Project 2 - Membrane trafficking in EBV malignancies: Implication of deubiquitinase UCH-L1

项目2 项目概要 摘要 细胞外膜囊泡（例如外泌体）和膜突起（例如 细胞间运输网络中的纳米管隧道为理解肿瘤开辟了新视角 发展和进步。泛素系统是生物发生和功能的中央调节因子之一 外泌体和纳米管的形成，以及包括 Epstein-Barr 病毒 (EBV) 在内的肿瘤病毒感染导致 在细胞转化过程中通过操纵该系统来解除细胞功能的管制。近年来的研究 多年来清楚地表明，进化上保守的小家族的潜在功能范围 泛素 C 末端水解酶 (UCH) 的范围比想象的要宽得多。其中，UCH-l1为 特别感兴趣：包括我们在内的最近研究表明，这种独特的去泛素酶与 不仅在细胞转化和原发性肿瘤形成中，而且是癌症进展的主要调节因子 好吧。直接根据我们之前的研究和大量初步数据，我们假设 UCH-L1 是 EBV 阳性癌症细胞内和细胞间运输的泛素依赖性过程的主要调节因子。 在目标 I 中，我们将分析外泌体生物发生如何需要 UCH-L1 的独特生化功能 和分选，以及 EBV 主要癌基因潜膜蛋白 1 (LMP1) 如何参与这些过程 流程。在 Aim II 中，基于我们最近发现 N-cadherin 在 EBV 驱动的 B 细胞中高表达 淋巴瘤，并与这些细胞中的 UCH-L1 共定位，我们将探讨基于 N-钙粘蛋白的作用 EBV 阳性癌细胞产生的促转移因子的细胞间运输中的复合物。的 该目标的结果将阐明 EBV 转化细胞产生的纳米管和外泌体是如何隧道化的 通过将促侵袭因子转移到肿瘤基质组织来改变肿瘤微环境。在目标 III 中，我们 将研究是否用具有抗-的特定小分子抑制剂抑制 UCH-L1 活性 细胞培养物中的致瘤作用也将在人源化体内针对 EBV B 细胞淋巴生成发挥作用 老鼠。我们将确定 EBV 诱导的正常永生化是否需要 UCH-L1 DUB 活性。 人类 B 细胞，以及抑制 UCH-L1 是否会潜在影响 EBV 基因的表达和功能 受感染的细胞以及病毒重新激活期间。 EBV 与多种高侵袭性恶性肿瘤密切相关 淋巴和上皮起源；患有这些恶性肿瘤的患者的治疗提出了独特的挑战，并且 结果仍然很差。最近的研究证明了这种 UCH-L1 具有深远的抗转移作用 作为浸润性癌小鼠模型中的抑制剂，UCH-L1 酶活性的特异性抑制剂可能提供 现有疗法的辅助。

Project 2 Project Summary Abstract The implication of extracellular membrane vesicles such as exosomes, and membrane protrusions such as tunneling nanotubes, in intercellular trafficking networks opens a new perspective in understanding tumor development and progression. The ubiquitin system is one of the central regulators of biogenesis and function of exosomes and nanotubes, and infection with tumor viruses including Epstein-Barr Virus (EBV) results in deregulation of cellular functions by manipulation of this system during cell transformation. Studies in recent years clearly demonstrate that the spectrum of potential functions of the small evolutionarily conserved family of Ubiquitin C-terminal Hydrolases (UCHs) is much wider than was suspected. Among them, UCH-l1 is of special interest: recent studies including ours demonstrate that this unique deubiquitinase is closely involved not only in cell transformation and in primary tumor formation, but is a main regulator of cancer progression as well. Based directly on our previous studies and substantial preliminary data, we hypothesize that UCH-L1 is a major regulator of ubiquitin-dependent processes of intra- and inter-cellular trafficking in EBV-positive cancers. In Aim I, we will analyze how distinct biochemical functions of UCH-L1 are required for exosome biogenesis and sorting, and how the EBV major oncogene, Latent Membrane Protein 1 (LMP1), is involved in these processes. In Aim II, based on our recent discovery that N-cadherin is highly expressed in EBV-driven B-cell lymphomas, and co-localizes with UCH-L1 in these cells, we will explore the role of N-cadherin-based complexes in intercellular trafficking of pro-metastatic factors produced by EBV-positive cancer cells. The results in this Aim will clarify how tunneling nanotubes and exosomes produced by EBV-transformed cells change the tumor microenvironment by transferring pro-invasive factors to tumor stromal tissues. In Aim III we will investigate whether inhibition of UCH-L1 activity with specific small-molecule inhibitors that have anti- tumorigenic effects in cell culture will also be active in vivo against EBV B-cell lymphogenesis in humanized mice. We will determine whether UCH-L1 DUB activity is required for EBV-induced immortalization of normal human B-cells, and whether inhibition of UCH-L1 affects expression and function of EBV genes in latently infected cells as well as during viral reactivation. EBV is tightly linked to several highly invasive malignancies of lymphoid and epithelial origin; treatment of patients with these malignancies poses unique challenges, and outcomes remain poor. Since recent study has demonstrated a profound anti-metastatic effect of such UCH-L1 inhibitor in a mouse model of invasive carcinoma, specific inhibitors of UCH-L1 enzymatic activity may offer an adjunct to existing therapies.