Presentation Authors: Yunze Xu*, Yiran Huang, Shanghai, China, People's Republic of
Introduction: Sphingosine kinase 1 (SphK1) is the major source of the bioactive lipid and GPCR (G-proteincoupledreceptor) agonist sphingosine 1-phosphate (S1P). Numerous studies have documented that SphK1-S1P signalling hasmultiple functions in tumorigenesis. The present study was conducted to investigate the expression of SphK1-S1P signalling and itsprognostic significance in renal cell carcinoma (RCC). Meanwhile, we set out to SphK1 as a novel molecular target in RCC andexamine its role in tumor cell growth and sunitinib resistance in vitro and in vivo.
Methods: In this study, SphK1 expression levels as well as clinicopathological significance were evaluated in RCCcell lines and primary RCC clinical specimens by immunohistochemistry, immunofluorescence assay, qRT-PCR and western blot.We assessed the levels of S1P and Sph in tumor tissues by HPLC analysis and in the plasma of RCC patients and healthy controls byLC-MS/MS analysis. In vitro and in vivo knockdown or overexpression of SphK1 in RCC cell lines was used to determine its effecton tumor growth, cellular proliferation, colony formation, migration, invasion, and apoptosis. We conducted a phosphoproteinantibody array to identify ectopic phosphorylated proteins induced in 786-O cells with/without SphK1 expression vector. In addition,we further evaluated the effect of SphK1 antagonist fingolimod (FTY720) in RCC in vitro and in vivo, as a single agent or incombination with sunitinib, and attempted to explore its anticarcinogenic mechanisms.
Results: We identify upregulation of SphK1 significantly associated with poor prognosis of RCC patients, which contributing torenal proliferation, colony formation, migration and survival. Suppression of SphK1 activity either by shRNA or pharmacologicinhibitior FTY720 suppresses cell growth in vitro and in vivo. A comprehensive phosphoprotein antibody array reveals that SphK1overexpression promoted RCC progression by regulating the Akt/mTOR pathway. SphK1 could act as a canonical regulator ofhypoxia-inducible HIF-1Î± and HIF-2Î± in RCC cells. Moreover, stem-like phenotype due to sunitinib administration via oncogenicactivation of SphK1 could be ameliorated by SphK1 shRNA and FTY720. FTY720 administration enhanced tumor growth inhibitioneffect of sunitinib treatment on RCC cells in vitro and in vivo.
Conclusions: Our results unraveled that increased SphK1 kinase activation defines an important mechanism for maintaining stemlikephenotype and sunitinib resistance, therefore contributes to tumour development and represents therapeutic targets for RCC.