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)：自我更新的肿瘤增殖细胞驱动肿瘤持续生长，并对复发负责。如果肿瘤细胞自我更新的过程能够被关闭，那么肿瘤就会退化，患者将保持无复发。该提案的目的是确定Notch在胚胎性横纹肌肉瘤(ERMS)中调节肿瘤传播潜力的作用，ERMS是一种毁灭性的儿科肌肉恶性肿瘤。复发是ERMS患者面临的主要临床问题，只有不到40%的复发患者存活下来，这突显了识别推动自我更新和复发时肿瘤重新生长的分子途径的必要性。我的假设是，Notch通路的激活通过改变细胞分裂后的细胞命运决定，增加了肿瘤传播细胞池，导致对称细胞分裂增加，从而增加了与复发相关的克隆的比例。Notch被认为是正常肌肉干细胞自我更新的重要调节器，通过控制对称和不对称分裂，该途径通常通过NOTCH1和3的过度表达在ERMS中激活。我建议的初步数据显示，RAS驱动的ERMS包含一个分子上不同的ERMS繁殖细胞群，这些细胞高水平表达myf5，但缺乏分化的肌肉标志物表达。这些细胞可以直接在活的、荧光转基因的斑马鱼中可视化，从而前所未有地获得了在活动物中可视化自我更新的机会。此外，我已经证明，与缺乏Notch通路激活的ERMS相比，Notch和RAS的协同作用使肿瘤大小和ERMS增殖细胞的总数增加了25倍。伴随着ERMS增殖能力的增强的是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因子。在这些观察的基础上，我的建议将确定Notch改变斑马鱼和人类ERM中肿瘤传播潜力的细胞和分子机制。具体地说，Aim 1a将评估Notch是否赋予分子上可定义的ERMS细胞亚群肿瘤增殖潜力。Aim1b将通过对活的荧光转基因FISH进行动态实时成像，评估Notch途径的激活是否改变了ERMS传播细胞亚组分中的对称和不对称分裂。AIM 2将把这些发现扩展到人类疾病。目的2a将通过在原代人和已建立的细胞系中使用球集落形成试验来评估Notch途径是否在体外提高肿瘤增殖细胞的频率。Aim 2b将利用Low的有限稀释细胞移植 将人类原代ERMS细胞导入免疫受损的小鼠，比较Notch活性高和低的肿瘤，并将肿瘤内ERMS增殖细胞的频率关联起来。目的3将评估在ERMS肿瘤细胞系和低代人原代ERMS中，Notch是否通过指导重要的肌肉转录调控因子PAX7和MYF5的表达来调节自我更新，这两个调控因子在人和斑马鱼的ERMS中都上调。总之，我的建议提供了一个全面的策略来询问Notch途径如何调节ERMS的自我更新，并可能具有巨大的治疗意义，因为临床相关的Notch途径抑制剂可能会降低肿瘤增殖细胞的频率，自我更新，并最终复发。