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，目前尚无治愈性治疗。在寻找新的治疗药物，我们已经表明，TRAIL（肿瘤坏死因子相关的凋亡诱导配体）可以诱导程序性细胞死亡（凋亡）的胶质母细胞瘤细胞，它可能会提供一个新的希望治愈。因此，TRAIL已进入癌症治疗的临床试验。不幸的是，来自这些试验的早期报告显示出有限的抗肿瘤活性，并表明大多数人类癌症包括胶质母细胞瘤对TRAIL具有抗性。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诱导的细胞凋亡通过与死亡受体DR 5结合并将半胱天冬酶-8募集至DR 5以形成DISC（死亡诱导信号传导复合物）而发生。在DISC中，胱天蛋白酶-8形成二聚体，然后在程序性细胞死亡的起始中变得具有蛋白水解活性。我们的初步研究表明，caspase-8的二聚化和切割在TRAIL耐药胶质母细胞瘤细胞中受到抑制。在寻找半胱天冬酶-8抑制的机制中，我们发现了在TRAIL处理之前形成的DR 5相关蛋白复合物，因此我们将其命名为PLAC（pre-ligand assembly complex）。我们的初步研究表明，PLAC成分定义了DISC的后续形成，从而控制caspase-8的二聚化和切割。具体而言，初步研究鉴定了RIP（受体相互作用蛋白）和A20（PLAC中的RIP泛素酶），并表明在TRAIL诱导的从PLAC到DISC的形成过程中，TRAIL处理导致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-8二聚化和切割。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，确定连接到RIP的聚泛素链，并确定caspase-8上的聚泛素结合结构域。此外，我们将从患者的胶质母细胞瘤中产生小鼠异种移植物和原代培养物，并确定A20是否在肿瘤中过表达，从而确定肿瘤中的TRAIL抗性。完成后，本研究将确定A20作为治疗靶点，从而通过开发可以靶向A20抑制机制的联合治疗来克服肿瘤对TRAIL治疗的耐药性，从而有益于人类健康。

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.