GFPT1 accelerates immune escape in breast cancer by modifying PD-L1 via O-glycosylation.

BACKGROUND: Immune escape is one of the causes of poor prognosis in breast cancer (BC). Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first speed-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is essential for the progression of BC. Nevertheless, the mechanism of the influence of GFPT1 in BC immune escape is not clear. METHODS: First, the level of GFPT1 in BC was analyzed by starbase, and GFPT1 expression in BC tissues was measured by qRT-PCR, western blot and IHC. Then, the O-GlcNAc levels were detected by western blot. Thereafter, Co-IP was applied to examine the relationship between GFPT1 and PD-L1. At last, a mouse model was constructed for validation in vivo. RESULTS: Firstly, we discovered that GFPT1 was obviously strengthened in BC. Knockdown or introduction of GFPT1 correspondingly degraded and elevated O-GlcNAc levels in cells. Further researches revealed that there was a reciprocal relationship between GFPT1 and PD-L1. Mechanistically, we disclosed that GFPT1 enhanced PD-L1 protein stability through O-glycosylation. More interestingly, GFPT1 accelerated BC cell immune escape via upregulation of O-glycosylation-modified PD-L1. In vivo, silencing of GFPT1 attenuated immune escape of BC cells by reducing PD-L1 levels. CONCLUSION: GFPT1 promoted BC progression and immune escape via O-glycosylation-modified PD-L1. GFPT1 may be a potential target for BC therapy.

Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation.

Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9, simulating that found in a patient with CMS. As Gfpt1 exon 9 is exclusively expressed in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (1) UDP-HexNAc, CMP-NeuAc and protein O-GlcNAcylation were reduced in skeletal muscles; (2) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (3) markers of the unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of endoplasmic reticulum (ER) stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.

Deamidation enables pathogenic SMAD6 variants to activate the BMP signaling pathway.

The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis. SMAD6 encodes a negative regulator of BMP, and rare variants of SMAD6 are recurrently found in individuals with birth defects. However, we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway. We sought to determine whether these SMAD6 variants have common pathogenic mechanisms. Here, we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation. Mechanistically, increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes, both of which lead to BMP signaling pathway activation. Specifically, two residues, N262 and N404, in SMAD6 were identified as the crucial sites of deamidation, which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2 (GFPT2). Additionally, treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway. Conversely, when wild-type SMAD6 was manually simulated to mimic the deamidated state, the reversed function of activating BMP signaling was reproduced. Taken together, these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity, which can be induced by a subset of various SMAD6 variants. Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation, which might prevent the off-target effects of gene editing.

Nutlin-3a induces KRAS mutant/p53 wild type lung cancer specific methuosis-like cell death that is dependent on GFPT2.

BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. CONCLUSION: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.

Cancer-associated fibroblast expression of glutamine fructose-6-phosphate aminotransferase 2 (GFPT2) is a prognostic marker in gastric cancer.

Glutamine fructose-6-phosphate aminotransferase 2 (GFPT2) is a rate-limiting enzyme in hexosamine biosynthesis involved in the occurrence and progress of many cancers. What role it plays in gastric cancer (GC) is still unclear. In this study, transcriptome sequencing data from the Harbin Medical University (HMU)-GC cohort and The Cancer Genome Atlas (TCGA) dataset were combined with the HMU-TCGA training cohort to analyze the biological function and clinical significance of GFPT2. The correlation of GFPT2 with immune cells and stromal cells was analyzed in the GC immune microenvironment through transcriptome sequencing data and a public single-cell sequencing database. In cell lines, GC tissues, and the tissue microarray, GFPT2 protein expression was confirmed by western blotting and immunohistochemistry. The mRNA of GFPT2 was highly expressed in the tumor (p < 0.001), and GC cells and tumors expressed high levels of GFPT2 protein. Compared to low expression, high GFPT2 mRNA expression was associated with higher levels of tumor invasion, higher pathological stages, and poor prognosis (p = 0.02) in GC patients. In a drug susceptibility analysis, GFPT2 mRNA expression was associated with multiple chemotherapeutic drug sensitivity, including docetaxel, paclitaxel, and cisplatin. Gene enrichment analysis found that GFPT2 was mainly primarily involved in the extracellular matrix receptor interaction pathway. The ESTIMATE, CIBERSORT, and ssGSEA algorithms showed that GFPT2 was associated with immune cell infiltration. In addition, GFPT2 was more likely to be expressed within cancer-associated fibroblasts (CAFs), and high levels of GFPT2 expression were highly correlated with four CAFs scores (all p < 0.05). Finally, a prognostic model to assess the risk of death in GC patients was constructed based on GFPT2 protein expression and lymph node metastasis rate. In conclusion, GFPT2 plays an essential role in the function of CAFs in GC. It can be used as a biomarker to assess GC prognosis and immune infiltration.

