PROJECT 1: Merkel Cell Carcinoma, Merkel Cell Polyomavirus and PP2A (James A. DeCaprio)

项目 1：默克尔细胞癌、默克尔细胞多瘤病毒和 PP2A (James A. DeCaprio)

• 批准号: 10227782
• 负责人: James A. DeCaprio
• 金额: $ 32.64万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227782
• 关键词: Address; Amino Acid Transporter; Amino Acids; Antigens; Cell division; Cells; Collaborations; DNA Double Strand Break; Development; Event; Goals; Human; In Vitro; Intention; Ionizing radiation; Knowledge; Lactate Transporter; Link; Malignant Neoplasms; Mediating; Merkel Cells; Merkel cell carcinoma; Neurosecretory Systems; Nonhomologous DNA End Joining; Oncogenes; Oncogenic; Output; PRKDC gene; Pathway interactions; Polyomavirus; Property; Protein phosphatase; Proteomics; Reporting; Role; Signal Transduction; Simian virus 40; Skin Cancer; Small T Antigen; Starvation; System; Testing; Uncertainty; behavior test; cancer genomics; cell transformation; cytokeratin 20; ds-DNA; effective therapy; insight; mTOR Signaling Pathway; metaplastic cell transformation; mouse polyomavirus; novel; precursor cell; programs; protein function; repaired; response; tumor; tumorigenesis

Summary Merkel cell polyomavirus (MCPyV) is a clear cause of Merkel cell carcinoma (MCC), a highly lethal skin cancer. MCPyV encodes a small T antigen (ST) that is essential for MCC, capable of transforming cells and interacting with protein phosphatase 2A (PP2A). This Project will search for novel insights into the mechanisms by which MCPyV contributes to MCC. A critical barrier to our understanding of how MCPyV contributes to the development of MCC is the lack of appreciation of whether MCPyV T antigens differ in their transforming activities from the canonical SV40 and murine polyomavirus T antigens. A second critical barrier is whether MCPyV ST itself and its interaction with PP2A are required in the development of MCC. A third critical barrier to our understanding of how MCPyV T antigens contribute to oncogenesis is the uncertainty regarding the cell- of-origin for MCC. To address these goals we propose the following Aims. Aim 1. Assess how MCPyV ST activates MTOR signaling pathways to promote transformation. The MTOR pathway is frequently activated in MCPyV-positive MCC. Furthermore, MTOR activation appears to contribute to survival of MCC tumors. Several reports have indicated that MCPyV ST can activate the MTOR downstream targets 4EBP1 and S6K although how it does this is not well understood. We have evidence that MCPyV ST induces expression of certain amino acid transporters. We propose that this effect contributes to activation of MTOR signaling especially during amino acid starvation. In addition, we have observed that MCPyV ST can specifically increase levels of lactate transporters reflecting an increase of intracellular lactate levels and may activate NFκB signaling. We will determine whether increased levels of the amino acid and lactate transporters contribute to MCPyV ST- mediated transformation. Aim 2. Determine the mechanism of inhibition of double stranded DNA break repair by MCPyV ST. We have observed that MCPyV ST can inhibit double stranded DNA break (DSB) repair in response to ionizing radiation (IR). We have also observed that MCPyV ST specifically inhibits non-homologous end joining (NHEJ) by interfering with the activation of DNAPK. We will determine if the ability of MCPyV ST to inhibit DSB repair by perturbing DNAPK and NHEJ activity is linked to perturbation of PP2A. We will determine the specific role of PP2A that is required by MCPyV ST to inhibit DNAPK and NHEJ activation and if this newly described property of MCPyV ST contributes to its overall transforming functions. Aim 3. Test the behavior of MCPyV T Antigens in Merkel cells. We have pursued isolation of primary human Merkel and precursor cells to determine if they are sensitive to transformation by MCPyV T antigens. We will establish an in vitro transformation system using Merkel cells or their precursors to test the role of the MCPyV T antigens in their natural background and clarify the specific oncogenic driver events required to generate an MCC tumor.

摘要 默克尔细胞多瘤病毒(MCPyV)是导致默克尔细胞癌(MCC)的明显原因，MCC是一种高度致命的皮肤 癌症。MCPyV编码一种对MCC至关重要的小T抗原(ST)，能够转化细胞和 与蛋白磷酸酶2A(PP2A)相互作用。这个项目将寻求对机制的新见解 通过MCPyV对MCC的贡献。我们理解MCPyV如何对 MCC的发展是缺乏对MCPyV T抗原在转化过程中是否存在差异的认识 规范的SV40和小鼠多瘤病毒T抗原的活性。第二个关键障碍是 MCPyV ST本身及其与PP2A的相互作用是MCC发展所必需的。第三个关键障碍 我们对MCPyV T抗原如何促进肿瘤发生的理解是关于细胞的不确定性- MCC的原产地。为了实现这些目标，我们提出了以下目标。 目的1.评估MCPyV ST如何激活MTOR信号通路促进转化。这个 在MCPyV阳性的MCC中，mTOR通路经常被激活。此外，MTOR激活似乎可以 有助于MCC肿瘤的生存。一些报告表明，MCPyV ST可以激活MTOR 下游目标是4EBP1和S6K，尽管它是如何做到这一点的还不是很清楚。我们有证据表明 MCPyV ST可诱导某些氨基酸转运蛋白的表达。我们认为，这种效应有助于 MTOR信号的激活，特别是在氨基酸饥饿期间。另外，我们观察到， MCPyV ST可以特异性地增加反映细胞内乳酸水平的乳酸转运体水平 并可能激活核因子κB信号通路。我们将确定增加的氨基酸水平和 乳酸转运蛋白参与了MCPyV-ST介导的转化。 目的2.探讨MCPyV ST抑制双链DNA断裂修复的机制。 我们观察到MCPyV ST可以抑制电离反应中的双链DNA断裂(DSB)修复 辐射(IR)。我们还观察到MCPyV ST特异性地抑制非同源末端连接(NHEJ) 通过干扰DNAPK的激活。我们将确定MCPyV ST是否具有抑制DSB修复的能力 通过干扰DNAPK和NHEJ，活性与PP2A的扰动联系在一起。我们将确定它的具体作用 MCPyV ST抑制DNAPK和NHEJ激活所需的PP2A，如果这一新描述的属性 MCPyV ST对其整体转化功能有一定贡献。 目的3.检测MCPyV T抗原在Merkel细胞中的行为。我们一直在寻求隔离主要的 以确定它们是否对MCPyV T抗原的转化敏感。 我们将建立一个使用默克尔细胞或其前体细胞的体外转化系统，以测试 MCPyV T抗原的自然背景，并阐明所需的特定致癌驱动事件 会产生MCC肿瘤。