Katsuhisa Noda

The α1-6-fucosyltransferase gene and its biological significance

Abstract GDP- L -Fuc: N -acetyl-β- D -glucosaminide α1-6-fucosyltransferase (α1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N -linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human α1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N -glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as α1-2FucT, α1-3FucT and α1-4FucT was found except for a region consisting of nine amino acids. The α1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The α1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. α1-6FucT is widely expressed in various rat tissues and the expression of α1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, α1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum α1-6-fucosylated α-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which α1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of α1-6FucT alone. Interestingly, when the α1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on α1-6FucT and summarize its properties, gene expression and biological significance.

Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma

In order to elucidate the clinical significance of the erbB family, epidermal growth factor receptor (EGF-R), c-erbB-2, c-erbB-3 and c-erbB-4 in hepatocellular carcinoma (HCC), we investigated the expression of these proteins by means of immunohistochemistry for HCC as well as adjacent noncancerous lesions. EGF-R was expressed in 68% of the HCC examined and showed correlation with the proliferating activity, stage, intrahepatic metastasis and carcinoma differentiation. c-erbB-2 was expressed in only 21% of the cases and showed no relationships with the clinicopathological parameters. c-erbB-3 protein was observed in 84% of the HCC and 38.1% of the noncancerous lesions. Its expression in HCC was equal to or greater than noncancerous lesions in 90.5% of the cases, and was related to the stage, portal invasion, cell proliferating activity, tumour size, intrahepatic metastasis and carcinoma differentiation. c-erbB-4 protein was expressed in 61.0% of HCC and in as much as 86.1% of the noncancerous lesions. Unlike the expression of c-erbB-3, that of c-erbB-4 in HCC was less than that of the adjacent noncancerous lesions in 51.2% of the cases. No statistical significance could be established between this protein expression in HCC and clinicopathological features. EGF-R and c-erbB-3 affected disease-free survival, but were not recognized as independent prognostic factors by multivariate analysis. The present study suggests that, of the four receptors, EGF-R and c-erbB-3 play important roles in the progression of HCC.

Regulation of the GnT-V promoter by transcription factor Ets-1 in various cancer cell lines.

Although the precise role of oligosaccharides in metastasis is presently unknown, numerous studies suggest that the β1–6 branching structure of N-linked oligosaccharides plays a role in tumor metastasis.N-Acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of the β1–6 branch, therefore appears to play a crucial role in tumor metastasis. Recently, we demonstrated that the expression of the GnT-V gene is regulated by a transcriptional factor, Ets-1 (Kang, R., Saito, H., Ihara, Y., Miyoshi, E., Koyama, N., Sheng, Y., and Taniguchi, N. (1996) J. Biol. Chem. 271, 26706–26712). In this study, we report an investigation of the general requirement for Ets-1 in the expression of GnT-V in cancer cell lines. In 16 cancer cell lines, the levels of GnT-V mRNA were closely correlated with ets-1 expression (r = 0.97; p < 0.0001). An increase in ets-1 levels by transfection of its cDNA led to an enhancement in GnT-V expression in cells that normally expressed low levels of ets-1. In contrast, the transfection of dominant negative ets-1 into cells that express high levels of ets-1 resulted in a decrease in GnT-V expression. Although Ets-1 cooperates with c-Jun in certain gene expressions, this was not the case in the regulation of theGnT-V gene. These results suggest that Ets-1 plays a significant role in regulating the expression of GnT-V in a variety of cancers and might be involved in the potential for malignancy via the action of GnT-V.

The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin.

The enzyme GnT-III (β1,4-N-acetylglucosaminyltransferase III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) residue on glycoproteins. Our previous study described that the transfection of GnT-lll into mouse melanoma cells results in the enhanced expression of E-cadherin, which in turn leads to the suppression of lung metastasis. It has recently been proposed that the phosphorylation of a tyrosine residue of β-catenin is associated with cell migration. The present study reports on the importance of bisecting GlcNAc residues by GnT-lll on tyrosine phosphorylation of β-catenin using three types of cancer cell lines. An addition of bisecting GlcNAc residues to E-cadherin leads to an alteration in cell morphology and the localization of β-catenin after epidermal growth factor stimulation. These changes are the result of a down-regulation in the tyrosine phosphorylation of β-catenin. In addition, tyrosine phosphorylation of β-catenin by transfection of constitutively active c-src was suppressed in GnT-III transfectants as well as in the case of epidermal growth factor stimulation. Treatment with tunicamycin abolished any differences in β-catenin phosphorylation for the mockvis à vis the GnT-lll transfectants. Thus, the addition of a specific N-glycan structure, the bisecting GlcNAc to E-cadherin-β-catenin complex, down-regulates the intracellular signaling pathway, suggesting its implication in cell motility and the suppression of cancer metastasis.

