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 原发性纤毛是功能充当细胞触角的多感官细胞器。我们发现睫状缺陷在 胆管细胞和/或原发性纤毛的丧失与多囊肝病等胆道疾病有关 （PLD）和胆管癌（CCA）。更好地理解纤毛和机制调节的信号 根据纤毛的恢复，即设计新疗法至关重要，在解剖的胆管细胞中纤毛损失至关重要。 纤毛疗法。我们目前的整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中显着持续且增强，两个 具有睫状功能障碍的疾病。此外，我们的目标是探索胆管细胞中睫状损失的机制 - 尤其是自噬机械的目标是如何解决该细胞器。该建议将评估如何 纤毛 - 自助噬菌的交流在胆管细胞中起作用，可降低睫状表达，从而减少睫状细胞，从而如何 纤毛的损失或功能障碍增强了EGFR信号传导。我们提出了病理诱导的纤毛噬菌 解释睫状损失/功能障碍，引起持续的EGFR信号传导。我们提出了三个具体目标： 在特定的目标1中：表征活化EGFR的睫状依赖性降解的分子机制 我们将评估纤毛对激活的EGFR降解的需求；表征EGFR的机制 转移到原发性纤毛；并评估E3泛素连接酶C-CBL易位的假设 EGF信号传导后的原发性纤毛，并驱动位于纤毛中的活化EGFR的降解。在特定目标中 2：要确定针对自噬机械靶向睫状成分的主要参与者，我们将评估 自噬和HDAC6/SIRT1在纤毛噬菌中的作用；评估HDAC6/SIRT1在纤毛磷中的作用 体内;研究睫状蛋白与自噬货物接收器之间的相互作用；并检验以下假设 在睫状缺陷的胆管细胞中，脱乙酰基酶的过表达诱导纤毛的赖氨酸脱乙酰基化 组件，导致通过相同的保留和靶向自噬机械的泛素化。 特定的自噬货物接收器。在特定目标3中：测试特定脱乙酰基酶的组合， 临床前啮齿动物模型中的自噬和EGFR抑制剂作为治疗方法，我们将评估效果 HDAC6抑制（Tubastatin-A或ACY-1215）和/或SIRT1抑制（Sirtinol）与自噬结合 具有或不具有EGFR抑制（Erlotinib，Afatinib）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的双重抑制剂）和EGFR抑制作用的体内效应；并评估最多 在患者衍生的Xenographictic中有希望的治疗方法。影响：确定新目标可能导致急需 这些毁灭性疾病的新治疗策略。我们的体外和临床前啮齿动物实验 模型将表征EGFR的睫状依赖性调节和主要纤毛之间的通信 和自噬过程，将为潜在的临床试验奠定基础。