A role for notch in self renewal in embryonal rhabdomyosarcoma

缺口在胚胎横纹肌肉瘤自我更新中的作用

• 批准号: 9485912
• 负责人: Myron Steve Ignatius
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-17 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9485912
• 关键词: Animals; Biological Assay; Candidate Disease Gene; Cell Fraction; Cell Line; Cell Transplantation; Cell division; Cells; Child; Childhood Soft Tissue Sarcoma; Clinic; Clinical; DNA Binding; Data; Disease; Embryonal Rhabdomyosarcoma; Family member; Fishes; Frequencies; Gene Chips; Genes; Genetic Epistasis; Goals; Growth; Human; Image; Immune; In Vitro; Label; Laboratories; Malignant Childhood Neoplasm; Methods; Modeling; Molecular; Mus; Muscle; Mutation; NOTCH1 gene; PAX7 gene; Pathologic; Pathway interactions; Patients; Population; Process; Regulatory Element; Relapse; Role; Sampling; Self-Direction; Signal Transduction; Therapeutic; Time; Tissues; Transgenic Model; Transgenic Organisms; Transplantation; Tumor Expansion; Update; Zebrafish; base; clinically relevant; diagnostic biomarker; experimental study; human disease; in vivo; in vivo imaging; inhibitor/antagonist; knock-down; neoplastic cell; notch protein; novel; outcome forecast; overexpression; relapse patients; satellite cell; self renewing cell; self-renewal; small hairpin RNA; tumor; tumor growth; tumor heterogeneity; tumor microenvironment

Self-renewing tumor-propagating cells drive continued tumor growth and are responsible for relapse. If the process by which tumor cells self-renew could be turned off, then tumors would regress and patients would remain relapse free. The goal of this updated proposal is to define the cellular and molecular mechanisms by which Notch regulates tumor-propagating potential and plasticity of the tumor propagating cell state in embryonal rhadomyosarcoma (ERMS), a devastating pediatric malignancy of the muscle. Relapse is the major clinical problem facing patients with ERMS, with less than 40% of relapse patients surviving their disease. Progress on this project using a combination of in vivo experiments in the zebrafish ERMS model and in in vitro experiments using ERMS cell lines and primary tissues has validated the hypothesis that Notch pathway activation increases the pool of tumor- propagating cells (TPCs), but rather surprisingly in vivo cell transplantation experiments finds that Notch enables the dedifferentiation of non-TPCs into TPCs. Using human patient samples, ERMS cell lines and correlative data in zebrafish has identified critical Notch regulated targets in human ERMS including SNAI1, MEF2C, PAX7 and MYF5. Preliminary data within my proposal shows that RAS-driven ERMS contain a molecularly distinct population of ERMS-propagating cells that express high levels of myf5 but lack differentiated muscle marker expression. These cells can be directly visualized in live, fluorescent- transgenic zebrafish, allowing unprecedented access to visualize self-renewal in live animals. Building on these observations, my proposal will determine the cellular and molecular mechanisms by which Notch alters tumor-propagating potential in both zebrafish and human ERMS. Specifically, Aim 1 will assess if Notch pathway activation alters symmetric vs. asymmetric divisions in the ERMS-propagating cell subfraction by dynamic real-time imaging of live, fluorescent transgenic fish. A sub aim will use lineage tracing methods to define the frequency and dynamics of dedifferentiation to make TPCs in ERMS. Aim 2 Will show that NOTCH1 expands TPCs in vivo in human ERMS by utilizing limiting dilution cell transplantation of low passage human primary ERMS cells into immune compromised mice. Aim 3 will assess the molecular mechanisms by which downstream NOTCH1 effector genes SNAI1, PAX7, MYF5 and MEF2C expands self-renewal, drives dedifferentiation and blocks terminal differentiation. In total, my proposal provides a comprehensive strategy to interrogate how the Notch pathway regulates ERMS self- renewal and will likely have immense therapeutic significance as clinically-relevant Notch pathway inhibitors would likely reduce tumor propagating cell frequency, dedifferentiation and ultimately relapse.

自我更新的肿瘤增殖细胞驱动肿瘤的持续生长，并导致复发。如果进程 通过它可以关闭肿瘤细胞的自我更新，然后肿瘤就会退化，患者就会继续复发 免费的。这项更新提案的目标是定义细胞和分子机制，通过这些机制 Notch调节肿瘤增殖潜能和肿瘤增殖细胞状态的可塑性 胚胎性横纹肌肉瘤(ERMS)，一种毁灭性的儿童肌肉恶性肿瘤。旧病复发是主要原因 ERMS患者面临的临床问题，不到40%的复发患者存活下来。进展 本项目采用斑马鱼ERMS模型的体内实验和体外实验相结合的方法 使用ERMS细胞系和原代组织证实了Notch途径激活增加了 肿瘤增殖细胞池(TPC)，但更令人惊讶的是，体内细胞移植实验发现 NOTCH使非TPC能够去分化为TPC。使用人类患者样本，ERMS细胞系和 斑马鱼的相关数据已经确定了人类ERMS中关键的Notch调控靶点，包括SNAI1， MEF2C、PAX7和MYF5。我的提案中的初步数据显示，RAS驱动的ERM包含 分子上不同的erms增殖细胞群，高水平表达myf5，但缺乏分化 肌肉标记物表达。这些细胞可以直接在活的、荧光转基因的斑马鱼中可视化，从而 史无前例地在活体动物中可视化自我更新。在这些观察的基础上，我的建议将 确定Notch改变两种斑马鱼肿瘤增殖潜能的细胞和分子机制 和人类的错误。具体地说，Aim 1将评估Notch通路的激活是否改变了对称性与非对称性 通过活的荧光转基因鱼的动态实时成像在erms繁殖的细胞亚组分中进行分裂。 一个子目标将使用血统追踪方法来定义去分化的频率和动态，以制造TPC 在急诊室。目的2将证明NOTCH1通过利用有限稀释细胞在人体内扩增TPC 低传代人原代ERMS细胞移植到免疫低下小鼠体内。目标3将评估 NOTCH1下游效应基因SNAI1、PAX7、MYF5和MEF2C扩增的分子机制 自我更新，推动去分化，阻止终末分化。总的来说，我的提案提供了一个 全面的策略，以询问Notch途径如何调节ERM自我更新，并可能 巨大的治疗意义，因为临床相关的Notch途径抑制剂可能会减少肿瘤 传播细胞频率，去分化，最终复发。