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.
Raw Protein RPAs
The relative peak area (RPA), i.e. peak area value of the 23 different activity levels of the 7 signal transducers after normalization to the HSP70 level analyzed in parallel in each sample (columns 2-24). This file contains one sample per row and one protein per column with the sample name in the first column and the protein name in the first row. Columns 25-27 contain the result of the mutation analysis of KRAS and BRAF. One in column 25 (MutationKRAS) indicate that KRAS is mutated in the sample, one in column 26 (MutationBRAF) indicates that BRAF is mutated, while one in column 27 (Wildtype) indicate that neither KRAS nor BRAF is mutated. A one in the binary variables in column 28-31 indicate the classification of each sample as normal mucosa, colorectal cancer (CRC) stage II, CRC stage IV, or metastasis. NaN is used to indicate that no measurement was done.
Padhan BMC Cancer 2016 Raw Protein RPAs.csv
Raw Protein RPAs Constructed features replicate corrected
File shows the relative peak area (RPA), i.e. peak area value of the measured protein after normalization to the HSP70 level analyzed in parallel in each sample (columns 2-24, 28-42). This file contains one sample per row and one protein per column with the sample name in the first column and the protein name in the first row. Column 25-27 contain the result of the mutation analysis of KRAS and BRAF. One in column 25 (MutationKRAS) indicate that KRAS is mutated in the sample, one in column 26 (MutationBRAF) indicates that BRAF is mutated, while one in column 27 (Wildtype) indicate that neither KRAS nor BRAF is mutated. Columns 28 to 42 contain the RPA values of the constructed features, i.e. features that are calculated based on the 23 different activity levels of the 7 signal transducers in column 2-24. The four replicates of each constructed feature contains the minimum, maximum, mean, and median value based on all possible ways to combine the replicates of the proteins used to construct the feature. A one in the binary variables in column 43-46 indicate the classification of each sample as normal mucosa, colorectal cancer (CRC) stage II, CRC stage IV, or metastasis. In the last column the classification is 1 = normal mucosa, 2 = colorectal cancer (CRC) stage II, 3 = CRC stage IV, or 5 = metastasis. NaN is used to indicate that no measurement was done.
Padhan BMC Cancer 2016 Raw Protein RPAs Constructed features replicate corrected.csv
Padhan BMC Cancer 2016 Supplemental Results
File contains the test statistic (T) for each possible combination of 1-3 features, including the constructed features (column 5 and 6). One combination is shown per row with the name of the feature combination in the first column and the header explaining the value in each column in the first row.
