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

Although the prognosis of patients with colorectal cancer (CRC) is steadily improving, the disease remains the second most common cause of cancer-related deaths in Europe [1]. The treatment of CRC is dependent on the disease stage and the location of the tumor. Conventional treatment includes surgery, radiation and chemotherapy (5-fluorouracil, irinotecan and/or oxaliplatin) [2], often combined with bevacizumab (a neutralizing antibody against vascular endothelial growth factor; VEGF) or cetuximab/panitumumab (neutralizing antibodies against epidermal growth factor receptor; EGFR), depending on disease stage and patient-related factors [3]. During the course of CRC, mutations accumulate in genes controlling cell survival and proliferation.

Several of the genes afflicted in CRC belong to the RAS pathway [4]. The RAS pathway involves at least 4 key protein families (RAS, RAF, mitogen-activated protein kinase kinase (MEK) and extracellular regulated kinase (ERK)) that are activated in a consecutive manner, creating a signaling cascade that eventually results in gene regulation. Approximately 50 % of metastatic CRCs have activating mutations in the KRAS or NRAS genes [5‐7]. Patients with RAS mutations do not respond favorably to treatment with neutralizing anti-EGFR antibodies [8]. BRAF is the best characterized of three closely related RAF proteins [9]. The BRAF gene harbors an activating mutation (V600E) in 5–12 % of all CRC [10]. Tumors may have mutations either in KRAS or BRAF though, as a rule, not in both [11]. Activation of certain protein kinase C (PKC) isoforms, such as PKCɛ, by phospholipase Cγ1 (PLCγ1), promotes RAF activation [12]. BRAF in turn activates the dual tyrosine and serine/threonine kinase MEK, which is mutated only very rarely in CRC [13]. The serine/threonine kinases ERK1/2, downstream of MEK, are also not mutated in CRC [13].

Cell proliferation is regulated also by the cytoplasmic tyrosine kinase c-SRC, which is activated when phosphorylated on tyrosine residue (Y) 418 in the kinase domain and which is inhibited when phosphorylated on the C-terminal Y527 [14]. c-SRC expression is reported to be 5–8 fold higher in premalignant colorectal polyps than in normal mucosa and a correlation between elevated c-SRC levels and CRC progression/metastatic potential has been suggested [15‐17]. c-SRC kinase inhibitors are being developed for therapeutic purposes [18, 19]. Resistance to BRAF inhibition in melanoma can be overcome by inhibiting c-SRC activity [20], indicating a convergence of the pathways.

Cell survival is regulated by the phosphoinositide 3-kinase (PI3K)/AKT pathway which, via mammalian target of rapamycin complex 1 (mTORC1), eventually results in activation of p70S6 kinase and gene induction [21]. The serine/threonine kinase AKT is activated by phosphorylation of threonine (T) 308 located in the kinase domain and serine (S) 473 in the C-terminal end, by phosphatidylinositol-dependent kinase 1 (PDK1) and mTORC2, respectively. The PI3K/AKT pathway is negatively regulated by the lipid phosphatase, phosphatase and tensin homolog (PTEN) [22], which has been identified as a tumor suppressor [23]. About 15 % of all CRCs have activating or suppressing mutations in the PI3KCA gene, encoding the p110α catalytic subunit of PI3K, as well as the PTEN gene [24]. Moreover, in wild type (non-mutated) KRAS gene tumors, the presence of PI3K and PTEN mutations indicates a poor prognosis [25].

To identify mutations in cancer is part of an effort to individualize each patient’s treatment. However, mutations may not result in changes in protein expression levels and/or activity, and the mutation status of a particular cancer may fail to convey information about additional events occurring during progression of the disease, which may override a particular mutation, e.g. compensatory upregulation of other proteins and pathways [26]. There is no doubt that the EGFR/RAS pathway and downstream ERK1/ERK2 activities are essential in CRC etiology and disease progression [27]. However, predicting RAS pathway activity is particularly complex as there are several different upstream and parallel activators on different levels and many alternative feedback loops [26]. Apart from the regulation of RAS activity through GTPase regulatory proteins (GAPs and GEFs), downstream signaling in the RAS pathway can be induced or modulated through activities in several other pathways, including the PLCγ/PKC, PI3K/AKT and c-SRC pathways. Another complicating aspect of RAS signaling in CRC is chromosomal fragility. 85 % of sporadic CRC cases display chromosomal instability, chromosome amplification and translocation leading to aneuploidy (see [28] and refs therein), whereas the remaining 15 % of patients have high-frequency microsatellite instability phenotypes i.e. frameshift mutations and base pair substitutions [29]. The chromosomal instability of CRC clearly influences the biological consequence of the mutations. Thus, taken together, the presence of a mutation in a signaling protein does not necessarily predict activity in the corresponding signaling pathway.

