Molecular Modeling of Pediatric Skeletal Muscle Tumors

描述（由申请人提供）：横纹肌肉瘤（RMS）是一种表现出不同程度骨骼肌分化的异质癌症集合。虽然占儿童恶性实体瘤的8%左右，但RMS是14岁以下儿童中最常见的软组织肉瘤。RMS的两种主要组织学亚型是胚胎型（eRMS）和肺泡型（aRMS）。高危患者的5年生存率为30%，肿瘤表达PAX3-FKHR融合基因的儿童预后很差；当转移时，他们的5年生存率<8%。这种标志性的遗传变化仅在aRMS中发现，被认为是肿瘤特异性癌基因，但没有分子靶向治疗。为了解决RMS知识的空白，我们已经创建了一个新的疾病模型，该模型基于原始人类骨骼肌细胞转化为其致瘤性对应物，使用一组定义的遗传变化。使用该模型，我们发现人类骨骼肌成肌细胞在免疫缺陷小鼠中作为异种移植物进行测试时可能转化为产生模拟RMS肿瘤的细胞。在确定骨骼肌来源的原代人细胞可以引起RMS后，我们研究了在其中表达PAX3-FKHR的影响，并发现了两种可能是其致癌行为基础的表型。首先，当PAX3-FKHR作为一种早期遗传变化稳定表达时，它可以绕过衰老检查点，并作为骨骼肌肿瘤发展的初始致癌打击。其次，当PAX3-FKHR作为晚期基因变化稳定表达时，可能通过激活Ras通路，将体内肿瘤形成的潜伏期从11周缩短到2周，因为在对照实验中PAX3-FKHR可以在功能上替代Ras癌基因。在这项提议中，我们希望了解PAX3-FKHR如何绕过衰老检查点，以及它如何加速先前转化细胞的肿瘤发生。为了实现这一目标，我们将(1)使用功能获得和功能丧失两种方法，检测PAX3-FKHR下游的候选蛋白在克服衰老检查点中的作用；(2)检测加速的肿瘤细胞在生长信号、凋亡和/或血管生成方面增强的自给自足，以及Ras通路在PAX3-FKHR增强的肿瘤发生中的作用。这些目标的实现将有助于深入了解小儿恶性肿瘤的发生机制，并为研究提供新的治疗靶点。此外，这种基因定义的模型将作为系统研究其他人类肉瘤的模板。

DESCRIPTION (provided by applicant): Rhabdomyosarcoma (RMS) is a heterogeneous collection of cancers demonstrating varying degrees of skeletal muscle differentiation. Although accounting for ~8% of pediatric malignant solid tumors, RMS is the most common soft tissue sarcoma in children younger than 14 years. The two major histologic subtypes of RMS are embryonal (eRMS) and alveolar (aRMS). High risk patients have a 5-year survival of 30%, and outcome is very poor for children whose tumors express the PAX3-FKHR fusion gene; when metastatic, their 5-year survival is <8%. This signature genetic change is found only in aRMS and considered a tumor-specific oncogene, but has no molecularly targeted treatment. To address gaps in knowledge of RMS, we have created a new model for this disease based on the conversion of primary human skeletal muscle cells to their tumorigenic counterpart, using a defined set of genetic changes. Using this model, we found that human skeletal muscle myoblasts may be converted to cells that generate tumors mimicking RMS when tested as xenografts in immunodeficient mice. Having established that primary human cells of skeletal muscle origin can give rise to RMS, we studied the repercussions of expressing PAX3-FKHR in them, and discovered two phenotypes that may underlie its oncogenic behavior. First, when PAX3-FKHR was stably expressed as an early genetic change, it enabled bypass of the senescence checkpoint and served as an initiating oncogenic hit for the development of skeletal muscle tumors. Second, when PAX3-FKHR was stably expressed as a late genetic change, it shortened the latency of in vivo tumor formation from 11 to 2 weeks, possibly through activation of the Ras pathway, since in control experiments PAX3-FKHR could functionally substitute for the RAS oncogene. In this proposal, we wish to understand how PAX3-FKHR enables bypass of the senescence checkpoint, and how it accelerates tumorigenesis in previously transformed cells. To accomplish this, we will (1) examine candidate proteins that are downstream of PAX3-FKHR for their role in overcoming the senescence checkpoint, using both gain-of-function and loss-of-function approaches, and (2) examine the accelerated tumor cells for enhanced self-sufficiency in growth signaling, apoptosis, and/or angiogenesis, and the role of the Ras pathway in this PAX3-FKHR-augmented tumorigenesis. The accomplishment of these aims will provide insight into the genesis of this pediatric malignancy, and provide new therapeutic targets for study. In addition, this genetically defined model will serve as a template for the systematic investigation of other human sarcomas. PUBLIC HEALTH RELEVANCE: This research uniquely models the series of oncogenic events causing the pediatric cancer rhabdomyosarcoma. It is expected to yield insight into the genesis of this cancer, provide new therapeutic targets for study, and serve as a template for the systematic analysis of other human sarcomas.