Mechanism of Action of the TBX3 Gene in Breast Cancer

TBX3 基因在乳腺癌中的作用机制

• 批准号: 10677744
• 负责人: Megan Agajanian
• 金额: $ 10.05万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677744
• 关键词: 3-Dimensional; Adaptor Signaling Protein; Adult; Amaze; Biochemistry; Biological Assay; Biology; Biotinylation; Bone Diseases; Cancer Model; Cell Fate Control; Cell Nucleus; Cell Proliferation; Cell Survival; Cell membrane; Cell surface; Cells; Cellular biology; Chemicals; Clathrin; Colorectal Cancer; Complex; Consensus; Data; Development; Docking; Down-Regulation; Drug Targeting; Embryonic Development; Endocytosis; Evaluation; Event; Excision; Family; Feedback; Flow Cytometry; Future; Genes; Genetic Transcription; Goals; Homeostasis; Human; Hyperactivity; Informatics; Ligands; Ligation; Literature; Longevity; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Measures; Mediating; Microscopy; Molecular; Nature; Neoplasm Metastasis; Normal Cell; Pathway interactions; Patient-Focused Outcomes; Pharmacology; Phosphorylation; Phosphotransferases; Physiological; Postdoctoral Fellow; Proliferating; Protein Family; Protein Isoforms; Protein Kinase; Proteins; Proteomics; Publications; Regulation; Reporter; Reporting; Repression; Research; Research Personnel; Research Project Grants; Role; Signal Transduction; Structure; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Training; Transcription Coactivator; Tumor Suppressor Proteins; Validation; WNT Signaling Pathway; Western Blotting; Work; attenuation; beta catenin; cancer therapy; career; casein kinase I; developmental disease; drug discovery; experience; experimental study; extracellular; functional genomics; gain of function; genetic manipulation; human disease; improved; in silico; inhibitor; kinase inhibitor; live cell imaging; loss of function; malignant breast neoplasm; member; migration; nervous system disorder; new therapeutic target; novel; phosphoproteomics; programs; protein protein interaction; protein structure; receptor; response; small molecule inhibitor; structural biology; therapeutic target; therapeutically effective

PROJECT SUMMARY WNT signaling is crucial for embryonic development and adult tissue homeostasis, with aberrant signaling resulting in developmental disorders and disease, including cancer. Although much is known, a deeper mechanistic understanding of this signaling cascade will improve our understanding of cancer formation, progression and metastasis, allowing for the development of more effective therapeutics. WNT/b-catenin signaling is driven by the stabilization of the transcriptional co-activator, b-catenin. In the absence of WNT ligand, a cytosolic destruction complex phosphorylates, ubiquitylates and degrades b-catenin. In the presence of WNT ligand, the WNT receptors, Frizzled and LRP6, and intracellular proteins form an alternative complex called the WNT signalosome. This results in b-catenin accumulation and activation of b-catenin target genes. Recent data demonstrate that upon WNT ligand engagement, the signalosome is endocytosed. Although conflicting data exist within the literature, a consensus is beginning to emerge that clathrin-dependent endocytosis of the signalosome results in signalosome degradation. This training proposal and my thesis project is devoted to elucidating the molecular events and dynamics of signalosome formation, stabilization and endocytosis in normal cells and in cancer, with an emphasis on kinases. In the first half of my graduate training, I utilized a gain-of- function screen of the kinome to identify AAK1 as a negative regulator of WNT signaling. I demonstrated that AAK1 activates a transcription independent negative feedback loop to promote LRP6 internalization, resulting in WNT signaling downregulation. In the course of these studies, we demonstrated that AAK1 promotes the phosphorylation of a clathrin adapter protein, AP2M1, 8-10 hrs post-WNT3A and that AAK1 and AP2M1 interact with the tumor suppressor, WTX. My lab previously discovered the WTX tumor suppressor as a component of the signalosome and b-catenin destruction complex. Therefore, I will define comprehensive WNT3A and WTX- dependent changes to the phosphoproteome by quantitative mass spectrometry and test whether WTX regulates signalosome endocytosis via AAK1. Additionally, CSNK1g is known to regulate phosphorylation of LRP6, an essential step for signalosome formation. CSNK1g has 3 isoforms, CSNK1g1/2/3, all identified as understudied kinases. My preliminary data suggest each isoform functions differently to activate WNT signaling and promote LRP6 internalization. A main focus for the remainder of my graduate work will be to functionally characterize the role of each CSNK1g isoform in regulating WNT signaling, define comprehensive protein-protein interaction networks and evaluate isoform specific changes to the WNT-driven phosphoproteome. Because this work is descriptive in nature, I expect it to be submitted for publication in 14 months. To summarize, the precise role of endocytosis in WNT signaling remains unclear, with numerous questions surrounding the mechanism(s) and components of endocytosis and its effects on signaling. This work, and my future postdoctoral work, will provide me training in and experience in the mechanisms of WNT signaling and feedback attenuation.

