Stem cell self-renewal programs in rhabdomyosarcoma

项目摘要 横纹肌肉瘤（RMS）是一种毁灭性的恶性肌肉，是诊断在数百名儿童 成年人每年在美国。在不可切除的、转移性的、或转移性的肿瘤患者中， 复发性RMS，肿瘤持续生长由少量自我更新的肿瘤细胞维持， 繁殖细胞（TPC）。然而，迄今为止，杀死TPC或将其分化为非TPC的靶向方法仍然存在。 尚未开发出增殖的、分化的RMS细胞类型。我们工作的长期目标是 揭示通过对TPC的调节作用驱动RMS生长的治疗相关途径。的 本申请的总体目标是确定非典型Wnt/平面电池极性 (Wnt/PCP）信号传导调节TPC自我更新，并且可以有针对性地抑制RMS生长。我们的中央 假设是货车高氏样蛋白2（Vangl 2），Wnt/PCP信号传导途径的核心调节物， RMS TPC的自我更新和增长。我们还假设Vangl 2表达仅限于TPC， 可用于分离和表征这些细胞。我们的初步数据表明，Vangl 2和Wnt/PCP 在斑马鱼和人类RMS中，信号传导调节TPC自我更新。VANGL 2失活导致降低的 在体外和体外培养的人RMS细胞中，TPC数量减少，肿瘤细胞生长减少，分化增加。 体内使用小鼠异种移植物。胚胎RMS的荧光转基因斑马鱼模型也表明， Vangl 2表达富集了自我更新的TPC，提供了动态可视化这些TPC的新方法。 活动物中的细胞，并量化改变Wnt/PCP信号传导对自我更新的影响。我们的理由是， VANGL 2在绝大多数人RMS中是活性的，并且是持续肿瘤生长所需的， 自我更新，这表明基于VANGL 2抑制的治疗策略将使大部分人受益。 高风险患者。目的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物，测试我们的假设，Vangl 2和 Wnt/PCP途径调节RMS中TPC自我更新和扩增。目标2将Vangl 2描述为 斑马鱼和人类RMS中TPC的标记物，提供了前所未有的动态可视化途径， Wnt/PCP途径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VANGL 2和Wnt/PCP信号传导的下游效应通路，测试我们的工作 VANGL 2通过激活RHOA小GT3信号传导驱动自我更新的假设。我们的工作 将揭示VANGL 2和Wnt/PCP途径驱动人类RMS生长的分子途径 和自我更新这些见解将为评估药物对TPC的影响提供新的生物标志物， 确定VANGL 2以外的新药物靶点用于治疗RMS。我们的工作预计会有很大的 积极的转化影响，推进我们对控制癌症自我更新过程的理解， 提供靶向VANGL 2以抑制患者来源的异种移植物生长的临床前功效。

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.