Mechanisms of Cell Cycle and Survival Regulation by Hedgehog Signaling

Hedgehog 信号传导的细胞周期和生存调节机制

• 批准号: 7993435
• 负责人: NATALIA ANDREA RIOBO-DEL GALDO
• 金额: $ 29.07万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-05 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993435
• 关键词: Adult; Apoptosis; Apoptotic; Binding; Biochemical; Biological; Biological Process; C-terminal; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Survival; Cells; Cilia; Complex; Cyclins; DNA Sequence Rearrangement; Data; Development; Embryonic Development; Epithelial; Erinaceidae; Family; Fibroblasts; G Protein-Coupled Receptor Genes; Gastrointestinal tract structure; Gene Targeting; Genetic Transcription; Growth; Guanine Nucleotide Exchange Factors; Health; Human; Immigration; Lead; Ligands; Literature; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Mammals; Membrane; Membrane Proteins; Mitochondria; Monomeric GTP-Binding Proteins; Mutation; Organ; Organelles; Pathway interactions; Peptides; Protein Dephosphorylation; Proteins; Publishing; Regulation; Role; Seminal; Signal Pathway; Signal Transduction; Structure; Testing; Up-Regulation; base; cancer cell; cell motility; cyclopamine; cytotoxicity; gain of function; genetic regulatory protein; hedgehog signal transduction; human SHH protein; human SMO protein; inhibitor/antagonist; insight; intervention effect; loss of function; migration; novel; overexpression; public health relevance; receptor; receptor coupling; response; rho; smoothened signaling pathway; therapeutic development; therapeutic target; transcription factor; tumor

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.

描述（由申请人提供）：Hedgehog（Hh）配体（Shh、Ihh和Dhh）通过两种膜受体（Ptc 1和Ptc 2）、调节膜蛋白Smoothened（SMO）和Gli转录因子发出信号。Ptc 1在配体不存在的情况下抑制SMO，而Hh结合导致Ptc 1抑制和激活SMO。Hh-SMO对Gli依赖性转录的激活被称为“经典”途径;相比之下，Hh蛋白的Gli非依赖性功能被归类为“非经典”。Gli激活需要共受体Cdo/Boc的存在和SMO重新定位到称为“初级纤毛”的结构。大多数人类癌症的特征在于由于Hh配体的上调、Ptc 1的功能丧失或SMO的功能获得而引起的“经典”Hh通路的超活化。值得注意的是，Hh蛋白的上调与上皮癌严格相关。我们确定了导致Gli激活的SMO下游的新型信号传导组分，并发现SMO作为经典的Gi偶联受体，刺激参与细胞周期调控、存活和迁移的信号。我们已经建立了新的范例，揭示了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变化。我们和其他人已经发现，Ptc 1通过其C-末端结构域（CTD）诱导细胞凋亡的Hhs的情况下，并独立于SMO。基于这一证据，我们假设，非典型信号，直接产生的Ptc 1或间接通过SMO，是主要的球员在HH-调节细胞周期的进展，生存和迁移。 我们将：1）表征增殖、存活和迁移中的非经典Hh信号传导的机制基础。我们将首先确定初级纤毛和共受体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机制。2)研究Ptc 1独立于SMO/Gli激活调控细胞周期进程和凋亡的机制。我们将首先确定Ptc 1 CTD的膜定位是否是驱动细胞死亡所必需的，并将确定细胞凋亡所需的CTD的最小片段。其次，我们将研究Ptc 1诱导Akt去磷酸化的机制，以及线粒体凋亡途径在Ptc 1 CTD诱导的细胞凋亡中的参与。最后，我们将研究Ptc 1导致细胞周期阻滞的机制。3)确定Ptc 1和SMO信号对Hh过表达癌细胞增殖和存活的贡献。基于Ptc 1 MCTD序列和环巴胺的肽将单独和组合应用于Hh过表达的上皮癌细胞，我们将评估它们对增殖和存活的影响。 总的来说，这些研究将为理解非经典Hedgehog通路提供见解，并将为开发靶向Ptc 1功能的新癌症疗法奠定机制基础。 公共卫生相关性：该提案旨在剖析由Hedgehog信号通路激活的参与调节细胞周期进展、存活和迁移的基本信号机制。我们提出了一个新的假设，即刺猬受体Ptc 1调节细胞周期的进展和生存独立的所谓的“经典”途径，其中涉及Smoothened（SMO）和Gli-dependent转录的靶基因。了解Ptc 1和SMO对正常细胞和癌细胞生长、存活和细胞周期调控信号通路的具体贡献，将为人类健康治疗应用的合理开发提供基础。