O-GlcNAc Transferase Expression Research Articles

Targeted Inhibition of O-Linked β-N-Acetylglucosamine Transferase as a Promising Therapeutic Strategy to Restore Chemosensitivity and Attenuate Aggressive Tumor Traits in Chemoresistant Urothelial Carcinoma of the Bladder.

Acquisition of acquired chemoresistance during treatment cycles in urothelial carcinoma of the bladder (UCB) is the major cause of death through enhancing the risk of cancer progression and metastasis. Elevated glucose flux through the abnormal upregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) controls key signaling and metabolic pathways regulating diverse cancer cell phenotypes. This study showed that OGT expression levels in two human UCB cell models with acquired resistance to gemcitabine and paclitaxel were significantly upregulated compared with those in parental cells. Reducing hyper-O-GlcNAcylation by OGT knockdown (KD) markedly facilitated chemosensitivity to the corresponding chemotherapeutics in both cells, and combination treatment with OGT-KD showed more severe growth defects in chemoresistant sublines. We subsequently verified the suppressive effects of OGT-KD monotherapy on cell migration/invasion in vitro and xenograft tumor growth in vivo in chemoresistant UCB cells. Transcriptome analysis of these cells revealed 97 upregulated genes, which were enriched in multiple oncogenic pathways. Our final choice of suspected OGT glycosylation substrate was VCAN, S1PR3, PDGFRB, and PRKCG, the knockdown of which induced cell growth defects. These findings demonstrate the vital role of dysregulated OGT activity and hyper-O-GlcNAcylation in modulating treatment failure and tumor aggression in chemoresistant UCB.

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.

Evaluation of the expression of fatty acid synthase and O-GlcNAc transferase in patients with liver cancer by exploration of transcriptome databases and experimental approaches.

Tumor occurrence and development are closely related to metabolism abnormalities. One of the metabolic networks that is dysregulated during carcinogenesis is the fatty acid synthesis pathway, which is mainly controlled by fatty acid synthase (FASN). We previously demonstrated in proliferating HepG2 liver cancer cells that FASN expression depends on the catalytic activity of O-GlcNAc transferase (OGT) and the activation of the mechanistic/mammalian target of rapamycin (mTOR) pathway. The aim of the present study was to go further in these investigations by analyzing datasets and tissues of patients with liver cancer. To that purpose, transcriptome databases were explored, and reverse transcription-quantitative PCR, western blotting and immunohistochemistry were used. Database analyses revealed that FASN and OGT gene expression was higher in certain cancer tissues, including liver hepatocellular carcinoma, compared with that in non-cancerous tissues. At the protein level, FASN expression was higher in the liver cancer-derived cell lines HepG2 and Hep3B compared with the immortalized human hepatocytes IHH cell line. However, neither the expression of OGT nor of its product O-GlcNAcylation showed any significant difference among the three hepatic cell lines. Subsequently, the expression of FASN and OGT at the protein and mRNA levels was evaluated in human liver cancer and non-tumoral tissues from the same patients with different liver lesions. The results from western blotting demonstrated a significant increase in OGT ands O-GlcNAcylation expression in liver cancer tissues independently of the type of lesion characterizing the non-tumoral counterpart. As previously reported for HepG2 proliferating cells, the protein level of FASN was positively correlated with the activation of mTOR and, although a rather upward trend, a high variability in its expression was monitored between patients. However, the results from immunohistochemistry showed no particular modification for OGT and O-GlcNAcylation expression and a significant increase in FASN expression in cancer tissues compared with that in adjacent non-tumoral tissues. Non-significant changes were observed for FASN and OGT mRNA levels between tumoral and non-tumoral samples, with a high variability between patients. Taken together, these results demonstrated that FASN expression was higher in hepatic cancer tissues in comparison with non-tumoral tissues. Furthermore, OGT expression and activity were shown to vary greatly between cell or cancer type, making any generalization difficult.

