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

Caspase-8 裂解 RIP1 对于 TRAIL 诱导的 NF-kB 激活至关重要

• 批准号: 7887290
• 负责人: HASEM HABELHAH
• 金额: $ 31.13万
• 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/7887290
• 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; Toxic effect; Work; cancer cell; caspase-8; cytotoxicity; in vivo; knock-down; knockout gene; mutant; neoplastic cell; overexpression; public health relevance; receptor; reconstitution; response

DESCRIPTION (provided by applicant): 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-?B 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. PUBLIC HEALTH RELEVANCE: TRAIL, a potential anti-cancer agent, has been found to enhance metastasis in TRAIL-resistant cancer cells by activating NF-kB in a caspase-8-dependent manner. We identified caspase-8 substrate that mediates this form of NF-kB activation. Our work will not only shed new light on the mechanism by which TRAIL activates NF- kB, but will also provide rationale for maximizing the potential effectiveness of TRAIL as an anti-cancer agent.

描述（由申请人提供）：TRAIL是死亡受体（DR）的配体，被认为是一种潜在的抗癌剂，因为它对肿瘤细胞显示出选择性的高细胞毒性，而对正常细胞几乎没有毒性。目前，针对DR的重组TRAIL和激动性抗体处于II期临床试验中。然而，最近的研究表明，许多类型的癌细胞对TRAIL具有内在或获得性抗性。此外，已经发现TRAIL应用激活NF-kB并增强抗肿瘤细胞中的转移。基因敲除研究表明，caspase-8的活性是必不可少的，不仅为TRAIL诱导的细胞死亡，但也为TRAIL诱导的NF-？B激活。目前，认为完全活化的半胱天冬酶-8诱导细胞凋亡，而部分活化的半胱天冬酶-8活化NF-κ B。然而，介导这种形式的NF-κ B活化的半胱天冬酶-8底物尚未被鉴定。我们已经鉴定了RIP 1作为介导TRAIL诱导的NF-κ B活化的半胱天冬酶-8底物，发现半胱天冬酶-8在三个位点切割RIP 1，并且发现这种切割在体内由cFLIP调节。在凋亡敏感细胞中，caspase-8在所有三个位点裂解RIP 1以响应TRAIL处理，导致RIP 1快速耗尽并诱导凋亡;然而，在凋亡抗性细胞中，TRAIL主要在一个位点诱导RIP 1裂解，产生组成型活性形式的RIP 1（p60 RIP 1 n），其激活NF-κ B通路。值得注意的是，cFLIP的过表达足以触发有限的RIP 1切割和p60 RIP 1 n的积累。重要的是，在霍奇金淋巴瘤中，cFLIP过表达，RIP 1的一部分组成性加工成p60 RIP 1 n。这些数据表明，cFLIP调节，caspase- 8介导的RIP 1的有限切割促进NF-κ B活化，并且这种切割在某些人类癌症中组成性发生。这些发现支持我们的中心假设，即cFLIP过表达将TRAIL诱导的caspase-8激活限制在中等水平，促进RIP 1加工为p60 RIP 1 n，从而促进NF-κ B激活。本研究的目的是评估cFLIP对caspase-8介导的RIP 1裂解的影响，分析RIP 1裂解调节NF-kB活化以响应TRAIL刺激的机制，并确定RIP 1裂解在癌细胞对TRAIL诱导的凋亡的抵抗中的病理作用。为了实现这些目标，我们提出了以下具体目标：1）确定caspase-8介导的RIP 1切割在促进TRAIL诱导的NF-κ B活化与细胞死亡中的作用; 2）表征caspase-8介导的RIP 1切割激活NF-κ B并抑制细胞死亡的机制; 3）评估RIP 1切割在癌细胞对TRAIL诱导的细胞凋亡的抗性中的病理生理学相关性。拟议的工作将定义TRAIL诱导的NF-kB激活的机制，并指导策略的发展，以最大限度地提高TRAIL作为抗癌药物的有效性。 公共卫生关系：TRAIL是一种潜在的抗癌剂，已发现其通过以半胱天冬酶-8依赖的方式激活NF-κ B来增强TRAIL抗性癌细胞的转移。我们确定了介导这种形式的NF-κ B激活的caspase-8底物。我们的工作不仅将揭示TRAIL激活NF-κ B的机制，而且还将为最大化TRAIL作为抗癌剂的潜在有效性提供理论依据。