The Hexosamine Biosynthesis Pathway: Regulation and Function.

The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate-N-acetyl glucosamine, UDP-GlcNAc, which is a key metabolite that is used for N- or O-linked glycosylation, a co- or post-translational modification, respectively, that modulates protein activity and expression. The production of hexosamines can occur via de novo or salvage mechanisms that are catalyzed by metabolic enzymes. Nutrients including glutamine, glucose, acetyl-CoA, and UTP are utilized by the HBP. Together with availability of these nutrients, signaling molecules that respond to environmental signals, such as mTOR, AMPK, and stress-regulated transcription factors, modulate the HBP. This review discusses the regulation of GFAT, the key enzyme of the de novo HBP, as well as other metabolic enzymes that catalyze the reactions to produce UDP-GlcNAc. We also examine the contribution of the salvage mechanisms in the HBP and how dietary supplementation of the salvage metabolites glucosamine and N-acetylglucosamine could reprogram metabolism and have therapeutic potential. We elaborate on how UDP-GlcNAc is utilized for N-glycosylation of membrane and secretory proteins and how the HBP is reprogrammed during nutrient fluctuations to maintain proteostasis. We also consider how O-GlcNAcylation is coupled to nutrient availability and how this modification modulates cell signaling. We summarize how deregulation of protein N-glycosylation and O-GlcNAcylation can lead to diseases including cancer, diabetes, immunodeficiencies, and congenital disorders of glycosylation. We review the current pharmacological strategies to inhibit GFAT and other enzymes involved in the HBP or glycosylation and how engineered prodrugs could have better therapeutic efficacy for the treatment of diseases related to HBP deregulation.

Abnormal decrement on high-frequency repetitive nerve stimulation in congenital myasthenic syndrome with GFPT1 mutations and review of literature.

Objectives: Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders characterized by neuromuscular junction defects. Mutations in GFPT1 have been shown to underlie CMS. An increasing number of patients with CMS due to mutations in GFPT1 have been reported. However, a comprehensive review of clinical and genetic analyses of GFPT-related CMS worldwide is lacking, especially, given that the common or hotspot mutations in GFPT1 have not been reported. Here, we described the clinical and genetic findings of three patients with GFPT1 mutations from southwestern China and reviewed the clinical and genetic features of patients with GFPT1-related CMS worldwide. Methods: Clinical, laboratory, electrophysiological, myopathological, and genetic analyses of three patients with GFPT1-related CMS from southwestern China were conducted, and a review of previously published or reported cases about congenital myasthenic syndrome with GFPT1 mutations in the PubMed database was made. Results: The clinical, laboratory, electrophysiological, and myopathological features by muscle biopsy of three patients with GFPT1-related CMS were consistent with those of previously reported patients with GFPT1 mutations. Additionally, an abnormal decrement in high-frequency RNS was found. Two different homozygous missense mutations (c.331C>T, p.R111C; c.44C>T, p.T15M) were detected by whole-exome sequencing (WES) or targeted neuromuscular disorder gene panels. Conclusion: A distinct decremental response to high-frequency RNS was found in three patients with GFPT1-related CMS from southwestern China, which has never been reported thus far. In addition, the location and degree of tubular aggregates (TAs) seemed to be associated with the severity of clinical symptoms and serum creatine kinase levels, further expanding the phenotypic spectrum of GFPT1-related CMS. Lastly, some potential hotspot mutations in GFPT1 have been found in GFPT1-CMS worldwide.

