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A proteomic analysis of cell signalling alterations in colorectal cancer

AuthorsMadoz-Curpide, J.; Cañamero, Marta; Sanchez, L.; Solano, Jennifer; Alfonso, Patricia; Casal, J. Ignacio
Issue DateDec-2007
PublisherAmerican Society for Biochemistry and Molecular Biology
CitationMolecular and Cellular Proteomics 6 (12) 2150-2164 (2007)
AbstractMultiple gene and protein alterations have been associated with every type of cancer. These alterations can be considered as biomarkers and might be useful in detecting cancer, determining prognosis, and monitoring disease progression or therapeutic response. Unfortunately finding new tumoral markers is not an easy task. Only 10 proteins have been described that are effectively used as biomarkers (e.g. prostate-specific antigen, carcinoembryonic antigen, and CA125) (1). Many of them show a lack of sensitivity or present high rates of false positives. New tools and strategies are needed to improve this situation. Protein microarrays in general and antibody arrays in particular offer a combination of high sensitivity and multiplexing possibilities that makes this strategy very attractive for tumor screening and biomarker identification (for a review, see Ref. 2; Ref. 3). Colorectal cancer (CRC)1 is the most abundant type of neoplasia in developed countries and the second cause of death among cancers. CRC has been well characterized from the genetics point of view (4, 5). It is a relatively slow process, which needs several successive mutations to be present in the tumoral cells and probably takes decades to develop completely. However, this knowledge about the genetic events that are necessary for the progression to carcinoma has not been translated into protein biomarkers. The current Dukes’ staging system for CRC is based on histopathological findings, such as the invasion of the intestinal muscular layer or the adjacent lymph nodes or the metastatic progression. In CRC, most of the tumors detected are already in advanced stages, such as Dukes’ C or D, lowering the estimated survival rate. Genomics studies of CRC involving DNA microarray analysis did not bring new classification tools or improved predictor panels (6–9).
Previously 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 CRC tissues, including some isoforms and post-translational modifications. Although DIGE is very sensitive, low abundance proteins are usually not detectable by mass spectrometry. To overcome this limitation and for a gain in sensitivity, we decided to test an antibody microarray strategy for detecting low abundance proteins. For antibody microarrays and depending on the affinity constant of the antibody, the detection limit can be 2 orders of magnitude below that for DIGE. In addition, the protein/antibody pairs are known “a priori.” Hence microarrays allow for a rapid identification of low expression proteins such as signaling molecules, cell cycle regulators, etc. 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). In addition, 12 of the antibodies were specific for particular post-translational modifications (i.e. eight antibodies were phosphospecific for different modifications of death-associated protein kinase, FAK, histone H3, MAPK, p38, PAK1, PYK2, and RAF; and four of them were acetyl-specific for histone H3). 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. In this study, 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 finally we observed global alterations in the cellular signaling machinery
Description15 p.-8 fig.- 3 tab.
Publisher version (URL)http://dx.doi.org/10.1074/mcp.M700006-MCP200
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