Cancer Cells Independently of Cell Surface Receptors Sphingosine-1-phosphate Inhibits Motility of Human Breast

Exogenous sphingosine-1-phosphate (SPP) inhibits chemotactic motility of several transformed cell lines. We have found that SPP at high micromolar concentrations decreased chemotaxis of estrogen-independent (MDA-MB-231 and BT 549) and estrogen-dependent (MCF-7 and ZR75-1) human breast cancer cells. Because SPP has been implicated as a lipid-signaling molecule with novel dual intraand intercellular actions, it was of interest to determine whether the effect of SPP on chemotactic motility of human breast cancer cells is mediated intracellularly or through the recently identified endothelial differentiation gene (EDG) family of G protein-coupled SPP receptors. There was no detectable specific binding of [P]SPP to MDA-MB-231 or MCF-7 cells; however, reverse transcription-PCR analysis revealed that both MDA-MB-231 and MCF-7 cells expressed moderate levels of EDG-3, neither expressed EDG-1, and EDG-5 mRNA was expressed in MCF-7 but not in MDAMB-231 cells. In contrast to SPP, sphinganine-1-phosphate, which binds to and signals through SPP receptors EDG-1, EDG-3, and EDG-5, had no effect on chemotactic motility of MDA-MB-231 or MCF-7 cells. To further discriminate between intracellular and receptor-mediated actions of SPP, we used caged SPP, a photolyzable derivative of SPP that elevates intracellular levels of SPP after illumination. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upon UV irradiation. In addition, in MCF-7 cells, overexpression of sphingosine kinase, the enzyme that produces SPP, inhibited chemotactic motility compared with vector-transfected cells and markedly increased cellular SPP levels in the absence of detectable secretion. Our results suggest that the inhibitory effect of SPP on chemotactic motility of human breast cancer cells is likely mediated through intracellular actions of SPP rather than through cell surface receptors.