Stem cell self-renewal programs in rhabdomyosarcoma

项目总结 横纹肌肉瘤(RMS)是一种毁灭性的肌肉恶性肿瘤，在数百名儿童中被诊断为 在美国每年都有成年人。不能切除、转移或转移的患者的存活率低于30%。 复发的RMS，持续的肿瘤生长由少数自我更新的肿瘤维持- 繁殖细胞(TPC)。然而，到目前为止，有针对性的方法来杀死TPC或将它们区分为非 增殖性、分化的RMS细胞类型尚未形成。我们工作的长期目标是 发现通过对TPC的调节作用来推动RMS增长的治疗相关途径。这个 此应用程序的总体目标是确定非规范WNT/平面细胞极性的程度 (WNT/PCP)信号调节TPC的自我更新，可以靶向抑制RMS的生长。我们的中央 假说是Wnt/PCP信号通路的核心调节因子梵高样蛋白2(Vangl2)调节 RMS TPC的自我更新和成长。我们还假设Vangl2的表达仅限于TPC和 可以用来分离和鉴定这些细胞。我们的初步数据显示Vangl2和Wnt/PCp 信号转导调节斑马鱼和人类RMS的TPC自我更新。Vangl2失活导致减少 在体外和体外培养的人RMS细胞中TPC数量、肿瘤细胞生长减少和分化增加 体内使用小鼠异种移植。转基因斑马鱼胚胎RMS模型也显示 Vangl2的表达丰富了自我更新的TPC，提供了动态可视化这些TPC的新方法 并量化改变Wnt/PCP信号对自我更新的影响。我们的理由是 研究表明，Vangl2在绝大多数人类RMS中都是活跃的，是肿瘤持续生长和 自我更新，表明基于Vangl2抑制的治疗策略将受益于很大一部分 高危患者。目标1将评估Wnt/PCP信号在RMS生长和自我更新中的作用 荧光转基因斑马鱼模型和患者来源的异种移植，验证了我们的假设Vangl2和 Wnt/PCP通路调节RMS中TPC的自我更新和扩张。目标2将把Vangl2描述为 斑马鱼和人类RMS中TPC的标记，提供前所未有的动态可视化访问 Wnt/PCP通路在调节细胞分裂后的自我更新和细胞命运选择中的作用。目标3 将阐明Vangl2和Wnt/PCP信号下游的效应通路，测试我们的工作 假设Vangl2通过激活RHOA小GTP酶信号来驱动自我更新。我们的工作 将揭示Vangl2和Wnt/PCP途径促进人类RMS生长的分子途径 和自我更新。这种洞察力将为评估药物对TPC的影响提供新的生物标志物，并可能 寻找治疗RMS的Vangl2以外的新药物靶点。我们的工作预计将有一个很大的 积极的翻译影响，促进我们对控制癌症和癌症自我更新的过程的理解 提供靶向Vangl2以抑制患者来源的异种移植物生长的临床前疗效。

PROJECT SUMMARY Rhabdomyosarcoma (RMS) is a devastating malignancy of muscle that is diagnosed in hundreds of children and adults annually in the USA. Survival rates are less than 30% in patients with unresectable, metastatic, or relapsed RMS, with continued tumor growth being maintained by a small number of self-renewing, tumor- propagating cells (TPCs). Yet, to date, targeted approaches to kill TPCs or to differentiate them into non- proliferative, differentiated RMS cell types have not been developed. The long-term goal of our work is to uncover therapeutically relevant pathways that drive RMS growth through their regulatory effects on TPCs. The overall objective of this application is to determine the extent to which non-canonical Wnt/Planar Cell Polarity (Wnt/PCP) signaling regulates TPC self-renewal and can be targeted to inhibit RMS growth. Our central hypothesis is that Van Gogh-like 2 (Vangl2), a core regulator of the Wnt/PCP signaling pathway, modulates self-renewal and growth of RMS TPCs. We also hypothesize that Vangl2 expression is confined to TPCs and can be used to isolate and characterize these cells. Our preliminary data indicate that Vangl2 and Wnt/PCP signaling regulate TPC self-renewal in both zebrafish and human RMS. VANGL2 inactivation leads to reduced TPC number, decreased tumor cell growth, and elevated differentiation in human RMS cells both in vitro and in vivo using mouse xenografts. Fluorescent transgenic zebrafish models of embryonal RMS also showed that Vangl2 expression enriches for self-renewing TPCs, providing novel approaches to dynamically visualize these cells in live animals and to quantify effects of altering Wnt/PCP signaling on self-renewal. The rationale for our work is that VANGL2 is active in a vast majority of human RMS and is required for continued tumor growth and self-renewal, suggesting that therapeutic strategies based on VANGL2 inhibition would benefit a large fraction of high-risk patients. Aim 1 will assess the role for Wnt/PCP signaling in RMS growth and self-renewal in a fluorescent-transgenic zebrafish model and patient-derived xenografts, testing our hypothesis that Vangl2 and the Wnt/PCP pathway regulate self-renewal and expansion of TPCs in RMS. Aim 2 will characterize Vangl2 as a marker of TPCs in both zebrafish and human RMS, providing unprecedented access to dynamically visualize roles for the Wnt/PCP pathway in regulating self-renewal and cell fate choices following cell division. Aim 3 will elucidate the effector pathways downstream of VANGL2 and Wnt/PCP signaling, testing our working hypothesis that VANGL2 drives self-renewal through the activation of RHOA small GTPase signaling. Our work will uncover the molecular pathways by which VANGL2 and the Wnt/PCP pathway drive human RMS growth and self-renewal. Such insights will provide new biomarkers for assessing drug effects on TPCs and will likely identify novel drug targets beyond VANGL2 for the treatment of RMS. Our work is predicted to have a large positive translational impact, advancing our understanding of processes that control self-renewal in cancer and providing pre-clinical efficacy of targeting VANGL2 to suppress growth of patient derived xenografts.