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(申请人提供)：这项研究的长期目标是开发通过调节拓扑异构酶I来抑制癌症的改进方法，拓扑异构酶I是喜树碱类化疗药物的重要靶点，并进一步阐明p14ARF/拓扑异构酶I复合体如何在癌症机制和治疗反应中起作用。P14ARF在P53途径中起重要作用，最近被发现与拓扑异构酶I发生一种新的相互作用。这种相互作用需要拓扑异构酶I的丝氨酸磷酸化，导致拓扑异构酶I活性的激活，并导致细胞对一类有效的针对拓扑异构酶I的喜树碱相关化疗药物的敏感性增强。癌症相关的异常减少了拓扑异构酶I的磷酸化，也取消了与p14ARF的复合体的形成，并与细胞培养中的耐药相关。虽然与癌症的机制和治疗高度相关，但对这种复合体的调节仍然知之甚少。因此，更好地了解这种相互作用是如何调节的，以及它是如何导致Topo I激活和治疗敏化的，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，以及改进逆转耐药性的治疗策略，这是治疗失败的主要原因。本项目的具体目标是(1)确定p14ARF/拓扑异构酶I相互作用的分子特征，(2)确定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I复合体形成的异常在癌症中发生的频率以及它们是否与肿瘤的临床属性统计相关，以及(4)确定p14ARF介导的治疗增敏的治疗潜力。这项研究将利用分子生物学技术来确定AIM I中拓扑异构酶I的结合结构域，并使用定义的合成DNA底物和纯化的p14ARF和Topo I蛋白进行生化分析，以解决AIM II中的机制问题。对于AIM III，研究将通过免疫沉淀/Western分析和免疫荧光分析正常组织和肿瘤组织的样本。目的IV将采用基于细胞培养的活性分析和裸鼠体内人肿瘤移植模型。这两种方法的结合旨在阐明p14ARF/拓扑异构酶I复合体的生物学功能，并确定其治疗和诊断潜力。与公共卫生相关：本研究将通过调节重要的细胞酶--拓扑异构酶I，为改进癌症治疗方法提供理论基础，拓扑异构酶I是一类高效化疗药物的靶标。拓扑异构酶I和p14ARF肿瘤抑制因子之间的一种新的细胞复合体是癌症的核心角色，它使癌细胞对拓扑异构酶I靶向药物敏感，而与癌症相关的形成复合体的异常与治疗耐药性有关，这是成功治疗癌症的主要障碍。通过阐明该复合体在癌症中的调节和功能，本研究将进一步深入了解这两种蛋白在癌症中的作用，并为该复合体的治疗和诊断开发奠定基础。