Overexpression Of O-GlcNAc Transferase Research Articles

Repeated sleep deprivation decreases the flux into hexosamine biosynthetic pathway/O-GlcNAc cycling and aggravates Alzheimer’s disease neuropathology in adult zebrafish

This study investigated chronic and repeated sleep deprivation (RSD)-induced neuronal changes in hexosamine biosynthetic pathway/O-linked N-acetylglucosamine (HBP/O-GlcNAc) cycling of glucose metabolism and further explored the role of altered O-GlcNAc cycling in promoting neurodegeneration using an adult zebrafish model. RSD-triggered degenerative changes in the brain led to impairment of memory, neuroinflammation and amyloid beta (Aβ) accumulation. Metabolite profiling of RSD zebrafish brain revealed a significant decrease in glucose, indicating a potential association between RSD-induced neurodegeneration and dysregulated glucose metabolism. While RSD had no impact on overall O-GlcNAcylation levels in the hippocampus region, changes were observed in two O-GlcNAcylation-regulating enzymes, specifically, a decrease in O-GlcNAc transferase (OGT) and an increase in O-GlcNAcase (OGA). Glucosamine (GlcN) treatment induced an increase in O-GlcNAcylation and recovery of the OGT level that was decreased in the RSD group. In addition, GlcN reversed cognitive impairment by RSD. GlcN reduced neuroinflammation and attenuated Aβ accumulation induced by RSD. Repeated treatment of zebrafish with diazo-5-oxo-l-norleucine (DON), an inhibitor of HBP metabolism, resulted in cognitive dysfunction, neuroinflammation and Aβ accumulation, similar to the effects of RSD. The pathological changes induced by DON were restored to normal upon treatment with GlcN. Both the SD and DON-treated groups exhibited a common decrease in glutamate and γ-aminobutyric acid compared to the control group. Overexpression of OGT in zebrafish brain rescued RSD-induced neuronal dysfunction and neurodegeneration. RSD induced a decrease in O-GlcNAcylation of amyloid precursor protein and increase in β-secretase activity, which were reversed by GlcN treatment. Based on the collective findings, we propose that dysregulation of HBP and O-GlcNAc cycling in brain plays a crucial role in RSD-mediated progression of neurodegeneration and Alzheimer’s disease pathogenesis. Targeting of this pathway may, therefore, offer an effective regulatory approach for treatment of sleep-associated neurodegenerative disorders.

The deubiquitinase EIF3H promotes hepatocellular carcinoma progression by stabilizing OGT and inhibiting ferroptosis

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal human malignancies, and with quite limited treatment alternatives. The proteasome is responsible for most of the protein degradation in eukaryotic cells and required for the maintenance of intracellular homeostasis. However, its potential role in HCC is largely unknown. In the current study, we identified eukaryotic translation initiation factor 3 subunit H (EIF3H), belonging to the JAB1/MPN/MOV34 (JAMM) superfamily, as a bona fide deubiquitylase of O-GlcNAc transferase (OGT) in HCC. We explored that EIF3H was positively associated with OGT in HCC and was related to the unfavorable prognosis. EIF3H could interact with, deubiquitylate, and stabilize OGT in a deubiquitylase-dependent manner. Specifically, EIF3H was associated with the GT domain of ERα via its JAB/MP domain, thus inhibiting the K48-linked ubiquitin chain on OGT. Besides, we demonstrated that the knockdown of EIF3H significantly reduced OGT protein expression, cell proliferation and invasion, and caused G1/S arrest of HCC. We also found that the deletion of EIF3H prompted ferroptosis in HCC cells. Finally, the effects of EIF3H depletion could be reversed by further OGT overexpression, implying that the OGT status is indispensable for EIF3H function in HCC carcinogenesis. In summary, our study described the oncogenic function of EIF3H and revealed an interesting post-translational mechanism between EIF3H, OGT, and ferroptosis in HCC. Targeting the EIF3H may be a promising approach in HCC.6aJiHquPswT5dhvMxQLovCVideo

