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.

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.

Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer.

PURPOSE: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm. EXPERIMENTAL DESIGN: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples. RESULTS: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-beta induced protein ig-h3 (betaIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78. CONCLUSIONS: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.

Expression of TGF-alpha in neuroendocrine tumours of the distal colon and rectum.

AIM: Transforming growth factor alpha (TGF-alpha) has been localized in neuroendocrine L-cells of the colon and rectum in previous studies. We examined whether neuroendocrine tumours with L-cell differentiation express TGF-alpha. EXPERIMENTAL DESIGN: Immunohistochemistry was performed for proglucagon- and pro-pancreatic polypeptide derivatives, as well as for TGF-alpha, and epidermal growth factor receptor (EGFR) using paraffin sections from 16 neuroendocrine tumours of the colon and rectum. Also, in situ hybridization for TGF-alpha and proglucagon was carried out. MAIN RESULTS: A strong expression of TGF-alpha at the protein level can be shown for neuroendocrine tumours of the hindgut. In one third of our cases we found a strong hybridization signal and in two thirds a moderate signal for TGF-alpha. The immunohistological phenotype concerning gut hormones is highly heterogeneous. Glucagon-like peptide 2 (GLP2) in our series was the most sensitive immunohistological hormone marker. MAJOR CONCLUSIONS: The immunophenotype of colorectal neuroendocrine tumours regarding hormone markers is heterogeneous. The expression of TGF-alpha corresponds to the immunohistological profile of normal L-cells. TGF-alpha, especially in the neuroendocrine L-cells, most probably acts as a multifunctional trophic factor responsible for cellular integrity and survival, and not as an oncogenic growth factor.