O-GlcNAc Transferase Expression Research Articles

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

SMAD4 promotes EMT in COPD airway remodeling induced by cigarette smoke through interaction with O-GlcNAc transferase

Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

The diverging role of O-GlcNAc transferase in corticotroph and somatotroph adenomas.

Molecular mechanisms involved in the pathogenesis and tumor progression of pituitary adenomas (PA) remain incompletely understood. Corticotroph and somatotroph PA are associated with a high clinical burden, and despite improved surgical outcomes and medical treatment options, they sometimes require multiple surgeries and radiation. Preliminary data suggested a role for O-GlcNAc Transferase (OGT), the enzyme responsible for the O-GlcNAcylation of proteins. O-GlcNAcylation and OGT have been found elevated in other types of tumors. We evaluated 60 functioning and nonfunctioning PA (NFPA) from operated patients and postmortem normal and tumoral pituitary tissue by immunohistochemistry. We performed transcriptomic analyses to explore the relevance of the O-GlcNAc Transferase (OGT) in PAs. We detected OGT in immunobiological analysis and define its level in PA tissue in patients. OGT was strongly associated with PA hormone secretory capacity in functioning PA and with tumor growth in NFPAs. In NFPAs, OGT was positively associated with tumor size but not with cavernous sinus invasion (Knosp grading). In GH-secreting PA, OGT expression was negatively correlated with circulating Insulin-like Growth Factor 1 level. In adrenocorticotropic hormone (ACTH)-secreting PA, OGT expression was positively associated with circulating ACTH levels. OGT did not correlate with tumor size in secreting PAs. OGT levels were higher in gonadotroph PA compared to normal glands. O-GlcNAcylation can be downregulated in non-cancerous tumors such as GH-secreting adenomas. Future studies are warranted to elucidate the role of OGT in the pathogenesis of PAs.

Dihydroartemisinin promotes tau O-GlcNAcylation and improves cognitive function in hTau transgenic mice

Tauopathy is a collective term for several neurodegenerative diseases characterized by the intracellular accumulation of hyperphosphorylated microtubule-associated protein Tau (P-tau). Our recent report has revealed the neuroprotective effect of dihydroartemisinin (DHA) on mice overexpressing human Tau (hTau) in the hippocampus by enhancing O-linked-N-Acetylglucosaminylation (O-GlcNAcylation) modification. However, whether DHA can improve synaptic and cognitive function in hTau transgenic mice by specifically promoting Tau O-GlcNAcylation is still unclear. Here, we introduced hTau transgenic mice, a more optimal tauopathy model, to study the effect of DHA on Tau O-GlcNAcylation. We reported that DHA treatment alleviated the deficits of hippocampal CA1 LTP and spatial learning and memory in the Barnes maze and context fear conditioning tests in hTau transgenic mice. Mechanically, we revealed that DHA exerted a significant protective effect by upregulating Tau O-GlcNAcylation and attenuating Tau hyperphosphorylation. Through molecular docking, we found a stable binding between DHA and O-GlcNAc transferase (OGT). We further reported that DHA treatment had no effect on the expression of OGT, but it promoted OGT nuclear export, thereby enhancing OGT-mediated Tau O-GlcNAcylation. Taken together, these results indicate that DHA exerts neuroprotective effect by promoting cytoplasmic translocation of OGT and rebuilding the balance of Tau O-GlcNAcylation/phosphorylation, enhancing O-GlcNAcylation of Tau, suggesting that DHA may be a potential therapeutic agent against tauopathy.

Do T2DM and Hyperglycaemia Affect the Expression Levels of the Regulating Enzymes of Cellular O-GlcNAcylation in Human Saphenous Vein Smooth Muscle Cells?

