Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE(2) in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF(2alpha) and 6-keto-PGF(1alpha) were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed rnRNA of EP1, EP3, and EN but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EN receptors coupled with G(s)-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-2384813, having highest affinity for EP1, EP1/EP2/DP, and EN receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE(2)-mediated migratory activity of these cells appeared to be associated predominantly with EN receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EN antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE(2) and EN agonist PGE(1) alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C31_5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE(2) or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis. (C) 2003 Elsevier Science (USA). All rights reserved.