High-throughput MRM assay to investigate the functional role and regulation of the androgen receptor signaling in models of prostate epithelial cells

The aim of the project is to establish a high-throughput/low-cost assay for the investigation and characterization of function role and the regulation of Androgen Receptor signaling in the normal prostate and the consequences of de-regulation for the physiological function of the prostate in the healthy organism. The prostate development and normal prostate function requires androgen signaling and action (1). The male androgens exert their action in the prostate through a nuclear hormone receptor, the Androgen Receptor (AR). It has been shown recently that androgen stimulation of a prostate cell lines in vitro changes the expression level of >600 genes (2). Recently, new potential AR genomic targets have been identified of which many show altered expression in prostate disease (3). The modulation of transcriptional activity of the AR appears to be influenced by a number of co-regulators and phosphorylation events. These involve for example signal transduction through e.g. TGF-?, IL-6, and IGF-I pathways (4). Despite all the progress in the field it is still unclear how exactly the key pathway (i.e., the Androgen pathway) in normal prostate development and growth is influenced and modulated by other cellular signaling pathways and - in particular - what type of de-function in the regulatory network of Androgen activity and regulation leads to the development of abnormal prostate behaviour (e.g., benign enlargements, malignancies etc). Therefore we aim to investigate the Androgen signaling network on a protein level in cellular models by systematically disturbing the members of the network (e.g., by siRNA) as well as known modulators of the network (e.g., EGF, TGF-?, IL-6, and IGF-I etc) followed by monitoring of the consequences on the protein levels of the members of the signaling network. By comparison of these types of experiments in different cellular models of the prostate epithelium we aim to unravel the most important members network, and/or the most important modulators of the of the Androgen signaling pathway for normal prostate function. As typically a high number of experimental conditions have to be tested to come to an understanding of the regulatory context of complex signaling networks, it is the goal of this project to establish a high-throughput multiplex MRM mass spectrometry assay that is able to measure a high number (>100) of the key proteins in the Androgen signaling network in a single experimental procedure. This requires the identification of the key members of the Androgen signaling network, the development and validation of an MRM assay for these proteins and the testing of multiplexing of many validated assays. Further, the establishment of cell culture of different prostate epithelial cell lines and testing/optimisation of conditions for 'network disturbance' experiments by e.g. siRNA. Year 1: Definition of the key members of the Androgen Receptor signaling pathway as well as main modulatory pathways (scientific literature). Cell culture conditions for prostate cell lines models. Years 2 & 3: Experimental conditions for siRNA investigations. MRM assay development of validation for key members of the Androgen Receptor signaling pathway. Multiplex assay optimisation for individually validated MRM assays. Validation of the developed multiplex MRM assay on cell line extracts. Years 4: 'Network disturbance' experiments by the introduction of commercially available and validated siRNA molecules to the identified key members of the Androgen Receptor signaling pathway as well as main modulatory pathways. Testing of the outcome by the developed MRM assay. Comment: the project will be supported by bioinformatics network modelling and analysis provided by Philips Research Key references (1) Roy, Vit Horm, 1999, 55, 309/352 (2) Valesco, Endocrinol, 2004, 145(8), 3913-3924 (3) Massie, EMBO Reports, 2007, AOP (4) Heinlein, 2002, Endocrine Reviews, 23(2), 175/200