Protein O-GlcNAcylation, a dynamic and reversible glucose-dependent post-translational modification of serine and threonine residues on target proteins, has been proposed to promote vascular smooth muscle cell proliferation and migration events implicated in vein graft failure (VGF). Therefore, targeting the enzymes (glutamine fructose-6P amidotransferase (GFAT), O-GlcNAc transferase (OGT), and O-GlcNAcase (OGA)) that regulate cellular O-GlcNAcylation could offer therapeutic options to reduce neointimal hyperplasia and venous stenosis responsible for VGF. However, it is unclear how type 2 diabetes mellitus (T2DM) and hyperglycaemia affect the expression of these enzymes in human saphenous vein smooth muscle cells (HSVSMCs), a key cell type involved in the vascular dysfunction responsible for saphenous VGF. Therefore, our aim was to assess whether T2DM and hyperglycaemia affect GFAT, OGT, and OGA expression levels in HSVSMCs in vitro. Expression levels of GFAT, OGT, and OGA were determined in low-passage HSVSMCs from T2DM and non-T2DM patients, and in HSVSMCs treated for 48 h with hyperglycaemic (10 mM and 25 mM) glucose concentrations, by quantitative immunoblotting. Expression levels of OGT, OGA, and GFAT were not significantly different in HSVSMC lysates from T2DM patients versus non-T2DM controls. In addition, treatment with high glucose concentrations (10 mM and 25 mM) had no significant effect on the protein levels of these enzymes in HSVSMC lysates. From our findings, T2DM and hyperglycaemia do not significantly impact the expression levels of the O-GlcNAcylation-regulating enzymes OGT, OGA, and GFAT in HSVSMCs. This study provides a foundation for future studies to assess the role of O-GlcNAcylation on VGF in T2DM.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

O-GlcNAcylation of NLRP3 Contributes to Lipopolysaccharide-Induced Pyroptosis of Human Gingival Fibroblasts.

Periodontitis is a leading chronic oral disorder and poses a serious burden on public health. O-GlcNAc glycosylation (O-GlcNAcylation) is regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the regulation of human gingival fibroblasts (HGFs) function. Hence, the purpose of this study is to investigate whether HGFs cell function and periodontitis pathogenesis are regulated by O-GlcNAcylation. Herein, we first established cell model of periodontitis induced by lipopolysaccharide (LPS). The cell viability was measured with CCK-8 assay. Pyroptosis was measured by flow cytometry and western blot. The inflammatory factors levels were detected with ELISA kits. Afterward, our findings indicated that LPS elevated the O-GlcNAcylation level of HGFs and inhibition of O-GlcNAcylation improved LPS-induced pyroptosis of HGFs. Mechanistically, LPS heightened the expression of OGT to induce the O-GlcNAcylation of NLRP3. Subsequently, we certified that Thr542 was the O-GlcNAcylation site of NLRP3. More importantly, upregulation of NLRP3 reversed the effects of OGT knockdown on LPS-induced pyroptosis. In general, the current research demonstrated that LPS contributed to the pyroptosis of HGFs by enhancing the OGT expression to promote O-GlcNAcylation of NLRP3, which suggested that O-GlcNAcylation of NLRP3 was a driving factor for periodontitis and offered a novel insight into the treatment of this disease.

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.

Quality control mechanism of mitochondria by 3,4-dihydroxybenzaldehyde through OGT-PINK1 pathway

Based on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1) pathway, the mechanism of 3,4-dihydroxybenzaldehyde(DBD) on mitochondrial quality control was investigated. Middle cerebral artery occlusion/reperfusion(MCAO/R) rats were established. SD rats were randomized into sham operation group(sham), model group(MCAO/R), DBD-L group(5 mg·kg~(-1)), and DBD-H group(10 mg·kg~(-1)). After 7 days of administration(ig), MCAO/R was induced in rats except the sham group with the suture method. Twenty-four h after reperfusion, the neurological function and the percentage of cerebral infarct area were measured. Based on hematoxylin and eosin(HE) staining and Nissl staining, the pathological damage of cerebral neurons was examined. Then the ultrastructure of mitochondria was observed under the electron microscope, and the co-localization of light chain-3(LC3), sequestosome-1(SQSTM1/P62), and Beclin1 was further detected by immunofluorescence staining. It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway. Therefore, Western blot was employed to detect the expression of OGT, mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin), and mitochondrial kinetic proteins dynamin-like protein 1(Drp1) and optic atrophy 1(Opa1). The results showed that MCAO/R group had neurological dysfunction, large cerebral infarct area(P<0.01), damaged morphological structure of neurons, decreased number of Nissl bodies, mitochondrial swelling, disappearance of mitochondrial cristae, decrease of cells with LC3 and Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 compared with the sham group(P<0.01). However, DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats, as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies. Moreover, DBD increased cells with LC3 and Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.

