Mechanism of Action of the TBX3 Gene in Breast Cancer

TBX3 基因在乳腺癌中的作用机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 WNT 信号传导对于胚胎发育和成人组织稳态至关重要，信号传导异常导致发育障碍和疾病，包括癌症。虽然知道的很多，但更深层次的对这种信号级联的机制理解将提高我们对癌症形成的理解，进展和转移，从而可以开发更有效的治疗方法。 WNT/b-连环蛋白信号传导是由转录辅激活因子 b-连环蛋白的稳定性驱动的。在没有 WNT 配体的情况下，胞质破坏复合物可磷酸化、泛素化并降解 β-连环蛋白。在 WNT 存在的情况下配体、WNT 受体、Frizzled 和 LRP6 以及细胞内蛋白形成另一种复合物，称为 WNT 信号体。这导致 b-连环蛋白积累并激活 b-连环蛋白靶基因。近期数据证明在 WNT 配体接合后，信号体被内吞。尽管数据相互矛盾文献中存在，一个共识开始出现，即网格蛋白依赖性内吞作用信号小体导致信号小体降解。这个培训计划和我的论文项目致力于阐明正常情况下信号体形成、稳定和内吞作用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，我利用了增益- 激酶组的功能筛选将 AAK1 识别为 WNT 信号传导的负调节因子。我证明了 AAK1 激活转录独立的负反馈环路以促进 LRP6 内化，从而导致 WNT 信号下调。在这些研究过程中，我们证明 AAK1 促进 WNT3A 后 8-10 小时，网格蛋白接头蛋白 AP2M1 磷酸化，并且 AAK1 和 AP2M1 相互作用与肿瘤抑制剂 WTX 一起使用。我的实验室之前发现 WTX 肿瘤抑制因子是信号体和β-连环蛋白破坏复合物。因此，我将综合定义WNT3A和WTX- 通过定量质谱分析磷酸化蛋白质组的依赖性变化并测试 WTX 是否调节通过 AAK1 进行信号小体内吞作用。此外，已知 CSNK1g 可以调节 LRP6 的磷酸化，LRP6 是一种信号小体形成的重要步骤。 CSNK1g 有 3 个亚型，CSNK1g1/2/3，均已确定为未充分研究的激酶。我的初步数据表明，每种亚型在激活 WNT 信号传导和促进 LRP6 内化。我余下的研究生工作的一个主要重点将是从功能上描述每个 CSNK1g 亚型在调节 WNT 信号传导中的作用，定义全面的蛋白质-蛋白质相互作用网络并评估 WNT 驱动的磷酸化蛋白质组的亚型特异性变化。因为这部作品是本质上是描述性的，我预计它会在 14 个月内提交出版。总而言之，确切的作用 WNT 信号传导中的内吞作用仍不清楚，围绕其机制和机制存在许多问题内吞作用的组成部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接受过 WNT 信号传导和反馈衰减机制方面的培训和经验。