Roessler Markus, Rollinger Wolfgang, Mantovani-Endl Liliana, Hagmann Marie-Luise, Palme Stefan, Berndt Peter, Engel Alfred M, Pfeffer Michael, Karl Johann, Bodenmüller Heinz, Rüschoff Josef, Henkel Thomas, Rohr Gerhard, Rossol Siegbert, Rösch Wolfgang, Langen Hanno, Zolg Werner, Tacke Michael
Centralized Diagnostics, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
Mol Cell Proteomics. 2006 Nov;5(11):2092-101. doi: 10.1074/mcp.M600118-MCP200. Epub 2006 Aug 6.
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.
本研究的目的是鉴定和验证人类结直肠癌（CRC）新的血清学蛋白质生物标志物。通过二维凝胶电泳分离来自匹配的CRC组织和相邻正常组织样本的蛋白质。从每块凝胶中切除所有斑点，并通过质谱鉴定包被的蛋白质。通过比较所得的蛋白质谱，可以鉴定失调的蛋白质。先前已报道了所有鉴定出的蛋白质列表以及对五种示例性选择的在CRC中升高的蛋白质的验证（罗斯勒，M.，罗林格，W.，帕尔梅，S.，哈格曼，M.L.，伯恩特，P.，恩格尔，A.M.，施奈德inger，B.，普费弗，M.，安德烈斯，H.，卡尔，J.，博登米勒，H.，鲁施霍夫，J.，亨克尔，T.，罗尔，G.，罗索尔，S.，罗施，W.，朗根，H.，佐尔格，W.，塔克，M.（2005年）烟酰胺N-甲基转移酶作为结直肠癌一种新的血清肿瘤标志物的鉴定。临床癌症研究。11，6550 - 6557）。在此，我们描述了另一种CRC潜在标志物蛋白的鉴定和初步验证。组织蛋白质谱的比较显示，蛋白酶体激活复合物亚基3（PSME3）在CRC组织中的表达显著升高。基于斑点模式无法检测到这种失调。肿瘤凝胶上含PSME3的斑点与匹配对照凝胶上的相应斑点没有明显差异。质谱分析显示，肿瘤凝胶上同一个斑点中存在两种蛋白质，即PSME3和膜联蛋白4（ANXA4），而对照凝胶上的匹配斑点仅包含一种蛋白质ANXA4。因此PSME3的失调被ANXA4掩盖，只能通过基于质谱的分析识别，而不能通过图像分析识别。为了验证这一发现，制备了PSME3抗体，并通过蛋白质印迹分析和免疫组织化学证实了其在CRC中的上调。最后，通过开发一种高灵敏度免疫测定法，可在人血清中检测到PSME3，并且与健康供体和良性肠道疾病患者相比，CRC患者血清中PSME3显著升高。我们建议将PSME3视为CRC的一种新型血清肿瘤标志物，其可能在该疾病患者的检测和管理中具有重要意义。需要进一步研究以全面评估这种候选标志物的潜在临床价值。