RELA∙8-Oxoguanine DNA Glycosylase1 Is an Epigenetic Regulatory Complex Coordinating the Hexosamine Biosynthetic Pathway in RSV Infection.

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.

Roles of GFPT2 Expression Levels on the Prognosis and Tumor Microenvironment of Colon Cancer.

Background: Recently, increasing evidence has suggested that Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) is related to carcinogenesis. However, the potential roles of GFPT2 in colon cancer still need to be fully investigated. Methods: We examined the protein levels of GFPT2 by immunohistochemistry (IHC) in tissues collected from 83 patients with colon cancer. We further detected GFBPT2 protein levels by Western Blot assay. We checked the relationship between GFPT2 expression levels and overall survival (OS), stromal and immune scores and immune components from The Cancer Gene Atlas (TCGA) database. GFBP2-related pathways were validated in the Cancer Cell Line Encyclopedia (CCLE) database. Expression of GFPT2 in single cell subpopulations was calculated from The Tumor Immune Single Cell Center (TISCH). The levels of GFPT2 and drug sensitivity data were performed from CellMiner dataset. Results: GFPT2 was highly expressed and correlated with poor pathological features in 83 colon cancer patients. Moreover, increased GFPT2 expression was significantly associated with poorer OS in 329 colon adenocarcinoma (COAD) patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the differentially expressed genes of GFPT2 were mostly enriched in focal adhesion, ECM receptor interaction, JAK/STAT signaling pathway and immune related pathways. In addition, GFPT2 expression was correlated with the tumor microenvironment (TME). GFPT2 expression was linked to cancer-associated fibroblasts (CAFs)-associated factors and epithelial-mesenchymal transition (EMT)-related factors. GFPT2 was positively correlated with immunosuppressive cells and regulated immunosuppressive factors and T-cell exhaustion. Finally, our data suggested that the expression of GFPT2 may be a judgment of the sensitivity of a certain class of drugs. Conclusions: Our work reveals the roles of GFPT2 in tumorigenesis, particularly in immune response, TME and drug resistance, which are crucial for the development of customized cancer therapies.

Deduction and exploration of the evolution and function of vertebrate GFPT family.

BACKGROUND: Glutamine-fructose-6-phosphate aminotransferase (GFPT) is a key factor in the hexosamine metabolism pathway. It regulates the downstream factor O-GlcNAc to change cell function and plays an important role in the metabolism and immune process of tissues and organs. However, the evolutionary relationship of GFPT family proteins in vertebrates has not been elucidated. OBJECTIVE: To deduce and explore the evolution and function of vertebrate GFPT family. METHODS: 18 GFPT sequences were obtained from Homo sapiens (H. sapiens), Trachypithecus francoisi (T. francoisi), Mus musculus (M. musculus), Rattus norvegicus (R. norvegicus), Gallus gallus (G. gallus), Zootoca vivipara (Z. vivipara), Xenopus tropicalis (X. tropicalis), Danio rerio (D. rerio), Rhincodon typus (R. typus), Plasmodium relictum from National Center for Biotechnology Information (NCBI). The physical and chemical characteristics and molecular evolution of GFPT family proteins and nucleic acid sequences were analyzed by ClustalX2, Gene Doc, MEGA-X, SMART, Datamonkey, R etc. RESULTS: Based on the neighbor-joining (NJ) phylogenetic tree and evolution fingerprints, GFPT family members of vertebrates can be divided into two groups: the GFPT1 group and the GFPT2 group. Seven positive selection sites were identified by IFEL and integrated methods mixed effects model of evolution (MEME) and fixed effects likelihood (REL). Finally, we predicted 28 phosphorylation sites and 18 ubiquitousness sites in the human GFPT1 sequence, 10 phosphorylation sites, and five ubiquitousness sites in GFPT2. Gene ontology (GO) analyzes the protein molecules and KEGG signaling pathways of vertebrates interacting with GFPT family proteins. CONCLUSIONS: Our work confirmed that higher animals GFPT family may have differentiated GFPT1 and GFPT2, which meets their own functional needs. This knowledge answers the question what the origin and evolution of GFPT family in vertebrates and provided the basis for disease treatment and function research of GFPT protein.

