EZH2 Loss Drives Resistance to Carboplatin and Paclitaxel in Serous Ovarian Cancers Expressing ATM.

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations.

A bead-based western for high-throughput cellular signal transduction analyses.

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.

Bead-Based Peptide Arrays for Profiling the Specificity of Modification State-Specific Antibodies.

Modification state-specific antibodies are powerful tools for investigating posttranslational modifications in proteins. The majority of these antibodies have been generated against peptide-antigen conjugates. They are useful in a plethora of methods, such as Western blotting, immunohistochemistry, sandwich immunoassay, immunoprecipitation, and immunoprecipitation coupled with mass spectrometry. Phosphorylation, acetylation, methylation, sulfation, nitrosylation, ubiquitination, and sumoylation are some of the modifications that can be studied using such antibodies. However, investigating the on- and off-target binding of antibodies is crucial to the interpretation of experimental data. Peptide arrays are excellent tools for such in-depth studies of off-target and on-target binding of antibodies. Dozens or even hundreds of modified peptides can be integrated into a single experimental setup to analyze the antibody's binding behavior. Here, we propose three different protocols for peptide bead array generation and describe their suitability for such types of assay.