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: 8037774
• 负责人: ANDREW DURRELL HOLLENBACH
• 金额: $ 28.58万
• 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/8037774
• 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）：腺泡状横纹肌肉瘤（ARMS）是一种预后不良的侵袭性儿童实体肌肉肿瘤，其特征通常是 t(2;13) 染色体易位，导致两种肌源性转录因子 Pax3 和 FOXO1 的融合。 Pax3 与 FOXO1 融合导致的转录活性改变被认为有助于 Pax3-FOXO1 依赖性肌源性分化抑制，这对于 ARMS 的发展至关重要。该提案的目的是阐明调节致癌 Pax3-FOXO1 融合蛋白转录活性的分子机制，该融合蛋白可能有助于抑制肌生成和 ARMS 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的调控已被广泛研究，不再进一步讨论。相比之下，由于对 Pax3 功能的调节知之甚少，因此该提案的重点是建立 Pax3 在正常肌生成中调节的分子机制。该模型随后应用于 Pax3-FOXO1 将为了解融合蛋白在抑制肌生成和 ARMS 发展中的作用提供关键信息。了解这些机制将有助于识别新的分子靶点，例如激酶和磷酸化位点，可用于合理开发治疗 ARMS 的潜在疗法。使用新型半体外激酶测定、二维磷酸肽分析和磷酸特异性抗体，我们证明酪蛋白激酶 II (CKII) 和糖原合酶激酶 32 (GSK32) 在体外磷酸化 Pax3 和 Pax3-FOXO1，有助于 Pax3 DNA 结合能力，Pax3 和 Pax3-FOXO1 在增殖的原代成肌细胞中，Ser205 被磷酸化，并且在诱导肌原性分化后，这种磷酸化在 Pax3 上迅速消失，而 Pax3-FOXO1 上则不会。我们假设 Pax3 在增殖的成肌细胞中被 CKII 和 GSK32 磷酸化，分化过程中磷酸化的丧失调节其转录活性，这对正常肌生成很重要，而分化过程中 Pax3-FOXO1 的异常磷酸化有助于 ARMS 的发展。我们将通过以下具体目标来解决这一假设：（1）确定磷酸化对原代成肌细胞增殖和分化中 Pax3 和 Pax3-FOXO1 转录活性调节的影响。 (2)体外分析CKII和GSK32对Pax3和Pax3-FOXO1的磷酸化，并研究这些激酶对原代成肌细胞中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 ARMS 相关的活性。公共卫生相关性：腺泡状横纹肌肉瘤 (ARMS) 是一种侵袭性儿童实体肌肉肿瘤，预后不良，其特征是致癌融合转录因子 Pax3-FOXO1。了解调节 Pax3-FOXO1 转录活性的分子机制以及这种调节对 ARMS 发展的贡献将确定新的分子靶点，例如激酶和磷酸化位点，可用于合理设计药物，以创造治疗 ARMS 的新疗法。