High Throughput Screen for Small Molecule Inhibitors of Colorectal Cancer Cell Pr

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DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is one of the leading causes of cancer mortality and morbidity in the United States. Studies indicate that CRC results from stepwise changes (mutations) in key genes with important cellular functions. These genes include tumor suppressor genes (TSGs) and oncogenes. For example, somatic or germline inactivation of TSGs such as adenomatous polyposis coli (APC) and p53; and oncogenic activation of KRAS and BRAF are crucial in the pathogenesis of CRC. However, despite this knowledge, therapies targeting to specific components of the altered signaling pathways in CRC remain at a relatively early stage. Our group previously demonstrated that a member of the Kr¿ppel-like factor (KLF) family of zinc finger transcription factors, KLF5, plays important roles in regulating proliferation of intestinal epithelial cells. KLF5 is predominantly expressed in the proliferating crypt cell compartment of the intestinal epithelium. Ectopic expression of KLF5 in transfected cells results in increased rates of proliferation and leads to anchorage independent growth. In addition, oncogenic activation of HRAS and KRAS in NIH3T3 and IEC6 cells, respectively, leads to transformation with a concomitant increase in KLF5 levels. Importantly, reduction of KLF5 by genetic or pharmacological means results in reduced rates of proliferation and anchorage-independent growth in oncogenic RAS-transformed cells. Moreover, CRC with activated KRAS are shown to contain high levels of KLF5. These results indicate that KLF5 is a key mediator for the pro-proliferative and transforming activities of activated KRAS, heretofore mutated in approximately 50% of CRC. Reduction of KLF5 expression in such CRC may offer a novel therapeutic approach in the treatment of CRC. The long-term GOAL of this research project is to understand the signaling pathways that modulate KLF5 expression in CRC. Our CENTRAL HYPOTHESIS is that KLF5 is a key mediator of proliferation of CRC containing activated KRAS. Our OBJECTIVE is to identify small molecule inhibitors of KLF5 expression in CRC cells using high throughput screening (HTS), with which to better understand the biological functions of KLF5 in modulating CRC proliferation and to develop potential therapeutic agents in the treatment of CRC. Using this R03 grant mechanism, we propose 2 SPECIFIC AIMS: (1) To perform HTS for small molecule inhibitors of KLF5 using cell-based luciferase reporter assays, and (2) To perform secondary and counter screening assays with which to validate the active compounds identified in specific aim 1. Although beyond the scope of this R03 grant, we will also attempt to develop strategies for further testing, in collaboration with the MLSCN center, to provide a final refinement of the structure and function of the active compounds. At the conclusion of the proposed project, we will be able to identify several highly active and specific inhibitors of KLF5 with which to further investigate the biological functions of KLF5 in mediating CRC proliferation. The identification of these compounds may also aid in the development of novel therapeutic approaches for CRC.