Requirement for sphingosine 1–phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference
Sung-Suk Chae, … , Henry Furneaux, Timothy Hla
Sung-Suk Chae, … , Henry Furneaux, Timothy Hla
Published October 15, 2004
Citation Information: J Clin Invest. 2004;114(8):1082-1089. https://doi.org/10.1172/JCI22716.
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Article Vascular biology

Requirement for sphingosine 1–phosphate receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference

Abstract

Angiogenesis, or new blood vessel formation, is critical for the growth and spread of tumors. Multiple phases of this process, namely, migration, proliferation, morphogenesis, and vascular stabilization, are needed for optimal tumor growth beyond a diffusion-limited size. The sphingosine 1–phosphate (S1P) receptor-1 (S1P1) is required for stabilization of nascent blood vessels during embryonic development. Here we show that S1P1 expression is strongly induced in tumor vessels. We developed a multiplex RNA interference technique to downregulate S1P1 in mice. The small interfering RNA (siRNA) for S1P1 specifically silenced the cognate transcript in endothelial cells and inhibited endothelial cell migration in vitro and the growth of neovessels into subcutaneous implants of Matrigel in vivo. Local injection of S1P1 siRNA, but not a negative control siRNA, into established tumors inhibited the expression of S1P1 polypeptide on neovessels while concomitantly suppressing vascular stabilization and angiogenesis, which resulted in dramatic suppression of tumor growth in vivo. These data suggest that S1P1 is a critical component of the tumor angiogenic response and argue for the utility of siRNA technology in antiangiogenic therapeutics.

Authors

Sung-Suk Chae, Ji-Hye Paik, Henry Furneaux, Timothy Hla

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Gene silencing by siRNA. (A) Scheme for the generation of multiplex siRN...
Gene silencing by siRNA. (A) Scheme for the generation of multiplex siRNA. ORF, open reading frame. (B) Gel electrophoresis of synthetic and multiplex siRNA. Synthetic siRNA or multiplex siRNA for S1P1 were analyzed by a non-denaturating gel electrophoresis and detected by ethidium bromide staining. Lane 1: DNA mol wt markers; lane 2: 21-bp synthetic siRNA for S1P1 (siS1P1); lane 3: RNaseIII-digested siRNA for S1P1. (C) Mouse endothelial cells were transfected with various siRNAs and total RNA was isolated and analyzed by Northern blot analysis using S1P1 or GAPDH probes as described. Data show the results of a representative experiment that was repeated at least 3 times. siβ-gal, siRNA for β-gal. (D) Mouse endothelial cells were transfected with various siRNAs and analyzed in a chemotaxis assay using S1P (100 nM) as a chemoattractant. Data represent mean ± SE from a representative experiment that was repeated twice. syn, synthetic; OF, oligofectamine alone.