Abstract
The leading cause of death in cancer patients is metastasis, for which an effective treatment is still necessary. During metastasis, cancer cells aberrantly express several glycans that are correlated with poor patient outcome. This study was aimed toward exploring the effects of O-GlcNAcylation on membranous N-glycans that are associated with the progression of cholangiocarcinoma (CCA). Global O-GlcNAcylation in CCA cells was depleted using specific siRNA against O-GlcNAc transferase (OGT), which transfers GlcNAc to the acceptor proteins. Using an HPLC-Chip/Time-of-Flight (Chip/TOF) MS system, the N-glycans associated with O-GlcNAcylation were identified by comparing the membranous N-glycans of siOGT-treated cells with those of scramble siRNA-treated cells. In parallel, the membranous N-glycans of the parental cells (KKU-213 and KKU-214) were compared with those of the highly metastatic cells (KKU-213L5 and KKU-214L5). Together, these data revealed that high mannose (Hex9HexNAc2) and biantennary complex (Hex5HexNAc4Fuc1NeuAc1) N-linked glycans correlated positively with metastasis. We subsequently demonstrate that suppression of O-GlcNAcylation decreased the expression of these two N-glycans, suggesting that O-GlcNAcylation mediates their levels in CCA. In addition, the ability of highly metastatic cells to migrate and invade was reduced by the presence of Pisum Sativum Agglutinin (PSA), a mannose-specific lectin, further indicating the association of high mannose type N-glycans with CCA metastasis. The molecular mechanism of O-GlcNAc-mediated progression of CCA was shown to proceed via a series of signaling events, involving the activation of Akt/Erk (i), an increase in FOXO3 phosphorylation (ii), which results in the reduction of MAN1A1 expression (iii) and thus the accumulation of Hex9HexNAc2 N-glycans (iv). This study demonstrates for the first time the association between O-GlcNAcylation, high mannose type N-glycans, and the progression of CCA metastasis, suggesting a novel therapeutic target for treatment of metastatic CCA.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Metastasis, a progressive process in which cancer cells develop the ability to colonize distant organs, is responsible for the majority of cancer deaths [1]
To our knowledge this study reports for the first time the molecular link between O-GlcNAcylation and membranous high mannose type N-glycans related to cancer progression
Expression of specific high mannose type N-glycans may be modulated by O-GlcNAcylation
Summary
The occurrence of metastasis is high for cholangiocarcinoma (CCA), a cancer of the bile duct. Few effective treatments for advanced CCA outside of surgical resection is available to prolong the survival of the patients. Aberrant glycosylation is commonly found in cancer, some of which associate with metastatic processes [5,6,7]. The correlation between the membranous glycosylation and the progression of cancer has been collectively reported [8,9,10,11]. In CCA, several aberrant glycans and glycoproteins are correlated with CCA progression, such as carbohydrate antigen 19-9, carcinoembryonic antigen, mucin (MUC), MUC2, MUC5AC, serum α1β-glycoprotein, and several lectin-binding glycans [12,13,14,15,16,17]
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