Molecular mechanisms of TRAIL resistance in glioblastoma

描述(申请人提供)：胶质母细胞瘤是最常见和致命的脑肿瘤，目前还没有根治方法。在寻找新的治疗药物的过程中，我们发现TRAIL(肿瘤坏死因子相关的凋亡诱导配体)可以诱导胶质母细胞瘤细胞的程序性死亡(凋亡)，这可能为治疗提供了新的希望。因此，TRAIL已进入癌症治疗的临床试验。不幸的是，早期的试验报告显示出有限的抗肿瘤活性，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，目前的研究方案将研究胶质母细胞瘤中TRAIL耐药的分子基础。TRAIL通过与死亡受体DR5结合，将caspase-8募集到DR5形成DISC(死亡诱导信号复合体)，从而诱导细胞凋亡。在视盘中，caspase-8形成二聚体，然后在启动程序性细胞死亡的过程中变得蛋白水解性活跃。我们的初步研究表明，在TRAIL耐药的胶质母细胞瘤细胞中，caspase-8的二聚化和切割受到抑制。在探索caspase-8抑制机制的过程中，我们发现了一个DR5相关的蛋白质复合体，它是在TRAIL处理之前形成的，因此我们将其命名为PLAC(配体前组装复合体)。我们的初步研究表明，plac组分定义了光盘的后续形成，从而控制了caspase-8的二聚化和切割。具体地说，初步研究确定了RIP(受体相互作用蛋白)和PLAC中的RIP泛素酶A20，并表明在TRAIL诱导的PLAC到盘中的形成过程中，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泛素化抑制了caspase-8的二聚化和切割。为了验证这一假说，我们将A20定义为RIP泛素连接酶，确定RIP上的多泛素链，并确定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产生小鼠异种移植和原代培养，并确定A20是否在肿瘤中过度表达，从而定义肿瘤中的TRAIL耐药。完成后，这项研究将确定A20为治疗靶点，从而通过开发针对A20抑制机制的联合疗法来克服肿瘤对TRAIL治疗的耐药性，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.