Sphingosine-1-phosphate (S1P), a potent sphingolipid mediator, regulates diverse cellular processes important for breast cancer progression, including cell proliferation, survival, migration, and angiogenesis. Two specific sphingosine kinase isoenzmes, SphK1 and SphK2, which have different subcellular localizations and distinct functions, produce S1P that mainly acts in autocrine and/or paracrine manners by binding to five specific cell surface G protein-coupled receptors, S1P1-5. SphK1 is overexpressed in breast cancers and promotes estradiol (E2)-dependent xenograft tumorigenesis of MCF7 breast cancer cells, yet it is not known how intracellularly generated S1P is released by tumor cells. In this work, we show that overexpression of SphK1, but not SphK2, increased S1P export from MCF7 cells, although both similarly increased its intracellular levels. Conversely, downregulation of SphK1, but not SphK2, with specific small interfering RNAs decreased export of S1P. Both E2 and epidermal growth factor (EGF) activated SphK1 and production of S1P but only E2 stimulated rapid release of S1P from MCF7 cells, as determined by labeling with [3H]sphingosine and differential extraction of [3H]S1P and by direct mass measurements using ESI-MS/MS. E2-mediated S1P release did not occur in ER\#945; negative MD-MBA-231 cells. Since ATP-binding cassette (ABC) transporters (also known as multi-drug resistant gene family) have been reported to be involved in secretion of chemotherapeutic drugs as well as lipids, we investigated their role in secretion of S1P from breast cancer cells. Release of S1P was inhibited by MK571, an inhibitor of ABCC1 (multi-drug resistant protein 1), and Fumitrimorgin C, an inhibitor of ABCG2, (breast cancer resistance protein), but not by the ABCB1 inhibitor, Verapamil. Moreover, downregulation of ABCC1 and ABCG2 with specific siRNAs markedly reduced E2-induced release of S1P. This is the first demonstration that the multi-drug resistant transporters ABCC1 and ABCG2 are involved in rapid release of S1P produced by SphK1 in response to E2. These findings may have important clinical implications since ABC transporters are upregulated in breast cancers and S1P release induced by E2 may play important roles in breast tumor progression. This work was supported by NCI grant R01CA61774 to SS and NIH grant 5K12HD055881 to KT.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 914.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO