Novel Fas/CD95 Signaling Mechanisms

描述（由申请人提供）：死亡受体Fas （CD95/APO-1）介导许多组织的细胞凋亡。在其配体FasL的刺激下，形成一个死亡诱导信号复合体（DISC），该复合体由接头分子FADD、启动体caspase 8和10以及caspase 8/10调节因子c-FLIP组成。最近发现，除了作为死亡受体的作用外，Fas还具有多种非凋亡活性，包括使Fas抗凋亡癌细胞更具移动性和侵袭性。这些新的活动也依赖于DISC成分，这就提出了如何选择参与凋亡或非凋亡信号通路的问题。我们最近表明，Fas需要内化到内体/溶酶体腔室中，以发出细胞凋亡的信号。然而，与之形成鲜明对比的是，我们发现受体内化并不需要通过Fas进行非凋亡信号传导。我们发现酪氨酸291参与调节Fas的内化，另一种翻译后修饰，半胱氨酸199的棕榈酰化，促进了高分子量DISC复合物（hiDISC）的形成。hiDISC形成于Fas内部化的场所。最近，我们发现Fas抑制了microRNA let-7的表达，我们获得了let-7通过靶向HMGA2和IMP-1驱动肿瘤进展的证据。我们假设Fas的两种翻译后修饰决定了Fas是否被内化，以及它是否激活凋亡或非凋亡信号通路，从而导致let-7的下调。研究Fas的内化、翻译后修饰调控及其对let-7表达的调控，可为抑制Fas的促瘤活性提供手段。我们提出以下三个具体目标：目的1：确定Fas信号是凋亡还是存活的调节机制。目的2：表征Fas和let-7之间的功能联系。目的3：阐明Fas信号突变体和Fas调控的mirna在卵巢癌小鼠模型中的功能。表征Fas非凋亡信号的新机制和功能后果可能有助于开发合理的策略和治疗方法，以干扰癌症和其他疾病中失调的Fas信号。公共卫生相关性：死亡受体Fas （CD95/APO-1）在许多组织中被视为细胞凋亡的介质，但很明显Fas具有多种非细胞凋亡活性，包括诱导增殖和肿瘤侵袭。我们将研究Fas信号的早期事件，包括受体内化，它决定了Fas信号是凋亡还是非凋亡途径，以及肿瘤抑制microrna的下调。这些研究将为了解Fas的不同信号活动提供基础，并设计新的治疗方法来干扰癌症和其他疾病中失调的Fas信号。

DESCRIPTION (provided by applicant): The death receptor Fas (CD95/APO-1) mediates apoptosis in many tissues. Upon stimulation by its ligand FasL, it forms a death inducing signaling complex (DISC) consisting of the adaptor molecule FADD, the initiator caspases 8 and 10, and the caspase-8/10 regulator c-FLIP. Recently it has become clear that, in addition to its role as a death receptor, Fas has multiple nonapoptotic activities including a role in rendering Fas apoptosis-resistant cancer cells more mobile and invasive. These novel activities also rely on DISC components, raising the question of how the choice to engage apoptotic or nonapoptotic signaling pathways is made. We have recently shown that Fas requires internalization into an endosomal/lysosomal compartment in order to signal apoptosis. However, in striking contrast, we found that receptor internalization is not required for nonapoptotic signaling through Fas. We found that tyrosine 291 is involved in regulating the internalization of Fas and that another postranslational modification, palmitoylation of cysteine 199, facilitates the formation of high molecular weight DISC complexes (hiDISC). hiDISC forms at the sites of Fas internalization. Recently, we found that Fas suppresses the expression of the microRNA let-7 and we obtained evidence that let-7 drives tumor progression through targeting HMGA2 and IMP-1. We hypothesize that the two posttranslational modifications of Fas determine whether Fas is internalized and whether it activates apoptotic or nonapoptotic signaling pathways resulting in downregulation of let-7. Identification of the role and the mechanisms of the internalization of Fas, its regulation by posttranslational modifications, and the regulation of let-7 expression by Fas could provide the means to neutralize the tumor promoting activities of Fas. We propose the following three specific aims: Aim 1: Determine the mechanisms that regulate whether Fas signals apoptosis or survival. Aim 2: Characterize the functional connection between Fas and let-7. Aim 3: Elucidate the function of Fas signaling mutants and of Fas regulated miRNAs in mouse models of ovarian cancer. Characterization of the novel mechanisms and functional consequences of nonapoptotic signaling of Fas may facilitate the development of rational strategies and therapeutic approaches for interfering with dysregulated Fas signaling in cancer and other diseases. PUBLIC HEALTH RELEVANCE: The death receptor Fas (CD95/APO-1) has been viewed as a mediator of apoptosis in many tissues but it has become clear that Fas has multiple nonapoptotic activities including the induction of proliferation and tumor invasiveness. We will study the early events of Fas signaling including receptor internalization that determine whether Fas signals apoptotic or nonapoptotic pathways and downregulation of tumor suppressing microRNAs. These studies will provide a basis for understanding the different signaling activities of Fas and to devise new therapeutic approaches for interfering with dysregulated Fas signaling in cancer and other diseases.