Heat shock-induced apoptosis

描述(申请人提供)：热疗或热休克疗法目前正在II/III期临床试验中单独使用或与放射或化疗联合使用，用于治疗各种癌症。不幸的是，尽管强烈的热休克可诱导细胞凋亡，但其潜在的机制仍存在争议，且尚不清楚。我们最近已经证明，热休克不需要任何已知的启动子半胱氨酸天冬氨酸酶或其激活复合体来诱导细胞凋亡。我们现在假设，热休克诱导细胞死亡，部分是通过刺激快速内溶酶体膜通透性(ELMP)，这与胞浆酸化和组织蛋白释放到细胞质中相一致，这两个过程都参与了Proaspase-3和促凋亡的BH3-Only蛋白的处理。重要的是，热休克诱导的线粒体外膜通透性(MOMP)在细胞死亡中是必不可少的，因为过度表达BID或缺乏BID的细胞或促凋亡的Bcl-2家族成员Bax和Bak-对细胞死亡具有抵抗力。MOMP对细胞死亡至关重要，很可能是因为它促进了凋亡抑制物(IAP)拮抗剂的释放，进而促进了caspase的活性。值得注意的是，热休克还需要c-jun氨基末端激酶(JNKs)来诱导MOMP，我们发现JNKs是通过一种非常新颖的途径激活的，涉及到所谓的细胞质“应激颗粒”(SGS)的形成。我们假设SGS的形成是通过热诱导RNA结合蛋白TIA-1的聚集启动的，SGS反过来通过肿瘤坏死因子受体相关因子2(TRAF2)和TGF2激活激酶1(TAK1)依赖的途径激活JNKs。我们进一步推测，JNKs通过激活额外的BH3-Only蛋白，如Bim，和/或通过抑制特定的抗细胞凋亡的Bcl2家族成员，如Bclxl或Mcl-1，使细胞对热休克诱导的MOMP敏感。在以下三个具体目标中，我们建议(1)确定ELMP是否引起胞浆酸化以及热休克蛋白70(Hsp70)是如何调节ELMP的；(2)确定组织蛋白在Proaspase-3和Bid激活中的作用；(3)表征独特的SG-TRAF2-TAK1-MAPKK-JNK途径，Hsp70对其调节，以及热休克后JNK的下游靶点。总而言之，这项拨款提案将确定热休克诱导细胞凋亡的基本机制，鉴于热疗作为一种临床有用的治疗选择重新引起人们的兴趣，这些机制非常相关。与公共健康相关：该赠款项目的重点是揭开介导热休克诱导细胞死亡的细胞机制。热休克或热疗目前正在临床试验中使用，要么单独使用，要么作为化疗或放射治疗的辅助手段用于各种癌症的治疗。不幸的是，人们对热休克是如何杀死细胞的知之甚少。我们在这方面的初步工作表明，热休克通过与其他类型的刺激非常不同的机制诱导细胞死亡。因此，预计本文提出的工作将显著提高我们对热休克在临床中的作用机制的理解，并可能为未来可用于治疗的新途径提供帮助。

DESCRIPTION (provided by applicant): Hyperthermia or heat shock therapy is currently being utilized in phase II/III clinical trials, either alone or in combination with radiation or chemotherapy, for the treatment of various cancers. Unfortunately, though intense heat shock induces apoptosis, the underlying mechanisms remain controversial and unclear. We have recently shown that heat shock does not require any of the known initiator caspases or their activating complexes to induce apoptosis. We now hypothesize that heat shock induces cell death, in part by stimulating rapid endo-lysosomal membrane permeabilization (ELMP), which coincides with cytosolic acidification and release of cathepsins into the cytoplasm, both of which participate in the processing of procaspase-3 and the proapoptotic BH3-only protein Bid. Importantly, heat shock-induced mitochondrial outer membrane permeabilization (MOMP) is essential for cell death, as cells that overexpress Bcl-2-or are deficient in Bid or the proapoptotic Bcl-2 family members Bax and Bak-are resistant to cell death. MOMP is critical for cell death most likely because it facilitates the release of inhibitor of apoptosis (IAP) antagonists, which in turn promote caspase activity. Remarkably, heat shock also requires c-Jun N-terminal kinases (JNKs) to induce MOMP, and we find that JNKs are activated through a highly novel pathway involving the formation of so-called cytoplasmic "stress granules" (SGs). We hypothesize that formation of SGs is initiated through heat-induced aggregation of the RNA binding protein TIA-1, and that SGs in turn activate JNKs through a tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and TGF2- activating kinase 1 (TAK1)-dependent pathway. We further speculate that JNKs sensitize cells to heat shock-induced MOMP by activating additional BH3-only proteins, such as Bim, and/or by inhibiting specific antiapoptotic Bcl-2 family members, such as Bcl-xL or Mcl-1. In the following three specific aims, we propose (1) to determine if ELMP elicits cytosolic acidification and to determine how ELMP is regulated by heat shock protein 70 (Hsp70), (2) to establish the roles of cathepsins in the activation of procaspase-3 and Bid, and (3) to characterize the unique SG-TRAF2-TAK1-MAPKK-JNK pathway, its regulation by Hsp70, and the downstream targets of JNK following heat shock. In summary, this grant proposal would determine the basic mechanisms of heat shock-induced apoptosis, which are highly relevant given the renewed interest in hyperthermia as a clinically useful treatment option. PUBLIC HEALTH RELEVANCE: The focus of this grant project is to unravel the cellular mechanisms that mediate heat shock-induced cell death. Heat shock, or hyperthermia, is currently being used in clinical trials, either alone or as an adjunct to chemo- or radiotherapy for the treatment of various cancers. Unfortunately, very little is known about how heat shock kills cells. Our preliminary work in this area suggests that heat shock induces cell death through mechanisms that are very different from other types of stimuli. Thus, it is anticipated that the work proposed herein will significantly improve our understanding of how heat shock works in the clinic and may shed light on new pathways that can be exploited therapeutically in the future.