A PTEN-regulated ubiquitin switch controlling TRAIL sensitivity in GBM

描述(由申请人提供)：这项建议的长期目标是通过确定对死亡配体试验的抵抗途径来改进多形性胶质母细胞瘤(GBM)的治疗。TRAIL是一种促凋亡多肽，可与TRAIL受体结合，诱导肿瘤选择性细胞死亡。尽管许多GBM对TRAIL敏感，但耐药性是常见的，并导致依赖TRAIL杀伤的治疗失败。GBM细胞对TRAIL的耐药与PTEN缺失、Akt激活和抗凋亡蛋白翻转的产生增加有关。然而，PTEN缺失和Akt激活也通过减少泛素化和Flips蛋白的破坏而导致TRAIL耐药。PTEN调控翻转蛋白泛素化的途径尚未被描述，尽管我们已经确定了两种相关蛋白：一种是名为AIP4的E3泛素连接酶，它直接与翻转蛋白相互作用、泛素化并破坏其稳定性；另一种是泛素去除酶USP8，它在PTEN丢失后降解，导致翻转蛋白的稳定。我们目前还不知道这些蛋白是否或如何相互关联并与PTEN相关，也不知道这些蛋白与TRAIL为基础的治疗耐药的相关性。然而，我们知道，在PTEN缺失后，USP8、AIP4和Flips都经历了泛素化的协同变化，并且这些泛素化的变化有可能改变蛋白质的功能(如果泛素作为单个肽或作为通过泛素的赖氨酸63链接的链添加)或稳定性(如果作为通过泛素的赖氨酸48位连接的链添加)。根据我们的初步数据，我们认为PTEN失活引发了一系列泛素反应，最终稳定了翻转并赋予TRAIL抗性。具体地说，我们假设PTEN缺失诱导Akt介导的磷酸化、K48多泛素化和USP8的降解。在缺乏USP8的去泛素化活性的情况下，USP8靶标和E3连接酶AIP4处于不活跃(单一或K63多泛素化)状态，无法泛素化/破坏其靶标翻转，从而导致GBM的TRAIL耐药。为了验证这一假设，我们将1：确定Akt介导的USP8直接磷酸化是否增加USP8泛素化、USP8降解和TRAIL耐药。2：鉴定负责USP8泛素化的Akt依赖的E3连接酶。3：确定Akt激活是否导致不同的泛素化模式，进而导致USP8降解、AIP4失活、翻转稳定和TRAIL抗性；4：确定USP8是否改变AIP4相互作用和/或活性，以及这是否对USP8介导的翻转失稳和TRAIL抗性降低至关重要。公共卫生相关性：由于TRAIL本身是一种很有前途的治疗剂，也是免疫系统和基于免疫的治疗如何消除GBM细胞的重要组成部分，本工作将为如何对患者进行TRAIL或基于免疫的治疗、如何规避GBM中常见的TRAIL抵抗以及如何创建更有效的基于TRAIL的治疗提供见解。本工作还将作为研究PTEN如何控制全球蛋白质稳定性的模板，并将为开发选择性靶向各种DuB/E3对的药物奠定基础，这些药物可以选择性地控制靶蛋白功能和PTEN的肿瘤抑制作用。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to improve the therapy of glioblastoma multiforme (GBM) by defining pathways of resistance to the death ligand TRAIL. TRAIL is a pro-apoptotic peptide that binds TRAIL receptors and induces a tumor-selective cell death. Although many GBM are susceptible to TRAIL, resistance is common, and contributes to the failure of therapies that rely on TRAIL-based killing. TRAIL resistance in GBM cells is associated with PTEN loss, Akt activation, and increased production of the anti-apoptotic protein FLIPs. PTEN loss and Akt activation, however, also contribute to TRAIL resistance by decreasing the ubiquitination and destruction of the FLIPs protein. The means by which PTEN regulates the ubiquitination of FLIPs have not been described, although we have identified two involved proteins; an E3 ubiquitin ligase called AIP4 that directly interacts with, ubiquitinates, and destabilizes FLIPs, and a ubiquitin-removing enzyme called USP8 whose degradation in response to PTEN loss leads to FLIPs stabilization. We do not currently understand if or how these proteins may be linked to each other and to PTEN, nor do we understand the relevance of these proteins for resistance to TRAIL-based therapies. We do know, however, that USP8, AIP4, and FLIPs all undergo co-ordinated changes in ubiquitination following PTEN loss, and that these changes in ubiquitination have the potential to alter protein function (if ubiquitin is added as a single peptide or as chains linked through lysine 63 of ubiquitin) or stability (if added as chains linked through the lysine 48 position of ubiquitin). Based on our preliminary data, we believe that PTEN inactivation sets off a cascade of ubiquitin reactions that ultimately stabilizes FLIPs and confers TRAIL resistance. Specifically we hypothesize that PTEN loss induces an Akt-mediated phosphorylation, K48 polyubiquitination, and degradation of USP8. In the absence of the deubiquitinating activity of USP8, the USP8 target and E3 ligase AIP4 is left in an inactive (mono- or K63-polyubiquitinated) state that is incapable of ubiquitinating/destroying its target FLIPS and that leads to TRAIL resistance in GBM. To test this hypothesis we will 1: Determine if direct Akt-mediated phosphorylation of USP8 increases USP8 ubiquitination, USP8 degradation, and TRAIL resistance. 2: Identify the Akt-dependent E3 ligase responsible for USP8 ubiquitination. 3: determine if Akt activation causes distinct ubiquitination patterns that in turn bring about USP8 degradation, AIP4 inactivation, FLIPS stabilization, and TRAIL resistance, and 4: Determine if USP8 alters AIP4 interactions and/or activity, and if this is critical for USP8-mediated FLIPs destabilization and reduced TRAIL resistance. PUBLIC HEALTH RELEVANCE: Because TRAIL is a promising therapeutic agent on its own, as well as an important component of how the immune system and immune-based therapies eliminate GBM cells, the present work will provide insight as to how to stratify patients for TRAIL- or immune-based therapies, how to circumvent the TRAIL resistance commonly seen in GBM, and in general how to create more effective TRAIL-based therapies. The present work will also serve as a template for investigating how PTEN controls global protein stability, and will set the stage for the development of agents that, by selectively targeting various DUB/E3 pairs, can selectively control target protein function and PTEN tumor suppressor action.