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

胆管细胞中初级纤毛的损失——与自噬机制的相互作用

• 批准号: 10605658
• 负责人: Sergio A Gradilone
• 金额: $ 49.38万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 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/10605658
• 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

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.

项目总结/摘要 初级纤毛是多感觉细胞器，起着细胞触角的作用。我们发现， 胆管细胞和/或初级纤毛的丧失与胆道疾病如多囊肝病有关 (PLD)和胆管癌（CCA）。更好地理解纤毛调控的信号传导及其机制 疾病胆管细胞中纤毛损失的研究对于设计基于纤毛恢复的新疗法至关重要，即， 纤毛疗法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 因子受体（EGFR）信号传导。EGFR信号传导在PLD和CCA中异常持续和增强， 睫状体功能障碍的疾病。此外，我们的目的是探讨胆管细胞纤毛缺失的机制， - 尤其是自噬机制是如何以细胞器的再吸收为目标的。该提案将评估如何 纤毛-自噬通讯在胆管细胞中起作用，以减少纤毛表达，因此， 纤毛的丧失或功能障碍增强EGFR信号传导。我们认为病理性食毛症 导致纤毛损失/功能障碍，诱导持续的EGFR信号传导。我们提出三个具体目标： 具体目标1：表征活化EGFR纤毛依赖性降解的分子机制， 我们将评估纤毛对活化的EGFR降解的需要;表征EGFR的机制， 易位到初级纤毛;并评估E3泛素连接酶c-CBL易位到初级纤毛的假设。 表皮生长因子受体（EGFR）是表皮生长因子受体（EGF）的一个重要组成部分，它通过表皮生长因子（EGF）信号传导作用于初级纤毛，并驱动位于纤毛中的活化EGFR的降解。具体目标 2：为了确定参与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HDAC 6/SIRT 1在体外纤毛表达中的作用;评估HDAC 6/SIRT 1在纤毛吞噬中的作用 在体内;研究纤毛蛋白和自噬货物受体之间的相互作用;并测试假设， 在纤毛缺陷的胆管细胞中，脱乙酰酶的过度表达诱导纤毛的赖氨酸脱乙酰化， 组分，这导致相同残基的泛素化和自噬机制的靶向， 特异性自噬货物受体。在具体目标3中：为了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 HDAC 6抑制（Tubastatin-A或ACY-1215）和/或SIRT 1抑制（Sirtinol）与自噬的组合 抑制剂（例如，HCQ、SAR 405）联合或不联合EGFR抑制（厄洛替尼、阿法替尼）;评估 Ciliomax（我们最近开发的一种新型双重抑制剂）加EGFR抑制的体内作用;并评估最 在患者来源的异种移植物中有希望的治疗。影响：确定新的目标可能导致急需的 针对这些毁灭性疾病的新的治疗策略。我们在体外和临床前啮齿动物中的实验 模型将表征EGFR的纤毛依赖性调节和初级纤毛之间的通信， 和自噬过程，这将为潜在的临床试验奠定基础。