We describe micro-western arrays, which enable quantitative, sensitive and high-throughput assessment of protein abundance and modifications following electrophoretic separation of micro-arrayed cell lysates. This method allowed us to measure 91 phosphosites on 67 proteins at six time points following stimulation with five EGF concentrations in A431 human carcinoma cells. We inferred the connectivities among 15 phosphorylation sites across 10 receptor tyrosine kinases (RTK) and 2 sites from Src kinase using Bayesian network modeling and two mutual information-based methods; the three inference methods yielded significant agreement on the network topology. These results imply multiple distinct RTK coactivation mechanisms and support the notion that small amounts of experimental data collected from phenotypically diverse network states may enable network inference.

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Androgen-dependent human LNCaP 104-S tumor xenografts progressed to androgen-independent relapsed tumors (104-Rrel) in athymic mice after castration. The growth of 104-Rrel tumors was suppressed by testosterone. However, 104-Rrel tumors adapted to androgen and regrew as androgenstimulated 104-Radp tumors. Androgen receptor expression in tumors and serum prostate-specific antigen increased during progression from 104-S to 104-Rrel but decreased during transition from 104-Rrel to 104-Radp. Expression of genes related to liver X receptor (LXR) signaling changed during progression. LXRA, LXRB, ATP-binding cassette transporter A1 (ABCA1), and sterol 27-hydroxylase decreased during progression from 104-S to 104-Rrel. These coordinated changes in LXR signaling in mice during progression are consistent with our previous findings that reduction of ABCA1 gene expression stimulates proliferation of LNCaP cells. To test if attenuation of LXR signaling may enhance prostate cancer progression from an androgen-dependent state to an androgen-independent state, castrated mice carrying 104-S tumors were given the synthetic LXR agonist T0901317 by gavage. T0901317 delayed progression from 104-S to 104-Rrel tumors. Based on our in vivo model, androgen is beneficial for the treatment of androgen-independent androgen receptorrich prostate cancer and modulation of LXR signaling may be a potentially useful therapy for prostate cancer. (Cancer Res 2006; 66(13): 6482-6)