Cross regulation between mTOR signaling and O ... - Springer Link

Journal of Bioenergetics and Biomembranes https://doi.org/10.1007/s10863-018-9747-y

Cross regulation between mTOR signaling and O-GlcNAcylation Ninon Very 1 & Agata Steenackers 2 & Caroline Dubuquoy 3 & Jeanne Vermuse 1 & Laurent Dubuquoy 3 & Tony Lefebvre 1 & Ikram El Yazidi-Belkoura 1 Received: 19 October 2017 / Accepted: 15 February 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the Nacetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global OGlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer. Keywords O-GlcNAcylation . mTOR signaling . Metabolism . Colon . Cancer . Obesity

Introduction O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification which occurs on serine (Ser) and threonine (Thr) residues of cytoplasmic, nuclear and mitochondrial proteins. OGT (O-GlcNAc transferase) transfers the N-acetylglucosamine (GlcNAc) residue from UDPGlcNAc onto target proteins and OGA (O-GlcNAcase) removes it. The nucleotide sugar donor uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is synthetized through

* Ikram El Yazidi-Belkoura ikram.el–yazidi@univ–lille1.fr 1

CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, F 59000 Lille, France

2

Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA

3

INSERM, U995, LIRIC – Lille Inflammation Research International Center, CHU Lille, Université de Lille, F 59000 Lille, France

the hexosamine biosynthesis pathway (HBP) at the crossroad of glucose, amino acids, fatty acids and nucleotides metabolisms (Hart and Akimoto 2009). O-GlcNAcylation competes with phosphorylation at the same or proximal sites to control many signaling pathways regulating cell proliferation and survival (Butkinaree et al. 2010; Hart et al. 2011). OGlcNAcylation is modulator of cellular processes in response to nutrients and stress; therefore this modification is considered a nutritional sensor. Dysregulation in O-GlcNAcylation processes was reported in neurodegeneration, cardiovascular diseases, diabetes and cancers (Hart and Akimoto 2009). Many studies involve O-GlcNAcylation in cancer biology by its critical roles in cell cycle, transcription, translation, ubiquitinproteasomal degradation and cellular nutrient/stress responses. Thus, this PTM is a pivotal regulator of cell proliferation, survival, migration and epithelial-mesenchymal transition in a context of oncogenesis (Ferrer et al. 2016). Many signaling pathways are controlled by OGlcNAcylation in response to nutrients and mitogenic signals. Under insulin stimulation, OGT O-GlcNAcylates insulin receptor substrate 1 (IRS1) (Park et al. 2005; Ball et al. 2006),

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phosphoinositide 3-kinase (PI3K) (Federici et al. 2002) and AKT (Park et al. 2005). OGT is also required for expression of the insulin receptor (Perez-Cervera et al. 2013). In turn, OGT expression is increased in response to insulin (Perez-Cervera et al. 2013) and its synthesis is controlled by the PI3K pathway (Olivier-Van Stichelen et al. 2012). The increase in OGlcNAcylation content results in mitogen-activated protein kinases (MAPK) pathway activation (Dehennaut et al. 2008); also the extracellular signal-regulated kinase 2 (Erk2) is O-GlcNAcylated during G2/M transition of cell cycle (Dehennaut et al. 2008). In colorectal cancer (CRC), the Wnt/β-catenin pathway is also under OGT control since βcatenin expression correlates with O-GlcNAcylation status (Olivier-Van Stichelen et al. 2012). O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at Thr41 and decreases global O-GlcNAcylation reduced β-catenin/α-catenin interaction and localization of β-catenin at the level of adherens junctions. Thus, one suggests that this glycosylation may regulate mucosa integrity and consequently play an important role in epithelial cancer development (Olivier-Van Stichelen et al. 2014). Recently, teams of Cho (Park et al. 2014) and Reginato (Sodi et al. 2015) demonstrated that mammalian target of rapamycin (mTOR) signaling regulates protein OGlcNAcylation in hepatic and breast cancer cells respectively through adjustment of OGT stability. In hepatic cancer cells, pharmacological inhibition of mTOR activity reduced global O-GlcNAcylation consequently to decreased OGT and increased OGA proteins (Park et al. 2014). In breast cancer cells, mTOR activation resulted in elevated OGT/O-GlcNAcylation levels through the control by the mTOR downstream transcription factor c-MYC in an RNA-independent manner (Sodi et al. 2015). mTOR senses the cell energy status in response to amino acids and growth factors. Once induced, mTOR increases cell growth and proliferation through phosphorylation of two downstream effectors: p70 ribosomal S6 protein kinase (p70S6K), a kinase implicated in ribosome biogenesis, and eukaryotic translation initiation factor 4E–binding protein (4EBP1). To push forward these observations, we aimed to draw a comparative view of the cross-talk between two metabolic state sensors, the O-GlcNAcylation and the mTOR signaling pathway in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells.

