A PTEN-regulated ubiquitin switch controlling TRAIL sensitivity in GBM

描述（由申请人提供）：本提案的长期目标是通过确定对死亡配体TRAIL的抗性途径来改善多形性胶质母细胞瘤（GBM）的治疗。TRAIL是结合TRAIL受体并诱导肿瘤选择性细胞死亡的促凋亡肽。尽管许多GBM对TRAIL敏感，但耐药性是常见的，并且导致依赖于基于TRAIL的杀伤的疗法失败。GBM细胞中的TRAIL抗性与PTEN损失、Akt活化和抗凋亡蛋白FLIPs的产生增加相关。然而，PTEN丢失和Akt活化也通过减少FLIPs蛋白的泛素化和破坏而导致TRAIL抗性。PTEN调控FLIP泛素化的方式尚未描述，尽管我们已经鉴定了两种相关蛋白;称为AIP 4的E3泛素连接酶直接与FLIP相互作用，泛素化并使其不稳定，以及称为USP 8的泛素去除酶，其响应于PTEN损失的降解导致FLIP稳定。我们目前还不了解这些蛋白质是否或如何相互联系以及与PTEN联系，我们也不了解这些蛋白质与对基于TRAIL的疗法的抗性的相关性。然而，我们确实知道，USP 8、AIP 4和FLIP在PTEN丢失后都经历了泛素化的协调变化，并且这些泛素化的变化有可能改变蛋白质功能（如果泛素作为单个肽或通过泛素的赖氨酸63连接的链添加）或稳定性（如果作为通过泛素的赖氨酸48位置连接的链添加）。基于我们的初步数据，我们认为PTEN失活引发了一系列泛素反应，最终稳定了FLIP并赋予TRAIL抗性。具体地说，我们假设，PTEN的损失诱导Akt介导的磷酸化，K48聚泛素化，和降解的USP 8。在缺乏USP 8的去泛素化活性的情况下，USP 8靶标和E3连接酶AIP 4处于无活性（单或K63-聚泛素化）状态，其不能泛素化/破坏其靶标FLIPS并导致GBM中的TRAIL抗性。为了验证这一假设，我们将1：确定直接Akt介导的USP 8磷酸化是否增加USP 8泛素化，USP 8降解和TRAIL抗性。2：鉴定负责USP 8泛素化的Akt依赖性E3连接酶。第三节：确定Akt激活是否引起不同的泛素化模式，其进而引起USP 8降解、AIP 4失活、FLIPS稳定化和TRAIL抗性，以及4：确定USP 8是否改变AIP 4相互作用和/或活性，以及这是否对于USP 8介导的FLIP去稳定化和降低的TRAIL抗性至关重要。公共卫生相关性：由于TRAIL本身是一种有前途的治疗剂，也是免疫系统和免疫疗法消除GBM细胞的重要组成部分，因此本研究将为如何对患者进行基于TRAIL或免疫疗法的分层，如何规避GBM中常见的TRAIL耐药性以及如何创建更有效的基于TRAIL的疗法提供见解。目前的工作也将作为一个模板，用于调查如何PTEN控制全球蛋白质的稳定性，并将设置阶段的开发代理，通过选择性地针对各种DUB/E3对，可以选择性地控制目标蛋白质的功能和PTEN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.