Alkaline Ceramidase and Sphingolipid Signaling

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DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P) mediates pleiotropic cellular responses such as cell proliferation, survival (anti-apoptosis), and motility. It has also been implicated in cardiovascular development, angiogenesis, inflammation, and tumorigenicity. S1P acts as a specific ligand for G-protein coupled receptors or an intracellular signaling molecule. It is now clear that S1P is only generated from sphingosine, which is in turn essentially derived from ceramide through the action of ceramidases. However, much remains unknown about the regulation of the levels of this important signaling molecule. The Pl's compelling preliminary data suggest that haCER2, one of three distinct human alkaline ceramidases that the PI recently cloned, has an important role in regulating the levels of S1P by controlling hydrolysis of ceramides, a rate-limiting step for the formation of S1P, and importantly, haCER2 is capable of regulating S1P-mediated tumor cell survival, angiogenesis, and tumor growth. The PI's long-term goals are to define the role of haCER2 in regulating the levels of S1P and S1P-mediated tumor growth and angiogenesis and to develop this concept into novel strategies for anti-cancer therapeutics. The Pl's central hypothesis is that haCER2 regulates the levels of S1P and S1P-mediated biological processes, particularly tumor angiogenesis and growth, which will be tested by the following specific aims. Aim 1 is to understand catalytic mechanisms of haCER2, haCER2 will be expressed in the yeast system, purified, reconstituted, and characterized biochemically. Aim 2 is to test the hypothesis that haCER2 regulates the generation of S1P in response to proangiogenic cytokines and growth factors, haCER2 regulation by these agonists will be studied in HeLa tumor cells and human umbilical vein endothelial cells by expression and activity studies, haCER2 promoter will be characterized and cis-acting elements responsive to cytokines, and growth factors will be identified. Aim 3 is to test the hypothesis that haCER2 has a role in regulating tumor growth and angiogenesis in vivo. Tumor cell lines that express the wild type, up-regulated, or down-regulated level of haCER2 will be established to define the roles of haCER2 in tumor angiogenesis and growth using Matrigel implant assay and tumor xenograft mouse models. These studies will firmly establish a role for haCER2 in regulating S1P and S1P-mediated biology and will pave the way for novel therapeutic approaches to cancer.