Role of EPAC Signaling in Melanoma Progression

摘要 皮肤黑色素瘤源于皮肤中的黑素细胞。暴露于太阳紫外线(UV)辐射是一种 重大风险因素。美国军事人员部署在世界各地紫外线指数较高的地区，从而 在服兵役期间和服兵役后增加患黑色素瘤的风险。如早期诊断，可早期切除原发灶 皮肤可以治愈。然而，随着黑色素瘤的转移，五年存活率急剧下降。 转移到局部和区域的淋巴结和远处的器官。尽管最近取得的成功和有针对性的承诺 治疗和免疫治疗、早期诊断和治疗局部原发癌仍然是最好的 降低癌症死亡率的策略。因此，对所涉及的分子机制有详细的了解 在黑色素瘤中，肿瘤进展是准确可靠的诊断和治疗侵袭性疾病的关键 皮肤黑色素瘤。环磷酸腺苷(CAMP)信号通路在黑素细胞增殖和凋亡中起关键作用 差异化。该途径是在腺苷酸催化活性升高细胞内cAMP的基础上启动的。 通过蛋白激酶A和转录因子CREB转导的环化酶最终导致 CAMP反应基因，包括黑素细胞主调节因子MITF。CAMP信号通路的作用 在黑色素瘤中的作用还不完全清楚。采用人类原发黑色素瘤组织微阵列(TMA)，小组 原代和转移性黑色素瘤细胞系及BRAFV600E/Pten-/-小鼠黑色素瘤模型 证明了cAMP信号在原代和转移性黑色素瘤细胞中扮演相反的角色，即作为一种促进- 原代细胞为增殖信号，转移细胞为抗增殖信号。我们还展示了这个开关 CAMP的作用是由涉及EPAC-RAP1(交换因子)的另一个cAMP信号轴介导的 由cAMP-RAS相关蛋白1直接激活)。更多的研究表明EPAC促进细胞周期 进展，激活mTORC1信号，调节细胞代谢和线粒体ROS的产生 选择性地存在于原代黑色素瘤细胞中。此外，使用一组原发和转移性黑色素瘤的研究 来自同一患者的细胞系显示EPAC功能的转换是渐进性的 提示EPAC是原发黑色素瘤生长的关键成分 肿瘤进展过程中信号网络的重新布线消除了转移性疾病对EPAC的依赖 黑色素瘤。在这里，我们建议检验如下假设：a)黑素细胞的致癌转化激活 EPAC信号和EPAC作为一个关键的信号节点抑制应激诱导的ROS的产生，促进 衰老逃逸和原发黑色素瘤的发展，以及b)基因表达的变化和代谢 在转移过程中发生的适应逐渐消除了对EPAC的依赖。这样做的具体目的是 应用是：1)建立EPAC在黑色素瘤发生和发展中的作用，2)确定 EPAC的生化和分子作用机制及3)EPAC在代谢中的作用 黑色素瘤进展过程中的适应。这里提出的实验的成功完成可能会导致 识别可靶向抑制黑色素瘤进展和治疗转移的途径和蛋白 黑色素瘤。因此，我们相信这个项目将在黑色素瘤领域产生持续的影响。

ABSTRACT Cutaneous melanoma arises from melanocytes in the skin. Exposure to solar ultraviolet (UV) radiation is a significant risk factor. Deployment of US military personnel around the world in areas of high UV index thus increases risk of melanoma during and after military service. If diagnosed early, excision of the primary tumor in the skin can be curative. However, the five-year survival rates decrease precipitously as melanoma metastasizes to local and regional lymph nodes and distant organs. Despite the recent successes and promise of targeted therapies and immunotherapy, early diagnosis and treatment of the localized primary cancer remain the best strategies to reduce cancer mortality. Therefore, a detailed understanding of the molecular mechanisms involved in melanoma tumor progression is critical for accurate and reliable diagnosis and treatment of aggressive cutaneous melanoma. Cyclic AMP (cAMP) signaling pathway plays a critical role in melanocyte proliferation and differentiation. This pathway is initiated upon elevation of intracellular cAMP by the catalytic activity of adenylyl cyclases and transduced through protein kinase A and transcription factor CREB culminating in expression of cAMP-responsive genes including the melanocyte master regulator, MITF. The role for cAMP signaling pathway in melanoma is not fully understood. Employing a human primary melanoma tissue microarray (TMA), a panel primary and metastatic melanoma cell lines and the BrafV600E/Pten-/- mouse model for melanoma, we recently demonstrated that cAMP signaling plays opposite roles in primary and metastatic melanoma cells, i. e., as a pro- proliferative signal in primary and as anti-proliferative signal in metastatic cells. We also showed that this switch in the cAMP of action is mediated by the alternative cAMP signaling axis involving EPAC-RAP1 (Exchange factor directly activated by cAMP- Ras-related protein 1). Additional studies showed that EPAC promotes cell cycle progression, activates mTORC1 signaling, and regulates cellular metabolism and mitochondrial ROS production selectively in primary melanoma cells. Furthermore, studies using a panel of primary and metastatic melanoma cell lines derived from the same patient revealed that the switch in the functions of EPAC occurs progressively during melanoma metastasis suggesting that EPAC is a critical component for the growth of primary melanoma and that rewiring of signaling networks during tumor progression abolish EPAC dependency in metastatic melanoma. Here, we propose to test the hypotheses that a) oncogenic transformation of melanocytes activates EPAC signaling and EPAC acts as a critical signaling node to suppress stress induced ROS generation, promote senescence escape and primary melanoma development, and b) gene expression changes and metabolic adaptions that occur during metastasis progressively abolish EPAC dependency. The specific aims of this application are: 1) Establish the role of EPAC in melanoma tumorigenesis and progression, 2) Determine the biochemical and molecular mechanisms of action of EPAC and 3) Investigate the role of EPAC in metabolic adaptation during melanoma progression. Successful completion of experiments proposed here could lead to identification of pathways and proteins that can be targeted to inhibit melanoma progression and treat metastatic melanoma. Therefore, we believe this project will have sustained impact in the field of melanoma.