摘要：

Advanced prostate cancers frequently overexpress epidermal growth factor receptor (EGFR) and aberrant activation of EGFR-mediated signal transduction plays an important role in prostate cancer progression. Therapeutic strategies targeting EGFR and its downstream signaling mechanisms have been hampered by 1) the heterogeneity of prostate cancer and 2) the complexity of intracellular signaling crosstalk. In the metastatic prostatic adenocarcinoma cell line, PC-3, EGF is known to activate the PI3K/Akt pathway as well as the classical EGFR signaling pathway (Ras/MAPK pathway) leading to increased proliferation, decreased apoptosis and enhanced migration. To profile EGF-induced intracellular signaling and crosstalk between pathways in PC3 cells, and the effects of kinase inhibitors on these pathways, we utilized a multiplex peptide-based immunoassay platform to detect 45 tyrosine phosphorylation sites on 34 different proteins simultaneously in a single sample (Ti-Tyr™ Chip, Epitome Biosystems, Inc). The resulting EGF stimulated molecular signatures were compared to the signatures generated when cells were pretreated with specific kinase inhibitors. We observed site-specific tyrosine phosphorylation on the EGF receptor and several proteins involved in tyrosine receptor kinase signaling, including Shc, Cbl, Erk1/2 and Crk. Pre-treatment of PC-3 cells with an EGFR-specific inhibitor (AG-1478), a MEK1/2-specific inhibitor (SL327), and a PI3K-specific inhibitor (LY294002), diminishes EGF-induced activation of ERK1/2 in these cells. In contrast, pre-treatment with a p38MAPK specific inhibitor (SB202190), results in enhanced EGF-induced activation of ERK1/2. We also observed increased tyrosine phosphorylation of Her2 in pretreatment with the PI3 kinase inhibitor LY294002. These results are indicative of altered pathway cross-talk upon pharmacological inhibition of specific pathways.

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