SMALL MOLECULE REGULATION OF BREAST CANCER EXPRESSION

Scripps Research Institute, La Jolla CA

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Abstract

The proposed research plan involves the development of new drugs for the treatment of human breast cancer. We plan to design small molecule ligands to target the promoter and enhancer elements of-the Her-2/neu oncogene and other genes that encode proteins involved in breast cancer proliferation. Since overexpression and/or mutation of Her-2/neu is correlated with a poor prognosis in breast and ovarian carcinomas, downregulation of the Her-2/neu gene (and protein) may prove effective in combination therapy for these cancers. Synthetic pyrrole-imidizole (Py-lm) polyamides have been shown to bind a wide range of DNA sequences with subnanomolar affinities; moreover, these molecules can inhibit transcription factor-DNA interactions and gene expression both in vitro and in cultured cells. A series of Py-Im polyamides will be designed and synthesized to target the region immediately flanking the TATA and CCAAT box elements and the binding sites for Sp1, AP-2, ESX, and RBPJkappa within the promoter and proximal enhancer of the human Her-2/neu oncogene. Using DNase footprinting methods and gel mobility shift assays, we will determine whether these polyamides inhibit binding of the TATA-box binding protein, TBP, to the Her-2/neu TATA element and other transcription factors to their cognate sites in this promoter. The effects of the polyamides on basal and activated transcription will be monitored using cell-free extracts from breast carcinoma cells and transcription assays with purified factors and RNA polymerase II. We will monitor the effects of the polyamides, individually and in combination, on Her-2/neu mRNA expression in human breast carcinoma cell lines. The levels of Her-2/neu protein in polyamide-treated and control cells will be monitored by Western blotting with a monoclonal antibody to Her-2/neu. The effects of down-regulation of Her-2/neu mRNA and protein levels on cell viability, cellular motility and tumorogenicity will be monitored in both cell-based assays and in small animal models. Toxicity of the polyamides will be assessed in cell-based assays and non-specific effects of the polyamides on genome-wide gene expression will be monitored using high density DNA array/hybridization methodology.