Hyperglycemia and O-GlcNAc transferase activity drive a cancer stem cell pathway in triple-negative breast cancer

BackgroundEnhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors.MethodsWe used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems.ResultsWe showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment.ConclusionsTaken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.

O-GlcNAcylation of RAB10 promotes hepatocellular carcinoma progression.

Ras-related protein Rab-10 (RAB10) is involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Here, we found RAB10, O-GlcNAc transferase (OGT), and O-GlcNAcylation were upregulated in HCC. In addition, RAB10 protein level was prominently positively correlated with the expression of OGT. O-GlcNAcylation modification of RAB10 was then investigated. Here we showed that RAB10 interacts directly with OGT in HCC cell lines, Meanwhile, O-GlcNAcylation enhanced RAB10 protein stability. Furthermore, knockdown of OGT suppressed aggressive behaviors of HCC in vitro and in vivo, while elevated RAB10 reversed these. Taken together, these results indicated that OGT mediated O-GlcNAcylation stabilized RAB10, thus accelerating HCC progression.

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-GlcNAc transferase in astrocytes modulates depression-related stress susceptibility through glutamatergic synaptic transmission.

Major depressive disorder is a common and devastating psychiatric disease, the prevalence and burden are substantially increasing worldwide. Multiple studies of depression patients have implicated glucose metabolic dysfunction in the pathophysiology of depression. However, the molecular mechanisms by which glucose and related metabolic pathways modulate depressive-like behaviors are largely uncharacterized. UDP-GlcNAc is a glucose metabolite with pivotal functions as a donor molecule for O-GlcNAcylation. O-GlcNAc transferase (OGT), a key enzyme in protein O-GlcNAcylation, catalyzes protein posttranslational modification by O-GlcNAc and acts as a stress sensor. Here, we show that Ogt mRNA was increased in depression patients and that astroglial OGT expression was specifically upregulated in the medial prefrontal cortex of susceptible mice after chronic social defeat stress. The selective deletion of astrocytic OGT resulted in antidepressant-like behaviors, moreover, astrocytic OGT in the mPFC bidirectionally regulated vulnerability to social stress. Furthermore, OGT modulated glutamatergic synaptic transmission through O-GlcNAcylation of glutamate transporter-1 (GLT-1) in astrocytes. OGT astrocyte-specific knockout preserved the neuronal morphology atrophy and Ca2+ activity deficits caused by chronic stress and resulted in antidepressant effects. Altogether, our study reveals that astrocytic OGT in the mPFC regulates depressive-like behaviors through the O-GlcNAcylation of GLT-1 and could be a potential target for antidepressants.

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 protein phosphatase 2A by okadaic acid induces translocation of nucleocytoplasmic O-GlcNAc transferase

Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.

Protein O-GlcNAcylation as a nutrient sensor signaling placental dysfunction in hypertensive pregnancy.

