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: 8808736
• 负责人: Myron Steve Ignatius
• 金额: $ 18.04万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-18 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8808736
• 关键词: Animals; Biological Assay; Cell Culture Techniques; Cell Fraction; Cell Line; Cell Transplantation; Cell division; Cells; Child; Childhood Soft Tissue Sarcoma; Clinical; DNA Binding; Data; Diagnostic; Disease; Embryonal Rhabdomyosarcoma; Fishes; Frequencies; Gene Chips; Genes; Genetic Epistasis; Goals; Growth; Health; Heterogeneity; Human; Image; Immune; In Vitro; Label; Laboratories; Life; Malignant Childhood Neoplasm; Malignant Neoplasms; Modeling; Molecular; Mus; Muscle; Muscle Development; Muscle satellite cell; NOTCH1 gene; Oncogenes; PAX7 gene; Pathologic; Pathway interactions; Patients; Play; Population; Process; Regulatory Element; Relapse; Role; Signal Transduction; Staging; Testing; Therapeutic; Time; Transgenic Model; Transgenic Organisms; Tumor Cell Line; Work; Zebrafish; base; clinically relevant; established cell line; human disease; in vivo; in vivo imaging; inhibitor/antagonist; meetings; muscle regeneration; neoplastic cell; notch protein; outcome forecast; overexpression; research study; satellite cell; self-renewal; stem cell population; tumor; tumor growth; tumor microenvironment

DESCRIPTION (provided by applicant): 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 proposal is to determine a role for Notch in regulating tumor-propagating potential 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, highlighting the need to identify molecular pathways that drive self-renewal and tumor re-growth at relapse. My hypothesis is that Notch pathway activation increases the pool of tumor-propagating cells by altering cell fate decisions following cell division, leading to increased symmetric cell divisions and subsequently larger fractions of relapse associated clones. Notch has been implicated as an important modulator of self-renewal in normal muscle stem cells by controlling symmetric versus asymmetric divisions and the pathway is commonly activated in ERMS through overexpression of NOTCH1 and 3. 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. Moreover, I have shown that Notch and RAS synergize to increase both tumor size and the overall pool of ERMS-propagating cells 25-fold when compared with ERMS that lack Notch pathway activation. Increased ERMS-propagating potential is accompanied by enhanced expression of pax7, myf5 and c-Met and in the context of muscle stem cells, both pax7 and myf5 are important transcriptional regulators. 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 1a will assess if Notch confers tumor-propagating potential to a molecularly definable subpopulation of ERMS cells. Aim1b 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. Aim 2 will extend these findings to human disease. Aim 2a will assess if Notch pathway enhances tumor-propagating cell frequencies in vitro through use of sphere colony forming assays in primary human and established cell lines. Aim 2b will utilize limiting dilution cell transplantation of low passage human primary ERMS cells into immune compromised mice, comparing tumors with high and low Notch activity and correlating the frequency of ERMS-propagating cells within the tumor mass. Aim 3 will assess in ERMS tumor cell lines and low passage human primary ERMS, if Notch regulates self-renewal by directing the expression of important muscle transcriptional regulators PAX7 and MYF5 both of which are upregulated in human and zebrafish ERMS. 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, self-renewal and ultimately relapse.

描述（由申请人提供）：自我更新肿瘤的细胞驱动肿瘤的持续生长，并负责复发。如果可以关闭肿瘤细胞自我更新的过程，那么肿瘤将消退，并且患者将保持无复发。该提案的目的是确定Notch在调节肿瘤传播潜力（ERMS）中的作用，这是肌肉的毁灭性儿科恶性肿瘤。复发是ERMS患者面临的主要临床问题，其中不到40％的复发患者幸存下来，强调需要鉴定出驱动自我更新和复发时肿瘤再增长的分子途径。我的假设是，Notch途径激活通过改变细胞分裂后的细胞命运决策来增加肿瘤传播细胞的池，从而导致对称细胞分裂增加，并随后更大的复发相关克隆分数。 Notch has been implicated as an important modulator of self-renewal in normal muscle stem cells by controlling symmetric versus asymmetric divisions and the pathway is commonly activated in ERMS through overexpression of NOTCH1 and 3. 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.这些细胞可以直接在活荧光转基因斑马鱼中直接可视化，从而使现场动物的自我更新前所未有。此外，我已经证明，与缺乏Notch途径激活的ERMS相比，Notch和RAS协同增强了肿瘤大小和ERMS传播细胞的整体库。 ERMS传播电位的增加伴随着PAX7，MyF5和C-MET的表达增强，在肌肉干细胞的背景下，PAX7和MyF5都是重要的转录调节剂。在这些观察结果的基础上，我的建议将确定斑马鱼和人类ERMS中缺口促进肿瘤潜在潜力的细胞和分子机制。具体而言，AIM 1A将评估Notch是否赋予ERMS细胞分子可定义的亚群的肿瘤传播潜力。 AIM1B将通过活荧光转基因鱼的动态实时成像来评估Notch途径激活是否改变ERMS传播细胞亚置折叠中的对称性和不对称分裂。 AIM 2将把这些发现扩展到人类疾病。 AIM 2A将通过在原代人和已建立的细胞系中使用球体集落形成测定法来评估Notch途径是否会在体外增强肿瘤传播细胞频率。 AIM 2B将利用限制稀释细胞移植 将人类原发性ERMS细胞传递到免疫损害的小鼠中，比较了较高和低凹槽活性的肿瘤，并将肿瘤肿块中ERMS传播细胞的频率相关联。如果Notch通过指导重要的肌肉转录调节剂PAX7和MYF5的表达来调节自我更新，AIM 3将在ERMS肿瘤细胞系和低通道人的原发性ERMS中评估。总的来说，我的建议提供了一项综合策略，以询问Notch途径如何调节ERMS自我更新，并且可能具有巨大的治疗意义，因为临床上与临床相关的Notch途径抑制剂可能会降低肿瘤传播细胞频率，自我更新并最终复发。