Materials and methods

stock solutions, respectively. Glucosamine was obtained from Sigma (G1514). Mouse monoclonal anti-O-GlcNAc (RL2) was purchased from Thermo Scientific (MA1–072), rabbit polyclonal anti-OGT (Ti-14) was purchased from SigmaAldrich (#O6014) and rabbit polyclonal anti-mTOR (#2983), rabbit polyclonal anti-phospho-mTOR (Ser2448, #5536), rabbit polyclonal anti-p70S6K (#2708), rabbit polyclonal anti-phospho-p70S6K (Thr389, #9234) and mouse clonal anti-GAPDH (#47724) were purchased from Cell Signaling. Rabbit monoclonal anti-OGA was from Abcam. Scrambled control siRNA (siCtrl) was obtained from Sigma (MISSION siRNA universal negative control #1) and siRNA targeting human OGA (siOGA) was purchased from Dharmacon (siGenome Smart Pool, Human MGEA5, M-012805-01).

Cell culture and synchronization Human fetal colon CCD841CoN and colon carcinoma HCT116 cells were obtained from the American Type Culture Collection (ATCC). CCD841CoN were cultured in Eagle’s minimum essential medium (EMEM, Lonza) with 5 mM glucose and HCT116 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) supplemented with 25 mM glucose. Both cell lines were maintained in medium supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS) and 2 mM L-glutamine at 37 °C, in a 5% (v/v) CO2enriched, humidified atmosphere. After been plated for 24 h, cells were washed twice with phosphate-buffered saline (PBS) and then were synchronized in serum-free medium for 24 h.

mTOR regulators treatment Twenty-four hours synchronized CCD841CoN and HCT116 cells were treated for 48 h with rapamycin (10 or 50 nM) or MHY1485 (10 μM) in DMEM medium containing 10% (v/v) FCS, 5 mM glucose and 1% (v/v) MACS bovine serum albumin (BSA) Stock Solution (Miltenyi Biotec). For time course experiments, cells were pre-treated 1 h with rapamycin (50 nM) before stimulation with 10% (v/v) FCS.

Hexosamine biosynthetic pathway (HBP) regulators treatment CCD841CoN and HCT116 cells were synchronized for 30 h and then treated with DMEM medium containing 25 mM glucose and 10% (v/v) FCS with or without azaserine (75 μM) and/or glucosamine (5 mM) for 24 h.

Reagents, antibodies and chemicals Small interfering RNA (siRNA) reverse transfection Rapamycin (Santa Cruz, sc-3504), MHY1485 (Sigma, SML0810) and azaserine (Sigma, A4142) were dissolved in dimethyl sulfoxide (DMSO) at 1 mM, 5 mM and 20 mM as

SiCtrl or siOGA (5 nM) were administered for 78 h to CCD841CoN and HCT116 cells with Lipofectamine™

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Fig. 1 Metabolism disruption induces O-GlcNAcylation increase correlated with activation of mTOR signaling in vivo and in vitro. a OGT, O-GlcNAcylation, phospho-mTOR and mTOR levels were analyzed by Western blot in proximal (P) and distal (D) tissues of four C57BL/6 and four ob/ob mice. b A correlation between ratios of phospho-mTOR/mTOR and OGT/GAPDH is found. The linear regression line, the R-square and the P value for the F-test are shown on. c Specificity of O-GlcNAc antibody (RL2) was confirmed by binding

competition of the antibody with 0.5 M free-GlcNAc prior to use on synchronized CCD841CoN and HCT116 proteins in WB analyses. d Synchronized normal CCD841CoN and cancer HCT116 colon cells were analyzed by using the same antibodies and e histograms of OGT/GAPDH and phospho-mTOR/mTOR ratios are represented. Data shown are the average ± standard derivation (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 Student’s t-test

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RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. Twenty four hours later, cells were synchronized for 30 h and then stimulated with DMEM containing 25 mM glucose and 10% (v/v) FCS.

