No abstract

RING finger protein 43 ( RNF43 ) is a ubiquitin E3 ligase that negatively regulates Wnt/β-catenin signalling. Mutation, inactivation and downregulation of RNF43 in cholangiocarcinoma (CCA) are associated with a less favourable prognosis. Since the functional role of RNF43 in CCA has not yet been demonstrated, the present study aimed to assess the effect of its overexpression in mediating CCA suppression via Wnt/β-catenin signalling pathway inhibition. Accordingly, RNF43 was overexpressed, and various malignant phenotypic changes studied, including cell proliferation, cell migration, chemotherapeutic sensitivity and the expression of several Wnt/β-catenin target genes. Overexpression of RNF43 in the CCA cell-line KKU-213B hindered activation of Wnt/β-catenin signalling, evidenced by: i) Accumulation of β-catenin in the cytoplasmic fraction and downregulation of several known Wnt target genes at the mRNA level [ AXIN2 , survivin ( BIRC5 ), CCND1, MMP-7, c-MYC and ABCB1 ( MDR1 )]; ii) a reduction of cell proliferation; iii) a significant decrease in KKU-213B cell migration with RNF43 overexpression via upregulation of E-cadherin ( CDH1 ); and iv) a reduction in N-cadherin ( CDH2 ), MMP-2, MMP-7 and MMP-9 . In addition, overexpression of RNF43 increased 5-fluorouracil sensitivity and downregulation of ABC transporter genes [including ABCB1 and ABCC1 (MRP1)]. The current results demonstrate a functional role for RNF43 in CCA by: i) Blocking β-catenin nuclear translocation; and ii) the subsequent downregulation of Wnt/β-catenin target genes (the latter being involved in the progression of CCA and chemotherapeutic drug susceptibility). Therefore, the present findings suggest that RNF43 could serve a tumour suppressive role in CCA.

Objective To investigate the expression of glycosphingolipids in serum and tissue from patients with cholangiocarcinoma compared with healthy controls. Methods Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used to demonstrate the comparative structural glycomics of glycosphingolipids in serum from patients with cholangiocarcinoma (n=15), compared with healthy controls (n = 15). GM2 expression in cholangiocarcinoma tissues (n = 60) was evaluated by immunohistochemistry. Results Eleven glycosphingolipids were detected by NSI-MSn: CMH (ceramide monohexose), Lac-Cer (galactose (Gal)β1-4 glucose (Glc)β1-1'-ceramide), Gb3 (Galα1-4Galβ1-4Glcβ1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)β1-3Galα1-4Galβ1-4Glcβ1-1'-ceramide/Galβ1-4 N-acetylglucosamine (GlcNAc)β1-3Galβ1-4Glcβ1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Galβ1-4Glcβ1-1'-ceramide), GM2 (GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), GM1 (Galβ1-3GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), hFA (hydroxylated fatty acid)-CMH, hFA-Lac-Cer, hFA-Gb3, and hFA-GM3. Lac-Cer was the most abundant structure among the lactosides and globosides (normal, 24.40% ± 0.11%; tumor, 24.61% ± 2.10%), while GM3 predominated among the gangliosides (normal, 29.14% ± 1.31%; tumor, 30.53% ± 4.04%). The two glycosphingolipids that significantly differed between healthy controls and patients with cholangiocarcinoma were Gb3 and GM2. High expression of GM2 was associated with vascular invasion in tissue from patients with cholangiocarcinoma. Conclusions Altered expression of glycosphingolipids in tissue and serum from patients with cholangiocarcinoma may contribute to tumor growth and progression. The ganglioside GM2, which significantly increased in the serum of patients with cholangiocarcinoma, represents a promising target as a biomarker for cholangiocarcinoma.

Alteration of mucin-type O-glycosylation is implicated in tumor progression and metastasis of cholangiocarcinoma (CCA). Core 1 β1-3 Galactosyltransferase (C1GALT1) is a primary enzyme that regulates the elongation of core 1-derived mucin-type O-glycans. Dysregulation of C1GALT1 has been documented in multiple cancers and is associated with aberrant core 1 O-glycosylation and cancer aggressiveness; however, the expression of C1GALT1 and its role in CCA progression remains unknown. Our study demonstrated that C1GALT1 was downregulated in CCA tissues at both the mRNA and protein levels. The biological function of C1GALT1 using siRNA demonstrated that suppression of C1GALT1 in the CCA cell lines (KKU-055 and KKU-100) increased CCA progression, evidenced by: (i) Induction of CCA cell proliferation and 5-fluorouracil resistance in a dose-dependent manner; (ii) up-regulation of growth-related genes, ABC transporter genes, and anti-apoptotic proteins; and (iii) an increase in the activation/phosphorylation of AKT and ERK in silencing C1GALT1 cells. We demonstrated that silencing C1GALT1 in CCA cell lines was associated with immature core 1 O-glycosylation, demonstrated by high expression of VVL-binding glycans and down-regulation of other main O-linked glycosyltransferases β1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) and ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) in C1GALT1 knockdown. Our findings demonstrate that down-regulation of C1GALT1 in CCA increases the expression of immature core 1 O-glycan, enhancing CCA progression, including growth and 5-fluorouracil resistance via the activation of the AKT/ERK signaling pathway.

Objective This study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue. Methods Sixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1. Results O-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II–IV), lymphatic invasion, and vascular invasion. Conclusion Our study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.