Abstract P2059: Perm1 Regulates O-glcnacylation Of Pgc-1alpha In The Heart

O-GlcNAcylation (O-GlcNAc) is a post-translational modification of proteins where N-acetylglucosamines are added to serine and threonine residues in proteins. This modification is regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. A recent study demonstrated that overexpression of OGT in the heart leads to excessive O-GlcNAcylation, resulting in mitochondrial dysfunction and the development of heart failure. However, what regulates OGT expression in the heart remains elusive. Perm1 is a striated-muscle-specific regulator of mitochondrial bioenergetics. Here, we found that Perm1 suppresses O-GlcNAcylation via gene repression of OGT. Adenovirus-mediated overexpression of Perm1 suppressed O-GlcNAcylation of proteins in H9c2 cells (47% of control, p<0.05), concurrent with downregulation of OGT (52% from control, p<0.05). Luciferase gene reporter assay revealed that Perm1 reduces the promoter activity of OGT, indicating that Perm1 acts as a gene repressor of OGT. Furthermore, phenylephrine-induced O-GlcNAcylation in cardiomyocytes was reversed by Perm1 overexpression through repressing OGT expression. Conversely, loss of Perm1 (Perm1-KO) in mice led to upregulation of OGT and the increase of O-GlcNAcylated proteins in the heart (176% and 144% from WT, respectively, both p<0.05, n=6 in WT, n=7 in KO). Concomitantly, Perm1-KO mice exhibited reduced contractility and mitochondrial function, manifested by a significant decrease in ejection fraction and succinate dehydrogenase activity, respectively (87% and 53% of WT, both p<0.05). Lastly, subcellular fractionation and co-immunoprecipitation assay of mouse hearts revealed that loss of Perm1 specifically increases O-GlcNAcylation of nuclear proteins, concurrent with excessive O-GlcNAcylation of PGC-1α, a master regulator of mitochondrial bioenergetics (2-fold increase in O-GlcNAcylated PGC-1α/total PGC-1α, p<0.05, n=3/group). Our results suggest that Perm1 is a novel regulator of O-GlcNAcylation in the heart that transcriptionally represses OGT expression, which presumably prevents excessive O-GlcNAcylation of PGC-1α and mitochondrial dysfunction under pathological stress.

Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF)

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 μM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 μM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.

Abstract 2442: KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death among women overall. One of the main challenges in fighting breast cancer is the intratumor heterogeneity as they are composed of different subpopulations of cancer cells. This heterogeneity of breast cancer is promoted and maintained by a subpopulation of cancer stem-like cells (CSC). CSC share properties of adult stem cells, capable of self-renewing, differentiating & repopulating a tumor. In addition to CSC role in promoting tumor growth, CSC are involved in metastasis, drug resistance and relapse of breast cancer. Therefore, understanding the mechanisms that drive and maintain CSC will help identify novel therapeutic targets in treating breast cancer. Functional activities of CSC are heavily influenced by modulations in the tumor microenvironment, such as alteration in nutrient status. One key component connecting signaling pathway and cancer cell metabolism is the enzyme O-GlcNAc Transferase (OGT), which utilize UDP-GlcNAc to modify nuclear and cytoplasmic proteins in a process termed O-GlcNAcylation. OGT and O-GlcNAc are upregulated in many cancers. Recently, we showed a direct connection between OGT/O-GlcNAc and CSC in breast cancer, in which OGT/O-GlcNAc is required and sufficient to promote the stemness of breast cancer cells in vitro, as well as tumorigenesis in vivo. Global transcriptomic analysis of CSCs-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in CSC. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE), reduced CSC population, and tumor growth in vivo. In contrast, KLF8 overexpression increased MFE and CSC population. Importantly, KLF8 knockdown blocked the effect of elevated OGT and O-GlcNAcylation in promoting MFE, while KLF8 overexpression rescued OGT inhibition in regulating in breast cancer cells. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level, while KLF8 overexpression increased expression of these CSC factors. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting CSC. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem-like cells population. Citation Format: Giang Le Minh, Emily Esquea, Tejsi Dhamelia, Neha Akella, Mauricio J. Reginato. KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2442.

O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells

To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2-ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2-ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2-ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2-ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2-ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2-ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2-ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2-ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2-ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2-ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2-ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2-ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2-ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.

Inhibition of O-GlcNAc transferase sensitizes prostate cancer cells to docetaxel.

