Novel Fas/CD95 Signaling Mechanisms

描述(申请人提供)：死亡受体Fas(CD95/APO-1)在许多组织中介导细胞凋亡。在其配体FasL的刺激下，它形成了一个由接头分子FADD、启动子caspase 8和10以及caspase-8/10调节因子c-flip组成的死亡诱导信号复合体(DISC)。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耐药癌细胞更具移动性和侵袭性。这些新的活动也依赖于视盘成分，这提出了一个问题，即如何选择参与凋亡或非凋亡信号通路。我们最近发现，Fas需要内化到内小体/溶酶体的小室中，以便发出细胞凋亡的信号。然而，与之形成鲜明对比的是，我们发现通过Fas的非凋亡信号并不需要受体内化。我们发现，酪氨酸291参与调节Fas的内化，另一个翻译后修饰，半胱氨酸199的棕榈酰化，促进了高分子量盘状复合体(HiDISC)的形成。HiDISC形成于Fas内化部位。最近，我们发现Fas抑制了microRNA let-7的表达，并获得了let-7通过靶向HMGA2和IMP-1而促进肿瘤进展的证据。我们推测，Fas的两个翻译后修饰决定了Fas是否内化，以及它是否激活了导致let-7下调的凋亡或非凋亡信号通路。识别Fas内化的作用和机制，翻译后修饰对其的调节，以及Fas对let-7表达的调节，可以为中和Fas的促肿瘤活性提供手段。我们提出了以下三个具体目标：目标1：确定Fas信号转导细胞凋亡或存活的机制。目的2：研究Fas和let-7之间的功能联系。目的：阐明Fas信号突变体及其调控的miRNAs在小鼠卵巢癌模型中的作用。对Fas非凋亡信号的新机制和功能后果的研究有助于开发合理的策略和治疗方法来干扰癌症和其他疾病中异常调节的Fas信号。公共卫生相关性：死亡受体Fas(CD95/APO-1)在许多组织中被认为是细胞凋亡的媒介，但已清楚地表明，Fas具有多种非凋亡活性，包括诱导增殖和肿瘤侵袭。我们将研究Fas信号的早期事件，包括决定Fas信号的受体内化、凋亡或非凋亡途径以及肿瘤抑制microRNAs的下调。这些研究将为了解Fas的不同信号活性和设计干扰癌症和其他疾病中失调的Fas信号的新的治疗方法提供基础。

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.