EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression

Abstract

Exchange Proteins directly Activated by cAMP (EPACs) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activate RAP1 and the alternative cAMP signaling pathway. We previously showed that the differential growth response of primary and metastatic melanoma cells to cAMP is mediated by EPAC. However, the mechanisms responsible for this differential response to EPAC signaling are not understood. In this study, we show that pharmacological inhibition or siRNA-mediated knockdown of EPAC selectively inhibits the growth and survival of primary melanoma cells by downregulation of cell cycle proteins and inhibiting the cell cycle progression independent of ERK1/2 phosphorylation. EPAC inhibition results in upregulation of AKT phosphorylation but a downregulation of mTORC1 activity and its downstream effectors. We also show that EPAC regulates both glycolysis and oxidative phosphorylation, and production of mitochondrial reactive oxygen species, preferentially in primary melanoma cells. Employing a series of genetically matched primary and lymph node metastatic (LNM) melanoma cells, and distant organ metastatic melanoma (MM) cells, we show that the LNM and MM cells become progressively less responsive and refractory to EPAC inhibition suggesting loss of dependency on EPAC signaling correlates with melanoma progression. Analysis of TCGA dataset showed that lower RAPGEF3, RAPGEF4 mRNA expression in primary tumor is a predictor of better disease-free survival of patients diagnosed with primary melanoma suggesting that EPAC signaling facilitates tumor progression and EPAC is a useful prognostic marker. These data highlight EPAC signaling as a potential target for prevention of melanoma progression. Implications: This study establishes loss of dependency on EPAC-mTORC1 signaling as hallmark of primary melanoma evolution and targeting this escape mechanism is a promising strategy for metastatic melanoma.

Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1

“…Rap1 Activity Assay: Rap1-GTP levels were quantified using the Rap1 Activation Assay Kit (NewEast Biosciences, 81 401) as described previously. [41] Briefly, tissue and cell samples were incubated with an antiactive Rap1 antibody and protein A/G agarose at 4 °C for 1 h. The agarose beads were precipitated by centrifugation at 5 000 g for 1 min, washed three times, and resuspended in 20 μL of 2 × loading buffer for 5 min before being collected for western blot detection.…”

cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment

“…The effects of cAMP signaling are even more complex in melanoma. cAMP stimulates primary melanoma growth through EPAC1/2-Rap1/mTORC1 pathways, while it does not promote metastatic melanoma cells growth [ 125 – 127 ]. Adenylate cyclase inhibitor impairs cAMP signaling and suppresses melanoma growth, while low levels of CREB phosphorylation correlates with melanoma metastasis and recurrence [ 126 ].…”