Targeting the APC/Cdc20 E3 ubiquitin ligase for chemoradiation sensitization

摘要：（不超过30行） 异常的泛素化过程与包括癌症在内的各种人类疾病密切相关。的 后期促进复合物（APC）通过与细胞膜结合来调节细胞周期进程 激活蛋白Cdh 1和Cdc 20在不同细胞周期阶段促进APC依赖性泛素化， 破坏其基质。最近的研究表明，APC Cdc 20是一个有吸引力的抗癌靶点， Cdc 20的缺失部分地通过触发有丝分裂停滞和随后的凋亡抑制体内肿瘤发生。 凋亡我们最近报道了促凋亡蛋白Bim作为一种新的APCCdc 20底物， 支持Cdc 20是致癌基因的观点。然而，Cdc 20如何由上游调节器控制 以及Cdc 20-Bim信号轴如何在体内促进放化疗抗性仍在很大程度上 没有地址我们的初步结果表明，CUL 3SPOP E3连接酶特异性靶向Cdc 20， 泛素化和随后的破坏以减弱其致癌活性。此外，我们证明 APCCdc 20以D-box依赖的方式控制Bim的泛素化和随后的破坏。 值得注意的是，通过表达Tax-病毒介导的APCCdc 20蛋白而获得升高的APCCdc 20活性的人成人T细胞白血病细胞， 癌蛋白，表现出降低的Bim水平和对抗癌试剂的相应抗性。相反地， 在各种人类癌细胞系中Cdc 20的耗尽使它们对各种化疗药物的反应敏感 毒品更重要的是，Cdc 20和多个APC核心组分在siRNA筛选中被鉴定， 在敲低后，以Bim依赖性方式使头颈癌细胞对γ-辐射敏感。基于 根据我们的初步数据，我们假设SPOP是Cdc 20稳定性的一种新的上游负调节因子， APCCdc 20负调控细胞凋亡途径的诱导，以响应抗- 有丝分裂剂和γ-辐射部分通过靶向Bim进行泛素化和破坏。我们将研究 通过实现以下三个具体目标来实现我们的假设。在目标#1中，我们将确定分子 SPOP介导的Cdc 20泛素化和降解的潜在机制，并检查是否 头颈部癌中SPOP缺乏导致肿瘤发生升高，部分原因是Cdc 20积累。我们 目标#2中提出的工作将确定APCCdc 20在控制细胞凋亡中的生理作用， 体内通过调节Bim和其他Cdc 20底物的丰度。这些研究将有助于 进一步了解抗有丝分裂剂诱导细胞凋亡的分子机制，并提供 开发特异性Cdc 20抑制剂作为有效抗癌疗法的基本原理。在目标#3中，我们将 验证APCCdc 20 E3连接酶作为新的放射增敏靶点。这些研究将 显著扩展了我们目前对Cdc 20缺失如何发挥其抗肿瘤生物学功能的认识 通过控制比姆的数量我们提出的研究将为设计 通过放射靶向Cdc 20以实现癌症患者更好的临床治疗的新策略。

Abstract: (no more than 30 lines) Aberrant ubiquitination processes are tightly associated with various human diseases including cancer. The Anaphase Promoting Complex (APC) has been documented to regulate cell cycle progression through binding activator proteins Cdh1 and Cdc20 at different cell cycle stages to promote APC-dependent ubiquitination and destruction of their substrates. Recent studies revealed that APCCdc20 is an attractive anti-cancer target and depletion of Cdc20 suppresses in vivo tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. We have recently reported the pro-apoptotic protein Bim as a novel APCCdc20 substrate, whichF supports the notion that Cdc20 is an oncogene. However, how Cdc20 is controlled by upstream regulator(s) and how the Cdc20-Bim signaling axis contributes to chemoradio-resistance in vivo remains largely unaddressed. Our preliminary results showed that the CUL3SPOP E3 ligase specifically targets Cdc20 for ubiquitination and subsequent destruction to attenuate its oncogenic activity. Furthermore, we demonstrated that APCCdc20 governs the ubiquitination and subsequent destruction of Bim in a D-box-dependent manner. Notably, human Adult T-cell Leukemia cells that acquire elevated APCCdc20 activity via expressing the Tax-viral- oncoprotein, exhibit reduced Bim levels and corresponding resistance to anti-cancer reagents. Conversely, depletion of Cdc20 in various human cancer cell lines sensitized their response to various chemotherapeutic drugs. More importantly, Cdc20 and multiple APC-core components were identified in an siRNA screen that upon knockdown sensitizes head-and-neck-cancer cells to γ-irradiation in a Bim-dependent manner. Based on our preliminary data, we hypothesize that SPOP is a novel upstream negative regulator of Cdc20 stability and oncogenic functions, and APCCdc20 negatively governs the induction of apoptotic pathway in response to anti- mitotic agents and γ-irradiation in part through targeting Bim for ubiquitination and destruction. We will examine our hypothesis by accomplishing the following three specific aims. In Aim #1, we will determine the molecular mechanisms underlying SPOP-mediated ubiqutination and degradation of Cdc20 and to examine whether SPOP deficiency in head and neck cancer leads to elevated tumorigenesis in part by Cdc20 accumulation. Our proposed work in Aim #2 will determine the physiological role of APCCdc20 in governing cellular apoptosis in vivo by regulating the abundance of Bim and other Cdc20 substrates. These proposed studies would help to further understand the molecular mechanisms underlying anti-mitotic agents-induced apoptosis, and provide the rationale for developing specific Cdc20 inhibitors as effective anti-cancer therapies. In Aim #3, we will validate the APCCdc20 E3 ligase as a novel radio-sensitizing target. Taken together, these studies will significantly extend our current knowledge of how depletion of Cdc20 exerts its anti-tumor biological functions through controlling the abundance of Bim. Our proposed studies will provide the molecular basis for designing novel strategies to target Cdc20 by radiation for achieving better clinical treatment of cancer patients.