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

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（喜树碱样化疗药物的重要靶点）来抑制癌症的改进方法，并进一步阐明p14 ARF/拓扑异构酶I复合物如何有助于癌症机制和治疗反应。p14 ARF在p53通路中发挥着重要作用，最近发现它与拓扑异构酶I有一种新的相互作用。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活性的活化，并导致细胞对靶向拓扑异构酶I的一类有效的喜树碱相关化疗剂的敏感性增强。减少拓扑异构酶I磷酸化的癌症相关异常也会破坏与p14 ARF的复合物形成，并与细胞培养中的治疗抗性相关。虽然与癌症的机制和治疗高度相关，但对这种复合物的调节仍然知之甚少。因此，更好地了解相互作用是如何调节的，以及它如何导致topo I的激活和治疗敏化，将进一步阐明这两种蛋白质在癌症中的作用，并可能导致改进的诊断策略，用于识别耐药肿瘤，以及改进的治疗策略，用于逆转耐药，这是治疗失败的主要原因。本项目的具体目的是（1）确定p14 ARF/拓扑异构酶I相互作用的分子特征，（2）确定p14 ARF介导的拓扑异构酶I活化的分子机制，（3）确定p14 ARF/拓扑异构酶I复合物形成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在统计学上相关，（4）确定p14 ARF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确定目标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p14 ARF和topo I蛋白进行生化分析，以解决目标II中的机制问题。对于目的III，本研究将通过免疫沉淀/蛋白质免疫分析和免疫荧光分析正常和肿瘤组织标本。目的IV将采用基于细胞培养的活力测定和裸鼠中的人肿瘤异种移植模型。这种方法的组合旨在阐明p14 ARF/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。公共卫生相关性：本研究将开发一个基本原理，通过调制的基本细胞酶，拓扑异构酶I，一个目标的一类非常有效的化疗药物，以改善治疗方法的癌症。拓扑异构酶I和p14 ARF肿瘤抑制因子之间的一种新型细胞复合物，是癌症的核心参与者，使癌细胞对拓扑异构酶I靶向药物敏感，并且复合物形成中的癌症相关异常与治疗抗性相关，这是成功治疗癌症的主要障碍。通过阐明该复合物在癌症中的调节和功能，本研究将进一步了解这两种蛋白在癌症中的作用，并为开发该复合物的治疗和诊断奠定基础。

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.