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial-Mesenchymal Transition and Responds to Oxidative Stress.

Breast cancer cells that have undergone partial epithelial-mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-ß. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.

Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2.

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.

FOXA2 inhibits doxorubicin-induced apoptosis via transcriptionally activating HBP rate-limiting enzyme GFPT1 in HCC cells.

Apoptosis plays an important role in both carcinogenesis and cancer treatment. Understanding the mechanisms through which resistance to apoptosis occurs in cancer cells has huge implications for cancer treatment. Although pieces of evidence have shown that elevated levels of global O-GlcNAcylation play an anti-apoptotic role in myriad cancers, the underlying mechanism is still ambiguous. In this study, we demonstrated that FOXA2, an essential transcription factor for liver homeostasis and hepatocellular carcinoma (HCC) development, inhibits doxorubicin (DOX)-induced apoptosis through elevating cellular O-GlcNAcylation in HCC cells. In response to DOX treatment, elevated FOXA2 and global O-GlcNAcylation level was observed in HCC cells, and higher FOXA2 levels indicated lower levels of DOX-induced apoptosis. Subsequently, we demonstrated that FOXA2 is a direct transcriptional activator of the hexosamine biosynthetic pathway (HBP) rate-limiting enzyme GFPT1. The upregulation of FOXA2 expression induced the synthesis of intracellular UDP-GlcNAc, which is the sugar substrate of O-GlcNAcylation produced by the HBP. The flux through the HBP elevated the global O-GlcNAcylation level and led to the activation of survival signaling pathways in HCC cells. Furthermore, GFPT1 was proved to be an important downstream regulator of FOXA2-mediated apoptotic suppression. These results provide insights into the molecular mechanism by which FOXA2 inhibits DOX-induced HCC cell apoptosis and suggest that targeting FOXA2 might offer a new strategy for HCC treatment.

Promoting roles of KLF5 in myocardial infarction in mice involving microRNA-27a suppression and the following GFPT2/TGF-ß/Smad2/3 axis activation.

Myocardial infarction (MI) is a major atherosclerotic cardiovascular disease which represents a leading cause of death worldwide. Kruppel-like factor 5 (KLF5) is a member of the kruppel-like transcription factor family which has been reported with pro-apoptotic functions in myocardial cells. This work focuses on the function of KLF5 in the pathogenesis of MI and the molecules involved. A mouse model with MI was established. Hypoxia/reoxygenation (H/R)-treated H9C2 cells were applied for in vitro experiments. A KLF5-specific inhibitor ML264 was administrated in cell and animal models. ML264 significantly reduced apoptosis, expression of fibrosis-related markers, reactive oxygen species in the H/R-treated H9C2 cells, and it reduced myocardial injury, infarct size, apoptosis and fibrosis in the myocardial tissues in model mice through specific downregulation of KLF5. A microRNA (miRNA) microarray analysis was performed, which suggested miR-27a as the most upregulated miRNA in the H/R-treated cells after ML264 treatment. miR-27a mimic reduced apoptosis and fibrosis in H/R-treated cells, while miR-27a inhibition blocked the protective roles of ML264. The integrated bioinformatic analyses and luciferase assays confirmed glutamine fructose-6-phosphate transaminase 2 (GFPT2) mRNA as a target of miR-27a. Overexpression of GFPT2 counteracted the protective functions of miR-27a against MI through the activation of the TGF-ß/Smad2/3 signaling pathway. To conclude, this study evidenced that KLF5 possibly induces cell and tissue damage in MI through downregulation of miR-27a and the subsequent activation of GFPT2/TGF-ß/Smad2/3 axis. This study may offer novel thoughts into MI treatment.

Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma.

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system XC- because of the impaired uptake of cystine. However, the outcome following the accumulation of endogenous glutamate in lung adenocarcinoma (LUAD) has not yet been determined. Yes-associated protein (YAP) is sustained by the hexosamine biosynthesis pathway (HBP)-dependent O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation), and glutamine-fructose-6-phosphate transaminase (GFPT1), the rate-limiting enzyme of the HBP, can be phosphorylated and inhibited by adenylyl cyclase (ADCY)-mediated activation of protein kinase A (PKA). However, whether accumulated endogenous glutamate determines ferroptosis sensitivity by influencing the ADCY/PKA/HBP/YAP axis in LUAD cells is not understood. Methods: Cell viability, cell death and the generation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the responses to the induction of ferroptosis following the inhibition of system XC-. Tandem mass tags (TMTs) were employed to explore potential factors critical for the ferroptosis sensitivity of LUAD cells. Immunoblotting (IB) and quantitative RT-PCR (qPCR) were used to analyze protein and mRNA expression. Co-immunoprecipitation (co-IP) assays were performed to identify protein-protein interactions and posttranslational modifications. Metabolite levels were measured using the appropriate kits. Transcriptional regulation was evaluated using a luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). Drug administration and limiting dilution cell transplantation were performed with cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. The associations among clinical outcome, drug efficacy and ADCY10 expression were determined based on data from patients who underwent curative surgery and evaluated with patient-derived primary LUAD cells and tissues. Results: The accumulation of endogenous glutamate following system XC- inhibition has been shown to determine ferroptosis sensitivity by suppressing YAP in LUAD cells. YAP O-GlcNAcylation and expression cannot be sustained in LUAD cells upon impairment of GFPT1. Thus, Hippo pathway-like phosphorylation and ubiquitination of YAP are enhanced. ADCY10 acts as a key downstream target and diversifies the effects of glutamate on the PKA-dependent suppression of GFPT1. We also discovered that the protumorigenic and proferroptotic effects of ADCY10 are mediated separately. Advanced-stage LUADs with high ADCY10 expression are sensitive to ferroptosis. Moreover, LUAD cells with acquired therapy resistance are also prone to higher ADCY10 expression and are more likely to respond to ferroptosis. Finally, a varying degree of secondary labile iron increase is caused by the failure to sustain YAP-stimulated transcriptional compensation for ferritin at later stages further explains why ferroptosis sensitivity varies among LUAD cells. Conclusions: Endogenous glutamate is critical for ferroptosis sensitivity following the inhibition of system XC- in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors.

Novel compound heterozygous variants in the GFPT1 gene leading to rare limb-girdle congenital myasthenic syndrome with rimmed vacuoles.

BACKGROUND:  Congenital myasthenic syndrome (CMS) is a heterogeneous group of rare disorders with impaired neuromuscular transmission caused by genetic defects, which is characterized by fatigable muscle weakness. CASE PRESENTATION:  Herein, we report a case of limb-girdle CMS (LG-CMS) in a 15-year-old Chinese girl with limb weakness and mild ptosis. The patient presented with well-defined clinical manifestations, muscle imaging, and electrophysiological features associated with CMS. On muscle biopsy, in addition to tubular aggregates identified, an extremely unusual pathological change of rimmed vacuoles in muscle fibers was observed. Whole-exome sequencing disclosed two novel heterozygous variants (c.14 T>A and c.581 T>C) in the human glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene, leading to the substitutions of phenylalanine to tyrosine (p.F5Y) and serine (p.F194S), respectively. Both variants were predicted to be likely pathogenic by SIFT, Polyphen-2, and Mutation Taster. Treatments with pyridostigmine bromide and albuterol produced a dramatic improvement. CONCLUSIONS:  Collectively, molecular genetic analysis and muscle biopsy play crucial roles in the diagnosis of GFPT1-related LG-CMS with rimmed vacuoles (a rare phenotype of CMS) and have important implications for treatment decision.

