Characterization And Potential Functions Of MGb2-Ag/TRAK1: A Novel Gastrointestinal Cancer Associated Antigen

【Background】MGb2 is a gastrointestinal cancer specific monoclonal antibody developed in our laboratory in 1980s. It can be used to detect gastric cancer with high sensitivity and specificity, especially for gastric signet-ring cell carcinoma. However, its target antigen MGb2-Ag has not been identified.【Objectives】The present study aimed: (a) to assess the distribution of gastrointestinal cancer associated antigen MGb2-Ag in human tissues, (b) to determine the clinical pathological significance of MGb2-Ag in gastric cancer, (c) to identify the physiochemicl nature of MGb2-Ag and to explore biological function of MGb2-Ag in gastric carcinogenesis.【Methods】Distribution of MGb2-Ag was detected by immunohistochemistry. MGb2-Ag was enriched using immunoprecipitation followed by confirmation with Western blotting and silver staining. The target protein band was detected by MALDI-TOF MS followed by bioinformatics analysis. Prokaryotic expression system and immunohistochemistry provided the further supporting evidences. siRNAs against Trak1 were subcloned into plasmid vector. Cell cycle analysis and apoptosis detection were used to explore the role of siRNAs in cancer cell line using transient transfection.【Results】This study provided further evidences on distribution characteristics of MGb2-Ag in human cancers and non-cancerous tissues. Immunostaining analysis demonstrated that the presence of over-expression of MGb2-Ag was correlated with an increased aggressiveness of the tumor, including poorer differentiation, deeper penetration and metastasis of gastric cancer cells. The positive rate of MGb2-Ag was 81.48% (66/81) in gastric cancer, 100% (23/23) in gastric signet-ring cell carcinoma and mucinous adenocarcinoma, 13.16 (5/38) in gastric precancerous conditions, and 0% (0/22) in chronic superficial gastritis. Approximately 106 kDa protein immunoreactive with MGb2 antibody in gastric cancer cell lines KATOIII, MKN-45 or gastric cancer tissues, was identified as Trafficking protein, kinesin-binding 1(TRAK1), which has been reported as a new molecular gained limited recognization. Both anti-TRAK1 antibody and MGb2 antibody could recognize prokaryotic expressed TRAK1. The immunostaining characteristics of TRAK1 in paraffin-embedded tissues of gastric cancer, intestinal metaplasia or chronic superfacial gastritis were highly identical with MGb2-Ag in the continuous sections of these samples. Transfection of two siRNAs targeting Trak1 into gastric cancer cell line MKN-45 decreased apoptosis and promoted cell cycle G1 to S stage transition.【Conclusions】The present study assessed the distribution of MGb2-Ag in human cancer and non-cancerous tissues and analyzed the clinical pathological significance of MGb2-Ag in gastric cancer. The results showed that the high expression of MGb2-Ag was associated with the poorer differentiation, deeper penetration and presence of lymphonde metastasis of gastric cancer cells. MGb2-Ag was identified as TRAK1, and MGb2-Ag/TRAK1 might be a promising gastrointestinal cancer biomarker and act as an anti-tumor target.