Proteomic Analysis of Neuroblastoma Subtypes in Response to Mitogen-Activated Protein Kinase Inhibition: Profiling Multiple Targets of Cancer Kinase Signaling

Abstract

Survival for high-risk neuroblastoma (NB) remains poor despite aggressive therapy. Novel therapies are vital for improving prognosis. We previously showed differential NB subtype sensitivity to p42/44 mitogen-activated protein kinase (ERK/MAPK) pathway inhibition. In this study, we investigated proteomic changes associated with resistance or sensitivity to MAPK kinase (MEK) inhibition in NB subtypes. SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type) were treated with MEK inhibitor U0126 (10 microM) for 1 and 24 h. Proteins were extracted from untreated and treated cells and analyzed for differential expression by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Selected polypeptides were extracted from the gel and identified by liquid chromatography-linked tandem mass spectrometry (LC-MS/MS). We identified 15 proteins that were decreased by 2.5-fold between untreated and 1 h treated cells and subsequently up-regulated 5-fold after 24 h drug treatment. N-type NB (MEK-resistant) showed the least altered proteomic profile whereas the I-type (MEK-sensitive) and S-type NB (MEK-intermediate) generated significant protein changes. The majority of proteins identified were induced by stress. Protein differences exist between MEK inhibitor-treated NB subtypes. Identified polypeptides all have roles in mediating cellular stress. These data suggest that inhibition of the ERK/MAPK in NB subtypes leads to an intracellular stress response. The most resistant NB cell line to MEK inhibitor treatment generated the least protective protein profile, whereas the intermediate and most sensitive NB cells produced the most stress response. These findings suggest stress related protein expression may be targeted in assessing a response to ERK/MAPK therapeutics.