Sphingolipid Signaling in Wilms' Tumor Cells

Connecticut Children'S Medical Center, Hartford CT

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Abstract

DESCRIPTION (provided by applicant): Pediatric and adult renal tumors are typically, angiogenic, invasive and potentially metatastic. Morbidity and mortality associated with Wilm's tumor is usually associated with metastases. Recently, the lysophospholipid, sphingosine-1-phosphate (S1P), and its associated G-protein-coupled receptors have emerged as putative regulators of angiogenesis, tumor proliferation, migration and metastasis. S1P receptor isotypic expression may be a determinant of tumor cell migration. We propose to gain mechanistic insights into the role of S1P receptor signaling in Wilm's tumor cells. In Aim 1 the expression of S1P receptors and S1P metabolizing enzymes in Wilm's tumor cell lines and clinical specimens will be evaluated using quantitative RT-PCR, western blot analysis, and immunohistochemical techniques. In Aim 2 the mechanisms of S1P signaling will be studied by S1P receptor overexpression and RNA interference. Cell lines that overexpress key SIP-metabolizing enzyme will be used to assess cell behavior in response to changes in S1P. S1P-receptor coupling to G proteins (Gi, GS, Gq and G12/13) and subsequent activation of Rho family GTPases (Rho, Rac, Cdc42) and Rho kinases have been implicated in S1P regulation of tumor cell motility. We will study the importance of these pathways and of the effectors downstream from Rho kinase, such as LIM-kinase (LIMK) and cofilin, by studying Rho and Rac activation, inhibition of Rho kinase, and the phosphorylation status of LIMK and cofilin. In Aim 3 growth, invasion and metastasis of naive cell lines and of S1P receptor-modified Wilm's cell lines in an established nude mouse model will be studied, thereby confirming and validating our ex vivo and in vitro results. Renal cell carcinoma cell lines from adult patients will be used for comparison. The results of these studies will contribute to our understanding of the processes of Wilm's tumor migration/metastasis and may offer novel therapeutic approaches for renal tumors.