Determining the Mechanism of Transformation for Rhabdomyosarcoma

摘要 横纹肌肉瘤（RMS）是一种侵袭性软组织肉瘤的儿童。虽然许多临床试验 尽管致力于改善患者的预后，但高危儿童的3年生存率在2009年没有超过20%。 过去三十年患有RMS的儿童接受手术，放疗和化疗，这会导致严重的 终身的后果。横纹肌肉瘤被认为是由于肌肉发生受阻， 肌肉发育导致肿瘤形成。然而，RMS可以在缺乏骨骼肌的组织中发展 如膀胱、前列腺和胆道系统，提示RMS可能有其他来源的细胞 而不是肌源性祖细胞我们实验室最近发表的一篇论文，Drummond等人，鉴定的内皮细胞 作为RMS起源的候选细胞。我们的基因工程小鼠模型表达了一种组成型活性的 在表达由脂肪蛋白2（aP 2）启动子驱动的Cre重组酶的细胞中的Smoothened（SmoM 2）等位基因。 这些小鼠中有50%在出生后28天发展出特别局限于颈部的RMS。aP 2-Cre表示为 多种细胞类型，包括内皮细胞，但不包括肌肉细胞。这表明RMS可以由非 肌原性细胞，但仍存在许多问题。我所提出的项目侧重于理解的作用， SmoM 2在肿瘤发生和增殖中的作用，以及引起肌源性命运转换的下游信号传导， 内皮祖细胞确定肿瘤维持是否需要持续的SmoM 2， 增殖，我将在体外培养肿瘤细胞作为肿瘤球，并评估肿瘤球的能力， SmoM 2被击倒。我的项目的第二个方面是确定细胞的机制， 重新编程导致RMS形成，特别是在颈部。我们实验室的初步数据， 来自其他小组的研究表明，头部和颈部肌肉特异性转录因子如Tbx 1， Pitx 2、Tcf 21和Musculin在SmoM 2表达后增加。为了确定细胞的机制 通过在aP 2谱系细胞中SmoM 2表达启动的重编程，我们的实验室产生了转基因小鼠， 有条件地过表达Tbx 1，并获得另一个有条件地缺失Tbx 1等位基因， 使我们能够确定Tbx 1是否足以和必要的导致肿瘤发生。了解 RMS重编程的机制基础不仅有助于我们为这些疾病创造更有针对性的治疗方法， 患者，但也表明类似的重编程事件可能有助于其他实体瘤。

ABSTRACT Rhabdomyosarcoma (RMS) is an aggressive, soft-tissue sarcoma in children. While many clinical trials have worked to improve patient outcomes, the 3-year survival rate for high-risk children hasn’t risen above 20% for the past three decades. Children with RMS undergo surgery, radiation, and chemotherapy, which lead to serious life-long consequences. RMS is thought to arise from a block in myogenesis which prevents normal skeletal muscle development leading to tumor formation. However, RMS can develop in tissues devoid of skeletal muscle such as the urinary bladder, prostate, and biliary tree, suggesting that RMS may have an alternative cell of origin other than a myogenic progenitor. A recent publication by our lab, Drummond et al., identified endothelial cells as a candidate cell of origin for RMS. Our genetically engineered mouse model expresses a constitutively active Smoothened (SmoM2) allele in cells expressing Cre recombinase driven by the adipose protein 2 (aP2) promoter. 50% of these mice develop RMS specifically restricted to the neck by 28 days of life. aP2-Cre is expressed by multiple cell types including endothelial cells, but not muscle cells. This indicates that RMS can arise from non- myogenic cells, however many questions still remain. My proposed project focuses on understanding the role of SmoM2 in tumorigenesis and proliferation, and the downstream signaling that causes a myogenic fate switch in endothelial progenitors. To determine whether continued SmoM2 is required for tumor maintenance and proliferation, I will culture tumor cells in vitro as tumor spheres and evaluate the ability of tumor spheres to passage with SmoM2 knocked down. The second aspect of my project is to identify the mechanism of cellular reprogramming leading to RMS formation specifically in the neck. Preliminary data from our lab, supported by research from other groups, shows that head and neck muscle specification transcription factors such as Tbx1, Pitx2, Tcf21 and Musculin are increased upon SmoM2 expression. To determine the mechanism of cellular reprogramming initiated by SmoM2 expression in aP2-lineage cells, our lab generated a transgenic mouse that conditionally overexpresses Tbx1 and acquired another that has conditional deletion of the Tbx1 allele, which allows us to ascertain whether Tbx1 is sufficient and necessary to cause tumorigenesis. Understanding the mechanistic basis for reprogramming in RMS will not only help us create more directed therapies for these patients, but also suggests that similar reprogramming events may contribute to other solid tumors.