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。这个 后期促进复合体(APC)通过结合调节细胞周期进程 激活蛋白CDH1和CDC20在不同细胞周期阶段促进APC依赖的泛素化和 破坏它们的底物。最近的研究表明，APCCDC20是一个有吸引力的抗癌靶点， 缺失CDC20抑制体内肿瘤的发生部分是通过触发有丝分裂停止和随后的 细胞凋亡。我们最近报道了促凋亡蛋白Bim作为一种新的APCCdc20底物，它是F 支持Cdc20是致癌基因的观点。然而，cdc20是如何被上游监管机构控制的(S) 而在体内，cdc20-bim信号轴是如何在化疗放射抵抗中起作用的仍很大程度上仍未得到证实。 未寻址。我们的初步结果表明，CUL3SPOP E3连接酶特异性地针对CDC20 泛素化和随后的破坏以减弱其致癌活性。此外，我们还演示了 APCCDc20以D-box依赖的方式调控Bim的泛素化和随后的破坏。 值得注意的是，成人T细胞白血病细胞通过表达Tax病毒而获得升高的APCCDc20活性。 癌蛋白，表现出降低的Bim水平和相应的抗癌试剂耐药性。相反， 不同人类癌细胞株中CDC20的缺失使其对不同化疗药物的反应敏感 毒品。更重要的是，在siRNA筛选中鉴定出了CDC20和多个APC-核心成分 基因敲除后，头颈部癌细胞以一种Bim依赖的方式对γ辐射敏感。基于 我们的初步数据，我们假设SPOP是一种新型的上游负调节分子，对CdC20的稳定性和 APCCDC20负向调控抗肿瘤诱导细胞凋亡途径 有丝分裂因子和γ辐射部分通过靶向BIM进行泛素化和破坏。我们将研究 我们的假设是通过实现以下三个具体目标来实现的。在目标1中，我们将确定分子 SPOP介导的CDC20泛量化和降解的潜在机制，并研究 头颈癌中SPOP缺乏导致肿瘤形成增加，部分原因是由于镉蓄积。我们的 Aim#2中提议的工作将确定APCCDc20在调控细胞凋亡中的生理作用。 通过调节Bim和其他CdC20底物的丰度。这些拟议的研究将有助于 进一步了解抗有丝分裂药物诱导细胞凋亡的分子机制，并提供 开发特定的cdc20抑制剂作为有效的抗癌疗法的基本原理。在目标3中，我们将 验证APCCDC20 E3连接酶作为新的放射增敏靶点的有效性。总而言之，这些研究将 极大地扩展了我们目前对耗尽CDC20如何发挥其抗肿瘤生物学功能的了解 通过控制Bim的丰度。我们提出的研究将为设计提供分子基础 通过辐射靶向cdc20的新策略，以实现癌症患者更好的临床治疗。

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.