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

描述（由申请人提供）：TRAIL是一种死亡受体（dr）的配体，被认为是一种潜在的抗癌药物，因为它对肿瘤细胞具有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和激动抗体正处于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，已发现TRAIL应用可激活NF-kB并增强凋亡抵抗癌细胞的转移。基因敲除研究表明，caspase-8活性不仅对TRAIL诱导的细胞死亡至关重要，而且对TRAIL诱导的NF-？B激活。目前认为，完全激活的caspase-8诱导细胞凋亡，而部分激活的caspase-8激活NF-kB。然而，介导这种形式的NF-kB激活的caspase-8底物尚未被确定。我们已经确定RIP1是介导TRAIL诱导的NF-kB激活的caspase-8底物，发现caspase-8在三个位点上切割RIP1，并发现这种切割在体内受cFLIP调节。在凋亡敏感细胞中，caspase-8响应TRAIL处理在所有三个位点切割RIP1，导致RIP1快速耗尽并诱导凋亡；然而，在抗凋亡细胞中，TRAIL主要在一个位点诱导RIP1切割，产生一种组成型的RIP1活性形式（p60RIP1n），激活NF-kB通路。值得注意的是，cFLIP的过表达足以触发RIP1的有限切割和p60RIP1n的积累。重要的是，在霍奇金淋巴瘤中，cFLIP过表达，部分RIP1被组成性地加工成p60RIP1n。这些数据表明，crisp调控的caspase- 8介导的RIP1的有限切割促进NF-kB的激活，并且这种切割在某些人类癌症中组成性地发生。这些发现支持了我们的中心假设，即cFLIP过表达将TRAIL诱导的caspase-8激活限制在中等水平，促进RIP1加工到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切割的影响，剖析RIP1切割在TRAIL刺激下调节NF-kB激活的机制，并确定RIP1切割在癌细胞抵抗TRAIL诱导的凋亡中的病理作用。为了实现这些目标，我们提出以下具体目标：1)确定caspase-8介导的RIP1切割在促进trail诱导的NF-kB活化和细胞死亡中的作用；2)描述caspase-8介导的RIP1切割激活NF-kB和抑制细胞死亡的机制；3)评估RIP1切割在癌细胞抵抗trail诱导的凋亡中的病理生理学相关性。这项工作将定义TRAIL诱导NF-kB激活的机制，并指导开发策略，以最大限度地提高TRAIL作为抗癌药物的有效性。

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.