Core fucosylation of E-cadherin enhances cell-cell adhesion in human colon carcinoma WiDr cells

α1,6‐Fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of α1,6 core fucose to the innermost N‐acetylglucosamine residue of the N‐glycan, has been implicated in the development, immune system, and tumorigenesis. We found that α1,6‐fucosyltransferase and E‐cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E‐cadherin appeared as well as normal sized E‐cadherin in cancer samples. To investigate the correlation between α1,6‐fucosyltransferase and E‐cadherin expression, we introduced α1,6‐fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E‐cadherin was significantly increased in α1,6‐fucosyltransferase‐transfected WiDr cells in dense culture, which resulted in an enhancement in cell–cell adhesion. The transfection of mutated α1,6‐fucosyltransferase with no enzymatic activity had no effect on E‐cadherin expression, indicating that core fucosylation is involved in the phenomena. In α1,6‐fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E‐cadherin and E‐cadherin dependent cell–cell adhesion was decreased. The introduction of α1,6‐fucosyltransferase into kidney epithelial cells from α1,6‐fucosyltransferase–/– mice restored the expression of E‐cadherin and E‐cadherin‐dependent cell–cell adhesion. Based on the results of lectin blotting, peptide N‐glycosidase F treatment, and pulse‐chase studies, it was demonstrated that the low molecular weight population of E‐cadherin contains peptide N‐glycosidase F insensitive sugar chains, and the turnover rate of E‐cadherin was reduced in α1,6‐Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E‐cadherin. These results suggest a possible role of core fucosylation in the regulation of cell–cell adhesion in cancer. (Cancer Sci 2009; 100: 888–896)

The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila

Notch is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. Endocytic trafficking of Notch plays important roles in the activation and downregulation of this receptor. A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. It was recently proposed that O-fut1 acts as a chaperon for Notch in the endoplasmic reticulum and is required for Notch to exit the endoplasmic reticulum. Here, we report that O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 was indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which we show was independent of the O-fucosyltransferase activity of O-fut1. We also found that O-fut1 promoted the turnover of Notch, which consequently downregulated Notch signaling. O-fut1 formed a stable complex with the extracellular domain of Notch. In addition, O-fut1 protein added to conditioned medium and endocytosed was sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells. Thus, an extracellular interaction between Notch and O-fut1 is essential for the normal endocytic transportation of Notch. We propose that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis.

High expression of α‐1‐6 fucosyltransferase during rat hepatocarcinogenesis

α‐1‐6 Fucosylated α‐fetoprotein (AFP) is known to be elevated in patients with primary hepatoma and has been suggested as being useful as an early indicator and predictor of the poor prognosis for hepatoma. Although GDP‐L‐fucosyl‐N‐acetyl‐β‐D‐glucosaminide α‐1‐6 fucosyltransferase (α‐1‐6 FucT), is the key enzyme involved in α‐1‐6 fucosylation of AFP, when and how the expression of α‐1‐6 FucT is enhanced during hepatocarcinogenesis is unknown. Recently, we established a convenient assay method for this enzyme and were successful in the purification and cDNA cloning of α‐1‐6 FucT from human gastric cancer, as well as from porcine brain. In the present study, levels of α‐1‐6 FucT activity and mRNA expression have been determined during hepatocarcinogenesis in LEC rats which spontaneously develop hereditary hepatitis and hepatoma. The fetal liver contained the highest enzymatic activity, which tended to increase in inverse proportion to gestation. The enzymatic activity was significantly increased in hepatoma tissues as compared with uninvolved adjacent tissues. Northern‐blot analysis revealed high expression of α‐1‐6 FucT mRNA in hepatoma tissues, whereas the expression was fairly low in normal, hepatitis and uninvolved adjacent liver tissues. While the fetal liver had the highest enzymatic activity, the expression of α‐1‐6 FucT mRNA was low, suggesting that another α‐1‐6 FucT is induced in fetal liver or that post‐translational modification occurs. High expression of α‐1‐6 FucT was also observed in 3′‐MeDAB‐induced rat hepatomas and some rat hepatoma cell lines. Collectively, α‐1‐6 FucT was strongly enhanced from an early stage of hepatocarcinogenesis and was maintained at a high level in rat hepatomas. Int. J. Cancer 75:444–450, 1998.

