Journal Article

High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer

Åkerud, Peter, Nordling, Torbjörn E. M., Nygren, Peter, Padhan, Narendra, Sundström, Magnus, Nelander, Sven, Birgisson, Helgi, Claesson-Welsh, Lena

2016

Overview

Background The progression of colorectal cancer (CRC) involves recurrent amplifications/mutations in the epidermal growth factor receptor (EGFR) and downstream signal transducers of the Ras pathway, KRAS and BRAF. Whether genetic events predicted to result in increased and constitutive signaling indeed lead to enhanced biological activity is often unclear and, due to technical challenges, unexplored. Here, we investigated proliferative signaling in CRC using a highly sensitive method for protein detection. The aim of the study was to determine whether multiple changes in proliferative signaling in CRC could be combined and exploited as a “complex biomarker” for diagnostic purposes. Methods We used robotized capillary isoelectric focusing as well as conventional immunoblotting for the comprehensive analysis of epidermal growth factor receptor signaling pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT, phospholipase Cγ1 (PLCγ1) and c-SRC in normal mucosa compared with CRC stage II and IV. Computational analyses were used to test different activity patterns for the analyzed signal transducers. Results Signaling pathways implicated in cell proliferation were differently dysregulated in CRC and, unexpectedly, several were downregulated in disease. Thus, levels of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV while total ERK1/2 expression remained unaffected. In addition, c-SRC expression was lower in CRC compared with normal tissues and phosphorylation on the activating residue Y418 was not detected. In contrast, PLCγ1 and AKT expression levels were elevated in disease. Immunoblotting of the different signal transducers, run in parallel to capillary isoelectric focusing, showed higher variability and lower sensitivity and resolution. Computational analyses showed that, while individual signaling changes lacked predictive power, using the combination of changes in three signaling components to create a “complex biomarker” allowed with very high accuracy, the correct diagnosis of tissues as either normal or cancerous. Conclusions We present techniques that allow rapid and sensitive determination of cancer signaling that can be used to differentiate colorectal cancer from normal tissue.

• Publisher: BioMed Central,BioMed Central Ltd
• Subject: Analysis / Biomarkers, Tumor / Biomedical and Life Sciences / Biomedicine / Biopsy / c-SRC / Cancer Research / Cell Line, Tumor / Cell Proliferation / Colorectal cancer / Colorectal Neoplasms - diagnosis / Colorectal Neoplasms - genetics / Colorectal Neoplasms - metabolism / CSK Tyrosine-Protein Kinase / Development and progression / Diagnosis / ErbB Receptors - genetics / ErbB Receptors - metabolism / ERK / Gene Expression Regulation, Neoplastic / Health Promotion and Disease Prevention / Humans / Isoelectric focusing / Isoelectric Focusing - methods / Medicine/Public Health / Mitogen-Activated Protein Kinase 3 - genetics / Mitogen-Activated Protein Kinase 3 - metabolism / Mutation / Neoplasm Staging / Oncology / Phospholipase C gamma - metabolism / Phosphorylation / post-genomic analysis and emerging technologies / Proliferation / Proto-Oncogene Proteins c-akt - metabolism / Research Article / Ribosomal Protein S6 Kinases, 70-kDa - metabolism / Sensitivity and Specificity / Signal Transduction / src-Family Kinases - metabolism / Surgical Oncology / Systems biology