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

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.