O-GlcNAcylation Regulates Cell Autophagy In Bladder Cancer And Its Mechanism

Background and Objective:O-Glc NAcylation is posttranslational modification by O-linked β-Nacetylglucosamine(O-Glc NAc) moiety at serine or threonine residues of proteins. O-Glc NAc can modify multiple cellular processes, such as cell cycle, transcription, translation and signal transduction. Aberrant O-Glc NAc modification has been implicated in cancer, yet the role of O-Glc NAcylation in bladder cancer has not been reported. Autophagy, which is associated with tumorigenesis, neurodegenerative diseases and so on, involves membrane formation and fusion, including autophagosome formation, autophagosome lysosome fusion, and the the degradation of intra-autophagosomal contents by lysosomal hydrolases. Since autophagy is induced by extracellular and intracellular signals, including oxidative stress, ceramide, and endoplasmic reticulum stress, we postulate that O-Glc NAc glycosylation may regulate cell autophagy.Methods:To address our hypothesis, firstly, we validated the impact of O-Glc NAc glycosylation on the induction of autophagy. We used drugs and genetic modification to regulate cell O-Glc NAc glycosylation, and then fluorescence microscope and western blotting were used to test cell autophagy. Secondly, we defined the mechanisms by which O-Glc NAc glycosylation regulates autophagy. We focused on the potential glycosylation of autophagy related genes, such as ULK1. Finally, we determined whether interfering O-Glc NAc glycosylation would affect cancer cell proliferation and metastasis in vitro and vivo. Cellular and molecular biology methods, such as MTT, clone formation, flow cytometry instrument, cell scratch experiment, transwell chambers, transplanted tumor in nude mice, gene manipulation, immunoprecipitation, and western blot, have been applied.Results:We found that autophagy flux was significantly elevated in bladder cancer cells with lower O-Glc NAcylation, inversely autophagy flux was significantly reduced in higher O-Glc NAcylation. Additionally, we demonstrated O-Glc NAcylation regulated autophagy in bladder cancer cells via AMPK and its target ULK1, which had autophagy regulatory roles. Reducing O-Glc NAcylation increased AMPK activation and elevated levels of phosphorylated ULK1-S555, and then increased autophagy flux. Increasing O-Glc NAcylation decreased AMPK activation and reduced levels of phosphorylated ULK1-S555, and then decreased autophagy flux. Finally, the roles of O-Glc NAcylation in bladder cancer were investigated in vitro and vivo. The results showed that lower O-Glc NAcylation markedly reduced cell growth and proliferation of bladder cancer; it also reduced bladder cancer invasion and metastasis. Higher O-Glc NAcylation markedly enhanced cell growth and proliferation of bladder cancer; it also enhanced bladder cancer invasion and metastasis.Conclusions:All together, this study suggests that O-Glc NAc glycosylation has its role to bladder cancer via the suppression of cell autophagy. O-Glc NAc glycosylation reversely regulates cell autophagy through AMPK-ULK1 pathway. Successful accomplishment of the project will provide important information to validate the role of O-Glc NAc glycosylation in cancer initiation and progression by disruption of autophagy, and eventually will aid in the development of novel, effective therapeutic approaches for bladder cancer.