GFPT2 promotes metastasis and forms a positive feedback loop with p65 in colorectal cancer.

As a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation, Glutamine fructose-6-phosphate amidotransferase 2 (GFPT2) is involved in human breast and lung tumorigenesis. However, whether GFTP2 is associated with tumor metastasis remains unclear. Here, we found that GFPT2 promoted the proliferation, migration, invasion and metastasis of colorectal cancer (CRC) cells. Mechanically, p65 acted as an upstream transcription factor of GFPT2 and regulated its expression and function. In turn, GFPT2 enhanced the glycosylation of p65, which led to the nuclear translocation of p65 and then activated NF-κB pathway. Thus, GFTP2 and p65 formed a positive feedback loop to promote the progression of CRC. In addition, GFPT2 was up-regulated in CRC tissues and closely related with liver metastasis (P<0.0001) and tumor stage (P=0.0184). High expression of GFPT2 predicted poor prognosis for CRC patients. Moreover, GFTP2 expression was positively linked with O-linked N-acetylglucosamine transferase in CRC tissues. Our study reveals a new mechanism of GFPT2 in CRC metastasis and provides a new target therapeutic target to deter metastasis.

LncRNA ELFN1-AS1 promotes esophageal cancer progression by up-regulating GFPT1 via sponging miR-183-3p.

Accumulating studies highlight the critical role of long non-coding RNAs (lncRNAs) in the development of various human cancers. Extracellular leucine rich repeat and fibronectin type III domain containing 1-antisense RNA 1 (ELFN1-AS1) was shown to be a newly found lncRNA that abnormally expressed in human tumors. However, till now the specific function of this lncRNA in esophageal cancer (ESCA) remains unknown. In this study, we discovered that higher ELFN1-AS1 expression indicated shorter patient survival in pan-cancer, including ESCA, using online The Cancer Genome Atlas (TCGA) tools. The lncRNA ELFN1-AS1 was significantly up-regulated in ESCA tissues and cell lines when compared with the counterparts. Down-regulation of ELFN1-AS1 restrained cell proliferation, migration, and invasion of ESCA in vitro. In addition, we found that the expression of microRNA-183-3p (miR-183-3p) and ELFN1-AS1 or glutamine-fructose-6-phosphate transaminase 1 (GFPT1) were inversely correlated in ESCA. Both ELFN1-AS1 and GFPT1 are direct targets of miR-183-3p in ESCA. The effects of ELFN1-AS1 knockdown on ESCA progression were partially rescued by inhibition of miR-183-3p or over-expression of GFPT1. In summary, the results of this study suggest that the lncRNA ELFN1-AS1 facilitates the progression of ESCA by acting as a competing endogenous RNA (ceRNA) to promote GFPT1 expression via sponging miR-183-3p.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) promotes the EMT of serous ovarian cancer by activating the hexosamine biosynthetic pathway to increase the nuclear location of ß-catenin.

The hexosamine biosynthetic pathway (HBP), a branch of glucose metabolism, provides a substrate for glycosylation modification, which has a wide-ranging effect on cellular functions. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) has been reported to regulate the HBP as the first and rate-limiting enzyme. Given the inverse association between GFPT2 expression and survival of patients with serous ovarian cancer (SOC) observed in The Cancer Genome Atlas (TCGA) database, we attempted to investigate the role of GFPT2 and its related mechanisms in SOC. The results showed that GFPT2 was over-expressed in SOC tissues, and positive correlations with advanced stage (FIGO III/IV), suboptimal removal rate and poor survival were observed in 90 SOC patients. Cell migration and invasion were also inhibited in GFPT2 knockdown SKOV3 and HEY cells. The levels of O-linked ß-N-acetylglucosamine (O-GlcNAc) and intranuclear ß-catenin were evaluated and the observed increase in O-GlcNAcylation induced by GFPT2 may contribute to epithelial-mesenchymal transition (EMT). These data provide novel insights into the function of GFPT2 and O-GlcNAcylation in the EMT and thus the invasiveness SOC.