Mechanisms of Cell Cycle and Survival Regulation by Hedgehog Signaling

描述(由申请人提供)：Hedgehog(HH)配体(Shh、Ihh和Dhh)通过两个膜受体(Ptc1和Ptc2)、调节膜蛋白Smoothned(SMO)和Gli转录因子发出信号。Ptc1在没有配体的情况下抑制SMO，而HH结合则导致Ptc1抑制和激活SMO。HH-SMO激活依赖Gli的转录被称为“规范”途径；相反，HH蛋白的Gli非依赖功能被归类为“非规范”。Gli的激活需要协同受体CDO/Boc的存在，并将SMO重新定位到被称为“初级纤毛”的结构中。大多数人类癌症的特征是由于HH配体的上调、Ptc1的功能丧失或SMO功能的获得而导致“典型的”HH途径的过度激活。值得注意的是，HH蛋白的上调与上皮性癌症密切相关。我们鉴定了SMO下游导致Gli激活的新的信号成分，并发现SMO作为一种经典的Gi偶联受体，刺激参与细胞周期调节、生存和迁移的信号。我们已经建立了新的范例，揭示Shh可以激活Rho家族的小G蛋白，并导致细胞骨架重排和细胞运动的改变。我们和其他人已经发现，在没有HHS的情况下，Ptc1通过其C末端结构域(CTD)诱导细胞凋亡，并且不依赖于SMO。基于这一证据，我们推测，由Ptc1直接或通过SMO间接产生的非规范信号是HH调节细胞周期进程、生存和迁移的主要参与者。我们将：1)描述非规范的HH信号在增殖、存活和迁移中的机制基础。我们将首先确定初级纤毛和辅助受体CDO/Boc在这些反应中的潜在意义。我们还将研究SMO激活小GTP酶的机制，以及它们参与细胞迁移的过程。2)研究Ptc1独立于SMO/Gli激活调控细胞周期进程和细胞凋亡的机制。我们将首先确定Ptc1 CTD的膜定位是否是驱动细胞死亡所必需的，并将确定凋亡所需的CTD的最小片段。其次，我们将探讨Ptc1诱导Akt去磷酸化的机制，以及线粒体凋亡通路在Ptc1 CTD诱导的细胞凋亡中的作用。最后，我们将研究Ptc1导致细胞周期停滞的机制。3)确定Ptc1和SMO信号在HH过表达癌细胞增殖和存活中的作用。基于Ptc1、MCTD序列和环胺的多肽将单独和联合应用于HH高表达的上皮性癌细胞，我们将评估它们对增殖和生存的影响。总体而言，这些研究将为理解非典型的Hedgehog途径(S)提供见解，并将为开发针对Ptc1功能的新的癌症治疗药物奠定机制基础。 与公共健康相关：这项建议旨在剖析Hedgehog信号通路激活的基本信号机制，这些机制参与调节细胞周期进程、生存和迁移。我们提出了一个新的假说，即Hedgehog受体Ptc1独立于所谓的“正则”途径调节细胞周期的进展和存活，该途径涉及靶基因的平滑(SMO)和Gli依赖的转录。了解Ptc1和SMO在正常细胞和癌细胞的生长、存活和细胞周期调控中的信号通路的具体作用，将为人类健康的治疗应用提供理论基础。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) ligands (Shh, Ihh, and Dhh) signal through two membrane receptors (Ptc1 and Ptc2), the regulatory membrane protein Smoothened (SMO), and the Gli transcription factors. Ptc1 represses SMO in the absence of ligand while Hh binding results in Ptc1 inhibition and activation of SMO. Activation of Gli-dependent transcription by Hh-SMO is referred to as the "canonical" pathway; in contrast, Gli-independent functions of Hh proteins are classified as "non-canonical". Gli activation requires the presence of the co-receptors Cdo/Boc and relocalization of SMO to a structure known as the "primary cilium". Most human cancers are characterized by hyperactivation of the "canonical" Hh pathway due to upregulation of Hh ligands, loss-of-function of Ptc1, or gain-of-function of SMO. Remarkably, upregulation of Hh proteins is strictly associated with epithelial cancers. We identified novel signaling components downstream of SMO that lead to Gli activation and uncovered that SMO acts as a classical Gi-coupled receptor that stimulates signals involved in cell cycle regulation, survival, and migration. We have established novel paradigms revealing that Shh can activate small G proteins of the Rho family and lead to cytoskeletal rearrangements and changes in cell motility. We and others have found that Ptc1 induces apoptosis in the absence of Hhs via its C-terminal domain (CTD), and independently of SMO. Based on this evidence, we hypothesize that non-canonical signals, generated directly by Ptc1 or indirectly through SMO, are the main players in Hh-regulation of cell cycle progression, survival and migration. We will: 1) Characterize the mechanistic basis of non-canonical Hh signaling in proliferation, survival, and migration. We will first determine the potential implication of the primary cilium and the co- receptors Cdo/Boc in these responses. We will also investigate the mechanistic aspects of activation of small GTPases by SMO, and their involvement in cell migration. 2) Study the mechanism of regulation of cell cycle progression and apoptosis by Ptc1 independently of SMO/Gli activation. We will first determine whether membrane localization of Ptc1 CTD is necessary to drive cell death, and will identify the minimal fragment of the CTD required for apoptosis. Secondly, we will investigate the mechanism of Ptc1- induced Akt dephosphorylation, and the participation of the mitochondrial apoptotic pathway in Ptc1 CTD- induced apoptosis. Lastly, we will study the mechanisms that lead to cell cycle arrest by Ptc1. 3) Establish the contribution of Ptc1 and SMO signals for proliferation and survival of Hh overexpressing cancer cells. Peptides based on Ptc1 MCTD sequence and cyclopamine will be applied alone and in combination to Hh-overexpressing epithelial cancer cells and we will evaluate their effects on proliferation and survival. Overall, these studies will provide insights into the understanding of non-canonical Hedgehog pathway(s) and will set a mechanistic basis for development of new cancer therapeutics targeting Ptc1 functions. PUBLIC HEALTH RELEVANCE: This proposal is aimed at dissecting the basic signaling mechanisms activated by the Hedgehog signaling pathway that are involved in regulation of cell cycle progression, survival and migration. We propose the novel hypothesis that the Hedgehog receptor Ptc1 regulates cell cycle progression and survival independently of the so-called "canonical" pathway, which involves Smoothened (SMO) and Gli-dependent transcription of target genes. Understanding the specific contributions of Ptc1 and SMO to signaling pathways implicated in growth, survival and cell cycle regulation in normal and cancer cells, will provide the basis for rationale development of therapeutic applications for human health.