Molecular Modeling of Pediatric Skeletal Muscle Tumors

描述(申请人提供)：横纹肌肉瘤(RMS)是一种表现为不同程度骨骼肌分化的异质性癌症集合。虽然约占儿童恶性实体肿瘤的8%，但RMS是14岁以下儿童最常见的软组织肉瘤。RMS的两个主要组织学亚型是胚胎型(ERMS)和肺泡型(ARM)。高危患者的5年存活率为30%，对于肿瘤表达PAX3-FKHR融合基因的儿童来说，预后非常差；当肿瘤转移时，他们的5年存活率为8%。这种标志性的基因变化只在手臂上发现，被认为是肿瘤特异性癌基因，但没有分子靶向治疗。为了解决有关RMS的知识空白，我们创建了一种新的模型，该模型基于原始人类骨骼肌细胞向其致瘤对应细胞的转换，使用了一组定义的基因变化。使用这个模型，我们发现人类骨骼肌成肌细胞可以转化为产生肿瘤的细胞，当作为免疫缺陷小鼠的异种移植进行测试时，这些细胞产生类似RMS的肿瘤。在确定骨骼肌源性的原代人类细胞可以产生RMS之后，我们研究了在其中表达PAX3-FKHR的反应，并发现了可能支持其致癌行为的两种表型。首先，当PAX3-FKHR作为早期基因改变稳定表达时，它能够绕过衰老检查点，并在骨骼肌肿瘤的发展中起到启动致癌作用。第二，当PAX3-FKHR作为一种晚期基因突变稳定表达时，它可能通过激活RAS途径，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。在这项提案中，我们希望了解PAX3-FKHR如何使衰老检查点绕过，以及它如何加速先前转化的细胞中的肿瘤形成。为此，我们将(1)使用功能获得和功能丧失的方法，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。这些目标的实现将为深入了解这种儿科恶性肿瘤的发病机制，并为研究提供新的治疗靶点。此外，这个基因定义的模型将作为系统研究其他人类肉瘤的模板。 公共卫生相关性：这项研究独特地模拟了导致儿童癌症横纹肌肉瘤的一系列致癌事件。它有望深入了解这种癌症的发生机制，为研究提供新的治疗靶点，并为其他人类肉瘤的系统分析提供模板。

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.