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

• 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
• 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

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.

项目摘要/摘要 初级纤毛是起细胞触角作用的多感官细胞器。我们发现纤毛缺陷存在于 胆管细胞和/或初级纤毛的丧失与多囊肝病等胆道疾病有关。 (PLD)和胆管细胞癌(CCA)。更好地理解纤毛调控的信号转导及其机制 对病变胆管细胞纤毛丢失的研究是设计基于纤毛修复的新疗法的关键。 纤毛疗法。我们目前的总体目标是了解纤毛在调节表皮生长中的作用。 因子受体(EGFR)信号传导。EGFR信号在PLD和CCA中异常持久和增强，两个 有睫毛功能障碍的疾病。此外，我们的目标是探索胆管细胞纤毛丢失的机制。 -特别是自噬机制是如何针对这种细胞器的再吸收的。这项提案将评估如何 纤毛-自噬通讯在胆管细胞中起作用，以减少纤毛的表达，因此，如何 纤毛缺失或功能障碍可增强EGFR信号转导。我们认为病理诱导的纤毛吞噬 导致纤毛丢失/功能障碍，诱导持续的EGFR信号传导。我们提出三个具体目标： 在特定目标1：表征活化的EGFR纤毛依赖降解的分子机制， 我们将评估纤毛对活化的EGFR降解的需求；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 初级纤毛对EGF信号的影响，并驱动位于纤毛中的活化的EGFR的降解。以特定的目标 2：为了确定将纤毛组件定向到自噬机制中的关键角色，我们将评估 自噬和HDAC6/SIRT1在体外纤毛表达中的作用评价HDAC6/SIRT1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；并检验假设 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 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一种治疗方法，我们将评估其效果 HDAC6抑制(Tubasatin-A或Acy-1215)和/或SIRT1抑制(Sirtinol)结合自噬 体外和体内有或没有EGFR抑制的抑制剂(如HCQ、SAR405)(Erlotinib、Afatinib)；评估 纤毛(我们最近开发的一种新的双重抑制剂)加EGFR抑制的体内效应；并评估最 在患者来源的异种移植中有希望的治疗方法。影响：确定新的目标可能会带来急需的 针对这些毁灭性疾病的新治疗策略。我们在体外和临床前啮齿类动物中的实验 模型将表征EGFR的纤毛依赖性调节和初级纤毛之间的通信 以及自噬过程，这将为潜在的临床试验奠定基础。