Abstract 5457: OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1

Abstract

O-GlcNAcylation is a reversible post-translational modification that adds an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety onto serine/threonine residues of target proteins. This modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser) in mammals. Recent studies have revealed that OGT expression and O-GlcNAc modifications are elevated in several cancers, but specific O-GlcNAc targets are not well defined. We conducted a global transcriptome profiling in MDA-MB-231 breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. We found significant up-regulation of genes involved in the NRF2-dependent stress response when OGT activity is inhibited in different tumor types. We also discovered a strong positive correlation of gene signatures between low OGT activity and NRF2 activation in multiple human tumor gene expression datasets. NRF2, the primary regulator of redox balance, is usually activated by oxidative stress and repressed under basal conditions by the KEAP1-CUL3 ubiquitin ligase complex. However, we found that OGT inhibition increases NRF2 protein level through reducing poly-ubiquitination of NRF2 in the absence of oxidative stress. By chemical sugar labeling and mass spectrometry assays, we identified that KEAP1 is directly O-GlcNAcylated by OGT especially within the BTB and Kelch motifs. Of all 11 putative O-GlcNAc sites on KEAP1, we found serine 104 is responsible for regulating NRF2 activity through affecting KEAP1-CUL3 interaction. Interestingly, we found the amount of intracellular glucose co-vary with KEAP1 O-GlcNAcylation and NRF2 protein, suggesting that glucose metabolites may utilize the nutrient sensing O-GlcNAcylation to fine-tune the antioxidant response. We propose that cancer cells could utilize O-GlcNAcylation, specifically on KEAP1, to regulate NRF2-mediated stress responses in response to the dynamics of intracellular glucose level. Since the NRF2 pathway is inappropriately activated in certain tumor types due to dysregulated function of KEAP1, such as non-small cell lung cancer, where it provides stress resistance and a growth advantage. Our study could provide new insight into redox cancer biology and provide novel strategy to modulate NRF2 activity during tumor development. Citation Format: Po-Han Chen, Timothy J. Smith, Jianli Wu, Michael Boyce, Jen-Tsan Ashley Chi. OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2017-5457

OGT-Mediated KEAP1 Glycosylation Accelerates NRF2 Degradation Leading to High Phosphate-Induced Vascular Calcification in Chronic Kidney Disease

“…Meanwhile, the protein KEAP1 has also garnered the interest of numerous researchers, with prior evidence demonstrating that its O-GlcNAcylation S104 can effectively cause ubiquitination and degradation of NRF2 (Chen et al, 2017b), which thus corroborates our findings. Moreover, (Chen et al, 2017a) identified a positive correlation between lowered OGT activity and NRF2 activation in several gene expression datasets of human tumors, while NRF2 in turn has been found to be activated in response to oxidative stress and repressed by KEAP1. Furthermore, the in vitro findings of our study were also validated by in vivo experimentation, which highlighted that OGT repressed autophagy through the KEAP1/NRF2 axis and accelerated high phosphateinduced VC in 5/6 Nx rats.…”

“…Recent studies have evidenced the promotive effects of OGT and KEAP1 in vascular damage, wherein the transcription factor NRF2 was found to restrict VC (Ha et al, 2014;Zhang et al, 2019). OGT induces O-glycosylation of KEAP1 at serine 104 (S104) and then facilitates ubiquitination and degradation of NRF2 (Chen et al, 2017a). Subsequently, our efforts were aimed at further validating their functions in VSMCs.…”