Proteomic analysis of cellular signaling networks has strongly gained in importance in the field of cancer research and treatment. In particular, there is a focus on the activation status of growth factor receptor related signaling cascades represented by phosphoproteins, as key mediators of cellular signaling. For example, the inhibition of the EGF-receptor pathway has been widely integrated into clinical practice for different tumor entities, e.g. non small lung cancer and colorectal cancer. Although, predictive genetic markers of response to targeted therapy such as KRAS testing are already in use, heterogeneity in responses to treatment has been clinically observed. In order to understand this heterogeneity on the proteome level, the activation status of cell signaling proteins has to be analyzed. The in-depth analysis of isoform phosphorylation can potentially reveal significant biological differences in activation patterns and might result in predictive signatures.

In this study, we differentially analyzed basal isoform phosphorylations of selected key proteins of two EGF-receptor downstream pathways, in four primary colorectal tumors and their corresponding metastases using the NanoPro1000 technology. Patient's biospecimen were collected according to standard operating procedures, ensuring a high quality of tissues characterized by ischemia times below 10 minutes, comprehensive clinical data and corresponding sets of body fluids. The detailed characterization of isoform phosphorylations was conducted using the recently introduced NanoPro1000 technology. This assay provides the separation of phospho protein isoforms by high-resolution isoelectric focusing (IEF) combined with a specific, antibody based detection. In addition to these data, the mutation status of selected, clinical relevant genes (KRAS, BRAF and PIK3CA) was determined in primary and metastatic tumor samples using Sanger sequencing. Among the analyzed primary tumors, there were two KRAS mutants, one BRAF mutant and one wild type tumor. Identical results were found in the matched metastasis.

NanoPro1000 analysis revealed that isoform phosphorylation of ERK1/2, AKT and MEK1/2 differed among patients, as well as primary tumors and metastases. For example, ERK1 showed a tendency to be higher mono- and dual-phosphorylated in the primary tumors, whereas mono-phosphorylated ERK2 was elevated in metastases. However, a correlation to the mutation status was not found.

These differences may explain the heterogeneity of individual responses to target anticancer treatment among patients, independent of their mutation status. Therefore, the NanoPro1000 technology can potentially be useful to identify predictive biomarker for the development of resistance to anti-EGF-receptor treatment and further improve personalized medicine.

Citation Format: Florian T. Unger, Jana Krueger, Janina Schaller, Cordula Dede, Alexandra Samsen, Hartmut Juhl, Kerstin A. David. Comparison of ERK, AKT and MEK isoform phosphorylations between primary and corresponding metastatic colorectal carcinoma lesions using the NanoPro1000 technology. [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 2408. doi:10.1158/1538-7445.AM2013-2408