Upregulation of the S1P receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE and EP/EP activation.

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca]), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E synthase expression, and consequently with increased PGE synthesis. PGE synthesis was decreased by antagonists and siRNA against S1P and S1P. Moreover, in parental MDA-MB-231 cells overexpression of S1P by cDNA transfection also increased PGE synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P antagonism, COX-2 inhibition or PGE receptor 2 (EP) and 4 (EP) antagonism, but not by S1P antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE signaling via EP and EP.