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

描述（由申请人提供）：本申请响应通知编号（NOT-OD-09-058），标题为：NIH宣布竞争性修订申请的恢复法案资金可用性。本研究的长期目标是通过调节喜树碱样化疗药物的重要靶点拓扑异构酶I（topo I）来开发改进的癌症治疗，并进一步阐明p14 ARF/topo I复合物如何有助于癌症的机制和治疗反应。p14 ARF在p53通路中起着公认的作用，并且还参与与topo I的新的p53非依赖性相互作用。这种相互作用需要topo I丝氨酸磷酸化，激活topo I，并增强细胞对喜树碱和相关topo I靶向化疗药物的敏感性。具有降低的topo I磷酸化的癌细胞缺乏p14 ARF/topo I复合物并且对喜树碱具有抗性。因此，更好地了解这种复合物的调节将阐明这两种蛋白质在癌症中的作用，并可能导致改善癌症的诊断和治疗策略。该基金的具体目标是：（1）确定p14 ARF/topo I相互作用的分子特征，（2）确定p14 ARF介导的topo I激活的分子机制，（3）确定癌症中p14 ARF/topo I复合物形成异常的频率以及它们是否与肿瘤的临床属性统计学相关，（4）确定p14 ARF介导的治疗敏化的治疗潜力。修订后的资助引入了一个额外的目标（5），研究基于精脒辅酶A的组蛋白乙酰化抑制剂的作用，这是一类癌细胞靶向化合物，可抑制双链断裂修复并使细胞对喜树碱敏感。目的是研究这些抑制剂如何影响拓扑异构酶I单独和与p14 ARF的组合，并提供了一个临床前评估其疗效作为治疗增敏剂的存在和不存在的ARF。本研究将采用分子生物学技术（Aim I）、生物化学试验（Aim II和V）、免疫沉淀/蛋白质免疫分析和免疫荧光（Aim III）以及基于细胞培养的活力试验和裸鼠人肿瘤异种移植模型（Aim IV和V）。 公共卫生相关性：这项研究开发了基于p14 ARF肿瘤抑制剂和基于Spermidine-CoA的组蛋白乙酰化抑制剂的新方法，以改善细胞对拓扑异构酶I靶向化疗的反应，并逆转治疗耐药性，这是成功治疗癌症的主要障碍。该研究还开发了用于识别治疗反应性肿瘤和优化治疗方案的诊断工具。 .

DESCRIPTION (provided by applicant): This application responds to Notice Number (NOT-OD-09-058) entitled: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The long term goal of this study is to develop improved cancer treatments through modulation of topoisomerase I (topo I), an important target for camptothecin-like chemotherapeutic drugs, and to further elucidate how the p14ARF/topo I complex contributes to the mechanism of cancer and to the therapy response. p14ARF plays a well-established role in the p53 pathway, and also engages in a novel p53-independent interaction with topo I. The interaction requires topo I serine phosphorylation, activates topo I, and enhances cellular sensitivity to camptothecin and related topo I-targeted chemotherapeutic drugs. Cancer cells with reduced topo I phosphorylation lack p14ARF/topo I complexes and are resistant to camptothecin. A better understanding of the regulation of this complex would therefore elucidate the roles of both proteins in cancer, and could lead to improved diagnostic and therapeutic strategies for cancer. The Specific Aims of the parent grant are to (1) Determine the molecular features of the p14ARF/topo I interaction, (2) Define the molecular mechanism of p14ARF-mediated activation of topo I, (3) Determine how frequently abnormalities in p14ARF/topo I complex formation occur in cancer and whether they correlate statistically with clinic attributes of tumors, and (4) Determine the therapeutic potential of p14ARF-mediated therapy sensitization. The revised grant introduces an additional Aim (5) that studies the effects of Spermidine-CoA-based inhibitors of histone acetylation, a class of cancer cell-targeted compounds that inhibit double strand break repair and sensitize cells to camptothecin. The Aim examines how these inhibitors affect topo I alone and in combination with p14ARF and provides a preclinical evaluation of their efficacy as therapy sensitizers in the presence and absence of ARF. The study will employs molecular biology techniques (Aim I), biochemical assays (Aims II and V), immunoprecipitation/western analysis and immunofluorescence (Aim III), and cell culture-based viability assays and human tumor xenograph models in nude mice (Aims IV and V). PUBLIC HEALTH RELEVANCE: This study develops novel approaches based on the p14ARF tumor suppressor and Spermidine-CoA-based inhibitors of histone acetylation to improve cellular responses to topoisomerase I-targeted chemotherapies and reverse therapy resistance, a major obstacle to successful cancer treatment. The study also develops diagnostic tools for identifying therapy responsive tumors and optimizing therapeutic regimens. .