title: Apical membrane expression of distinct sulfated glycans represents a novel marker of cholangiolocellular carcinoma.
abstract: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver neoplasm, followed by hepatocellular carcinoma. ICC can be further subclassified as (i) perihilar and (ii) peripheral types, the latter histologically resembling small-sized intrahepatic bile ducts, such as interlobular bile ducts, cholangioles/ductules and the canals of Hering. Cholangiolocellular carcinoma (CoCC), now classified by the World Health Organization as a subtype of combined hepatocellular-cholangiocarcinoma, is currently regarded as a subtype of peripheral-type ICC. The present study was undertaken to determine whether sulfated glycans recognized by the MECA-79 monoclonal antibody could serve as a CoCC marker. Using immunohistochemistry, we show that MECA-79 sulfated glycans are preferentially expressed at the apical membrane of cholangiocytes found in small-sized intrahepatic bile ducts in normal liver and in canalicular structures formed in CoCC. We also report that apical membrane MECA-79 sulfated glycan expression colocalizes with that of mucin 1 (MUC1) core proteins. We also present immunoblotting of Chinese hamster ovary cells overexpressing FLAG-tagged MUC1 to show that MUC1 serves as a MECA-79 scaffold. Furthermore, we report that SSP-25 human ICC cells overexpressing N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), but not GlcNAc6ST-1, exhibit membrane expression of MECA-79 sulfated glycans, suggesting that GlcNAc6ST-2 catalyzes MECA-79 epitope biosynthesis in cholangiocytes. Moreover, both wild-type and GlcNAc6ST-1 knockout mice exhibit apical membrane MECA-79 expression in small-sized intrahepatic bile ducts, namely interlobular bile ducts, whereas MECA-79 expression was completely absent in comparable tissues from GlcNAc6ST-1 and GlcNAc6ST-2 double knockout mice. These data collectively indicate that apical membrane localization of MUC1 proteins decorated with GlcNAc6ST-2-dependent MECA-79 sulfated glycans may mark cholangiocytes with cholangiolar/ductular differentiation and could serve as a useful CoCC marker.
authors.affiliation: Department of Tumor Pathology, Faculty of Medical Sciences, University of Fukui, Eiheiji, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Hoshino
authors.affiliation: Department of Pathology, Fukui Red Cross Hospital, Fukui, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohta
authors.affiliation: Department of Pathology and Laboratory Medicine, Kariya Toyota General Hospital, Kariya, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
authors.affiliation: Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Uchimura
authors.affiliation: Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Sakai
authors.affiliation: Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uehara
authors.affiliation: Department of Tumor Pathology, Faculty of Medical Sciences, University of Fukui, Eiheiji, Japan.
authors.forename: Shulin
authors.initials: S
authors.lastname: Low
authors.affiliation: Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Fukushima
authors.affiliation: Department of Tumor Pathology, Faculty of Medical Sciences, University of Fukui, Eiheiji, Japan.
authors.forename: Motohiro
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis*; D001650:Bile Duct Neoplasms / Q000378:metabolism* / Q000473:pathology; D001653:Bile Ducts, Intrahepatic / Q000378:metabolism* / Q000473:pathology; D014408:Biomarkers, Tumor / Q000378:metabolism; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000473:pathology; D016764:Cell Polarity; D018281:Cholangiocarcinoma / Q000378:metabolism* / Q000473:pathology; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry; D018345:Mice, Knockout; D018396:Mucin-1 / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D000097811:Carbohydrate Sulfotransferases
chemical_list: D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D014408:Biomarkers, Tumor; D007527:Isoenzymes; D008565:Membrane Proteins; D018396:Mucin-1; D011134:Polysaccharides; D011994:Recombinant Proteins; D015238:Sulfotransferases; D013463:Sulfuric Acid Esters; D000097811:Carbohydrate Sulfotransferases; C069886:L-selectin counter-receptors; C060192:MUC1 protein, human