Molecular mechanisms of TRAIL resistance in glioblastoma

描述（由申请人提供）：胶质母细胞瘤是最常见和致命的脑肿瘤，目前尚无治愈方法。为了寻找新的治疗药物，我们已经证明TRAIL （tumor necrosis factor-related apoptosis-inducing ligand，肿瘤坏死因子相关凋亡诱导配体）可以诱导胶质母细胞瘤细胞的程序性细胞死亡（apoptosis），这可能为治愈胶质母细胞瘤提供了新的希望。TRAIL因此进入了癌症治疗的临床试验阶段。不幸的是，早期的试验报告显示，TRAIL的抗肿瘤活性有限，并表明包括胶质母细胞瘤在内的大多数人类癌症对TRAIL具有耐药性。因此，目前的研究计划将研究胶质母细胞瘤中TRAIL耐药的分子基础。trail诱导的细胞凋亡是通过与死亡受体DR5结合并募集caspase-8到DR5形成DISC（死亡诱导信号复合体）而发生的。在DISC中，caspase-8形成二聚体，然后在程序性细胞死亡的启动过程中变得具有蛋白水解活性。我们的初步研究表明，在trail抗性胶质母细胞瘤细胞中，caspase-8二聚体和裂解被抑制。为了寻找caspase-8抑制的机制，我们发现了在TRAIL治疗之前形成的dr5相关蛋白复合物，因此我们将其命名为placc（预配体组装复合物）。我们的初步研究表明，placc组分定义了DISC的后续形成，从而控制了caspase-8的二聚化和裂解。具体来说，初步研究在PLAC中发现了RIP（受体相互作用蛋白）和A20（一种RIP泛素酶），并表明TRAIL处理在TRAIL诱导的PLAC到DISC形成过程中导致RIP泛素化和caspase-8抑制。因此，我们假设a20介导的RIP泛素化抑制caspase-8二聚体和切割。为了验证这一假设，我们将定义A20作为RIP泛素连接酶的作用，确定RIP附着的多泛素链，鉴定caspase-8上的多泛素结合域。此外，我们将从患者的胶质母细胞瘤中生成小鼠异种移植物和原代培养物，确定A20在肿瘤中是否过表达，从而确定肿瘤中TRAIL的耐药性。本研究完成后，将确定A20为治疗靶点，开发针对A20抑制机制的联合疗法，克服肿瘤对TRAIL治疗的耐药，从而造福人类健康。

DESCRIPTION (provided by applicant): Glioblastoma is the most common and lethal brain tumor for which there is no curative treatment. In search for new therapeutic agents, we have shown that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can induce programmed cell death (apoptosis) in glioblastoma cells and it may offer a new hope for a cure. TRAIL has therefore entered the clinical trials for cancer treatment. Earlier reports from the trials, unfortunately, have shown a limited antitumor activity and indicated that the majority of human cancers including glioblastomas are resistant to TRAIL. The current research proposal will therefore examine the molecular basis of TRAIL resistance in glioblastomas. TRAIL-induced apoptosis occurs through binding to the death receptor DR5 and the recruitment of caspase-8 to DR5 for the formation of DISC (death-inducing signaling complex). In the DISC, caspase-8 forms dimers and then becomes proteolytically active in the initiation of the programmed cell death. Our Preliminary Studies show that the caspase-8 dimerization and cleavage are inhibited in TRAIL-resistant glioblastoma cells. In search for the mechanism in the caspase-8 inhibition, we have discovered a DR5-associated proteins complex that is formed prior to TRAIL treatment and we therefore name the PLAC (pre-ligand assembly complex). Our Preliminary Studies show that the PLAC components define the subsequent formation of the DISC and thus control caspase-8 dimerization and cleavage. Specifically, the Preliminary Studies identify RIP (receptor-interacting protein) and A20, a RIP ubiquitin enzyme in the PLAC and show that TRAIL treatment leads to the RIP ubiquitination and caspase-8 inhibition during the TRAIL-induced formation from the PLAC to the DISC. We therefore hypothesize that A20-mediated RIP ubiquitination inhibits caspase-8 dimerization and cleavage. To test this hypothesis, we will define the role of A20 as the RIP ubiquitin ligase, determine the polyubiquitin chain attached to RIP and identify the polyubiquitin binding domain on caspase-8. In addition, we will generate mouse xenografts and primary cultures from patient's glioblastomas and determine whether A20 is overexpressed in the tumors and thus defines the TRAIL resistance in the tumors. Upon the completion, this study will identify A20 as a therapeutic target and thus benefit human health by developing the combination therapy that can target A20 inhibitory mechanisms to overcome the tumor resistance to TRAIL treatment.