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

总结 默克尔细胞多瘤病毒（MCPyV）是默克尔细胞癌（MCC）的明确病因，MCC是一种高致死性皮肤癌， 癌MCPyV编码MCC所必需的小T抗原（ST），能够转化细胞， 与蛋白磷酸酶2A（PP 2A）相互作用。该项目将寻找新的见解的机制 MCPyV对MCC的贡献。这是我们理解MCPyV如何促进 MCC的发展是缺乏对MCPyV T抗原在它们的转化中是否不同的评价， 来自典型SV 40和鼠多瘤病毒T抗原的活性。第二个关键障碍是， MCPyV ST本身及其与PP 2A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- MCC的原产地。为了实现这些目标，我们提出以下目标。 目标1.评估MCPyV ST如何激活MTOR信号通路以促进转化。的 MTOR通路在MCPyV阳性MCC中频繁激活。此外，MTOR激活似乎 有助于MCC肿瘤的存活。多份报告表明，MCPyV ST可激活MTOR 下游靶向4 EBP 1和S6 K，但其如何做到这一点还不清楚。我们有证据表明 MCPyV ST诱导某些氨基酸转运蛋白的表达。我们认为，这种影响有助于 MTOR信号转导的激活，特别是在氨基酸饥饿期间。此外，我们注意到， MCPyV ST可以特异性地增加乳酸转运蛋白的水平，反映细胞内乳酸的增加 并可能激活NFκB信号。我们将确定是否增加的氨基酸水平， 乳酸转运蛋白有助于MCPyV ST介导的转化。 目标二。确定MCPyV ST抑制双链DNA断裂修复的机制。 我们已经观察到MCPyV ST可以抑制双链DNA断裂（DSB）修复， 辐射（IR）。我们还观察到MCPyV ST特异性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抑制DSB修复的能力是否 通过干扰DNAPK和NHEJ活性，与PP 2A的干扰有关。我们将确定具体的作用， MCPyV ST抑制DNAPK和NHEJ活化所需的PP 2A，并且如果这种新描述的特性 的MCPyV ST有助于其整体转化功能。 目标3。检测MCPyV T抗原在默克尔细胞中的行为。我们一直在寻求初级隔离 人默克尔和前体细胞，以确定它们是否对MCPyV T抗原的转化敏感。 我们将利用默克尔细胞或其前体细胞建立体外转化系统，以测试 MCPyV T抗原在其天然背景中的表达，并阐明了 产生MCC肿瘤。

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.