1. Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress
- Author
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Qiong Wang, Sigurdur Trausti Karvelsson, Aristotelis Kotronoulas, Thorarinn Gudjonsson, Skarphedinn Halldorsson, and Ottar Rolfsson
- Subjects
Glutamine ,claudin-low breast cancer ,GlcNAc-P, N-acetylglucosamine phosphate ,LAMA3, Laminin subunit alpha 3 ,Biochemistry ,Analytical Chemistry ,RT-qPCR, Quantitative reverse transcription PCR ,Glycogen Synthase Kinase 3 ,PCSK1N, Proprotein convertase subtilisin/kexin type 1 Inhibitor ,GLUT4, Glucose transporter type 4 ,SERPINB5, Serpin family B member 5 ,Cell Movement ,SD, Standard deviation ,BP, Biological process ,GO, Gene ontology ,TWIST, Twist family BHLH transcription factor 1 ,AKAP12, A-kinase anchor protein 12 ,EMT ,BRENCs, Breast endothelial cells ,CADM3, Cell adhesion molecule 3 ,EDTA, Ethylenediaminetetraacetic acid ,PBS, Phosphate-buffered saline ,PYGB, Glycogen phosphorylase, brain form ,SLP-2, Stomatin-like protein 2 ,UGDH, UDP-glucose 6-dehydrogenase ,EGF, Epidermal growth factor ,ERBB2 (HER2), Erb-B2 receptor tyrosine kinase 2 ,FGFBP1, Fibroblast growth factor-binding protein 1 ,TFA, Trifluoroacetic acid ,RCN3, Reticulocalbin 3 ,KRT1, Keratin 1 ,UDP-Glc, UDP-glucose ,ERK/MAPK, Mitogen-activated protein kinase ,MMS, Mesenchymal metabolic signature ,S100A14, S100 calcium binding protein A14 ,TNFα, Tumor necrosis factor alpha ,TCA, Tricarboxylic acid cycle ,AKR1B1, Aldo-keto reductase family 1, member B1 (aldose reductase), isoform CRA_a ,KEGG, Kyoto Encyclopedia of Genes and Genomes ,IL18, Interleukin-18 ,proteomics ,GALE, UDP-glucose 4-epimerase ,GFPT2, Glutamine-fructose-6-phosphate transaminase 2 ,UAP1, UDP-N-acetylhexosamine pyrophosphorylase ,RT, Room temperature ,PVDF, Polyvinylidene fluoride ,Humans ,DSP, Desmoplakin ,ALDH1A3, Aldehyde dehydrogenase 1 family, member A3, isoform CRA_a ,Molecular Biology ,Transaminases ,FLNC, Filamin-C ,Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) ,HPDL, 4-hydroxyphenylpyruvate dioxygenase-like protein ,sXBP1, Spliced X-box binding protein 1 ,RELA, RELA Proto-Oncogene, NF-κB subunit, transcription factor p65 ,TAGLN, Transgelin ,POMC, Pro-opiomelanocortin ,PRSS23, Serine protease 23 ,DCD, Dermicidin ,FDR, False discovery rate ,GALNT7, N-acetylgalactosaminyltransferase 7 ,CID, Collision-induced dissociation ,iBAQ, Intensity-based absolute quantification ,SIRT6, Sirtuin 6 ,GSSG, Oxidized glutathione ,GES, Gene expression studies ,KRAS, KRAS proto-oncogene, GTPase ,NSCLC, Non-small-cell lung cancer ,CCLE, Cancer Cell Line Encyclopedia ,LFQ, Label-free quantification ,H2O2, Hydrogen peroxide ,MYL9, Myosin light chain 9 ,S100A2, S100 calcium binding protein A2 ,FASP, Filter-aided sample preparation ,SILAC, Stable isotope labeling by amino acids in cell culture ,EMT, Epithelial-mesenchymal transition ,CDH2, Cadherin-2 ,PKP2, Plakophilin-2 ,oxidative stress ,PGM2L1, Glucose 1,6-bisphosphate synthase ,GFPT1, Glutamine-fructose-6-phosphate aminotransferase 1 ,HER2 (ERBB2), Human epidermal growth factor receptor 2 ,PFA, Paraformaldehyde ,ITGB4, Integrin subunit alpha 4 ,UDP, Uridine diphosphate ,Fructosephosphates ,RTK, Receptor tyrosine kinase ,CDH1, Cadherin-1 ,NDRG1, N-Myc downstream regulated 1 ,MGST1, Microsomal glutathione S-transferase 1 ,IGF, Insulin like growth factor ,TGF-β, Transforming growth factor beta ,ITGA6, Integrin subunit alpha 6 ,GSK3-β, Glycogen synthase kinase 3 beta ,HBP, Hexosamine biosynthesis pathway ,NF-κB, Nuclear factor kappa B ,Female ,DDA, Data-dependent acquisition ,Epithelial-Mesenchymal Transition ,GLUT1, Glucose transporter 1 ,ODC, Ornithine decarboxylase ,UDP-GlcNAc, UDP-N-acetylglucosamine ,Breast Neoplasms ,UDP-GlcA, UDP-glucuronate ,LKB1, Serine/threonine kinase 11 (STK11) ,DTT, Dithiothreitol ,SQOR, Sulfide:quinone oxidoreductase ,GSH, Reduced glutathione ,UTP, Uridine-5′-triphosphate ,NT5E, 5′-Nucleotidase Ecto ,Cell Line, Tumor ,K5/6/8/14/19, Keratin 5/6/8/14/19 ,SLC2A4, Solute carrier family 2 member 4 ,PKCα, Protein kinase C alpha ,IPA, Ingenuity Pathway Analysis ,PGM3, Phosphoacetylglucosamine mutase ,IGF1R, Insulin like growth factor 1 receptor ,PPP, Pentose phosphate pathway ,LAMB3, Laminin subunit beta 3 ,VIM, Vimentin ,OGT, O-Linked N-Acetylglucosamine (GlcNAc) Transferase ,Research ,SDS, Sodium dodecyl sulfate ,STRING, Search Tool for the Retrieval of Interacting Genes/Proteins ,EPCAM, Epithelial cell adhesion molecule ,SERPINE1, Serpin family E member 1 ,GFPT2 ,H2S, Hydrogen sulfide ,LAD1, Ladinin-1 ,CTGF, Connective tissue growth factor ,FBS, Fetal bovine serum ,HUVECs, Human umbilical vein endothelial cells ,TCGA, The Cancer Genome Atlas ,ANXA3, Annexin A3 ,IAA, Iodoacetamide ,AKR1C1, Aldo-keto reductase family 1, member C1 ,HMS LINCS, Harvard Medical School Library of Integrated Network-based Cellular Signatures - Abstract
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-β. 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., Graphical Abstract, Highlights • GFPT2 is upregulated following EMT. • GFPT2 is a marker for claudin-low breast cancer. • GFPT2 affects vimentin, cell proliferation, and cell invasion. • GFPT2 responds to oxidative stress. • GFPT2 is regulated by insulin and EGF., In Brief Epithelial–mesenchymal transition (EMT) is a cellular process inherent to cancer cell metastasis. Metabolic reprogramming is a driver of EMT. We performed proteomic profiling of three isogenic cell lines from human breast epithelium representing the epithelial, mesenchymal, and “partial” mesenchymal states of EMT to identify metabolic vulnerabilities associated with cell invasion. Bioinformatic and functional analysis revealed that the metabolic enzyme GFPT2 is a marker of claudin-low breast cancer, responds to oxidative stress, and impacts EMT, cell growth, and cell invasion.
- Published
- 2022
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