Abstract
To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.
Highlights
Multiple gene and protein alterations have been associated with every type of cancer
We have carried out studies on differential protein expression analysis based on two-dimensional DIGE gels [10], which enabled us to identify the most abundant proteins in Colorectal cancer (CRC) tissues, including some isoforms and post-translational modifications
12 of the antibodies were specific for particular post-translational modifications
Summary
The antibody microarray used in these studies contained 224 different antibodies [11], representing markers for eight biological pathways of interest (apoptosis, cell cycle, neurobiology, cytoskeleton, signal transduction, and nuclear proteins). Other antibodies were able to discriminate active versus non-active states in functional proteins. These capabilities represent an enormous value in cell signaling characterization, enabling the microarray system to track consecutive nodes of a given cellular pathway, helping to annotate functional variations in those proteins responsible for triggering a cascade of events related to the onset, modification, or conclusion of a cellular process. We used an antibody microarray to monitor the changes in the protein expression pattern of tumoral cells from biopsies of patients with colorectal carcinoma as compared with the pattern of normal cells from the surrounding non-affected mucosa. We identified a specific signature for CRC, new markers based on less abundant proteins that might have a potential use in diagnosis, and we observed global alterations in the cellular signaling machinery
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