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

项目摘要/摘要： TRAIL是一种死亡受体(Drs)的配体，被认为是一种潜在的抗癌药物，因为它显示出高选择性。 对肿瘤细胞有细胞毒性，对正常细胞几乎没有毒性。目前，一项重组的试验和 针对DRS的激动型抗体正在进行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具有内在或获得性耐药。此外，试验应用程序具有 已发现可激活核因子-kB，促进抗凋亡癌细胞的转移。基因敲除研究 已经证明，caspase-8活性不仅在TRAIL诱导的细胞死亡中是必不可少的，而且在TRAIL-2诱导的细胞死亡中也是必不可少的。 诱导核因子-kB活化。目前认为，完全激活的caspase-8可诱导细胞凋亡，而 部分激活的caspase-8激活了核因子-kB。然而，介导这种形式的核因子-kB的caspase-8底物 激活状态尚未确定。我们已经确定RIP1是一种caspase-8底物，它介导TRAIL- 诱导核因子-kB激活，发现caspase-8在三个位点切割RIP1，并发现这种切割是 在体内受cFLIP调控。在对凋亡敏感的细胞中，caspase-8在所有三个位置都切割RIP1，以响应 TRAIL治疗，导致RIP1迅速耗尽并诱导细胞凋亡；在耐凋亡细胞中， 然而，TRAIL主要在一个位点诱导RIP1的裂解，产生一种结构性的RIP1活性形式 (P60RIP1n)激活核因子-kB途径。值得注意的是，cFLIP的过度表达足以触发有限的 RIP1的裂解和p60RIP1n的积累。重要的是，在霍奇金淋巴瘤中，cFLIP过度表达 并且RIP1的一部分被构成地加工成p60RIP1n。这些数据表明，cFLIP调控的caspase- 8介导的RIP1的有限切割促进了NF-kB的激活，并且这种切割发生在 某些人类癌症。这些发现支持我们的中心假设，即cFLIP过表达限制TRAIL- 诱导caspase-8适度激活，将RIP1处理促进到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裂解调节核因子-kB活化以响应TRAIL的机制 刺激，并确定RIP1裂解在肿瘤细胞对TRAIL诱导的抵抗中的病理作用 细胞凋亡。为了实现这些目标，我们建议进行以下具体目标：1)确定角色 Caspase-8介导的RIP1裂解促进TRAIL诱导的NF-kB激活与细胞死亡的比较；2) Caspase-8介导的RIP1裂解激活核因子-kB并抑制细胞的机制 3)评估RIP1裂解在肿瘤细胞对TRAIL诱导的耐药性中的病理生理学相关性 细胞凋亡。这项拟议的工作将定义TRAIL诱导的核因子-kB激活的潜在机制，以及 指导制定策略，最大限度地发挥TRAIL作为抗癌剂的有效性。

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.