounds from Specific Aim 3B, and D) determine the role of SK/S1P in vivo angiogenesis and tumor growth and vascularity by using the molecular approaches from Specific Aim 3A and the compounds from Specific Aim 3B. These studies will place the SK/S1P pathway at the heart of the angiogenic process, which is initiated by hypoxia and mediated by COX-2 and VEGF. These studies will also have novel and important therapeutic implications.