The expression of O-GlcNAc transferase (OGT) and its catalytic product, O-GlcNAcylation (O-GlcNAc), are elevated in many types of cancers, including prostate cancer (PC). Inhibition of OGT serves as a potential strategy for PC treatment alone or combinational therapy. PC is the second common cancer type in male worldwide, for which chemotherapy is still the first-line treatment. However, the function of inhibition of OGT on chemotherapeutic response in PC cells is still unknown. In this study, we show that inhibition of OGT by genetic knockdown using shRNA or by chemical inhibition using OGT inhibitors sensitize PC cells to docetaxel, which is the most common chemotherapeutic agent in PC chemotherapy. Furthermore, we identified that microRNA-140 (miR-140) directly binds to OGT mRNA 3' untranslated region and inhibits OGT expression. Moreover, docetaxel treatment stimulates miR-140 expression, whereas represses OGT expression in PC cells. Overexpression of miR-140 enhanced the drug sensitivity of PC cells to docetaxel, which could be reversed by overexpression of OGT. Overall, this study demonstrates miR-140/OGT axis as therapeutic target in PC treatment and provides a promising adjuvant therapeutic strategy for PC therapy.

OGT/CDK5/ACSS2 Axis Regulates Breast Cancer Brain Metastasis Growth

Onset of brain metastases (BMs) in breast cancer patients is considered an end‐stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Thus, there is an urgent need to develop novel treatment strategies. Metastatic breast cancer cells colonizing the brain encounter adverse ‘nutritional environment’, as the levels of key metabolic fuels such as glucose are much lower, in part because they are avidly consumed by neurons. To overcome this challenge, brain‐metastatic cancer cells rely on acetate as a fuel source and convert it into acetyl‐CoA via upregulation of acetyl‐CoA synthetase 2 (ACSS2) enzyme. We have previously shown that brain tumors increase the nutrient sensing post‐translational modification O‐GlcNAcylation and O‐GlcNAc transferase (OGT) levels to regulate acetate metabolism into acetyl‐CoA via increased phosphorylation of ACSS2 on Ser‐267 in a cyclin dependent kinase 5 (CDK5)‐dependent manner, increasing brain tumor growth. Here, we show that human breast cancer cells selected to metastasize to the brain contain increased levels of O‐GlcNAc, OGT and ACSS2‐Ser267 phosphorylation compared to parental cells and show that human breast cancer brain metastatic patient samples contain elevated ACSS2‐Ser‐267 levels. Moreover, overexpression of OGT or ACSS2‐S267D phospho‐mimetic mutant confer a growth advantage on brain metastatic breast cancer cells in an in vivo intracranial xenograft model, may be due to an increase in lipogenic proteins. Additionally, we show that pharmacologically targeting CDK5 and ACSS2 with small molecule inhibitors reduces tumor growth in a novel orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling in transducing nutritional state to regulate acetate metabolism in breast cancer BM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastasis.

KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells

One of the main challenges in treating breast cancer is the intra‐tumor heterogeneity, which, partly, is maintained and promoted by a sub‐population of breast cancer cells called breast cancer stem‐like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self‐renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O‐GlcNAc transferase (OGT). OGT is responsible to adding O‐GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP‐GlcNAc – final product of the hexosamine biosynthetic pathway. This modification is termed O‐GlcNAcylation and both OGT and O‐GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O‐GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA‐seq analysis of BCSC‐enriched mammospheres with OGT overexpression revealed Kruppel‐like‐factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O‐GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O‐GlcNAcylation, suggesting a possible feed‐forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple‐negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem‐like cells population.

VPRBP Functions Downstream of the Androgen Receptor and OGT to Restrict p53 Activation in Prostate Cancer.

In conclusion, we have shown that VPRBP/DCAF1 promotes prostate cancer cell proliferation by restraining p53 activation under the influence of the AR and OGT.

TET3- and OGT-Dependent Expression of Genes Involved in Epithelial-Mesenchymal Transition in Endometrial Cancer.

