RIP1 Cleavage by Caspase-8 is Essential for TRAIL-induced NF-kB Activation

Caspase-8 对 RIP1 的切割对于 TRAIL 诱导的 NF-kB 激活至关重要

• 批准号: 8403529
• 负责人: HASEM HABELHAH
• 金额: $ 28.38万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-23 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403529
• 关键词: Antibodies; Antineoplastic Agents; Apoptosis; Cancer cell line; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Cleaved cell; Data; Development; Effectiveness; Exhibits; Hodgkin Disease; Housing; Human; Induction of Apoptosis; Ligands; Light; Malignant Neoplasms; Mediating; N-terminal; NF-kappa B; Neoplasm Metastasis; Normal Cell; Pathway interactions; Phase II Clinical Trials; Process; Proteins; Recombinants; Resistance; Role; Site; TNFSF10 gene; TRAF2 gene; Toxic effect; Work; abstracting; cancer cell; caspase-8; cytotoxicity; in vivo; knock-down; knockout gene; mutant; neoplastic cell; overexpression; receptor; reconstitution; response

Project Summary/Abstract: TRAIL, a ligand for death receptors (DRs), is considered a potential anti-cancer agent, as it shows selective high cytotoxicity toward tumor cells and little or no toxicity against normal cells. Currently, a recombinant TRAIL and agonistic antibodies directed at DRs are in phase-II clinical trials. However, recent studies have demonstrated that many types of cancer cells possess intrinsic or acquired resistance to TRAIL. Moreover, TRAIL application has been found to activate NF-kB and enhance metastasis in apoptosis-resistant cancer cells. Gene knockout studies have demonstrated that caspase-8 activity is essential not only for TRAIL-induced cell death, but also for TRAIL- induced NF-kB activation. At present, it is believed that fully activated caspase-8 induces apoptosis whereas partially activated caspase-8 activates NF-kB. However, the caspase-8 substrates that mediate this form of NF-kB activation have not been identified. We have identified RIP1 as a caspase-8 substrate that mediates TRAIL- induced NF-kB activation, discovered that caspase-8 cleaves RIP1 at three sites, and found that this cleavage is regulated in vivo by cFLIP. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 at all three sites in response to TRAIL treatment, resulting in rapid RIP1 depletion and the induction of apoptosis; in apoptosis-resistant cells, however, TRAIL induces RIP1 cleavage mainly at one site, producing a constitutively active form of RIP1 (p60RIP1n) that activates the NF-kB pathway. Notably, overexpression of cFLIP is sufficient to trigger limited RIP1 cleavage and the accumulation of p60RIP1n. Importantly, in Hodgkin's lymphoma, cFLIP is overexpressed and a portion of RIP1 is constitutively processed to p60RIP1n. These data suggest that cFLIP-regulated, caspase- 8-mediated limited cleavage of RIP1 promotes NF-kB activation, and that such cleavage occurs constitutively in certain human cancers. These findings support our central hypothesis that cFLIP overexpression restricts TRAIL- induced caspase-8 activation to a moderate level, promoting RIP1 processing to p60RIP1n and, thereby, NF-kB activation. The objective of the proposed study is to evaluate the influence of cFLIP on caspase-8-mediated RIP1 cleavage, dissect the mechanisms by which RIP1 cleavage modulates NF-kB activation in response to TRAIL stimulation, and determine the pathological role of RIP1 cleavage in cancer cell resistance to TRAIL-induced apoptosis. To achieve these objectives, we propose to carry out the following specific aims: 1) determine the role of caspase-8-mediated RIP1 cleavage in promoting TRAIL-induced NF-kB activation versus cell death; 2) characterize the mechanisms by which caspase-8-mediated RIP1 cleavage activates NF-kB and inhibits cell death; 3) assess the pathophysiological relevance of RIP1 cleavage in cancer cell resistance to TRAIL-induced apoptosis. The proposed work will define the mechanisms that underlie TRAIL-induced NF-kB activation, and guide the development of strategies to maximize the effectiveness of TRAIL as an anti-cancer agent.

项目摘要/摘要： TRAIL 是死亡受体 (DR) 的配体，被认为是一种潜在的抗癌药物，因为它表现出高选择性 对肿瘤细胞有细胞毒性，对正常细胞几乎没有毒性。目前，重组 TRAIL 和 针对 DR 的激动性抗体正在进行 II 期临床试验。然而，最近的研究表明 许多类型的癌细胞对 TRAIL 具有内在的或获得性的抗性。此外，TRAIL应用程序还具有 已被发现可激活 NF-kB 并增强抗凋亡癌细胞的转移。基因敲除研究 已经证明 caspase-8 活性不仅对于 TRAIL 诱导的细胞死亡至关重要，而且对于 TRAIL- 诱导 NF-kB 激活。目前认为，完全激活的caspase-8可以诱导细胞凋亡，而完全激活的caspase-8则可以诱导细胞凋亡。 部分激活的 caspase-8 会激活 NF-kB。然而，介导这种形式 NF-kB 的 caspase-8 底物 激活尚未确定。我们已将 RIP1 鉴定为介导 TRAIL- 的 caspase-8 底物。 诱导 NF-kB 激活，发现 caspase-8 在三个位点切割 RIP1，并发现这种切割是 体内受 cFLIP 调节。在凋亡敏感细胞中，caspase-8 在所有三个位点裂解 RIP1，以响应 TRAIL 治疗，导致 RIP1 快速耗竭并诱导细胞凋亡；在抗凋亡细胞中， 然而，TRAIL 主要在一个位点诱导 RIP1 裂解，产生 RIP1 的组成型活性形式 (p60RIP1n) 激活 NF-kB 通路。值得注意的是，cFLIP 的过度表达足以触发有限的 RIP1 裂解和 p60RIP1n 积累。重要的是，在霍奇金淋巴瘤中，cFLIP 过度表达 RIP1的一部分被组成型加工成p60RIP1n。这些数据表明 cFLIP 调节的 caspase- 8 介导的 RIP1 有限切割促进 NF-kB 激活，并且这种切割持续发生在 某些人类癌症。这些发现支持我们的中心假设，即 cFLIP 过度表达限制 TRAIL- 诱导 caspase-8 激活至中等水平，促进 RIP1 加工为 p60RIP1n，从而促进 NF-kB 激活。本研究的目的是评估 cFLIP 对 caspase-8 介导的 RIP1 的影响 裂解，剖析 RIP1 裂解响应 TRAIL 调节 NF-kB 激活的机制 刺激，并确定 RIP1 裂解在癌细胞对 TRAIL 诱导的抵抗中的病理作用 细胞凋亡。为了实现这些目标，我们建议开展以下具体目标：1）确定角色 caspase-8 介导的 RIP1 裂解促进 TRAIL 诱导的 NF-kB 激活与细胞死亡的关系； 2） 描述 caspase-8 介导的 RIP1 裂解激活 NF-kB 并抑制细胞的机制 死亡; 3) 评估 RIP1 裂解在癌细胞对 TRAIL 诱导的耐药性中的病理生理学相关性 细胞凋亡。拟议的工作将定义 TRAIL 诱导 NF-kB 激活的机制，以及 指导策略的制定，以最大限度地发挥 TRAIL 作为抗癌药物的有效性。