O-GlcNAcylation of MORC2 at threonine 556 by OGT couples TGF-\u03b2 signaling to breast cancer progression

MORC family CW-type zinc finger 2 (MORC2) is a newly identified chromatin-remodeling enzyme involved in DNA damage response and gene transcription, and its dysregulation has been linked with Charcot-Marie-Tooth disease, neurodevelopmental disorder, and cancer. Despite its functional importance, how MORC2 is regulated remains enigmatic. Here, we report that MORC2 is O-GlcNAcylated by O-GlcNAc transferase (OGT) at threonine 556. Mutation of this site or pharmacological inhibition of OGT impairs MORC2-mediated breast cancer cell migration and invasion in vitro and lung colonization in vivo. Moreover, transforming growth factor-β1 (TGF-β1) induces MORC2 O-GlcNAcylation through enhancing the stability of glutamine-fructose-6-phosphate aminotransferase (GFAT), the rate-limiting enzyme for producing the sugar donor for OGT. O-GlcNAcylated MORC2 is required for transcriptional activation of TGF-β1 target genes connective tissue growth factor (CTGF) and snail family transcriptional repressor 1 (SNAIL). In support of these observations, knockdown of GFAT, SNAIL or CTGF compromises TGF-β1-induced, MORC2 O-GlcNAcylation-mediated breast cancer cell migration and invasion. Clinically, high expression of OGT, MORC2, SNAIL, and CTGF in breast tumors is associated with poor patient prognosis. Collectively, these findings uncover a previously unrecognized mechanistic role for MORC2 O-GlcNAcylation in breast cancer progression and provide evidence for targeting MORC2-dependent breast cancer through blocking its O-GlcNAcylation.

MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase.

Simple SummaryThe identification of biomarkers that predict the metastatic potential of tumors is a current area of interest in cancer research. A previous study from our laboratory identified numerous microRNA (miRNA) biomarkers that are differentially expressed in pathologic stage I clear cell renal cell carcinoma (ccRCC) tumors that progress to metastatic disease. This study investigated the role of aberrant expression of one of these miRNA, miR-424-5p, and one of its associated protein targets, O-GlcNAc-transferase (OGT). We examined the influence of miR-424-5p and OGT expression on the proliferation, migration, and invasion of ccRCC cells, and confirmed the direct interaction between miR-424-5p and OGT. These findings suggest that the decrease in miR-424-5p expression observed in these small renal masses leads to an increase in OGT, which facilitates metastasis.MicroRNAs (miRNAs) are non-coding post-transcriptional regulators of gene expression that are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in tumor progression. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors, including miR-424-5p, that are associated with an aggressive phenotype. We investigate the impact of this dysregulated miRNA and its protein target O-GlcNAc-transferase (OGT) to better understand the mechanisms behind aggressive stage I ccRCC. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of miR-424-5p and OGT. Cells were transfected with pre-miR-424-5p, a lentiviral anti-OGT shRNA, or were treated with the demethylating agent 5-Aza-2′-deoxycytidine. Cell proliferation was measured via MT cell viability assay. Cell migration and invasion were analyzed using Transwell assays. The expression of miR-424-5p was determined through qRT-PCR, while OGT protein expression was evaluated through Western blotting. The interaction between miR-424-5p and OGT was confirmed via luciferase reporter assay. The transfection of ccRCC cells with pre-miR-424-5p or anti-OGT shRNA significantly inhibited cell proliferation, migration, and OGT expression, while miR-424-5p also attenuated cell invasion. Addition of the demethylating agent significantly reduced cell proliferation, migration, invasion, and OGT expression, while significantly increasing the expression of miR-424-5p. Altogether, these findings suggest that epigenetic downregulation of miR-424-5p, which in turn augments OGT expression, contributes to the creation of aggressive forms of stage I ccRCC.

O-GlcNAcylation inhibits hepatic stellate cell activation.

Protein O-GlcNAcylation is a critical post-translational modification regulating gene expression and fundamental cell functions. O-GlcNAc transferase (OGT) emerged as a key regulator of liver pathophysiology and disease. In this study, we aimed to evaluate the role of OGT in hepatic stellate cells (HSCs) and its consequent role in liver fibrosis. Primary HSCs were isolated from C57/B6 mice. Cell morphology and α-SMA immunofluorescence staining were observed under scanning confocal microscope. Transcriptomic profile was evaluated by RNAseq (Illumina). Promoter activity was examined by luciferase and β--Galactosidase reporter assays. Liver fibrosis mouse models were induced either by intraperitoneal injection of CCl4 at 3 times/week for 4 weeks or by feeding with methionine and choline deficient (MCD) diet for 4 weeks. OGT protein expression and protein O-GlcNAcylation were significantly decreased in CCl4 - or MCD diet-induced liver fibrosis as compared with normal liver in mice. OGT expression and protein O-GlcNAcylation were also decreased in primary HSCs isolated from liver with CCl4 -induced fibrosis compared with those from normal liver. RNA-seq showed that OGT knockdown in HSCs modulated key signaling pathways involved in HSC activation. Promoter sequence analysis of the differentially expressed genes predicted serum response factor (SRF) as a key transcription factor regulated by OGT. Luciferase reporter assay confirmed that OGT repressed activity of SRF to induce α-SMA transcription. Mutations of specific O-GlcNAcylation sites on SRF increased its transcriptional activity, validating negative regulation of SRF by OGT-mediated O-GlcNAcylation. Our results suggest that OGT functions as a negative regulator of HSC activation by promoting SRF O-GlcNAcylation to protect against liver fibrosis.

