Department of Dermatology, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Wisconsin.
William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin.
Mol Cancer Res. 2022 Oct 4;20(10):1548-1560. doi: 10.1158/1541-7786.MCR-22-0026.
Exchange proteins directly activated by cAMP (EPAC) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activates 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 pharmacologic 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 cells, we show that the LNM and metastatic melanoma cells become progressively less responsive and refractory to EPAC inhibition suggesting loss of dependency on EPAC signaling correlates with melanoma progression. Analysis of The Cancer Genome Atlas 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.
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.
环磷酸腺苷（cAMP）直接激活的交换蛋白（EPAC）属于 RAP 鸟苷酸交换因子（RAPGEF）家族。EPAC1/2（RAPGEF3/4）激活 RAP1 和替代 cAMP 信号通路。我们之前表明，原代和转移性黑素瘤细胞对 cAMP 的不同生长反应是由 EPAC 介导的。然而，导致这种对 EPAC 信号差异反应的机制尚不清楚。在这项研究中，我们表明，通过下调细胞周期蛋白和抑制细胞周期进程（不依赖于 ERK1/2 磷酸化），药理学抑制或 siRNA 介导的 EPAC 敲低选择性地抑制原代黑素瘤细胞的生长和存活。EPAC 抑制导致 AKT 磷酸化上调，但 mTORC1 活性及其下游效应物下调。我们还表明，EPAC 调节糖酵解和氧化磷酸化以及线粒体活性氧的产生，在原代黑素瘤细胞中更优先。我们使用一系列遗传匹配的原发和淋巴结转移（LNM）黑素瘤细胞和远处器官转移黑素瘤细胞，表明 LNM 和转移性黑素瘤细胞对 EPAC 抑制的反应性和耐药性逐渐降低，这表明对 EPAC 信号的依赖性丧失与黑素瘤进展相关。对癌症基因组图谱数据集的分析表明，原发肿瘤中 RAPGEF3、RAPGEF4mRNA 表达降低是原发性黑素瘤患者无病生存更好的预测因子，提示 EPAC 信号促进肿瘤进展，EPAC 是一个有用的预后标志物。这些数据强调了 EPAC 信号作为预防黑素瘤进展的潜在靶点。
本研究确立了对 EPAC-mTORC1 信号的依赖性丧失作为原代黑素瘤进化的标志，靶向这种逃避机制是转移性黑素瘤的一种有前途的策略。
