Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells

Lu Jin,Boxin Xue,Han Xiang,Lixia Wang,Xiaolong Liu,Yuxi Shan,Feng Yuan,Qiu Yao,Guangcheng Dai

doi:10.1186/s11658-020-00208-x

Abstract

BackgroundHigh levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms.MethodsO-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified.ResultsCellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy.ConclusionWe characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK.Graphical abstractBlockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

Highlights

High levels of the post-translational modification O-GlcNAcylation (OGlcNAc) are found in multiple cancers, including bladder cancer
O-GlcNAcylation negatively regulates autophagy in bladder cancer cells To determine whether autophagy is regulated by O-GlcNAcylation at basal levels in bladder cancer cell lines, we examined the autophagic flux in cells treated with DON or TG
As an inhibitor for different glutamine utilizing enzymes, DON inhibits the formation of UDP-GlcNAc, thereby reducing O-GlcNAcylation [26, 27]

Summary

Introduction

High levels of the post-translational modification O-GlcNAcylation (OGlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of OGlcNAcylation on autophagy in bladder cancer remains unclear. Bladder cancer is the second most common genitourinary malignancy, accounting for approximately 20% of the cases and mortality in this class worldwide [1]. Jin et al Cellular & Molecular Biology Letters (2020) 25:17 nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDPGlcNAc is the donor sugar for protein glycosylations, including the post-translational modification of nuclear and cytoplasmic proteins with O-linked-β-N-acetylglucosamine, a process mediated by the enzyme O-GlcNAc transferase (OGT) [4, 5]. O-GlcNAcylation can regulate protein functions by competing with phosphorylation on the same or proximal sites in proteins [7]

Methods

Results

Conclusion