TET3 is a member of the TET (ten-eleven translocation) proteins family that catalyzes the conversion of the 5-methylcytosine into 5-hydroxymethylcytosine. TET proteins can also affect chromatin modifications and gene expression independently of their enzymatic activity via interactions with other proteins. O-GlcNAc transferase (OGT), the enzyme responsible for modification of proteins via binding of N-acetylglucosamine residues, is one of the proteins whose action may be dependent on TET3. Here, we demonstrated that in endometrial cancer cells both TET3 and OGT affected the expression of genes involved in epithelial to mesenchymal transition (EMT), i.e., FOXC1, TWIST1, and ZEB1. OGT overexpression was caused by an increase in TWIST1 and ZEB1 levels in HEC-1A and Ishikawa cells, which was associated with increased O-GlcNAcylation of histone H2B and trimethylation of H3K4. The TET3 had the opposite effect on gene expressions and histone modifications. OGT and TET3 differently affected FOXC1 expression and the migratory potential of HEC-1A and Ishikawa cells. Analysis of gene expressions in cancer tissue samples from endometrial cancer patients confirmed the association between OGT or TET3 and EMT genes. Our results contribute to the knowledge of the role of the TET3/OGT relationship in the complex mechanism supporting endometrial cancer progression.

O-GlcNAcylation in Ventral Tegmental Area Dopaminergic Neurons Regulates Motor Learning and the Response to Natural Reward.

Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways, and its disturbance has been found in neurodegenerative diseases and metabolic disorders. However, its role in the mesolimbic dopamine (DA) system, especially in the ventral tegmental area (VTA), needs to be elucidated. Here, we found that injection of Thiamet G, an O-GlcNAcase (OGA) inhibitor, in the VTA and nucleus accumbens (NAc) of mice, facilitated neuronal O-GlcNAcylation and decreased the operant response to sucrose as well as the latency to fall in rotarod test. Mice with DAergic neuron-specific knockout of O-GlcNAc transferase (OGT) displayed severe metabolic abnormalities and died within 4-8 weeks after birth. Furthermore, mice specifically overexpressing OGT in DAergic neurons in the VTA had learning defects in the operant response to sucrose, and impaired motor learning in the rotarod test. Instead, overexpression of OGT in GABAergic neurons in the VTA had no effect on these behaviors. These results suggest that protein O-GlcNAcylation of DAergic neurons in the VTA plays an important role in regulating the response to natural reward and motor learning in mice.

Abstract 86: O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Abstract Cancer cells alter their metabolism to increase cell growth. A subset of the glucose taken up is shunted into the hexosamine biosynthetic pathway where it is used to synthesize UDP-GlcNAc, a substrate of O-GlcNAc transferase (OGT), which modifies cytoplasmic and nuclear proteins with O-linked sugar moieties. Here, we show that OGT and O-GlcNAcylation are elevated in glioblastoma (GBM) cancer cells and in GBM patient samples that correlates with disease progression. Reduction of OGT expression in GBM cells led to significant reduction in anchorage-independent growth, acetyl-CoA levels, and decrease in free fatty acids. Conversely, overexpressing OGT in GBM cells had the opposite effect. Reducing OGT expression in GBMcells transplanted in an orthotopic intracranial mouse model reduced tumor growth and extended survival. Mechanistically, we show that OGT overexpression increases carbon-flux of acetate to acetyl-CoA, a reaction carried by the enzyme acetyl-CoA synthetase 2 (ACSS2). Indeed, OGT regulates ACSS2 protein levels and O-GlcNAcylation increases ACSS2phosphorylation on Ser-267 in a cyclin dependent kinase 5 (CDK5)-dependent manner, which regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Using an ex vivo GBM brain slice model we show that treatment of GBM-transplanted slices with OGT inhibitor Ac-GlcNAc-5S or pan-cdk inhibitor dinaciclib reduced growth of pre-formed tumors in cultured brain slices. These results suggest a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate acetate metabolism and identify OGT and CDK5 as novel therapeutic targets for treatment of glioblastoma. Citation Format: Lorela Ciraku, Zachary Bacigalupa, Rebecca Moeller, Jing Ju, Rusia H. Lee, Christina Ferrer, Sophie Trefely, Nathaniel W. Snyder, Luca D'Agostino, Christos D. Katsetos, Wenyin Shi, Mauricio J. Reginato. O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 86.

