Mechanism of regulation for the oncogenic Pax3-FOXO1 in Alveolar Rhabdomyosarcoma

腺泡状横纹肌肉瘤中致癌 Pax3-FOXO1 的调控机制

• 批准号: 7740485
• 负责人: ANDREW DURRELL HOLLENBACH
• 金额: $ 29.47万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740485
• 关键词: Address; Affect; Alveolar Rhabdomyosarcoma; Apoptosis; Biological; Biological Assay; Cell Proliferation; Childhood; Chimeric Proteins; Chromosomal translocation; DNA Binding; Development; Drug Design; Event; Glycogen Synthase Kinases; Goals; In Vitro; Modeling; Molecular; Molecular Target; Myoblasts; Myomatous neoplasm; Oncogenic; Pathology; Phospho-Specific Antibodies; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Proliferating; Proteins; Regulation; Reporting; Role; Small Interfering RNA; Solid; Survival Rate; Testing; Transcriptional Regulation; Tumor Cell Line; casein kinase II; cell type; genetic regulatory protein; inhibitor/antagonist; kinase inhibitor; mimetics; mutant; myogenesis; neoplastic cell; novel; outcome forecast; protein protein interaction; public health relevance; research study; transcription factor; two-dimensional

DESCRIPTION (provided by applicant): Alveolar Rhabdomyosarcoma (ARMS), an aggressive childhood solid muscle tumor with a poor prognosis, is frequently characterized by a t(2;13) chromosomal translocation resulting in the fusion of two myogenic transcription factors, Pax3 and FOXO1. Alterations in Pax3 transcriptional activity resulting from its fusion to FOXO1 are believed to contribute to the Pax3-FOXO1-dependent inhibition of myogenic differentiation important for the development of ARMS. The goal of this proposal is to elucidate the molecular mechanisms regulating the transcriptional activities of the oncogenic Pax3-FOXO1 fusion protein that may contribute to the inhibition of myogenesis and the development of ARMS. We will "deconstruct" the fusion protein to understand the molecular mechanisms regulating the functions of the wild-type transcription factors. The regulation of FOXO1 has been extensively studied and will not be discussed further. In contrast, since very little is known about the regulation of the functions of Pax3, this proposal focuses on establishing a molecular mechanism for the regulation of Pax3 in normal myogenesis. The subsequent application of this model to Pax3-FOXO1 will provide critical information to understand the role of the fusion protein in the inhibition of myogenesis and the development of ARMS. Understanding these mechanisms will allow the identification of novel molecular targets, such as kinases and sites of phosphorylation, which can be exploited for the rational development of potential therapies for the treatment of ARMS. Using a novel semi-in vitro kinase assay, two-dimensional phosphopeptide analysis, and a phospho-specific antibody, we demonstrate that casein kinase II (CKII) and glycogen synthase kinase 32 (GSK32) phosphorylate Pax3 and Pax3-FOXO1 in vitro contributing to Pax3 DNA binding ability, Pax3 and Pax3-FOXO1 are phosphorylated at Ser205 in proliferating primary myoblasts, and that this phosphorylation is rapidly lost on Pax3 but not Pax3-FOXO1 upon the induction of myogenic differentiation. We hypothesize that Pax3 is phosphorylated by CKII and GSK32 in proliferating myoblasts that the loss of phosphorylation during differentiation regulates its transcriptional activity important for normal myogenesis, and the aberrant phosphorylation of Pax3-FOXO1 during differentiation contributes to the development of ARMS. We will address this hypothesis through the following specific aims: (1) Determine the consequences of phosphorylation on the regulation of the transcriptional activities of Pax3 and Pax3-FOXO1 in proliferating and differentiating primary myoblasts. (2) Analyze the phosphorylation of Pax3 and Pax3-FOXO1 by CKII and GSK32 in vitro and investigate the effects of these kinases on Pax3 and Pax3-FOXO1 transcriptional activities in primary myoblasts. (3) Determine how phosphorylation of Pax3-FOXO1 regulates its activity as it relates to ARMS. PUBLIC HEALTH RELEVANCE: Alveolar Rhabdomyosarcoma (ARMS) is an aggressive childhood solid muscle tumor with a poor prognosis that is characterized by the oncogenic fusion transcription factor Pax3-FOXO1. Understanding the molecular mechanisms regulating the transcriptional activities of Pax3-FOXO1 and the contributions this regulation makes to the development of ARMS will identify novel molecular targets, such as kinases and sites of phosphorylation, which can be exploited for the rational design of drugs to create novel therapies for the treatment of ARMS.

描述（由申请人提供）：肺泡横纹肌肉瘤（ARM）是一种侵略性的儿童固体肌肉肿瘤，预后较差，经常以T（2; 13）染色体易位为特征，导致两个肌原源性转录因子融合，PAX3和FOXO1。据信，PAX3转录活性的变化被认为有助于PAX3-FoxO1依赖性抑制肌原性分化对臂发育很重要。该提案的目的是阐明调节致癌性PAX3-FOXO1融合蛋白转录活性的分子机制，该蛋白可能有助于抑制肌发生和臂的发展。我们将“解构”融合蛋白，以了解调节野生型转录因子功能的分子机制。 FOXO1的调节已经进行了广泛的研究，不会进一步讨论。相反，由于对PAX3功能的调节知之甚少，因此该提案着重于在正常肌发生中建立用于调节PAX3的分子机制。随后将该模型应用于PAX3-FOXO1将提供关键信息，以了解融合蛋白在抑制肌发生和臂发育中的作用。了解这些机制将允许鉴定新的分子靶标，例如激酶和磷酸化的位点，可以利用这些靶标，以合理地发展潜在的疗法来治疗臂的治疗。使用一种新型的半体外激酶测定，二维磷酸肽分析和磷酸特异性抗体，我们证明了酪蛋白激酶II（CKII）和糖原合酶激酶32（GSK32）磷酸酯PAX3和PAX3-Foxo1中的磷酸化酶激酶32（GSK32），并PAX3 DNA结合能力，PAX3和PAX3-FOXO1在增殖的原代成肌细胞中在Ser205处磷酸化，并且这种磷酸化在PAX3上迅速丢失，但在诱导肌原性分化后的PAX3-FoxO1上不会损失。我们假设PAX3在增生的肌细胞中被CKII和GSK32磷酸化，即分化过程中磷酸化的丧失调节其转录活性对正常肌发生重要的转录活性，并且在分化过程中PAX3-Foxo1的异常磷酸化有助于分化臂的发育。我们将通过以下特定目的解决这一假设：（1）确定磷酸化对PAX3和PAX3-FOXO1转录活性在增殖和区分原代成肌细胞中的转录活性的后果。 （2）分析CKII和GSK32在体外分析PAX3和PAX3-FOXO1的磷酸化，并研究了这些激酶对原代肌细胞中PAX3和PAX3-FOXO1转录活性的影响。 （3）确定PAX3-FoxO1的磷酸化如何调节其与手臂相关的活性。公共卫生相关性：肺泡横纹肌肉瘤（ARM）是一种侵略性的儿童固体肌肉肿瘤，预后较差，其特征在于致癌融合转录因子PAX3-FOXO1。了解调节PAX3-FoxO1转录活性的分子机制以及该调节对武器发展的贡献将识别出新的分子靶标，例如激酶和磷酸化的位点，可以利用这些靶标的，这些靶标可以利用这些靶标，以创建新颖的药物设计新的分子靶标治疗武器的疗法。