Abstract MP48: Smooth Muscle Cell-Specific Deletion Of O-GlcNAc Transferase Impedes Atherosclerotic Lesion Formation In Western Diet-fed Apoe -/- Mice

Attachment of O-linked N-acetylglucosamine (O-GlcNAc) to nucleocytoplasmic proteins or ‘O-GlcNAcylation’ is a ubiquitous post-translational modification affecting numerous cellular processes. We and others have previously reported that augmented protein O-GlcNAcylation mediates upregulation of numerous genes associated with atherosclerosis. Recent studies suggest the role of O-GlcNAc transferase (OGT), a key regulator of O-GlcNAc signaling, in diabetic vascular calcification and wound healing. However, the role of OGT in the etiology of atherosclerosis is elusive. The goal of the current study was to interrogate whether OGT plays a direct role in the development of atherosclerosis. For this, we crossed tamoxifen-inducible male Myh11-CreERT2;OGT fl/y ;ApoE -/- mice with female OGT fl/+ ;ApoE -/- to generate SMC-specific OGT knockout on ApoE -/- background. To induce Cre recombinase activity, mice genotypes were injected i.p. with 60mg/Kg/day tamoxifen (peanut oil-vehicle control) once daily for 5 consecutive days beginning at 6 wks age. This was followed by a Western diet feeding regimen for additional 6-7 wks. Mice were harvested at 14 wks age after overnight fasting; plasma, aorta, and heart were collected for biochemical, molecular, and lesion studies. Immunoblotting confirmed loss of OGT expression in aortic vessels of tamoxifen-treated Cre tg ;OGT fl/y ;ApoE -/- mice (smOGT KO ;ApoE -/- ) vs. age-matched tamoxifen-treated Cre tg ;OGT +/y ;ApoE -/- littermates (smOGT WT ;ApoE -/- , with intact OGT). Aortic root morphometry revealed a significant reduction (2.5-fold) in lesion lipid burden in smOGT KO ;ApoE -/- mice compared with smOGT WT ;ApoE -/- . This was accompanied by attenuated PCNA (proliferation marker), osteopontin (SM synthetic marker), pERK (SM signaling regulator), YY1 (transcriptional repressor of SM contractile genes), and SRF (transcriptional regulator of SMC proliferation) expression in smOGT KO ;ApoE -/- vs. smOGT WT ;ApoE -/- aortic lysates, shown via immunoblotting. Interestingly, SMC-specific OGT deletion had no effect on plasma total cholesterol and total triglyceride levels. Taken together, these results demonstrate a protective role of SMC-specific loss of OGT on atherosclerotic lesion formation.

Upregulation of OGT by Caveolin-1 promotes hepatocellular carcinoma cell migration and invasion.

Caveolin-1 (CAV1), a major structural protein of caveolae, is reported to exert a positive regulatory effect on tumor growth and to play a crucial role in hepatocellular carcinoma (HCC) cell metastasis by regulating glycosyltransferase expression and cellular glycosylation. However, the role of CAV1 in modulating protein glycosylation and tumor metastasis remains to be further elucidated. In the present study, we showed that CAV1 promoted the expression of O-GlcNAc transferase (OGT), which catalyzed the addition of O-GlcNAc residues to a variety of nuclear and cytoplasmic proteins. In human HCC cell lines with different metastatic potentials, high levels of OGT and cellular O-GlcNAcylation were associated with CAV1 expression and cell metastasis. Overexpression of CAV1 increased the levels of OGT and O-GlcNAcylation, and cell migration was also increased. Furthermore, CAV1 was found to reduce the expression of Runt-related transcription factor 2 (RUNX2) in HCC cells. Subsequently, this effect resulted in the attenuation of the RUNX2-induced transcription of microRNA24 (miR24), a microRNA previously shown to suppress OGT mRNA expression by targeting its 3' untranslated regions (UTR). Finally, we demonstrated that CAV1 induced cellular O-GlcNAcylation and HCC cell invasion. This study provides evidence of CAV1-mediated increases in OGT expression and O-GlcNAcylation. These data provide insight into a novel mechanism underlying HCC metastasis and suggest a novel strategy for the treatment of HCC.

DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers with rapid progression and a high mortality rate. Our previous study demonstrated that DNA polymerase iota (Pol ι) is overexpressed in ESCC tumors and correlates with poor prognosis. However, its role in ESCC proliferation remains obscure. We report here that Pol ι promotes ESCC proliferation and progression through Erk- O-GlcNAc transferase (OGT) regulated Glucose-6-phosphate dehydrogenase (G6PD) overactivation. Cell clonogenic ability was assessed by colony formation assay. Cell proliferation was assessed by EdU incorporation assay. Our transcriptome data was reanalyzed by GSEA and validated by analysis of cellular metabolism, G6PD activity, and cellular NADPH concentration. The level of Pol ι, OGT, G6PD and O-GlcNAcylation in ESCC cells and patient samples were analyzed. The MEK inhibitor PD98059 was applied to confirm OGT expression regulation by the Erk signaling. The G6PD inhibitor polydatin was used to examine the role of G6PD activation in Pol ι promoted proliferation. We found that Pol ι promotes ESCC proliferation. It shunted the glucose flux towards the pentose phosphate pathway (PPP) by activating G6PD through OGT-promoted O-GlcNAcylation. The expression of OGT was positively correlated with Pol ι expression and O-GlcNAcylation. Notably, elevated O-GlcNAcylation was correlated with poor prognosis in ESCC patients. Pol ι was shown to stimulate Erk signaling to enhance OGT expression, and the G6PD inhibitor polydatin attenuated Pol ι induced tumor growth in vitro and in vivo. In conclusion, Pol ι activates G6PD through Erk-OGT-induced O-GlcNAcylation to promote the proliferation and progression of ESCC, supporting the notion that Pol ι is a potential biomarker and therapeutic target of ESCC.

RANBP2 Activates O-GlcNAcylation through Inducing CEBPα-Dependent OGA Downregulation to Promote Hepatocellular Carcinoma Malignant Phenotypes.

Simple SummaryHepatocellular carcinoma (HCC) is characterized by a poor prognosis and high mortality rate, with complex molecular alterations, including glycosylation. O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic modification process jointly controlled by the “writer” O-GlcNAc transferase (OGT) and “eraser” O-GlcNAcase (OGA), and an increase in total O-GlcNAc correlates with advanced malignant HCC phenotypes. The aim of our study was to explore the potential regulatory patterns underlying the OGT/OGA imbalance that contributes to HCC malignancies. We confirmed that RANBP2, one of the SUMO E3 ligases, downregulates OGA transcription while not affecting OGT. As a transcriptional factor positively regulating OGA, CEBPα was also SUMOylated and destabilized by RANBP2. Our in vitro and in vivo studies provide evidence of RANBP2 downregulating OGA and thus triggering O-GlcNAcylation in a CEBPα-dependent manner. The subsequent hyper-O-GlcNAcylation of protein substrates such as PGC1α via the RANBP2–CEBPα–OGA pathway may represent a pharmaceutical strategy for HCC treatment.O-GlcNAcylation is an important post-translational modification (PTM) jointly controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant hyper-O-GlcNAcylation is reported to yield hepatocellular carcinoma (HCC) malignancy, but the underlying mechanisms of the OGT/OGA imbalance responsible for HCC tumorigenesis remain largely unknown. Here, we report that RAN-binding protein 2 (RANBP2), one of the small ubiquitin-like modifier (SUMO) E3 ligases, contributed to malignant phenotypes in HCC. RANBP2 was found to facilitate CCAAT/enhancer-binding protein alpha (CEBPα) SUMOylation and degradation by direct interplay with CEBPα. As a transcriptional factor, CEBPα was verified to augment OGA transcription, and further experiments demonstrated that RANBP2 enhanced the O-GlcNAc level by downregulating OGA transcription while not affecting OGT expression. Importantly, we provided in vitro and in vivo evidence of HCC malignant phenotypes that RANBP2 triggered through an imbalance of OGT/OGA and subsequent higher O-GlcNAcylation events for oncogenic proteins such as peroxisome proliferative-activated receptor gamma coactivator 1 alpha (PGC1α) in a CEBPα-dependent manner. Altogether, our results show a novel molecular mechanism whereby RANBP2 regulates its function through CEBPα-dependent OGA downregulation to induce a global change in the hyper-O-GlcNAcylation of genes, such as PGC1α, encouraging the further study of promising implications for HCC therapy.

