Role of Polyamines in MYCN-Amplified Neuroblastoma

多胺在 MYCN 扩增神经母细胞瘤中的作用

• 批准号: 7667635
• 负责人: ANDRE S BACHMANN
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667635
• 关键词: Accounting; Adenosylmethionine Decarboxylase; Affect; Apoptosis; Biological Assay; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cell Survival; Cells; Cessation of life; Child; Childhood; Cloning; Co-Immunoprecipitations; Communication; DL-alpha-Difluoromethylornithine; DNA; Development; Disease; Employee Strikes; Enzymes; Evaluation; Event; Flow Cytometry; Gene Expression; Genetic Transcription; Half-Life; High Pressure Liquid Chromatography; Hydrogen Peroxide; In Vitro; Individual; Investigation; Laser Microscopy; MYCN gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Mammalian Cell; Measures; Messenger RNA; Metabolic; Metabolism; Molecular; Mutation; Neuroblastoma; Nitric Oxide; Northern Blotting; Nuclear; Oncogenes; Ornithine Decarboxylase; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Polyamines; Production; Protein Overexpression; Proteins; Putrescine; Rate; Regulation; Research; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Running; SAM486A; SAT gene; Sepiapterin reductase; Series; Signal Pathway; Signal Transduction; Signaling Protein; Site-Directed Mutagenesis; Spermidine; Spermidine/Spermine N1-Acetyltransferase; Spermine; Staging; System; Techniques; Testing; Tetracycline; Tetracyclines; Translating; Western Blotting; Work; Yeasts; base; enzyme activity; human FRAP1 protein; inhibitor/antagonist; insight; migration; novel; novel therapeutics; outcome forecast; response; tumor; tumorigenesis; urea cycle; yeast two hybrid system

DESCRIPTION (provided by applicant): Neuroblastoma (NB) is the third most common malignancy in childhood and accounts for about 15% of cancer-related deaths in children. One of the most striking abnormalities found in NB tumors is the amplification of the MYCN oncogene, which strongly correlates with more aggressive tumors and poor prognosis of late stage disease. The complete absence of effective treatments for high-risk (late stage) NB patients indicates the need for novel therapeutic approaches, including drugs that block MYCN expression. The primary objective of our previous work has been to evaluate the polyamine biosynthetic pathway as an alternative target for NB therapy. Central to our investigation was the evaluation of two clinically tested polyamine inhibitors, DFMO and SAM486A, which specifically inhibit the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), respectively. Since MYCN has been shown to activate ODC gene expression, we hypothesized that ODC and polyamines might play a role in MYCN-amplified NB tumorigenesis. Our initial results showed that the depletion of polyamines in MYCN-amplified NB cells caused G1 cell cycle arrest and affected p27Kip1 levels, retinoblastoma protein Rb phosphorylation, and most intriguingly, blocked MYCN expression, thus providing a rationale for the potential use of polyamine inhibitors in late-stage NB patients. In the current proposal we outline studies that examine how MYCN, ODC, and polyamines regulate important cell cycle and cell survival pathways in NB cells. In Specific Aim 1 we will study whether MYCN overexpression in NB cells impacts endogenous ODC and other metabolic enzymes of polyamine metabolism, and whether this translates into changes in polyamine pools, cell cycle progression, and cell proliferation. These studies will be performed using three unique NB cell lines, in which MYCN expression can be switched on/off by adding tetracycline. In Specific Aim 2 we will examine the role of polyamines in cell cycle regulation and PI3K/Akt cell survival activation in NB cells. Specifically, we will determine the differential effects of individual polyamines (putrescine, spermidine, and spermine) on the regulation and phosphorylation/activation of key signaling proteins including MYCN, p27Kip1, Cdk2, Akt, and mTOR. In Specific Aim 3 we will elucidate the role of ODC in cell signaling by examining its interaction with two new cellular proteins. The physiological relevance of these interactions will be verified by mutational analyses and a series of cell-based biological assays. The proposed methods and techniques to pursue this work are: Northern blot, RT-PCR, nuclear run-ons, messenger RNA half-life studies, Western blot, yeast two-hybrid, co-immunoprecipitation, GST-pull down, site-directed mutagenesis, DNA cloning, confocal laser microscopy, enzyme and in vitro kinase assays, flow cytometry, and HPLC. At the conclusion of this research, we will have uncovered how ODC and individual polyamines regulate important molecular events that control cell cycle and cell survival pathways of NB cells. Our research will be useful in the development of novel treatments for the most aggressive forms of the childhood cancer neuroblastoma.

描述（由申请人提供）：神经母细胞瘤（NB）是儿童期第三常见的恶性肿瘤，约占儿童癌症相关死亡的15%。在NB肿瘤中发现的最显著的异常之一是MYCN癌基因的扩增，其与更具侵袭性的肿瘤和晚期疾病的不良预后密切相关。对于高危（晚期）NB患者完全缺乏有效的治疗方法，表明需要新的治疗方法，包括阻断MYCN表达的药物。我们以前工作的主要目的是评估多胺生物合成途径作为NB治疗的替代靶点。我们研究的中心是评价两种临床测试的多胺抑制剂DFMO和SAM 486 A，它们分别特异性抑制多胺生物合成酶鸟氨酸脱羧酶（ODC）和S-腺苷甲硫氨酸脱羧酶（S-adenosylmethionine decarboxylase，ODC）。由于MYCN已被证明激活ODC基因表达，我们假设ODC和多胺可能在MYCN扩增的NB肿瘤发生中发挥作用。我们的初步研究结果表明，在MYCN扩增的NB细胞中多胺的耗尽导致G1细胞周期停滞，并影响p27 Kip 1水平，视网膜母细胞瘤蛋白Rb磷酸化，最有趣的是，阻断MYCN表达，从而为多胺抑制剂在晚期NB患者中的潜在用途提供了理论基础。在目前的提案中，我们概述了研究MYCN，ODC和多胺如何调节NB细胞中重要的细胞周期和细胞存活途径。在具体目标1中，我们将研究NB细胞中MYCN过表达是否影响内源性ODC和多胺代谢的其他代谢酶，以及这是否转化为多胺库，细胞周期进程和细胞增殖的变化。这些研究将使用三种独特的NB细胞系进行，其中MYCN表达可以通过添加四环素来打开/关闭。在具体目标2中，我们将研究多胺在NB细胞中的细胞周期调节和PI 3 K/Akt细胞存活激活中的作用。具体来说，我们将确定个别多胺（腐胺，亚精胺和精胺）的调节和磷酸化/激活的关键信号蛋白，包括MYCN，p27 Kip 1，Cdk 2，Akt和mTOR的差异影响。在具体目标3中，我们将通过研究ODC与两种新的细胞蛋白的相互作用来阐明ODC在细胞信号传导中的作用。这些相互作用的生理相关性将通过突变分析和一系列基于细胞的生物测定来验证。建议的方法和技术进行这项工作是：北方印迹，RT-PCR，核run-ons，信使RNA半衰期的研究，蛋白质印迹，酵母双杂交，免疫共沉淀，GST-下拉，定点诱变，DNA克隆，激光共聚焦显微镜，酶和体外激酶测定，流式细胞术，和HPLC。在这项研究的结论中，我们将揭示ODC和单个多胺如何调节控制NB细胞的细胞周期和细胞存活途径的重要分子事件。我们的研究将有助于开发针对最具侵袭性的儿童癌症神经母细胞瘤的新疗法。