Due to the existing challenges in CRC therapy, the development of rapid and sensitive screens to measure the biological activity of key signal transducers, which could serve as drug targets or as predictive or prognostic biomarkers, is warranted. Previously, the CRC proteome has been investigated using mass spectrometry to identify up- and downregulation of proteins, using mostly cell lines but also, to some extent, patient samples [30]. However, this is the first study to comprehensively address the proliferative signaling proteome in CRC tissues. For this purpose, we have developed protocols for highly sensitive, robotized isoelectric focusing, to show that signaling in the RAS pathway is dysregulated in human CRC primary tumors compared with normal mucosa. Moreover, by computational and geometric assessment of the signal transduction patterns in the different tissues examined (normal, stage II and stage IV CRC), we show that combinations of patterns from several pathways could serve as biomarkers and be exploited for the classification of tissues as normal or cancerous. We suggest that further refinement of complex signatures can be exploited for prognostic purposes.

Substantial research efforts over the last decades have resulted in increased understanding of CRC mutations and molecular consequences; still, due to the complexity of the tumor biology and the heterogeneity of the cancer, CRC remains a fatal disease. Here, we show that signaling pathways regulating cell survival and proliferation were differently regulated in CRC tissues compared to normal mucosa. Expression of ERK1 and SRC appeared significantly suppressed in CRC tissues compared with normal mucosa while expression of AKT and PLCγ1 were upregulated. See Table 1 for a summary of the pattern of proliferative CRC signaling identified in this study.

Signaling was analyzed using capillary isoelectric focusing, which we found to be superior to conventional immunoblotting in sensitivity and resolution. After loading of samples and antibodies, the processing was robotized, resulting in highly reproducible and sensitive detection. For example, ERK1/2 protein was detected in 2.5 ng of CRC lysate per capillary (corresponding to 6.25 μg/ml total lysate). Moreover, protein variants, phosphorylated at different residues, could be separated and quantified independently. For ERK1/2 proteins, six of the isoforms (pERK1, ppERK1, ERK1, pERK2, ppERK2, ERK2) could be identified and quantified in relation to the house keeping proteins analyzed in parallel. In comparison, conventional immunoblotting run on the same samples required much more protein for each analysis. It often failed to resolve protein phospho-variants and reproducibility was low, in part due to problems with transfer of proteins to the filter. Ongoing efforts include adapting the isoelectric focusing protocol for the detection of signal transducers in formalin-fixed, paraffin-embedded samples to make the procedure applicable in clinical routines.

Using the isoelectric focusing strategy, several important observations were made that can be related to earlier reports on CRC signaling (see also summary in Table 1):

Several decades of ambitious basic and clinical research have demonstrated the challenges in identifying reliable biomarkers in cancer. Challenges include the complexity of the primary tumor tissue consisting of, apart from the tumor cells, a range of host-derived endothelial, fibroblast and inflammatory cells; potential differences between the primary tumor and metastasis; and the possibility that biopsies may not be representative. In this study, the proportion of tumor cells ranged from about 30–60 % in most samples, based on the estimation of mutated DNA/total DNA in the samples (data not shown). An important conclusion from the current study is that the combination of several features from the conducted analyses allows a very high confidence in classifying the tissues as normal or cancerous. The particular combination of pERK1, SRC peak 6, and p70S6K peak 3 selected here to distinguish cancer tissue from normal tissue, may or may not indicate convergence of the included pathways in CRC signaling. The main objective of the selection was to allow unbiased diagnosis. Thus, we propose that reliable prognostic and diagnostic biomarkers should be designed using complex patterns rather than a single molecular or genetic marker. For clinical translation, the isoelectric focusing analyses can easily be made routine and scaled up. For example, 96 unique samples could be run in parallel to yield information on three or more selected pathways (by mixing several appropriate antibodies yielding non-overlapping patterns) in a 10 h run in a robotized set-up. Combined with the powerful computational evaluation to identify sets of signaling components showing significant characteristics, this strategy could prove to be clinically feasible for diagnostic purposes beyond the treatment of CRC. Based on the results obtained this far, we predict that the measurement of seven protein forms (i.e. selected peaks from the electropherograms) would be sufficient for the correct classification of both non-cancerous versus cancerous tissue as well as for the CRC grade. Moreover, analysis of a larger cohort of samples, combined with information on chosen therapy and disease outcomes, would allow the use of supervised learning for identification of clinically relevant subtypes.

The study suggests that such combinations of different signalling activities could serve as predictive or prognostic complex biomarkers.