Figure 4a shows that AH23848 enhanced NK-mediated lysis in a concentration-dependent fashion. metastatic properties in a murine model of metastatic breast cancer [9, 16C18]. Due to recent concerns regarding the safety of COX-2 inhibitors, we have initiated studies to test the hypothesis that PGE2 directly affects tumor cell Eriodictyol behavior in an autocrine manner and that these direct effects are mediated by one or more EP receptor expressed on the tumor cell. Further, that inhibition of EP receptor signaling could, like inhibition of PGE2 synthesis, limit metastasis. Cellular effects of PGE2 are mediated through a family of G-protein-coupled receptors designated ARNT EP1, EP2, EP3 and EP4 . We characterized EP receptor expression and function in two murine mammary tumor cell lines (66.1, 410.4) as well as the immortalized murine mammary epithelial cell line EpH4. Both murine breast tumor and mammary epithelial cells express EP1, EP2, EP3 and EP4 (Fig. 1). There is considerably less EP1 detected in comparison to EP2, EP3 and EP4. Open in a separate window Fig. 1 Flow cytometric analysis of EP staining on two murine mammary tumor cell lines (410.4, 66.1) and immortalized mammary epithelial cells (EpH4). Shaded peak is Eriodictyol specific EP staining, open peak is staining with isotype-control antibody COX inhibitors are highly effective at reducing murine mammary tumor metastasis [9, 16, Eriodictyol 18]. Murine mammary tumor cells spontaneously secrete high levels of PGE2. We have hypothesized that production of PGE2 by tumor cells contributes to metastatic ability in an autocrine fashion in which tumor-PGE2 signals through EP receptors on the tumor cells to enhance tumor dissemination. We further hypothesized that blockade of PGE-mediated signaling, downstream of PGE2 synthesis, might have therapeutic effects similar to those observed when PGE2 synthesis is prevented with COX inhibitors. To test this hypothesis, Eriodictyol we employed both a pharmacologic antagonist of EP4 as well as a gene-silencing approach to determine the role of EP4 in tumor metastasis. Figure 2a shows the reduced EP4 expression in 66.1 cells transfected with a plasmid expressing shRNA directed to murine EP4. Ligand binding to EP2 and EP4 is coupled to PKA/adenyl cyclase and mediates elevations in intracellular cAMP. The reduction in EP4 expression in 66.1 cells compromised their ability to elevate cAMP in response to the EP4 selective agonist PGE1-OH in comparison to 66.1vector cells (Fig. 2b). The EP4 antagonists AH23848 or ONO208 inhibited the cAMP response to PGE1-OH in 66.1vector cells but had no impact on the cAMP response in 66.1shEP4 cells. When 66.1vector or 66.1shEP4 cells were introduced into Balb/cByJ mice, lung colonizing ability of cells expressing less EP4 was significantly compromised (Fig. 2c, = 0.008). We derived four additional independent clones expressing reduced levels of EP4 and lung colonization was reduced by 43%, 53%, 53% and 84% when these cells were injected into mice. Likewise, systemic treatment with the EP4 antagonist AH23848 (10 mg/kg) inhibited lung colonization of parental 410.4 or 66.1 cells by 88% and 32%, respectively (Fig. 2d, = 0.008, = 0.02, respectively). When tumor cells were transplanted to the mammary gland of mice, EP4 gene silencing did not inhibit local tumor growth (data not shown), however spontaneous metastases were reduced by 77% (= 0.01). Depletion of NK cells leads to loss of endogenous control of tumor dissemination leading to a fourfold increase in lung metastases and in these mice, AH23848 no longer inhibited metastasis. The reduction of lung metastasis achieved by EP4 silencing (Fig. 3b) was also severely compromised in NK-depleted mice. In this experiment, EP4 silencing reduced lung colonization by 58% in immunologically intact mice (= 0.0003); in NK-depleted mice, lung colonies were reduced by 16% in mice injected with 66.1shEP4 versus 66.1vector cells,.