Abstract 3084: KLF8 is a required downstream effector of OGT/O-GlcNAcylation in regulating breast cancer stem-like cells

Abstract One of the main challenges in treating breast cancer is the intra-tumor heterogeneity, which, partly, is maintained and promoted by a sub-population of breast cancer cells called breast cancer stem-like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self-renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O-GlcNAc transferase (OGT). OGT is responsible to adding O-GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP-GlcNAc - final product of the hexosamine biosynthetic pathway. This modification is termed O-GlcNAcylation and both OGT and O-GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O-GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA-seq analysis of BCSC-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we showed that KLF8 expression in breast cancer cells is regulated by OGT at protein and mRNA level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and CD44H/CD24L BCSC population. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O-GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, breast cancer patient survival was lower with high KLF8 or OGT levels. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer[LM1] . Together, our results suggested that KLF8 is a downstream target of OGT/O-GlcNAcylation in promoting breast cancer stem-like cells population. Citation Format: Giang Le Minh, Neha M. Akella, Tejsi Dhameliya, Mauricio J. Reginato. KLF8 is a required downstream effector of OGT/O-GlcNAcylation in regulating breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3084.

O-GlcNAc Transferase - An Auxiliary Factor or a Full-blown Oncogene?

The β-linked N-acetyl-d-glucosamine (GlcNAc) is a posttranslational modification of serine and threonine residues catalyzed by the enzyme O-GlcNAc transferase (OGT). Increased OGT expression is a feature of most human cancers and inhibition of OGT decreases cancer cell proliferation. Antiproliferative effects are attributed to posttranslational modifications of known regulators of cancer cell proliferation, such as MYC, FOXM1, and EZH2. In general, OGT amplifies cell-specific phenotype, for example, OGT overexpression enhances reprogramming efficiency of mouse embryonic fibroblasts into stem cells. Genome-wide screens suggest that certain cancers are particularly dependent on OGT, and understanding these addictions is important when considering OGT as a target for cancer therapy. The O-GlcNAc modification is involved in most cellular processes, which raises concerns of on-target undesirable effects of OGT-targeting therapy. Yet, emerging evidence suggest that, much like proteasome inhibitors, specific compounds targeting OGT elicit selective antiproliferative effects in cancer cells, and can prime malignant cells to other treatments. It is, therefore, essential to gain mechanistic insights on substrate specificity for OGT, develop reagents to more specifically enrich for O-GlcNAc-modified proteins, identify O-GlcNAc "readers," and develop OGT small-molecule inhibitors. Here, we review the relevance of OGT in cancer progression and the potential targeting of this metabolic enzyme as a putative oncogene.

Proteomics studies to investigate alterations in the tau interactome under conditions of elevated cellular O‐GlcNAcylation

AbstractBackgroundNeurofibrillary tangles consist of tau protein aggregates and represent a hallmark pathology of Alzheimer’s disease. Tau aggregation, specifically the formation of early aggregates, is believed to be neurotoxic. O‐linked glycosylation of proteins with β‐linked N‐acetylglucosamine (O‐GlcNAcylation) is exclusively mediated by two enzymes in cells, O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). Importantly, increasing O‐GlcNAcylation attenuates tau aggregation in cell‐free systems and cells and reduces tau pathology in various animal models, but the underlying molecular mechanism is still unclear. We aimed to determine how protein‐protein interactions of tau are affected by altered O‐GlcNAcylation levels in cells.Method(1) Immunoprecipitation of tau complexes and nano LC‐MS/MS analysis (IP‐MS) were performed to detect changes in tau binding proteins in HEK293T cells, upon elevated O‐GlcNAcylation by OGT overexpression and OGA inhibition. (2) Proximity labeling by TurboID, an engineered biotin ligase, fused to either wild‐type or O‐GlcNAcylation‐resistant mutant tau and nano LC‐MS/MS analysis were performed to assess changes in the proximity of tau to other proteins in iPSC‐derived human cortical neurons (iN), after increasing O‐GlcNAcylation by OGA inhibition.Result(1) We detected proteins that constitutively or differentially interact with tau from IP‐MS experiments in HEK293T cells. Microtubule and cytoskeletal proteins were consistently bound to tau regardless of changes in cellular O‐GlcNAcylation. RNA binding proteins and ribosomal proteins increased but cytosolic/exosome proteins and chaperone proteins decreased their interaction with tau upon elevated cellular O‐GlcNAcylation. (2) Proximity labeling of proteins by TurboID‐tau in iN successfully identified microtubule‐associated proteins and proteins that were previously reported to interact with tau in the brain. Interestingly, six proteins significantly changed their proximity to O‐GlcNAcylation‐resistant mutant tau, whereas increased O‐GlcNAcylation by OGA inhibition did not change the proximity labeling profile of wild‐type or mutant tau proteins.ConclusionThese results demonstrate that increasing cellular O‐GlcNAcylation does not significantly alter the general subcellular localization of tau, but affects its interaction with specific binding partners, some of which were previously implicated to play important a role in neurodegeneration. These data may provide novel mechanistic insights into how O‐GlcNAcylation impacts tau pathology.

