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 I V 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.
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. ? ? ? ? ?
