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

计划2 项目摘要 细胞外膜囊泡如外来体和膜突起如 细胞间运输网络中的隧道纳米管为理解肿瘤开辟了新的视角 发展和进步。泛素系统是生物发生和功能的中心调节器之一 外泌体和纳米管，以及包括EB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而使细胞功能失调。最近的研究 多年的研究清楚地表明，小进化保守家族的潜在功能谱 泛素C-末端水解酶（UCH）的作用范围比人们所怀疑的要广得多。其中，UCH-11为 特别感兴趣：最近的研究，包括我们的研究表明，这种独特的去泛素化酶密切参与， 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.直接基于我们以前的研究和大量的初步数据，我们假设UCH-L1是一个 EBV阳性癌症中细胞内和细胞间运输的泛素依赖性过程的主要调节剂。 在目的一，我们将分析如何不同的生化功能的UCH-L1所需的外来体生物发生 以及EBV主要癌基因潜伏膜蛋白1（LMP 1）如何参与这些过程。 流程.在Aim II中，基于我们最近发现N-钙粘蛋白在EBV驱动的B细胞中高度表达， 淋巴瘤，并与UCH-L1在这些细胞中共定位，我们将探讨基于N-钙粘蛋白的作用。 在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 本研究的结果将阐明EBV转化细胞如何产生隧道纳米管和外泌体 通过将促侵袭因子转移到肿瘤间质组织来改变肿瘤微环境。在Aim III中， 将研究是否抑制UCH-L1活性与特定的小分子抑制剂，具有抗- 在细胞培养中的致瘤作用也将在体内对人源化细胞中的EBV B细胞淋巴发生有活性。 小鼠我们将确定UCH-L1 DUB活性是否是EBV诱导的正常人永生化所必需的。 人B细胞，以及UCH-L1的抑制是否影响潜伏性EBV基因的表达和功能。 感染细胞以及病毒再激活期间。EBV与几种高度侵袭性的恶性肿瘤密切相关， 淋巴和上皮来源;治疗这些恶性肿瘤患者面临独特的挑战， 结果仍然很差。由于最近的研究已经证明了这种UCH-L1的深刻的抗转移作用 抑制剂在侵袭性癌小鼠模型中，UCH-L1酶活性的特异性抑制剂可提供 辅助现有疗法。

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.