Cross Interaction Between OGT and mTOR in T Cells and its Consequences in Autoimmunity

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that has been studied extensively, yet an understanding of the mechanisms of this disease remains unclear. It is known that epigenetic mechanisms are involved in the disease. We found O‐GlcNAc Transferase (OGT), an X‐linked gene, overexpressed due to hypomethylation in T cells from lupus patients (J. Autoimmunity, 2013). Furthermore, it was reported that the mechanistic target of rapamycin (mTOR) modifies T cell cytokine patterns and contributes to the TReg defect in patients with lupus. (J. Immunology, 2014).PurposeTo investigate the role OGT has in T cell signaling in order to understand the contribution of its overexpression to lupus. In particular, we investigated the relationship between OGT and the mTOR pathway in T cells to understand their participation in the disease.MethodsHealthy female donors were recruited by advertising. The research was reviewed and approved by the University of South Alabama Review Board for Human Subject Research. Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation, then T cells were isolated by magnetic beads. One fraction was transiently transfected for 48 h with 2μg of OGT siRNA using a nucleofector device (Amaxa). Other fractions were subjected to different treatments. Cells were stimulated after treatments with PMA/Ionomycin for 4h and the whole cell extracts were separated by SDS‐PAGE electrophoresis. Protein expression was analyzed by immunoblotting using specific antibodies. ß actin was used as loading control.ResultsIncreasing the levels of glycosylated proteins with 100 mM Pugnac, a competitive inhibitor of O‐GlcNAc‐β‐N‐acetylglucosaminidase (OGA), or with 5mM Glucosamine, increased the phosphorylation of p70S6K, substrate of mTORC1, which indicates an increase in mTOR activity. On the other side, when OGT is knocked down by siRNA, as expected, the protein expression of OGT levels as well as glycosylated protein levels decreased. Interestingly, the activity of mTOR also decreased. Similar results were observed with 10 nM MHY1485, potent activator of mTOR, or with 50 nM rapamycin and 10 nM Torin‐1, both inhibitors of mTOR, which increased or decreased the phosphorylation of its substrate respectively. However, they were unable to modify the levels of glycosylation.ConclusionsThese results strongly suggest that mTOR activation is OGT‐dependent and is located downstream of OGT in T cells. Overexpression of OGT could be responsible for the increase in mTOR activity in T cells from lupus patients.Support or Funding InformationNIH 5R03 AR067518‐02Summer undergraduate research fellowship (SURF) University of South AlabamaDepartment of Biomedical Science, College of Allied Health, University of South Alabama

The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.

O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity

Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.

O-linked β-N-acetylglucosamine transferase is involved in pro-opiomelanocortin gene expression in mouse pituitary corticotroph AtT-20 cells

Glucocorticoids and glucocorticoid receptors (GRs) suppress pituitary pro-opiomelanocortin (Pomc) gene expression. O-linked β-N-acetylglucosamine (O-GlcNAc) modification, mediated by O-GlcNAc transferase (OGT), plays an important role during gene transcription. However, whether OGT is involved in the GR-mediated transrepression that occurs in pituitary corticotroph cells is currently unknown. Here, we report that OGT regulates Pomc expression in the mouse corticotroph cell line AtT-20. The overexpression of OGT has an additive effect on the GR-mediated negative transcription pathway. Both the knockdown of OGT by RNA interference and the use of a chemical OGT inhibitor abolished the repressive effects of Pomc expression induced by GRs. OGT inhibition leads to both the decreased recruitment of GRs and the increased recruitment of RNA polymerase II to the Pomc locus. O-GlcNAc modification is involved in the negative regulation of Pomc transcription in corticotroph cells. OGT may be a promising therapeutic target for the treatment of Cushing’s disease.