Sphingosine-1-Phosphate Regulates Glioblastoma Cell Invasiveness through the Urokinase Plasminogen Activator System and CCN1/Cyr61

Glioblastoma multiforme (GBM) is an aggressively Invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid sphingosine-1-phosphate (S1P) stimulates In vitro Invasiveness of GBM cells and that high expression levels of the enzyme that forms SIP, sphingosine kinase-1 (SphK1), correlate with shorter survival time of GBM patients. We also recently showed that SIP induces expression of CCN1 (also known as Cyr61), a matricellular protein known to correlate with poor patient prognosis, In GBM cells. In this study, we further explored the role of CCN1 as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, In S1P-Induced invasion using a spheroid Invasion assay. We also investigated the roles of various SIP receptors in stimulating Invasiveness through these pathways. SIP Induced expression of uPA and its receptor, uPAR, In GBM cells. Whereas S1P(1), S1P(2), and S1P(3) receptors all contribute, at least partially, SIP, overexpression led to the most dramatic induction of the uPA system and of spheroid Invasion, even In the absence of added S1P. Furthermore, neutralizing antibodies directed against uPA or CCN1 significantly decreased both basal and S1P-stimulated GBM cell Invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment SIP receptor-mediated enhancement of uPA activity or Invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell Invasion, whereas SIP receptor signaling enhances invasion, partially through uPA and CCN1. (Mol Cancer Res 2009;7(1):23-32)