Oncogene c-Myc-dependent pathways in cutaneous melanoma

描述（由申请人提供）：恶性黑色素瘤是最具侵袭性的人类癌症类型之一。它的转移能力，加上对传统抗癌化疗的耐药性，使得黑色素瘤极难治愈，因此，转移性黑色素瘤患者的中位生存期为8.5个月。黑色素瘤进展的分子机制尚不完全清楚，与肿瘤进展的特定阶段相关的良好标志物尚未确定。据报道，在黑色素瘤中致癌基因C-MYC表达升高，尽管关于该事件发生在哪个阶段仍存在争议。我们发现，在大多数转移性黑色素瘤样本和黑色素瘤细胞系中，大量的C-MYC维持在蛋白质水平。我们建议的总体假设是恶性黑色素瘤的生存能力取决于高水平的C-MYC。我们证明，在一些黑色素瘤细胞系中，高C-MYC水平和蛋白质稳定性不能完全通过激活MAPK-ERK途径来解释，并且抑制参与C-MYC蛋白体降解的因素可能参与了这些细胞中C-MYC水平和蛋白质稳定性的上调。表达分析发现，在促进C-MYC蛋白降解的蛋白中，与正常黑色素细胞相比，PP2A磷酸酶复合物的B56a亚基在所有测试的黑色素瘤细胞系中表达不足。此外，黑色素瘤细胞中PP2A-B56a亚基的抑制导致c-MYC水平上调。为了确定myc靶基因对黑色素瘤细胞活力的限制率，我们通过慢病毒为基础的shRNA稳定地抑制了几种人类黑色素瘤细胞系中的C-MYC。C-MYC的缺失导致细胞凋亡和/或增殖抑制，同时细胞内脱氧核糖核苷三磷酸酯（dNTPs）的数量减少，以及参与dNTP代谢的几种限速酶的下调。重要的是，在细胞培养基中添加核苷酸生物合成前体可以部分减轻C-MYC缺失引起的凋亡/细胞抑制效应。因此，我们提出i)确定升高的C-MYC蛋白水平是否与黑色素瘤进展的特定阶段相关，ii)功能表征PP2A-B56a在C-MYC依赖性黑素细胞转化和C-MYC介导的黑色素瘤细胞活力维持中的作用。iii)功能表征所鉴定的酶参与myc介导的核苷酸代谢控制，iv)使用生化分析方法鉴定myc缺失细胞中受影响的核苷酸代谢的限速步骤。

DESCRIPTION (provided by applicant): Malignant melanoma is 1 of the most aggressive types of human cancer. Its ability to metastasize, in combination with resistance to conventional anticancer chemotherapy, makes melanoma extremely difficult to cure and consequently, the median survival of metastatic melanoma patients is 8.5 months. Molecular mechanisms underlying melanoma progression are not completely understood, and good markers associated with specific stage(s) of tumor progression are yet to be determined. Elevated expression of the oncogene C-MYC was reported in melanoma, although a controversy remains as to what stage(s) this event occurs. We discovered that high amounts of C-MYC are maintained at the protein level in the majority of metastatic melanoma samples and melanoma cell lines. The overall hypothesis of our proposal is that viability of malignant melanoma depends on high levels of C-MYC. We demonstrated that high C-MYC levels and protein stability in several melanoma cell lines could not be fully accounted for by activation of MAPK-ERK pathway, and that inhibition of factors involved in proteosomal degradation of C-MYC is likely to participate in upregulation of C-MYC levels and protein stability in these cells. Expression analysis identified that among proteins promoting C-MYC proteolitic degradation, B56a subunit of PP2A phospahtase complex was underexpressed in all tested melanoma cell lines compared to normal melanocytes. Additionally, inhibition of PP2A-B56a subunit in melanoma cells caused upregulation of c-MYC levels. To identify MYC-target genes rate-limiting for melanoma cell viability, we stably inhibited C-MYC in several human melanoma cell lines by lentivirus-based shRNA. Depletion of C-MYC resulted in apoptosis and/or inhibition of proliferation accompanied by decreased amounts of intracellular deoxyribonucleoside triphopshates (dNTPs) and downregulation of several rate-limiting enzymes involved in dNTP metabolism. Importantly, apoptotic/cytostatic effects caused by C-MYC depletion were partially alleviated by supplementing cell media with precursors of nucleotide biosynthesis. We therefore propose to i) determine whether elevated C-MYC protein levels are associated with a specific stage of melanoma progression and ii) functionally characterize the role of PP2A-B56a in C-MYC-dependent melanocytic transformation and in C-MYC-mediated maintenance of viability of melanoma cells. iii) functionally characterize involvement of the identified enzymes in MYC-mediated control of the nucleotide metabolism and iv)identify rate-limiting steps of nucleotide metabolism affected in MYC-depleted cells using methods of biochemical analysis.