项目摘要 WNT信号传导对于胚胎发育和成体组织稳态至关重要， 导致发育障碍和疾病，包括癌症。虽然知道得很多，但更深层次的 对这种信号级联的机械理解将提高我们对癌症形成的理解， 进展和转移，从而允许开发更有效的治疗剂。WNT/b-连环蛋白 信号传导由转录辅激活因子β-连环蛋白的稳定化驱动。在不存在WNT配体的情况下， 胞质破坏复合物磷酸化、泛素化和降解β-连环蛋白。在WNT存在的情况下 配体、WNT受体、Frizzled和LRP 6以及细胞内蛋白质形成了一种替代性复合物，称为 WNT信号体。这导致β-连环蛋白积累和β-连环蛋白靶基因的激活。最近的数据 证明在WNT配体接合时，信号体被内吞。尽管数据相互矛盾 存在于文献中，一个共识开始出现，网格蛋白依赖的内吞作用的细胞， 信号体导致信号体降解。这个培训计划和我的论文项目致力于 阐明了正常细胞中信号体形成、稳定和内吞的分子事件和动力学， 细胞和癌症，重点是激酶。在我的研究生培训的前半部分，我利用了一个收益- 功能筛选的激酶组，以确定AAK 1作为WNT信号的负调节。我证明了 AAK 1激活转录非依赖性负反馈环以促进LRP 6内化，导致 WNT信号下调。在这些研究过程中，我们证明了AAK 1促进了 WNT 3A后8-10小时网格蛋白衔接蛋白AP 2 M1的磷酸化，以及AAK 1和AP 2 M1相互作用 肿瘤抑制因子WTX我的实验室以前发现WTX肿瘤抑制因子是 信号体和B-连环蛋白破坏复合体。因此，我将定义全面的WNT 3A和WTX- 通过定量质谱法测定磷酸化蛋白质组的依赖性变化，并测试WTX是否调节 通过AAK 1进行信号体内吞。此外，已知CSNK 1g调节LRP 6的磷酸化， 信号体形成的重要步骤。CSNK 1g有3种亚型，CSNK 1g 1/2/3，均被确定为研究不足 激酶。我的初步数据表明，每种异构体的功能不同，以激活WNT信号传导，并促进 LRP 6内化。我的研究生工作的其余部分的主要重点将是功能上的特点， 每个CSNK 1g亚型在调节WNT信号传导中的作用，定义了全面的蛋白质-蛋白质相互作用 网络，并评估WNT驱动的磷酸化蛋白质组的同种型特异性变化。因为这项工作是 由于这是一份描述性的报告，我预计它将在14个月内提交出版。总而言之， WNT信号传导中的内吞作用仍不清楚，围绕其机制和 内吞作用的组分及其对信号传导的影响。这项工作，以及我未来的博士后工作，将提供 我在WNT信号传导和反馈衰减机制方面的培训和经验。