Abstract Background: We previously reported that Epac, a guanine nucleotide exchange factor directly activated by cAMP, increases melanoma cell migration through Ca2+ elevation. Both Epac-induced cell migration and Ca2+ elevation were reduced by pharmacological inhibition of phospholipase C (PLC) and inositol triphosphate (IP3) receptor. Accordingly, we studied the involvement of PLC/IP3 receptor in Epac-induced Ca2+ elevation by knockdown of these molecules. However, the mechanism how Epac-induced Ca2+ elevation mediates melanoma cell migration is unknown. Here, we investigate the effect of Epac induced-Ca2+ elevation on actin assembly, which plays a major role in cell migration. Methods: SK-Mel-2, a cell line from human metastatic melanoma, was used in this study. Intracellular Ca2+ was measured with Fura-2AM fluorescent dye. Cell migration was examined with the Boyden chambers. IP3 production was examined by IP3 radioimmunoassay. Actin assembly was examined by immunocytochemistry. In some experiments, ablation of a target protein with lentivirus-based shRNA induction system was performed. Results: 8-pMeOPT-2′-O-Me-cAMP (8-pMeOPT), an Epac-specific agonist, increased Ca2+ and cell migration in SK-Mel-2. Such Ca2+ elevation and cell migration was negated by ablation of PLCε or IP3 receptor 1. In addition, Epac-induced IP3 production was inhibited by U73122, a PLC inhibitor, and ablation of PLCε. These data suggest that Epac increases Ca2+, and thus cell migration, via the PLCε/IP3 receptor 1 signaling pathway. Cytochalasin D, an actin assembly inhibitor, reduced 8-pMeOPT-induced cell migration. In addition, immunocytochemistry showed that 8-pMeOPT increases actin assembly. When U73122 or Xestopongin C, an IP3 receptor inhibitor, was used, Epac-induced actin assembly was decreased. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly. Further, immunoprecipitation showed that 8-pMeOPT increased physical interaction of myosin heavy chain IIA (MHCIIA), which enhances actin assembly, with S100A4, a Ca2+-binding protein which activates MHCIIA. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly via the S100A4/MHCIIA signaling pathway. Conclusion: Epac increases Ca2+ via the PLCε/IP3 receptor 1 pathway. Also, Epac-induced Ca2+ elevation mediates melanoma cell migration via actin assembly. These data suggested that Epac could be a target for treating patients with melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5280.