O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.

Dysfunctions in Wnt signaling increase β‐catenin stability and are associated with cancers, including colorectal cancer. In addition, β‐catenin degradation is decreased by nutrient‐dependent O ‐GlcNAcylation. Human colon tumors and colons from mice fed high‐carbohydrate diets exhibited higher amounts of β‐catenin and O ‐GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O ‐GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β‐catenin. By ETD‐MS/MS, we identified 4 O ‐GlcNAcylation sites at the N terminus of β‐catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β‐catenin reduced O ‐GlcNAcylation by 75%. Interestingly, elevating O ‐GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β‐catenin stability. Analyses of β‐catenin O ‐GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β‐catenin degradation rate. Finally, inhibiting O ‐GlcNAcylation decreased the β‐catenin/α‐catenin interaction necessary for mucosa integrity, whereas O ‐GlcNAcase silencing improved this interaction. These results suggest that O ‐GlcNAcylation regulates not only the stability of β‐catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O ‐GlcNAcylation of β‐catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.—Olivier‐Van Stichelen, S., Dehennaut, V., Buzy, A., Zachayus, J.‐L., Guinez, C., Mir, A‐M., El Yazidi‐Belkoura, I., Copin, M.‐C., Boureme, D., Loyaux, D., Ferrara, P., Lefebvre, T. O ‐GlcNAcylation stabilizes β‐catenin through direct competition with phosphorylation at threonine 41. FASEB J. 28, 3325–3338 (2014). www.fasebj.org

During the past two decades, O ‐GlcNAc modification of cytosolic and nuclear proteins has been intensively studied. Nevertheless, the function of this post‐translational modification remains unclear. It has been recently speculated that O ‐GlcNAc could act as a protective signal against proteasomal degradation, both by modifying target substrates and/or by inhibiting the proteasome itself. In this work, we have investigated the putative relation between O ‐GlcNAc and the ubiquitin pathway. First, we showed that the level of both modifications increased rapidly after thermal stress but, unlike ubiquitinated proteins, O ‐GlcNAc‐modified proteins failed to be stabilized by inhibiting proteasome function. Increasing O ‐GlcNAc levels, using glucosamine or PUGNAc, enhanced ubiquitination. Inversely, when O ‐GlcNAc levels were reduced, using forskolin or glucose deprivation, ubiquitination decreased. Targeted‐RNA interference of O ‐GlcNAc transferase also reduced ubiquitination and moreover halved cell thermotolerance. Finally, we demonstrated that the ubiquitin‐activating enzyme E1 was O ‐GlcNAc modified and that its glycosylation and its interaction with Hsp70 varied according to the conditions of cell culture. Altogether, these results show that O ‐GlcNAc and ubiquitin are not strictly antagonistic post‐translational modifications, but rather that the former might regulate the latter, and also suggest that E1 could be one of the common links between the two pathways. —Guinez, C., Mir, A.‐M., Dehennaut, V., Cacan, R., Harduin‐Lepers, A., Michalski, J.‐C., Lefebvre, T. Protein ubiquitination is modulated by O ‐GlcNAc glycosylation. FASEB J . 22, 2901–2911 (2008)

Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in lung cancer metastasis. The class III deacetylase sirtuin 1 (SIRT1) possesses both pro- and anticarcinogenic properties. The role of SIRT1 in lung cancer EMT is largely undefined. The effect of SIRT1 on migration of lung cancer cells was evaluated by wound healing assay in vitro and metastasis assay in nude mice in vivo. Protein expression in human lung cancers and cultured lung cancer cells was assessed by western blotting and immunohistochemistry. Interaction between protein and DNA was measured by chromatin immunoprecipitation assay. SIRT1 promoter activity was determined by reporter assay. SIRT1 activation antagonized migration of lung cancer cells by suppressing EMT in vitro. Activation of SIRT1 by resveratrol also statistically significantly hampered (by 68.33%; P < .001, two-sided test) lung cancer cell metastasis in vivo. Hypoxia repressed SIRT1 transcription through promoting the competition between Sp1 and HIC1 on the SIRT1 proximal promoter in a SUMOylation-dependent manner. Disruption of SUMOylation by targeting either Ubc9 or PIASy restored SIRT1 expression in and favored an epithelial-like phenotype of cancer cells, thereby preventing metastasis. Decreased SIRT1 combined with elevated PIASy expression was implicated in more-invasive types of lung cancers in humans. We have identified a novel pathway that links SIRT1 down-regulation to hypoxia-induced EMT in lung cancer cells and may shed light on the development of novel antitumor therapeutics.

There are increasing evidences that dietary components and metabolic disorders affect gene expression through epigenetic mechanisms. These observations support the notion that epigenetic reprogramming-linked nutrition is connected to the etiology of metabolic diseases and cancer. During the last five years, accumulating data revealed that the nutrient-sensing O-GlcNAc glycosylation (O-GlcNAcylation) may be pivotal in the modulation of chromatin remodeling and in the regulation of gene expression by being part of the “histone code”, and by identifying OGT (O-GlcNAc transferase) as an interacting partner of the TET family proteins of DNA hydroxylases and as a member of the Polycomb group (PcG) proteins. Thus, it is suggested that O-GlcNAcylation is a post-translational modification that links nutrition to epigenetic. This review summarizes recent findings about the interplay between O-GlcNAcylation and the epigenome and enlightens the contribution of the glycosylation to epigenetic reprogramming.