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PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Georgetown University Washington,DC 20057
E-MAIL:fwang02@gumedlib.dm 1 .georgetown.edu Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogenindependent MDA-MB-231 human breast cancer cell line. Similar to many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase 2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness, also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397 transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility. Since SPP has been implicated as a lipid signaling molecule with novel dual intra and 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 EDG family of G protein-coupled SPP receptors. In contrast to SPP, dihydro-SPP, which binds to and signals through SPP receptors, EDG-1, -3, and -5, had no effect on chemotactic motility of MDA-MB-231 or MCF-7 cells. Caged SPP inhibited chemotactic motility of MDA-MB-231 cells only upon ultraviolet irradiation. In addition, in MCF-7 cells, overexpression of sphingosine kinase, the enzyme that produces SPP, inhibited chemotactic motility compared to vector-transfected cells, and markedly increased ...