ABSTRACT
Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)ß1-3Galß1-4GlcNAc(6-SO3-)ß1-3Galß1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.
Subject(s)
Colonic Neoplasms/genetics , Polysaccharides/biosynthesis , RNA, Messenger/chemistry , Sulfotransferases/chemistry , Antigens, Surface/chemistry , Antigens, Surface/genetics , Cell Adhesion/genetics , Cell Line, Tumor , Colonic Neoplasms/chemistry , Colonic Neoplasms/pathology , Epithelial Cells/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Polysaccharides/genetics , RNA, Messenger/genetics , Substrate Specificity , Sulfates/chemistry , Sulfotransferases/genetics , Tandem Mass Spectrometry , Carbohydrate SulfotransferasesABSTRACT
BACKGROUND: Although ascites cytology is important for therapeutic strategies, it is difficult to distinguish cancer infiltration from reactive mesothelial proliferation in some patients. In this study, the authors applied CDX2 immunocytochemistry to improve diagnostic accuracy. METHODS: The authors examined the distribution of CDX2 expression in carcinoma specimens using paraffin-embedded tissues from various organs from 549 cancer patients. CDX2 immunostaining was applied to the 116 ascites specimens. RESULTS: CDX2 expression was detected in a restricted range of cancers, with the vast majority of them originating from the gastrointestinal tract and pancreas. When applied to ascites specimens, no positive reactions were detected in any of the 81 cytology-negative and molecular genetic analysis-negative specimens. By contrast, 28 of 35 specimens diagnosed as suspicious for malignancy or malignancy showed a positive reaction. Furthermore, the authors found that a nuclear-positive reaction was easily evaluated, even with a high level of background staining, and single cancer cells in 10(6) normal cells could be detected. CONCLUSION: Results suggest that CDX2 is a specific and sensitive marker to detect gastrointestinal and pancreatic malignancies in ascites cytology.