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