Cells lysis and western blot analysis One hour before lysis, cells were incubated with 100 μM sodium orthovanadate (Sigma-Aldrich) to inhibit tyrosine phosphatases. Then, cells were washed with 10 mL cold PBS, then lysed on ice for 20 min in cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% SDS (w/v), 1% Triton-X100 (v/v), pH 7.4) containing the complete proteases inhibitors (protease cocktail inhibitors from Roche Diagnostics, Meylan, France), 50 mM sodium fluoride (Sigma-Aldrich), 100 μM orthovanadate; pH 7.4). The cell lysates were centrifuged at 20,000×g for 10 min at 4 °C. The supernatants were collected and protein concentrations were measured using the microBCA protein assay kit (Pierce, Fisher Scientific, Illkirch, France). Proteins (30 μg) were resolved by 8% SDS-PAGE and transferred to nitrocellulose membranes (Hybond™-C EXTRA, GE Healthcare Technology, Orsay, France). Membranes were blocked in 5% (w/v) nonfat dry milk or BSA in Tris-Buffered Saline (TBS) containing 0.05% Tween 20 (v/v) (TBS-T) and probed overnight at 4 °C with primary antibodies diluted in milk blocking solution: anti-RL2 (1:4000), anti-Ti14 (1:2000) and anti-GAPDH (1:5000) or BSA blocking solution: anti-OGA (1:10000), anti-mTOR (1:1000), anti-phospho-mTOR (1:1000), antip70S6K (1:1000) and anti-phospho-p70S6K (1:1000). After 3 TBS-T washes, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-mouse/rabbit IgG-HRP linked, 1:10000, GE Healthcare) for 1 h at room temperature. After 4 washes in TBS-T, blots were developed using enhanced chemiluminescence (ECL plus Reagent, GE Healthcare). After detection of phosphoproteins, blots were stripped in the antibody stripping buffer ( G e n e B i o - A p p l i c a t i o n LT D , E u r o m e d e x , Souffelweyersheim, France) for 15 min at room temperature, extensively washed in TBS-T and re-probed using the antibodies against the non-phosphorylated corresponding protein forms. Densitometry measurements were obtained using a chemiluminescence imaging system Fusion Solo (Vilber Lourmat) including the Fusion-Capt® software for image acquisition and analysis.

In vivo experiments Four C57BL/6 and four ob/ob obese male mice were purchased from the provider Charles River Elevage (Saint-Germain sur l’Arbresle, France). Procedures were carried out according to the French guidelines for the care of experimental animals. Mice were adapted to the

environment for 1 week prior to study and maintained in a 12 h light/dark cycle with water and standard diet (65% carbohydrate, 11% fat and 24% protein; SAFE, Augy, France). After three weeks, animals were sacrificed and proximal/distal colon tissues were flash-frozen and stored at −80 °C until protein extraction.

Results Effect of metabolism disruption on mTOR signaling activation in vivo and in vitro First, we questioned whether a global metabolism disruption can modulate mTOR signaling in colons of the obese ob/ob mice model in comparison with normal C57BL/6. Proximal and distal colons were isolated, and levels of O-GlcNAc, OGT, phospho-mTOR and mTOR were analyzed by Western Blot (Fig. 1a). Western Blot analysis of phosphorylation of mTOR was monitored to evaluate the activation status of the mTOR pathway. As we expected, compared to C57BL/ 6 mice, ob/ob mice express higher levels of OGT and O-GlcNAcylation. However, there is no correlation between these expression levels and proximal or distal tissue localization (Fig. 1a). On the other hand, there is a strong positive correlation (p = 0,002) between phospho-mTOR/mTOR and OGT/GAPDH ratios (Fig. 1a and b). Thus, C57BL/6 mice with low levels of OGT exhibit weakly activated mTOR signaling. On the contrary, ob/ob mice with higher levels of OGT show increased activation of the mTOR pathway. We confirmed the specificity of the RL2 antibody against the OGlcNAcylation modification by preincubating it with free GlcNAc (Fig. 1c). In parallel, in vitro experiments were performed in normal CCD841CoN and in highly metabolically active HCT116 colon cancer cells (Steenackers et al. 2016). HCT116 cells exhibits higher levels of OGT and O-GlcNAc when compared to CCD841CoN cells (Fig. 1d and e). Furthermore, aberrant O-GlcNAcylation in cancer cells is also associated with an increased expression of phospho-mTOR (Fig. 1d and e). These data support the hypothesis that a