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-Glcnacylation Modulates Erythropoiesis: O-Glcnac Transferase (OGT) Is an Essential Gene

Post-translational modifications (PTMs) can regulate protein activity; one such modification is O-GlcNAcylation. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. The O-GlcNAc PTM is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A balance in protein levels exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may be exploited therapeutically to alter gene expression. We previously demonstrated that O-GlcNAc plays a role in regulating human γ-globin gene transcription during development in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice and derivative immortalized bone marrow cells. OGT and OGA interact with Mi2β, a component of the GATA-1-FOG-Mi2b repressor complex that binds the -566 GATA site of the Aγ-globin promoter when γ-globin gene expression is silent. O-GlcNAcylation of Mi2βstimulates formation of this repressor complex, whereas removal of this PTM results in activation of Aγ-globin gene expression. Thus, O-GlcNAc cycling is a novel γ-globin regulatory mechanism, which might be modulated to increase fetal hemoglobin (HbF).Since O-GlcNAcylation involves input from multiple metabolic pathways, the modification acts as a general sensor of a cellular homeostasis. Thus, in response to environmental cues, the addition and removal of O-GlcNAc from proteins may be variably altered with potential effects on biochemical and transcriptional pathways essential for erythropoiesis. To better understand the mechanism as to how O-GlcNAcylation regulates β-like globin gene switching and whether it plays a role in erythropoiesis, we developed several new, innovative mouse models. These include erythroid-specific OGT conditional knockout mice, and transgenic mice with erythroid-specific enforced expression of human OGT or OGA to examine the effect of OGT loss-of-function or OGT/OGA gain-of-function on erythroid development in vivo . We utilized erythropoietin receptor promoter-Cre (ErGFPcre) mice or produced new tamoxifen-inducible μLCR-β-globin promoter-Cre (μLCR-β promoter-ERT2CreERT2) mice that were bred with floxed OGT mice to produce early erythroid progenitor-specific or tamoxifen-inducible erythroid-specific knockouts of OGT, respectively. In all instances, the final mice also contained the human β-YAC reporter transgene. Knockout of OGT using ErGFPcre results in fetal death during embryogenesis. Although conceptuses may be recovered up through embryonic day E12, they completely lack red blood cells and some organ anomalies are observed. An intermediate, anemic phenotype is observed when the knockout is incomplete. Adult floxed OGT, μLCR-β promoter-ERT2CreERT2 β-YAC mice treated with tamoxifen develop an anemia that is dose-time-dependent and ultimately correlates with the level of OGT still present. We present phenotypic and molecular data related to the hematopoietic system, including anemia, blood cell histology and morphology, standard blood indices and β-like globin gene expression during embryonic, fetal and adult stages of erythropoiesis for both the LOF and GOF mice.Understanding the molecular and cellular components of erythropoiesis is critical for treating anemia and other hematopoietic diseases which affect roughly 3 million Americans and 28% of the population worldwide. In a more general sense, our work may provide insight into the role of O-GlcNAc in regulating developmental and differentiation processes. DisclosuresNo relevant conflicts of interest to declare.

The crosstalk network of XIST/miR-424-5p/OGT mediates RAF1 glycosylation and participates in the progression of liver cancer.

Liver cancer is a major public health concern, but the mechanistic actions of biomarkers contributing to liver cancer remain to be determined. In this study, we aimed to investigate the regulatory cascade of microRNA-424-5p (miR-424-5p), X-inactive-specific transcript (XIST) and O-GlcNAc transferase (OGT) in liver cancer. Differentially expressed miRNAs and target genes related to liver cancer were predicted by bioinformatics analyses, and their expression was determined in liver tissues of patients with liver cancer and liver cancer cells. The RNA immunoprecipitation (RIP), RNA pull-down and dual luciferase reporter assay were used to examine the binding affinity among XIST and miR-424-5p and OGT. Then, gain- and loss-of-function assays were conducted to evaluate the effects of the XIST/miR-424-5p/OGT axis on malignant phenotypes. A nude mouse model of liver cancer was further established for in vivo substantiation. XIST and OGT were up-regulated in liver cancer tissues and cells, responsible for poor prognosis in patients with liver cancer, while miR-424-5p was down-regulated. XIST competitively bound to miR-424-5p to increase OGT expression. XIST silencing inhibited malignant phenotypes of liver cancer cells, while miR-424-5p down-regulation negated its effect. miR-424-5p suppressed RAF1 glycosylation by negatively regulating OGT expression and promoted its ubiquitination/degradation. Furthermore, XIST knockdown inhibited tumour growth and metastasis in nude mice, while ectopic OGT reversed its effect. These results reveal a novel mechanism by which the interaction of XIST/miR-424-5p/OGT participates in the malignancy and metastasis of liver cancer.

