Primary cilia loss in bile duct cells- the interplay with the autophagy machinery

胆管细胞初级纤毛损失——与自噬机制的相互作用

基本信息

批准号：
10898187
负责人：
Sergio A Gradilone
金额：
$ 6.79万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10898187
关键词：
Acetylation Autophagocytosis Biliary Tract Diseases Biogenesis CBL gene Cell Line Cells Cholangiocarcinoma Cilia Clinical Trials Communication Cytoplasm Data Deacetylase Deacetylation Defect Development Disease Ductal Epithelial Cell EGF gene Environment Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Epithelial Cells Erlotinib Family member Foundations Functional disorder Goals HDAC6 gene Histone Deacetylase In Vitro Inhibition of Cell Proliferation Leucine Zippers Liver Lysine Molecular Mus Needs Assessment Normal Cell Organelles Pathogenesis Pathologic Process Proteins Receptor Inhibition Receptor Signaling Regulation Rodent Model Role SIRT1 gene Sensory Signal Pathway Signal Transduction Structure Testing Therapeutic Ubiquitination Work alpha Tubulin bile duct biliary tract cell growth cell motility cholangiocyte combinatorial design experimental study in vivo inhibition of autophagy inhibitor malformation migration multisensory novel novel therapeutic intervention novel therapeutics overexpression patient derived xenograft model polycystic liver disease pre-clinical receptor restoration transcription factor ubiquitin-protein ligase

项目摘要

Project Summary/Abstract Primary cilia are multisensory organelles that function as cellular antennae. We found that ciliary defects in cholangiocytes and/or the loss of primary cilia are associated with biliary tract diseases like polycystic liver disease (PLD) and cholangiocarcinoma (CCA). A better understanding of the signaling regulated by cilia and mechanisms of ciliary loss in diseased cholangiocytes is critical to design new therapies based on the restoration of cilia, i.e. ciliotherapies. Our current overall objective is to understand the role of cilia in the regulation of epidermal growth factor receptor (EGFR) signaling. EGFR signaling is abnormally persistent and enhanced in PLD and CCA, two diseases with ciliary dysfunction. Furthermore, we aim to explore the mechanisms of ciliary loss in cholangiocytes – especially how the autophagy machinery is targeted to resorption of this organelle. This proposal will assess how cilia-autophagy communication works in cholangiocytes to reduce ciliary expression and, consequently, how the loss or dysfunction of cilia enhances EGFR signaling. We propose that pathologically-induced ciliophagy accounts for ciliary loss/dysfunction, inducing sustained EGFR signaling. We propose three Specific Aims: In Specific Aim 1: To characterize molecular mechanisms of the ciliary-dependent degradation of activated EGFR, we will assess the need of cilia for activated EGFR degradation; characterize the mechanisms of EGFR translocation to primary cilia; and assess the hypothesis that the E3 ubiquitin ligase c-CBL translocates to the primary cilia upon EGF signaling and drives the degradation of activated EGFR located in the cilia. In Specific Aim 2: To identify the key players involved in targeting ciliary components to the autophagy machinery, we will assess the role of autophagy and HDAC6/SIRT1 in ciliary expression in vitro; assess the role of HDAC6/SIRT1 in ciliophagy in vivo; study the interaction between ciliary proteins and autophagy cargo receptors; and test the hypothesis that in ciliary-defective cholangiocytes, overexpression of deacetylases induces lysine deacetylation of ciliary components, which leads to ubiquitination of the same residues and targeting of the autophagy machinery by specific autophagy cargo receptors. In Specific Aim 3: To test the combination of specific deacetylases, autophagy, and EGFR inhibitors in pre-clinical rodent models as a therapeutic approach, we will assess the effect of HDAC6 inhibition (Tubastatin-A or ACY-1215), and/or SIRT1 inhibition (Sirtinol) in combination with autophagy inhibitors (e.g., HCQ, SAR405) with or without EGFR inhibition (Erlotinib, Afatinib) in vitro and in vivo; assess the in vivo effects of Ciliomax (a novel dual inhibitor we recently developed) plus EGFR inhibition; and assess the most promising treatments in patient-derived xenografts. Impact: identifying novel targets could lead to much-needed new therapeutic strategies for these devastating diseases. Our experiments in in vitro and pre-clinical rodent models will characterize the ciliary-dependent regulation of EGFR and the communication between primary cilia and the autophagy process, which will lay the foundation for potential clinical trials.

项目概要/摘要初级纤毛是具有细胞触角功能的多感觉细胞器，我们发现纤毛缺陷。胆管细胞和/或初级纤毛的丧失与多囊肝等胆道疾病有关（PLD）和胆管癌（CCA）更好地了解纤毛调节的信号传导和机制。患病胆管细胞中纤毛损失对于设计基于纤毛恢复的新疗法至关重要，即我们目前的总体目标是了解纤毛在表皮生长调节中的作用。 EGFR 信号传导在 PLD 和 CCA 中异常持续和增强，这两种情况此外，我们的目标是探索胆管细胞纤毛损失的机制。 – 特别是自噬机制如何针对该细胞器的吸收该提案将评估如何进行。纤毛自噬通讯在胆管细胞中发挥作用，减少纤毛表达，从而减少纤毛表达我们认为，纤毛的缺失或功能障碍会增强 EGFR 信号传导。解释纤毛损失/功能障碍，诱导持续的 EGFR 信号传导。具体目标 1：表征活化 EGFR 纤毛依赖性降解的分子机制，我们将评估纤毛对激活 EGFR 降解的需求；表征 EGFR 的机制；易位至初级纤毛；并评估 E3 泛素连接酶 c-CBL 易位至初级纤毛受 EGF 信号传导影响，并驱动位于纤毛中的活化 EGFR 的降解。 2：为了确定将纤毛成分靶向自噬机制的关键参与者，我们将评估自噬和 HDAC6/SIRT1 在体外纤毛表达中的作用评估 HDAC6/SIRT1 在纤毛自噬中的作用；体内；研究纤毛蛋白和自噬货物受体之间的相互作用；并检验以下假设：在纤毛缺陷的胆管细胞中，脱乙酰酶的过度表达会诱导纤毛的赖氨酸脱乙酰化成分，这导致相同残基的泛素化和自噬机制的靶向特定的自噬货物受体在特定目标 3 中：测试特定脱乙酰酶的组合，自噬和 EGFR 抑制剂在临床前啮齿动物模型中作为治疗方法，我们将评估其效果 HDAC6 抑制（Tubastatin-A 或 ACY-1215）和/或 SIRT1 抑制（Sirtinol）与自噬相结合具有或不具有 EGFR 抑制作用（厄洛替尼、阿法替尼）的抑制剂（例如 HCQ、SAR405）在体外和体内评估； Ciliomax（我们最近开发的一种新型双重抑制剂）加 EGFR 抑制的体内效果并评估最显着的效果；患者来源的异种移植的有希望的治疗：确定新的靶点可能会带来急需的结果。我们在体外和临床前啮齿动物中进行的针对这些破坏性疾病的新治疗策略。模型将表征 EGFR 的纤毛依赖性调节以及初级纤毛之间的通讯以及自噬过程，这将为潜在的临床试验奠定基础。