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.

项目概要/摘要： TRAIL是一种死亡受体（DR）的配体，被认为是一种潜在的抗癌剂，因为它显示出高选择性， 对肿瘤细胞的细胞毒性和对正常细胞的很少或没有毒性。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， 许多类型的癌细胞具有对TRAIL的内在或获得性抗性。此外，TRAIL的应用 已发现其可激活NF-kB并增强抗肿瘤细胞的转移。基因敲除研究 已经证明，caspase-8活性不仅对于TRAIL诱导的细胞死亡是必需的，而且对于TRAIL诱导的细胞死亡也是必需的。 诱导NF-κ B活化。目前，认为完全活化的caspase-8诱导细胞凋亡， 部分活化的caspase-8活化NF-κ B。然而，介导这种形式的NF-κ B的caspase-8底物， 激活尚未确定。我们已经确定RIP 1是一种半胱天冬酶-8的底物，它介导TRAIL-1的表达。 诱导NF-kB活化，发现caspase-8在三个位点切割RIP 1，并发现这种切割是 通过cFLIP在体内调节。在凋亡敏感细胞中，caspase-8在所有三个位点切割RIP 1， TRAIL处理，导致RIP 1快速耗尽和诱导凋亡;在凋亡抗性细胞中， 然而，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）激活NF-κ B通路。值得注意的是，cFLIP的过表达足以引发有限的 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过度表达， 并且RIP 1的一部分被组成型加工成p60 RIP 1 n。这些数据表明，cFLIP调节的，caspase- 8-RIP 1介导的有限切割促进NF-kB活化，并且这种切割组成性地发生在 某些人类癌症这些发现支持了我们的中心假设，即cFLIP过表达限制了TRAIL的表达。 诱导caspase-8活化至中等水平，促进RIP 1加工为p60 RIP 1 n，从而促进NF-kB activation.本研究的目的是评估cFLIP对caspase-8介导的RIP 1的影响。 切割，剖析RIP 1切割调节NF-kB激活的机制，以响应TRAIL 刺激，并确定RIP 1切割在癌细胞对TRAIL诱导的肿瘤细胞耐药性中的病理作用。 凋亡为了实现这些目标，我们建议执行以下具体目标：1）确定角色 caspase-8介导的RIP 1切割促进TRAIL诱导的NF-kB活化与细胞死亡; 2） 描述caspase-8介导的RIP 1裂解激活NF-κ B并抑制细胞增殖的机制。 3）评估RIP 1切割在癌细胞对TRAIL诱导的肿瘤细胞凋亡的抗性中的病理生理学相关性。 凋亡拟议的工作将定义TRAIL诱导的NF-κ B激活的机制， 指导策略的发展，以最大限度地提高TRAIL作为抗癌剂的有效性。