O-GlcNAcylation promotes the migratory ability of hepatocellular carcinoma cells via regulating FOXA2 stability and transcriptional activity.

O-GlcNAcylation is a posttranslational modification that regulates numerous nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes, such as transcription, signal transduction, and cell motility. Although increasingevidence has shown that elevated levels of global O-GlcNAcylation are linked to the metastasis in hepatocellular carcinoma (HCC) cells, the underlying mechanism is still ambiguous. In this study, we demonstrated that forkhead box protein A2 (FOXA2), an essential transcription factor for liver homeostasis and HCC developing, was O-GlcNAcylated by O-GlcNAc transferase (OGT) and regulates HCC cells migration and invasion. Opposite FOXA2 and OGT expression tendency were observed in HCC tissues, and lower FOXA2 levels predicted a poor prognosis in HCC patients. The reduction of FOXA2 in HCC cells was found to be inversely correlated with the cellular O-GlcNAcylation and cell migratory ability. Notably, we found that FOXA2 was modified by O-GlcNAcylation and that O-GlcNAcylation activated the ubiquitination degradation of FOXA2 in highly metastatic HCC cells. Although this modification did not affect FOXA2 nuclear localization capability, O-GlcNAcylation on FOXA2 was key for attenuating FOXA2-mediated transcription. O-GlcNAcylation decreased the transcription of FOXA2 downstream target gene E-cadherin and it ultimately promoted O-GlcNAcylation-mediated HCC cell migration and invasion. The results provide insights into the role of O-GlcNAcylation in regulating FOXA2 activity and suggest its important implications in HCC metastasis.

Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.

Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

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.

OGT Is Crucial for Hepatocyte Survival and Liver Homeostasis Through the Control of Oxidative Stress

To address the role of O-GlcNAc transferase (OGT) in hepatocytes, the key enzyme of O-GlcNAcylation, we generated mice with a hepatocyte-specific deletion of OGT (OGT∆). Four weeks after birth, OGT∆ mice presented liver inflammation, oxidative stress, increased expression of ER stress and DNA damage markers, together with a decrease in O-GlcNAcylation of NRF1 and FoxO1, two key modulators of oxidative stress. In addition, OGT∆ hepatocytes were more sensitive to apoptosis when treated with staurosporine, an inducer of oxidative stress. The OGT∆ phenotype worsened with the presence of liver fibrosis septa surrounding regeneration nodules, associated with a significant increase in proliferation and inflammation markers. Interestingly, OGT expression was recovered in liver of 8 week-old OGT∆ mice suggesting counter-selection against OGT deficient cells. OGT recovery correlated with improvement of the hepatic oxidative phenotype, suggesting that OGT plays an essential role in hepatocyte survival and liver homeostasis through the control of oxidative stress.

Pregnancy decreases O-GlcNAc-modified proteins in systemic arteries of normotensive and spontaneously hypertensive rats

AimWe determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. Main methodsO-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration–response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 μmol/L) in the presence of L-NAME (NOS inhibitor, 100 μmol/L). Key findingsThe content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SignificanceIncreased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.

Exosomal O-GlcNAc transferase from esophageal carcinoma stem cell promotes cancer immunosuppression through up-regulation of PD-1 in CD8+ T cells.

Esophageal carcinoma stem cells (ECSCs) are responsible for the initiation and therapy-resistance of esophageal cancer. Nutrient sensor O-GlcNAc transferase (OGT) promoted the growth and metastasis of cancer cells. However, the contributions of OGT to the tumorigenesis of ECSCs remain largely uncover. In the present study, as compared to matched non-stem cancer cells, the expression of OGT was higher in ALDH+ ECSCs. Knock down of OGT by lentivirus system reduced the self-renewal capacities and tumorigenicity of ALDH+ ECSCs. In addition, OGT in exosome derived from ALDH+ ECSCs was taken up by neighboring CD8+ T cells and increased the expression of PD-1 in CD8+ T cells. Down-regulation of OGT increased the apoptosis of ALDH+ ECSCs induced by CD8+ T cells, which could be blocked by overexpression of PD-1 in CD8+ T cells. Together, OGT in exosome from ECSCs protects ECSCs from CD8+ T cells through up-regulation of PD-1.