Fig. 2 Activated mTOR signaling increases OGT and O-GlcNAc levels„ in colon cell lines. a OGT, O-GlcNAcylation, phospho-mTOR, mTOR, phospho-p70S6K and p70S6K levels were measured by Western Blot in synchronized normal CCD841CoN and HCT116 colon cancer cells treated for 48 h with a MHY1485 (10 μM) or c rapamycin (10 or 50 nM). b and d Histograms of OGT/GAPDH, phospho-mTOR/mTOR and phospho-p70s6k/p70s6k ratios are represented. Data shown are the average ± standard derivation (SD) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 Student’s t-test. e OGT, OGlcNAcylation, phospho-p70S6K and p70S6K levels were measured by Western Blot in synchronized CCD841CoN and HCT116 cells pretreated with or without rapamycin (50 nM) and stimulated by fetal calf serum (FCS 10% (v/v), from 4 to 32 h)

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disruption in metabolism associated with obesity or cancer phenotype induces an increase in O-GlcNAcylation correlated with activation of the mTOR pathway in vivo and in vitro.

Effect of mTOR signaling on in vitro O-GlcNAcylation We next studied the effect of pharmacological regulation of mTOR pathway on OGT and O-GlcNAc levels in vitro. CCD841CoN and HCT116 cells were treated with vehicle control or the mTOR pharmacological activator (MHY1485) (Fig. 2a) or inhibitor (Rapamycin) (Fig. 2c and e). Western Blot analyses of phosphorylation of mTOR and of its target p70S6K were monitored to evaluate the activation status of mTOR pathway. Treatment with MHY1485 results in mTOR pathway activation with a slight increased phosphorylation of mTOR and a significant increased phosphorylation of p70S6K in CCD841CoN and HCT116 cells (Fig. 2a and b). Furthermore, this signaling activation was accompanied by increased levels of OGT and O-GlcNAcylation (Fig. 2a and b). On the contrary, cell treatment with increasing concentrations of rapamycin (10 or 50 nM) decreases phosphorylation of mTOR in a dose-dependent manner and totally inhibits phosphorylation of p70S6K in both cell lines (Fig. 2c and d). At the same time, rapamycin induced a dose-dependent decrease of OGT expression and of global O-GlcNAcylation in both cell lines but with a different sensitivity to the inhibitor: 50 nM are required for HCT116 cells to reach a significant diminution of OGT and O-GlcNAc levels, whereas 10 nM is sufficient for normal cells (Fig. 2c and d). A time-course experiment of mTOR signaling induces by stimulation with 10% (v/v) FCS shows that FCS activates mTOR signaling in a time-dependent manner in both cell lines with a maximum of p70S6K phosphorylation reaches at 8 h (Fig. 2e). This signaling activation was accompanied by increased levels of OGT and OGlcNAcylation up to 4 h for CCD841CoN cells and 15 h for HCT116 cells. In parallel, rapamycin treatment inhibits activation of mTOR (as shown by total inhibition of p70S6K phosphorylation) and diminished OGT and OGlcNAcylation global levels compared to non-treated cells (Fig. 2e). Together, these results indicate that activated mTOR signaling increases OGT and O-GlcNAcylation levels in normal and colon cancer cells.

Effect of O-GlcNAcylation on mTOR activation in vitro Lastly, we studied the effect of O-GlcNAcylation on mTOR activation in vitro. In the one hand, we wondered whether the activation of mTOR signaling is dependent on HBP flux. The HBP pathway was explored using azaserine, an inhibitor of its rate-limiting enzyme glutamine: fructose-6-phosphate amidotransferase (GFAT), and glucosamine that upregulates HBP by bypassing GFAT

(Fig. 3a). We note that O-GlcNAc levels are decreased in CCD841CoN and HCT116 cells treated with azaserine and are rescued after co-treatment with glucosamine (Fig. 3b). In CCD841CoN cells, azaserine does not significantly affect phosphorylation of mTOR but decreases the total level of mTOR protein. In the same time, azaserine increases activation of p70S6K (phospho-p70S6K). However, co-treatment with azaserine and glucosamine does not modify the regulation induced by azaserine alone. In contrast, in HCT116 cancer cells, azaserine induces a decrease in phosphorylation of mTOR and p70S6K which are rescued after conjunction treatment with glucosamine (Fig. 3b and d). These results support a role of HBP flux and likely O-GlcNAcylation in activation of mTOR signaling in cancer cells. To confirm this hypothesis, we next up-regulated O-GlcNAcylation levels by transfecting cells with siRNA to down-regulate OGA expression. In normal CCD841CoN cells, increased OGlcNAcylation has no significant effect on expression of total and phosphorylated forms of mTOR while phosphorylated form and total p70S6K protein levels are diminished (Fig. 3c and e). On the contrary, in cancer HCT116 cells, OGA knockdown enhances phosphorylation of mTOR and p70S6K as well as corresponding total protein levels (Fig. 3c and e). These results demonstrate that O-GlcNAcylation participates in mTOR signaling activation in a pathological context of colon cancer cells.

