Basis of PAX3-FKHR oncogenesis in rhabdomyosarcoma

描述（申请人提供）：横纹肌肉瘤（Rhabdomyosarcoma， RMS），肿瘤或骨骼肌，是最常见的儿童软组织癌症。RMS的发展与环境因素没有已知的联系，因此了解RMS的关键必须来自研究RMS特有的致病变化。本研究的重点是肺泡RMS (aRMS)，这是最具侵袭性的RMS形式，肿瘤复发率和死亡率最高。ARMS携带t2；13染色体易位产生了一种嵌合蛋白，该蛋白结合了肌肉调节因子Pax3的DNA结合域和无处不在表达的FKHR的激活域。PAX3-FKHR是一种有效的转录因子，可导致肌肉细胞失去细胞周期和细胞分化控制。从aRMS细胞中去除PAX3-FKHR可导致细胞死亡。这些观察结果支持PAX3-FKHR是aRMS发生和发展的关键病原体。然而，PAX3-FKHR介导的肿瘤发生的分子基础仍然难以捉摸。从以前的资助期获得的数据为揭示融合蛋白的致癌基础提供了重要线索。PAX3-FKHR获得了改变通常不受亲本蛋白调节的基因表达的新机制。此外，PAX3-FKHR的致癌作用主要取决于这些转录特异性的增加。我们假设PAX3-FKHR特异性破坏的分子通路代表了aRMS发育中最关键的事件。这项资助的目的是研究pax3 - fkhr特异性调控机制，促进肌肉细胞的增殖和分化缺陷。本文提出了三个具体目标：Aim 1旨在鉴定和表征Pax3 -FKHR不依赖于Pax3和FKHR直接靶向表达的基因的功能作用。这项研究将通过PAX3-FKHR确定肌肉细胞转化的早期事件。Aim 2旨在定义PAX3-FKHR参与G1细胞周期缺陷的分子步骤，特别关注E2F1-Skp2-p27kip1调控轴。Aim 3的重点是描述PAX3-FKHR阻断肌肉分化的分子事件，重点是Myogenin/MEF2C活性失调在促进肌肉特异性基因（如Myf6）中的作用。目标2和3中概述的研究将为aRMS细胞不受控制的生长和分化表型提供分子基础。拟议研究的结果将填补aRMS开发的整个途径，从t2开始；13介导的PAX3-FHR的产生导致下游靶标对转化途径的异常激活。阐明肿瘤特异性激活/失活途径的基础将为患者的诊断或预后以及在不损害正常细胞功能的情况下特异性中断肿瘤功能的新治疗设计提供重要的决定因素。

DESCRIPTION (provided by applicant): Rhabdomyosarcoma (RMS), tumor or skeletal muscle, is the most common pediatric soft tissue cancer. RMS development has no known association with environmental factors, thus the key to understanding RMS must come from studying pathogenic changes that are unique to RMS. This application focuses on alveolar RMS (aRMS), the most aggressive RMS form with the highest tumor recurrence and mortality rate. ARMS carries a t2;13 chromosomal translocation that creates a chimeric protein that combines the DNA binding domain of a muscle regulatory factor Pax3 and the activation domain of a ubiquitously expressed FKHR. PAX3-FKHR is a potent transcription factor that causes muscle cells to lose both cell cycle and cell differentiation controls. Obliterating PAX3-FKHR from aRMS cells leads to cell death. These observations support PAX3-FKHR as a critical pathogen in the initiation and progression of aRMS development. Yet, the molecular basis of PAX3-FKHR mediated oncogenesis remains elusive. Data obtained from previous funding periods provided major clues for unveiling the oncogenic basis of the fusion protein. PAX3-FKHR gains novel mechanisms to alter expression of genes that are not normally regulated by the parent proteins. Moreover, the PAX3-FKHR oncogenic effect depends critically on these gains in transcription specificity. We hypothesize that molecular pathways specifically disrupted by PAX3-FKHR represent the most critical events involved in aRMS development. The objective of this grant is to investigate PAX3-FKHR-specific regulatory mechanisms that contribute to the proliferation and differentiation defects in muscle cells. Three specific aims are proposed: Aim 1 is designed to identify and characterize the functional role of genes whose expression is directly targeted by PAX3-FKHR independent of Pax3 and FKHR. This study will define early events in muscle cell transformation by PAX3-FKHR. Aim 2 is designed to define the molecular steps involved in G1 cell cycle defect by PAX3-FKHR, with a specific focus on E2F1-Skp2-p27kip1 regulatory axis. Aim 3 focuses on delineate the molecular events responsible for blockage in muscle differentiation by PAX3-FKHR, with an emphasis on the dysregulated Myogenin/MEF2C activity in promoting muscle specific genes such as Myf6. Studies outlined in Aims 2 and 3 will provide a molecular basis for the uncontrolled growth and differentiation phenotypes in aRMS cells. Results of the proposed studies will fill in the entire pathway in aRMS development, from t2;13 mediated creation of PAX3-FHR to aberrant activation of downstream targets to transformation pathways. Elucidating the basis of tumor-specific activation/inactivation pathways will provide important determinants in patient diagnosis or prognosis and in novel therapeutic design that specifically interrupt tumor function without damaging normal cell function.