Abstract 5521: Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies.
Authors: Chen Jinqiu , Lee Jih-Hsiang , Herrmann Michelle , Park Kang-Seo , Heldman Madeleine , Goldsmith Paul K. , Wang Yisong , Giaccone Giuseppe 
Published: August 2014

Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC) and genomic analysis has been commonly used to identify patients who may benefit from targeted therapies. Nevertheless, the accuracy of predicting responses to targeted therapies is only 50-80% based on sequencing or FISH. Developing proteomic biomarkers is valuable for evaluating therapeutic effects and better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, low throughput or/and lack of assay reproducibility, accuracy and sensitivity. In this study, a novel capillary isoelectricfocusing (IEF) immunoassay system (ProteinSimple, Santa Clara, CA) was used to study the dynamic phosphorylation status of signaling molecules in tyrosine kinase or/and MEK inhibitor treated NSCLC cells. NanoPro showed the same dynamic Erk phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation enables multiple protein phosphorylation isoforms to be resolved and detected using pan-reactive antibodies. In PD325901 MEK inhibitor treated cells, a specific MEK response pattern to drug treatment was revealed by NanoPro. This pattern demonstrated on-target effect of the drug, which was not detectable by Western blotting. NanoPro also identified a MEK2 response signature that may be associated with NSCLC cell sensitivity to the tyrosine kinase inhibitor, erlotinib, and distinguished erlotinib-sensitive from erlotinib-resistant cells. This MEK2 signature was further confirmed in cells with acquired resistance to erlotinib when compared to respective parental cells. In an HCC827 xenograft study, Western blotting was not able to differentiate human cancer cell signals from mouse stromal cell interference. In contrast, NanoPro detected and distinguished human Erk1 isoforms from the mouse isoforms based on their pI differences and demonstrated that erlotinib effectively inhibited Erk phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. We further used NanoPro to monitor responses of protein phosphorylations to erlotinib and AZD6244 (a MEK1/2 inhibitor) in a NSCLC patient by profiling 18 signaling molecules with as little as 8 μg tumor material. Signals from baseline and inhibitor treated samples were precisely quantified and compared. In conclusion, NanoPro can assess protein phosphorylation both qualitatively and quantitatively to provide more information about the activation status of proteins, which is not readily assessed by traditional Western blotting. Its higher sensitivity, better reproducibility and reduced tissue requirement are appealing for drug development and evaluation of drug effects in targeted therapies.

Citation Format: Jinqiu Chen, Jih-Hsiang Lee, Michelle Herrmann, Kang-Seo Park, Madeleine Heldman, Paul K. Goldsmith, Yisong Wang, Giuseppe Giaccone. Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5521. doi:10.1158/1538-7445.AM2013-5521