Fig. 3 O-GlcNAcylation activates mTOR signaling in colon cancer cells.„ a Schematic representation of the HBP and O-GlcNAcylation dynamic. GFAT is the rate-limiting step of the HBP and can be pharmacologically inhibited by azaserine or bypassed by glucosamine. The nucleotide sugar UDP-GlcNAc is synthetized through the HBP at the crossroad of glucose, amino acids, fatty acids and nucleotides metabolisms. UDP-GlcNAc is used by OGT as substrate to add GlcNAc to serine and threonine residues of target proteins and OGA removes O-GlcNAc moiety. Glc-6-P: glucose-6-phosphate; Fruc-6-P: fructose-6-phosphate, GlcN-6-P: glucosamine-6-phosphate; GlcNAc-6-P: N-acetylglucosamine-6-phosphate, GlcNAc-1-P: N-acetylglucosamine-1-phosphate. b OGlcNAcylation, phospho-mTOR, mTOR, phospho-p70S6K and p70S6K levels were analyzed by Western blot in synchronized normal CCD841CoN and HCT116 colon cancer cells treated for 24 h with or without azaserine (75 μM) and/or glucosamine (5 mM). d Histograms of O-GlcNAc/GAPDH, phospho-mTOR/mTOR and phospho-p70s6k/ p70s6k ratios are represented. Data shown are the average ± standard derivation (SD) of three independent experiments. NS P > 0.5, *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA test. c OGA, OGlcNAcylation, phospho-mTOR, mTOR, phospho-p70S6K and p70S6K levels were monitored by Western blot in synchronized CCD841CoN and HCT116 cells transfected with siCtrl or siOGA (5 nM) for 78 h. e) Histograms of O-GlcNAc/GAPDH, phosphomTOR/mTOR and phospho-p70s6k/p70s6k ratios are represented. Data shown are the average ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 Student’s t-test

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Discussion Nutrient excess is mainly responsible for obesity and diabetes whose incidences are progressing rapidly in industrialized countries. Notably, after being metabolized by HBP, increasing concentrations of circulating glucose and free fatty acids can participate to insulin resistance associated with type 2 diabetes (Ruan et al. 2013). In diabetic humans and animals, a significant increase in O-GlcNAcylation level is detected in many tissues including liver (Ruan et al. 2012; Baldini et al. 2016), heart (Fricovsky et al. 2012), leukocytes (Springhorn et al. 2012) and erythrocytes (Park et al. 2010). Here, we show that levels of OGT and O-GlcNAcylation are increased in ob/ob mice colon tissues and are associated with a higher activation of the nutrient sensing pathway mTOR. Several studies showed that activation of mTOR signaling can contribute to the development of insulin resistance (Tremblay and Marette 2001; Berg et al. 2002; Shah et al. 2004; Tremblay et al. 2005; Tzatsos and Kandror 2006). Elevated levels of glucose and leucine decrease AMP-activated protein kinase (AMPK) activity and activate mTOR signaling that leads to phosphorylation of IRS-1 by a negative feedback loop and reduction of insulin signaling (Saha et al. 2010). Moreover, Ishimura et al. demonstrated that AMPK is OGlcNAcylated in colon cancer cells and that increase of this PTM reduces AMPK phosphorylation and results in mTOR pathway activation (Ishimura et al. 2017). Thus, by this way and by targeting several other actors of insulin signaling (Federici et al. 2002; Park et al. 2005; Ball et al. 2006), O-GlcNAcylation could directly contribute to insulin resistance. In addition, we show here that OGT, O-GlcNAcylation and mTOR signaling are also increased in HCT116 colon cancer cells compared to CCD841CoN normal cells. A glucose metabolism dysregulation is also described in cancer (Ruan et al. 2013). The Warburg effect, an adaptive metabolic shift from oxidative phosphorylation to aerobic glycolysis, allows cancer cells to favor energy production and macromolecules biosynthesis by increasing glucose and glutamine consumption (Warburg 1956a, b). Two-to-3 % of glucose entering the cell is metabolized by HBP to produce the nucleotide sugar UDP-GlcNAc (Marshall et al. 1991). Elevation of OGT and O-GlcNAcylation levels was described in many cancer types including colorectal (Mi et al. 2011), breast (Caldwell et al. 2010) or hepatic (Zhu et al. 2012) tumors. O-GlcNAcylation can target several tumor suppressors such as p53 (Yang et al. 2006) and the retinoblastoma protein (Rb) (Wells et al. 2011), or oncoproteins like β-catenin (Olivier-Van Stichelen et al. 2012; a; 2014; Zhou et al. 2016), c-Myc (Kamemura et al. 2002; Itkonen et al. 2013) and forkhead box protein M1 (FoxM1) (Caldwell et al. 2010). We show that OGT and

