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Molecular & Cellular Proteomics 5:2092-2101, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification of PSME3 as a Novel Serum Tumor Marker for Colorectal Cancer by Combining Two-dimensional Polyacrylamide Gel Electrophoresis with a Strictly Mass Spectrometry-based Approach for Data Analysis^*

Markus Roessler, Wolfgang Rollinger, Liliana Mantovani-Endl, Marie-Luise Hagmann, Stefan Palme, Peter Berndt, Alfred M. Engel, Michael Pfeffer, Johann Karl, Heinz Bodenmüller, Josef Rüschoff^¶, Thomas Henkel^¶, Gerhard Rohr^||, Siegbert Rossol^**, Wolfgang Rösch, Hanno Langen, Werner Zolg and Michael Tacke^,

From the Centralized Diagnostics, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany, Roche Center of Medical Genomics, Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland, ^¶ Institute of Pathology and Biomedical Research, Klinikum Kassel GmbH, D-34125 Kassel, Germany, ^|| Institute of Gastroenterology, Hochtaunus-Kliniken gGmbH, D-61348 Bad Homburg vor der Höhe, Germany, ^** I. Medizinische Abteilung, Stadtkrankenhaus Rüsselsheim, D-65428 Rüsselsheim, Germany, and Krankenhaus Nordwest, D-60488 Frankfurt am Main, Germany

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550–6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

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