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Sphingosine-1- Phosphate 2 Receptor Induces Ccl2 Expression In Neuroblastoma/ A Targeted Inhibition Strategy
Mei-Hong Li1, Timothy Hla2, Fernando Ferrer1
1Connecticut Children's Medical Center/University of Connecticut Health Center, Hartford/Farmington, CT;2Weill Cornell Medical College/Center for Vascular Biology, New York, NY
Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The bioactive lipid sphingosine-1-phosphate (S1P) and its five specific receptors (S1P1-5) and known to impact tumor growth and progression. Preliminary data derived from human angiogenesis array showed that S1P induced the secretion of angiogenesis-related proteins VEGF and monocyte chemoattractant protein 1(MCP-1; CCL2), an important inflammatory chemokine in NB. Recently, we have shown that S1P/S1P2 signaling mediates VEGF expression and thus promotes NB growth. In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB.
Materials & Methods: Quantitative real-time PCR detected mRNA levels of S1PRs and CCL2 in NB SK-N-AS cells and tissues. CCL2 ELISA detected CCL2 protein secretion in SK-N-AS cells. Gain and loss of functions studies were performed using S1PR antagonists, adenoviral transduction and siRNA. NB murine xenograft models were used to test the efficacy of a selective S1P2R inhibitor in-vivo.
S1PR1-3 and CCL2 mRNA were abundantly expressed in NB tissues. In NB SK-N-AS cells S1P induced CCL2 mRNA expression and protein secretion in time- and concentration-dependent manners. Antagonism of S1P2 by specific S1P2 antagonist JTE-013 blocked S1P-induced CCL2 mRNA expression and protein secretion. Overexpression of S1P2 by adenoviral transduction into SK-N-AS cells increased CCL2 secretion while knockdown of S1P2 by S1P2 siRNA transfection decreased CCL2 secretion. The S1P2R inhibitor JTE-013 suppressed tumor growth in NB xenograft models.
Conclusions: Taken together, our data demonstrate that S1P induced CCL2 expression in NB cells via S1P2 and maybe a potential therapeutic target.
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