Regulation and Function of the p14ARF/topoisomerase I Complex in Cancer

p14ARF/拓扑异构酶 I 复合物在癌症中的调节和功能

• 批准号: 7653663
• 负责人: RUTH A GJERSET
• 金额: $ 37.35万
• 依托单位: TORREY PINES INST FOR MOLECULAR STUDIES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-10 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7653663
• 关键词: Address; Affect; Antibodies; Antineoplastic Agents; Binding; Biochemical; Biological; Biological Assay; Biological Process; C-terminal; CDKN2A gene; Camptothecin; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Nucleolus; Chromatin; Clinical; Complex; DNA; DNA Binding; Defect; Diagnostic; Down-Regulation; Drug Delivery Systems; Enzymes; Freezing; Frequencies; Goals; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Lead; Malignant Neoplasms; Maps; Mediating; Modeling; Molecular; Molecular Biology Techniques; Mutation; Nonhomologous DNA End Joining; Nude Mice; Pathway interactions; Pensions; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Play; Protein Kinase C; Proteins; Regulation; Resistance; Role; Serine; Small Interfering RNA; Specimen; Surrogate Markers; TP53 gene; Testing; Therapeutic; Tissues; Treatment Failure; Tumor Suppressor Proteins; Type I DNA Topoisomerases; base; cancer cell; cancer therapy; casein kinase II; chemotherapeutic agent; design; human TOP1 protein; improved; in vitro Assay; in vivo; insight; irinotecan; neoplastic; neoplastic cell; novel; overexpression; public health relevance; response; synthetic construct; tumor

DESCRIPTION (provided by applicant): The long term goal of this study is to develop improved ways to suppress cancer through modulation of topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, and to further elucidate how the p14ARF/topoisomerase I complex contributes to the mechanism of cancer and to the therapy response. p14ARF, which plays a well-established role in the p53 pathway, has recently been found to engage in a novel interaction with topoisomerase I. The interaction requires topoisomerase I serine phosphorylation, results in activation of topoisomerase I activity, and leads to enhanced cellular sensitivity to a potent class of camptothecin-related chemotherapeutic agents that target topoisomerase I. Cancer-associated abnormalities that reduce phosphorylation of topoisomerase I, also abolish complex formation with p14ARF and correlate with therapy resistance in cell culture. Though highly relevant to the mechanism and treatment of cancer, the regulation of this complex remains poorly understood. A better molecular understanding of how the interaction is regulated and how it leads to activation of topo I and therapy sensitization will therefore further elucidate the roles of both proteins in cancer, and could lead to improved diagnostic strategies for identifying resistant tumors as well as improved therapeutic strategies for reversing resistance, a major cause of treatment failure. The Specific Aims of this project are to (1) Determine the molecular features of the p14ARF/topoisomerase I interaction, (2) Define the molecular mechanism of p14ARF-mediated activation of topoisomerase I, (3) Determine how frequently abnormalities in p14ARF/topoisomerase I complex formation occur in cancer and whether they correlate statistically with clinical attributes of tumors, and (4) Determine the therapeutic potential of p14ARF-mediated therapy sensitization. The study will employ molecular biology techniques to identify binding domains on topoisomerase I in Aim I, and biochemical assays with defined synthetic DNA substrates and purified p14ARF and topo I proteins to address mechanistic issues in Aim II. For Aim III, the study will analyze specimens of normal and neoplastic tissue by immunoprecipitation/western analysis and immunofluorescence. Aim IV will employ cell culture-based viability assays and human tumor xenograph models in nude mice. Together this combination of approaches is designed to clarify the biological function of the p14ARF/topoisomerase I complex and determine its therapeutic and diagnostic potential. PUBLIC HEALTH RELEVANCE: The present study will develop a rationale for improved therapeutic approaches to cancer through modulation of the essential cellular enzyme, topoisomerase I, a target for a highly potent class of chemotherapeutic drugs. A novel cellular complex between topoisomerase I and the p14ARF tumor suppressor, a central player in cancer, sensitizes cancer cells to topoisomerase I-targeted drugs, and cancer associated abnormalities in complex formation correlate with therapy resistance, a major obstacle to successful treatment of cancer. By elucidating the regulation and function of this complex in cancer, this study will provide further insight into the roles of these two proteins in cancer, and establish a basis for exploiting the complex therapeutically and diagnostically.

描述（由申请人提供）：本研究的长期目标是开发通过调节拓扑异构酶 I（喜树碱类化疗药物的重要靶标）来抑制癌症的改进方法，并进一步阐明 p14ARF/拓扑异构酶 I 复合物如何发挥作用癌症的机制和治疗反应。 p14ARF 在 p53 通路中发挥着明确的作用，最近被发现与拓扑异构酶 I 发生新的相互作用。这种相互作用需要拓扑异构酶 I 丝氨酸磷酸化，导致拓扑异构酶 I 活性激活，并导致细胞敏感性增强一种针对拓扑异构酶 I 的有效的喜树碱相关化疗药物。减少拓扑异构酶 I 磷酸化的癌症相关异常，还消除了 p14ARF 的复合物形成，并与细胞培养中的治疗耐药性相关。尽管与癌症的机制和治疗高度相关，但对该复合物的调节仍知之甚少。因此，更好地了解相互作用如何调节以及它如何导致拓扑I激活和治疗敏化将进一步阐明这两种蛋白在癌症中的作用，并可能导致改进识别耐药肿瘤的诊断策略以及改进治疗逆转耐药性的策略，耐药性是治疗失败的主要原因。该项目的具体目标是 (1) 确定 p14ARF/拓扑异构酶 I 相互作用的分子特征，(2) 定义 p14ARF 介导的拓扑异构酶 I 激活的分子机制，(3) 确定 p14ARF/拓扑异构酶异常的频率I 复合物的形成发生在癌症中，以及它们是否与肿瘤的临床属性有统计相关性，以及 (4) 确定 p14ARF 介导的治疗敏化的治疗潜力。该研究将采用分子生物学技术来识别 Aim I 中拓扑异构酶 I 上的结合域，并使用确定的合成 DNA 底物和纯化的 p14ARF 和拓扑 I 蛋白进行生化测定，以解决 Aim II 中的机制问题。对于目标 III，该研究将通过免疫沉淀/蛋白质印迹分析和免疫荧光分析正常和肿瘤组织样本。 Aim IV 将在裸鼠中采用基于细胞培养的活力测定和人类肿瘤异种移植模型。这种方法组合旨在阐明 p14ARF/拓扑异构酶 I 复合物的生物学功能并确定其治疗和诊断潜力。公共健康相关性：本研究将通过调节必需的细胞酶拓扑异构酶 I（一类高效化疗药物的靶点）来开发改进癌症治疗方法的基本原理。拓扑异构酶 I 和 p14ARF 肿瘤抑制因子之间的新型细胞复合物（癌症的核心参与者）使癌细胞对拓扑异构酶 I 靶向药物敏感，而癌症相关的复合物形成异常与治疗耐药性相关，而治疗耐药性是成功治疗癌症的主要障碍。通过阐明这种复合物在癌症中的调节和功能，这项研究将进一步深入了解这两种蛋白质在癌症中的作用，并为利用该复合物进行治疗和诊断奠定基础。