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(Background and Aim) Oligosaccharides are one of the most important factors in the posttranslational modification of proteins and lipids. Glycomics, the systematic study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the post-genomics and post-proteomics era. In particular, fucosylation is one of the most important glycosylation involved in cancer and inflammation. Fucosylation, which comprises the transfer of a fucose residue to oligosaccharides and proteins, is regulated by many kinds of molecules, including several kinds of fucosyltranferases, GDP-fucose synthetic enzymes, and GDP-fucose transporter(s). Fucosylation is increased during carcinogenesis of many cancers through the up-regulation of expression of fucosylation-regulatory genes as described above. In contrast, we found a colon cancer cell line HCT116, which had complete loss of fucosylation due to the mutation of a GDP-fucose synthetic enzyme, GMDS (GDP-mannose-4, 6-dehydratase) a few years ago. While knockout mice of a GDP-fucose synthetic enzyme are lethal (Smith et al J Cell Biol, 158, 2002), HCT116 cells showed more aggressive pheno-type, including in vivo tumor growth and metastasis. The underlying mechanisms were found to be an escape from TRAIL-mediated immuno-surveillance of NK cells (Moriwaki et al, Gastroenterology 137, 2009). In the present study, we investigated whether or not the GMDS mutation was really found in clinical samples of colorectal cancer. (Subjects and Methods) Thirteen cases of liver metastasis, 6 cases of lymph node metastasis and 81 cases of the original cancer tissues derived from patients with colorectal cancer were included in this study. All projects were approved by the ethical committee of Osaka University. GMDS mutation was analyzed by RT-PCR using primers, which include all exons of GMDS gene. Immunohistochemical experiments were performed using AAL lectin, which recognizes all types of fucosylation. (Results and Discussion) Approximately 10-15% of GMDS mutation was found in the original cancer tissue as well as their metastatic liver cancer and lymph node metastasis. However no mutation was found in normal colon tissue around cancer. Most GMDS mutation was a hetero-type, but not a homo-type, which was found in HCT 116 cells. Immunohistochemical analysis showed complete loss of fucosylation in a few cases of metastatic cancer. These data indicate the involvement of GMDS mutation in the progression of colorectal cancer. Citation Format: Kotarosumitomo Nakayama, Kenta Moriwaki, Mayuka Shimomura, Hironobu Fujii, Shinji Takamatsu, Yoshihiro Kamada, Kohei Murata, Eiji Miyoshi. Clinical significance of GDP-mannose-4,6-dehydratase mutation and loss of fucosylation in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2684. doi:10.1158/1538-7445.AM2013-2684

Background: Fucosylation is a crucial oligosaccharide modification in cancer and inflammation. Total cellular proteins of cancer cells and the sera of patients with cancer both show increased fucosylation levels. Certain kinds of fucosylated proteins can be applied as novel cancer biomarkers in glyco-proteomic analyses. We previously identified fucosylated haptoglobin (Fuc-Hpt) as a serologic marker for pancreatic cancer, and recently developed a lectin-antibody enzyme-linked immunosorbent assay system for quantifying Fuc-Hpt. In the present study, we investigated the clinical outcome of Fuc-Hpt levels in patients with colorectal cancer (CRC), and examined the mechanisms underlying Fut-Hpt production using a murine tumor transplantation model. Methods: The relationship between Fuc-Hpt levels and clinical parameters was investigated in 77 patients with CRC, all of whom underwent primary resection. Serum Fuc-Hpt levels were examined in athymic nude mice injected with colon cancer cell lines that lacked fucosylation. Results: Fuc-Hpt levels were significantly associated with overall and relapse-free survival, distant metastasis, clinical stage, and curability. Multivariate analysis revealed that distant metastasis was an independent factor for increased Fuc-Hpt levels. The combination of Fuc-Hpt and CEA might be a better serologic marker to predict prognosis. Fuc-Hpt levels were higher in mice with direct injection of tumor cells into the spleen than in those injected subcutaneously. Conclusions: Fuc-Hpt might be a useful marker for the prognosis of CRC. Fuc-Hpt could be produced by the tissue surrounding tumor cells, which might be the mechanism underlying Fuc-Hpt elevation associated with distant metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4531. doi:1538-7445.AM2012-4531

A growing body of evidence has suggested that tumors are frequently composed of heterogeneous cell types, and that tumor initiation and growth are driven by a small subset of cells, termed cancer stem cells (CSCs) or tumor-initiating cells. CSCs can self-renew and also give rise to more differentiated progeny that comprise the bulk of a tumor. Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to CSCs have remained unknown. In this study, we examined glycan structures in CD133+CD13+ CSCs, which were recently found to have a high CSC ability, by means of lectin microarray. Seven sialylated glycan-recognizing lectins, MAL-I, SNA, SSA, TJA-I, ACG, ABA, and MAH, showed higher affinity to CD133+CD13+ CSCs than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in NOD/SCID mice and spheres under serum-free conditions than CD133+SSA- cells. These results suggest that hepatic CSCs highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSCs. This study is the first report to demonstrate the characteristic glycan structures in CSCs and to indicate a new methodology involving lectins for isolating CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4356. doi:10.1158/1538-7445.AM2011-4356

Oligosaccharides are some of the most important factors in the posttranslational modification of proteins and lipids. It is well known that oligosaccharide structures change during malignant transformation. Fucose is a type of constituent of oligosaccharides, and is notably associated with cancer and inflammation. Fucosylation is catalyzed by several kinds of fucosyltransferases, the GDP-fucose synthetic enzymes, and GDP-fucose transporter. Recently, we found a mutation of the GMDS gene, which involved in the synthesis of GDP-fucose, in a colon cancer cell line, HCT116, leading to a virtually complete deficiency of fucosylation. Down-regulation of cellular fucosylation rendered the cells resistant to tumor necrosis factor-related apotosis-inducing ligand (TRAIL) -induced apoptosis, resulting in the escape from natural killer (NK) cell-mediated tumor immune surveillance. Thus, GMDS and fucosylation are thought to be regulatory factors for TRAIL-induced apoptosis and tumor immune surveillance. In the present study, we searched mutation of the GMDS gene in human cancer cell lines and various kinds of cancer tissues. The aberrant transcripts with deletions of exons 2-4 and 5-7 were detected in the cDNA of LS174T and NCI-H716 cells, respectively. Since these mutations were heterozygous, the deficiency of fucosylation was not observed. In a gastric choriocarcinoma cell line, SCH, only short transcript with deletion of exons 2-4 was detected. This cell line showed a marked decrease in the fucosylation level. PCR and subsequent direct sequencing analyses using 100 human colon cancer tissues identified mutations in two cases. One of the two was c.739 C>T (p.R247X) in exon7, and the other was c.345+5 G>A (IVS4+5) in intron 4. Furthermore, RT-PCR analysis identified aberrant transcripts in 5 of 10 ovarian cancer tissues. All mutant GMDS genes found in this study had no enzymatic activity. Subsequently, the level of fucosylation in many colon cancer tissues was investigated by immunohistochemical analysis using two types of fucosylated oligosaccharide-specific lectin. The decrease of fucosylation was observed in certain cases. Especially, core-fucosylation was decreased in colon cancer with lymph node metastasis. These results suggest that cancer progression in certain patients with colon cancer is dependent on de-fucosylation. Thus, fucosylation is an attractive target for cancer diagnosis and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 800.