The lack of biomarkers for assessment of which patients may respond to pathway-targeted therapy creates a profound need for the application of integrated technologies for the discovery and translational validation of such biomarkers. Recent advances in proteomic methods and computational bioinformatics processing have enabled application of integrated proteomic technologies to the discovery of biomarkers. If used by ex vivo nano-sensor devices and in vivo nanoparticle imaging methods such biomarkers may provide effective new tools to cancer therapy development and use. We propose to discover candidate markers by integrating two directed and two comprehensive proteomic technologies: a) intracellular-signaling protein chips consisting of a directed analysis method which quantitates known, intracellular signaling proteins;b) living-cell capture sensor arrays which represent a nanosensor approach for directed analysis of the cell-surface and secretory proteomes;c) biotin-capture-based cell-surface profiling methods consisting of a comprehensive analysis method that identifies and quantitates the abundance of cell-surface proteins;d) solid-phase extraction of glycoprotein (SPEG) profiling, which is a comprehensive analysis method for the study of secreted proteins and blood. Biomarker discovery will initially be disease- (prostate cancer) and pathway- (human epidermal growth factor receptor (Her)-kinase axis) focused and will provide a foundation our CCNE-TR Center will use to produce, evaluate and validate nanosensors and nanoparticle-based imaging. We will first define the cell-surface and secretory proteomes of androgen-independent prostate cancer and identify proteins within these and the intracellular proteome that are indicative of the perturbations to the Her-kinase axis. We will analyze these sub-proteomes in primary culture models of androgen-independent prostate cancer by treatment with the Her-kinase targeted therapeutics, 2C4 (a humanized monoclonal antibody that binds epitopes on Her-2 that prevent ligand-mediated Her-2 heterodimerization) and geftinib (a small molecule inhibitor that competes for the ATP binding site on epidermal growth factor receptor). We will additionally stimulate with ligands targeting each of the receptor-dimer partners of this axis. Axis-response-informative proteins will be evaluated for their Her-kinase and prostate cancer specificity, by comparison with a database of gene and protein expression in other cell lines of different tissue origin available to the investigator group. Next we will integrate our in vitro results with information of the protein expression patterns of human xenograft models to identify a panel of markers with utility for predicting and/or for monitoring response. The bank of blood and viable tissue samples developed by the UCLA Prostate SPORE for use in this project represent different pre and post-treatment time points and a diverse collection of androgen-independent xenograft models characterized for sensitivity to the Her-kinase-targeted therapeutics. Lastly we will validate the utility of the biomarkers discovered in Specific Aims 1 and 2 for use with ex vivo nanosensor devices and in vivo nanoparticle imaging by generating affinity reagents for protein candidates;We will also validate the panel's ability to guiding human therapeutic intervention by using SPORE tissue and serum samples of prostate cancer patients treated with Her-kinase-directed therapies.
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