PT  - JOURNAL ARTICLE
AU  - Jing Zhang
AU  - Zejian Zhang
AU  - Stephanie Holst
AU  - Constantin Blöchl
AU  - Katarina Madunic
AU  - Manfred Wuhrer
AU  - Peter ten Dijke
AU  - Tao Zhang
TI  - Dissection of <em>N</em>-, <em>O</em>- and glycosphingolipid glycosylation changes in PaTu-S pancreatic adenocarcinoma cells upon TGF-β challenge
AID  - 10.1101/2021.05.14.444203
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.14.444203
4099  - http://biorxiv.org/content/early/2021/05/17/2021.05.14.444203.short
4100  - http://biorxiv.org/content/early/2021/05/17/2021.05.14.444203.full
AB  - Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-β (TGF-β) plays a key role in tumor progression, which is often associated with aberrant glycosylation. How PDAC cells respond to TGF-β and the role of glycosylation therein is, however, not well known. Here, we investigated the TGF-β-mediated response and glycosylation changes in SMAD4-deficient PaTu-8955S (PaTu-S) cell line. PaTu-S cells responded to TGF-β by upregulating SMAD2 phosphorylation and target gene expression. TGF-β induced expression of the mesenchymal marker N-cadherin, but did not significantly affect epithelial marker E-cadherin expression. The differences of N-glycans, O-glycans and glycosphingolipid (GSL) glycans in PaTu-S cells with TGF-β stimulation were examined. TGF-β treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in line with TGF-β-induced changes in the expression of glycosylation-related genes. In addition, we observed differences in O- and GSL-glycosylation profiles after TGF-β treatment, including lower levels of sialylated Tn antigen, and neoexpression of globosides. Furthermore, SOX4 expression was upregulated upon TGF-β stimulation, and its depletion blocked the TGF-β-induced N-glycomic changes. Thus, our study provides a mechanism by which TGF-β-induced N-glycosylation changes in SOX4 dependent and SMAD4 independent manner in pancreatic cancer cells. Our results open up avenues to study the relevance of glycosylation in TGF-β signaling in SMAD4 inactivated PDAC.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPDACpancreatic ductal adenocarcinomaTGF-βTransforming growth factor-βGSLglycosphingolipidTP53tumor suppressors tumor protein p53SMADSma and Mad relatedCDKN2Acyclin dependent kinase inhibitor 2ATβRIITGF-β type II receptorTβRITGF-β type I receptorMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNKc-Jun amino terminal kinaseIKKIκB kinasePI3Kphosphatidylinositol-3 kinaseEMTepithelial-to-mesenchymal transitionEMPepithelial–mesenchymal plasticitySOX4SRY-related HMG box 4TTF-1thyroid transcription factor-1IGFBP3insulin-like growth factor binding proteinLGALS3BPgalectin-3-binding proteinTNFtumor necrosis factorNF-κ-Bnuclear factor κ-light-chain-enhancer of activated BGALNT3polypeptide N-acetylgalactosaminyltransferase 3ECMextracelular matrixCAcancer antigenqRT-PCRquantitative real-time-polymerase chain reactionPGCnano-LC-ESI-MS/MS porous graphitized carbon nano-liquid chromatography coupled to a tandem mass spectrometerTFAtrifluoroacetic acidACNacetonitrileRPreverse phasePNGase FPeptide N-glycosidase FEGCase IEndoglycoceramidase IIBimmunoblottingIFimmunofluorescenceHEKhuman embryonic kidneyVIMvimentinRFPred fluorescent proteinATCCAmerican Type Culture CollectionDMEMDulbecco’s modified Eagle mediumFBSfetal bovine serumSTRshort tandem repeatRIPAradio immune precipitation assayMinminuteSDS-PAGEsodium dodecyl sulphate-polyacrylamide gel elelectrophoresisPVDFpolyvinylidene difluorideGAPDHglyceraldehyde 3-phosphate dehydrogenaseshRNAshort hairpin RNAVSVGvesicular stomatitis virushhourPESpolyethersulfoneBSAbovine serum albuminPBSphosphate buffered salineDAPI4’,6-diamidino-2-phenylindolehpfhour-post-fertilizationRTroom temperatureSPSsmart parameter settingCFGConsortium of Functional GlycomicsCCN2cellular communication network factor 2SERPINE1serpin family E member 1PTHLHparathyroid hormone like hormoneCDHgene cadherinSNAI2snail family transcriptional repressor 2F-actinfilamentous actinCHTcaudal hematopoietic tissueMAGTN-acetylglucosaminyltransferaseFUTfucosyltransferaseST3GALβ-galactoside alpha-2,3-sialyltransferaseST6GAL1β-galactoside alpha-2,6-sialyltransferase 1CRCcolorectal cancerDCsdendritic cellsHaCaThuman immortalized keratinocytesHSCshepatic stellate cellssiRNAsmall interfering RNAEICsextracted ion chromatograms