Elevated expression of UDP‐N‐acetylglucosamine: αmannoside β1,6 N‐acetylglucosaminyltransferase is an early event in hepatocarcinogenesis

Previous reports have suggested that changes in oligosaccharide structures, especially β1–6 branching in N‐glycans, which are biosynthesized by UDP‐N‐acetylglucosamine:α mannoside β1,6 N‐acetylglucosaminyltransferase (GnT‐V), are linked to tumor metastasis and invasion. In the present study, we investigated GnT‐V expression in human hepatocellular carcinoma (HCC) tissues. High expression of GnT‐V mRNA was observed in both HCC and the surrounding tissues but not in normal liver. Immunohistochemical study using a newly established monoclonal antibody against GnT‐V revealed that positive staining of GnT‐V was observed in 75% of HCC tissues and 60% of surrounding tissues and that liver cirrhosis showed much stronger staining of GnT‐V than chronic hepatitis without liver cirrhosis (p = 0.0035). In contrast, all of 12 cases of atypical adenomatous hyperplasia diffusely expressed GnT‐V. β1–6 branching in N‐glycans, products of GnT‐V, was increased in HCC tissues with high expression of GnT‐V, as judged by lectin blotting. Levels of GnT‐V expression in HCC tissues were positively correlated with a low Ki‐67 labeling index (p = 0.0009), small size (p < 0.0001), poor differentiation (p < 0.0001) and absence of portal invasion (p = 0.018). Furthermore, HCC cases with low or no expression of GnT‐V were more likely to show recurrence than cases with high expression (p = 0.0373). These findings strongly suggest that GnT‐V expression is concerned mainly with an early phase of hepatocarcinogenesis.

Deficiency of GMDS leads to escape from NK cell-mediated tumor surveillance through modulation of TRAIL signaling.

BACKGROUND & AIMS Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes apoptosis in cancer cells, but not normal cells, and is critically involved in tumor rejection through natural killer (NK) cell-mediated immune surveillance. Oligosaccharides are involved in various aspects in carcinogenesis, and fucosylation is one of the most important oligosaccharide modifications in cancer. Here, we report for the first time mutations of the GDP-mannose-4,6-dehydratase (GMDS) gene, which plays a pivotal role in fucosylation, in human colon cancer. The mutations resulted in resistance to TRAIL-induced apoptosis followed by escape from immune surveillance. METHODS The mock and GMDS-rescued HCT116 cells were investigated in terms of NK cell-mediated tumor surveillance by TRAIL signaling both in vitro and in vivo. The mutational analysis for GMDS was performed with kinds of cancer cell lines and tissues. RESULTS The mutation found here led to a virtually complete deficiency of cellular fucosylation, and transfection of the wild-type GMDS into HCT116 cells restored the cellular fucosylation. When mock and GMDS-rescued cells were transplanted into athymic mice, tumor growth and metastasis of the GMDS-rescued cells were dramatically suppressed through NK cell-mediated tumor surveillance. Furthermore, the GMDS-rescued cells showed high susceptibility to TRAIL-induced apoptosis, and anti-TRAIL blocking antibody suppressed the accelerated direct cell lysis of the GMDS-rescued cells by splenocytes. Similar mutations of the GMDS were found in certain human cancer tissues and other cell lines. CONCLUSIONS This pathway by GMDS mutation could be a novel type of cancer progression through cellular fucosylation and NK cell-mediated tumor surveillance.

Activation of c-Src gene product in hepatocellular carcinoma is highly correlated with the indices of early stage phenotype

BACKGROUND/AIMS The aim of this study was to investigate whether c-Src is involved in carcinogenesis and progression of human hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma. METHODS We designed an immunohistochemical study using Clone 28, an antibody that specifically recognizes the activated form of c-Src. RESULTS Hepatocytes in normal liver, chronic hepatitis with or without cirrhosis, atypical adenomatous hyperplasia, as well as bile ductular cells, and infiltrating mononuclear cells were all negative for immunohistochemical staining for the activated c-Src. Among 87 cases of HCC tested, 40 (46%) were positively stained for the activated c-Src, and this positive staining was inversely correlated with the Ki-67 labeling index (LI) (P = 0.0031), intrahepatic metastasis (P = 0.0099), TNM stage (P = 0.0062), alpha-fetoprotein (P = 0.0103) and epidermal growth factor-receptor expression (P = 0.0153). Positive staining for the activated c-Src was more frequently observed in well- or moderately-differentiated carcinoma (P = 0.0256). In multivariate analysis, the activated c-Src expression was independently related to the Ki-67 LI (P = 0.0197). In contrast to positive staining in HCC, cholangiocarcinoma were classified as negative in all 19 cases examined. CONCLUSIONS These results strongly suggest the involvement of activated c-Src in early stages of HCC, and suggest that cholangiocarcinoma might employ different signaling mechanisms.