During pregnancy, arterial hypertension may impair placental function, which is critical for a healthy baby's growth. Important proteins during placentation are known to be targets for O-linked β-N-acetylglucosamine modification (O-GlcNAcylation), and abnormal protein O-GlcNAcylation has been linked to pathological conditions such as hypertension. However, it is unclear how protein O-GlcNAcylation affects placental function and fetal growth throughout pregnancy during hypertension. To investigate this question, female Wistar and spontaneously hypertensive rats (SHR) were mated with male Wistar rats, and after pregnancy confirmation by vaginal smear, rats were divided into groups of 14, 17, and 20 days of pregnancy (DOPs). On the 14th, 17th, and 20th DOP, rats were euthanized, fetal parameters were measured, and placentas were collected for western blot, immunohistochemical, and morphological analyses. SHR presented a higher blood pressure than the Wistar rats (p=0.001). Across all DOPs, SHR showed reduced fetal weight and an increase in small-for-gestational-age fetuses. While near-term placentas were heavier in SHR (p=0.006), placental efficiency decreased at 17 (p=0.01) and 20 DOPs (p<0.0001) in this group. Morphological analysis revealed reduced junctional zone area and labyrinth vasculature changes on SHR placentas in all DOPs. O-GlcNAc protein expression was lower in placentas from SHR compared with Wistar at 14, 17, and 20 DOPs. Decreased expression of O-GlcNAc transferase (p=0.01) and O-GlcNAcase (p=0.002) enzymes was found at 14 DOPs in SHR. Immunohistochemistry showed reduced placental O-GlcNAc content in both the junctional zone and labyrinth of the placentas from SHR. Periodic acid-Schiff analysis showed decreased glycogen cell content in the placentas from SHR at 14, 17, and 20 DOPs. Moreover, glucose transporter 1 expression was decreased in placentas from SHR in all DOPs. These findings suggest that decreased protein O-GlcNAcylation caused by insufficient placental nutritional apport contributes to placental dysfunction during hypertensive pregnancy, impairing fetal growth.

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.

HIF-1α Expression Increases Preoperative Concurrent Chemoradiotherapy Resistance in Hyperglycemic Rectal Cancer.

Simple SummaryPreoperative Concurrent Chemoradiotherapy (CCRT) is one of the standard treatments for patients with locally advanced rectal cancer. However, the efficacy of CCRT in hyperglycemic rectal cancer patients does not seem to be as expected. Therefore, we used clinical specimens, ectopic hyperglycemia animal models, and rectal cancer cells in a high-glucose environment to confirm how the high-glucose environment causes radiation resistance. The results confirmed that the high-glycemic environment could induce the overexpression of HIF-1α to cause CCRT tolerance in rectal cancer and suggest that combining HIF-1α inhibitors could reverse radioresistance in a high glucose environment. In future, HIF-1α inhibitors might be used as sensitizers to improve the efficacy of hyperglycemic rectal cancer patients receiving CCRT.Purpose: Preoperative concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced rectal cancer patients. However, the poor therapeutic efficacy of CCRT was found in rectal cancer patients with hyperglycemia. This study investigated how hyperglycemia affects radiochemotherapy resistance in rectal cancer. Methods and Materials: We analyzed the correlation between prognosis indexes with hypoxia-inducible factor-1 alpha (HIF-1α) in rectal cancer patients with preoperative CCRT. In vitro, we investigated the effect of different concentrated glucose of environments on the radiation tolerance of rectal cancers. Further, we analyzed the combined HIF-1α inhibitor with radiation therapy in hyperglycemic rectal cancers. Results: The prognosis indexes of euglycemic or hyperglycemic rectal cancer patients after receiving CCRT treatment were investigated. The hyperglycemic rectal cancer patients (n = 13, glycosylated hemoglobin, HbA1c > 6.5%) had poorer prognosis indexes. In addition, a positive correlation was observed between HIF-1α expression and HbA1c levels (p = 0.046). Therefore, it is very important to clarify the relationship between HIF-1α and poor response in patients with hyperglycemia receiving pre-operative CCRT. Under a high glucose environment, rectal cancer cells express higher levels of glucose transport 1 (GLUT1), O-GlcNAc transferase (OGT), and HIF-1α, suggesting that the high glucose environment might stimulate HIF-1α expression through the GLUT1-OGT-HIF-1α pathway promoting tolerance to Fluorouracil (5-FU) and radiation. In the hyperglycemic rectal cancer animal model, rectal cancer cells confirmed that radiation exposure reduces apoptosis by overexpressing HIF-1α. Combining HIF-1α inhibitors was able to reverse radioresistance in a high glucose environment. Lower HIF-1α levels increased DNA damage in tumors leading to apoptosis. Conclusions: The findings here show that hyperglycemia induces the expression of GLUT1, OGT, and HIF-1α to cause CCRT tolerance in rectal cancer and suggest that combining HIF-1α inhibitors could reverse radioresistance in a high glucose environment. HIF-1α inhibitors may be useful for development as CCRT sensitizers in patients with hyperglycemic rectal cancer.