O-GlcNAcylation levels are under mTOR control in both normal and colon cancer cells. The underlying molecular mechanism of such a regulation has to be further deciphered. mTOR is a central regulator of cell growth, differentiation and survival and consequently its dysregulation plays a critical role in cancer proliferation, migration, invasion and metastasis (Zhou and Huang 2011). In this sense, O-GlcNAc regulation by mTOR signaling could be a conserved mechanism between normal and cancer cells. Finally, we show that down-regulation and up-regulation of O-GlcNAcylation by GFAT inhibition or OGA knockdown respectively decreases and increases mTOR signaling activation. Our team (Steenackers et al. 2016) previously demonstrated that OGT expression is necessary for proliferation, survival, adhesion and migration of CCD841CoN and HCT116 cells. In this sense, we speculate that OGlcNAcylation participate in colon oncogenesis, in part, by regulating biological properties of cancer cells via mTOR signaling. Actually, no study has proved that mTOR is directly targeted by O-GlcNAcylation; however some up-stream regulators such as IRS-1 (Park et al. 2005; Ball et al. 2006), PI3K (Federici et al. 2002) and AKT (Park et al. 2005) are OGlcNAcylated. Moreover, p70S6K is also modified by OGlcNAc (Zeidan et al. 2010). O-GlcNAcylation of these proteins could interplay with their phosphorylation status and therefore modify their folding, stability, subcellular localization, partners interaction or biological activity. Together our data indicate that a crosstalk between O-GlcNAcylation and mTOR signaling exists in contexts of metabolism dysregulation associated to obesity or cancer. Recently, Wani et al. described this crosstalk in primary neurons (Wani et al. 2017). These authors demonstrate that enhanced OGlcNAcylation by pharmacological inhibition of OGA with Thiamet-G increases mTOR signaling activation, attenuates autophagic flux and increases α-synuclein accumulation characteristic in neurodegenerative diseases such as Parkinson. Reciprocally, mTOR inhibition by rapamycin decreased basal levels of protein O-GlcNAcylation. Because of evidence of O-GlcNAcylation and mTOR implications in cancer development and their established cross-regulation, one could propose targeting one or these two components to enhance efficiency of drug therapy treatments in CRC. Thus, inhibition of mTOR with pharmacological inhibitor Torin-1 (Francipane and Lagasse 2013) or reduced OGT level with RNA interference (Caldwell et al. 2010) have been shown to suppress tumor growth in vivo. Investigation in this field could lead to the development of better anti-mTOR therapeutic strategies such as more effective pharmacological inhibitors in combination with adapted diet. Indeed, Torin-2, which is synthesized with improved synthetic route and which has better pharmacokinetic properties (Liu et al. 2011), could be used more successfully in cancer than current clinical anti-mTOR treatments.

J Bioenerg Biomembr Acknowledgements This work was supported by the BLigue Contre le Cancer/Comité du Nord^ the BFondation ARC (Association pour la Recherche sur le Cancer),^ the Région Nord-Pas de Calais (Cancer Regional Program), the University of Lille and the BCentre National de la Recherche Scientifique^. The authors are also grateful to the BSIte de Recherche Intégré sur le Cancer^ (SIRIC) ONCOLille and to FR 3688 FRABio. N.V. is the recipient of a fellowship from the BMinistère de l'Enseignement Supérieur et de la Recherche^.

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