Dissection of N-, O- and glycosphingolipid glycosylation changes in PaTu-S pancreatic adenocarcinoma cells upon TGF-β challenge

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 Statement The authors have declared no competing interest. * Abbreviations : PDAC : pancreatic ductal adenocarcinoma TGF-β : Transforming growth factor-β GSL : glycosphingolipid TP53 : tumor suppressors tumor protein p53 SMAD : Sma and Mad related CDKN2A : cyclin dependent kinase inhibitor 2A TβRII : TGF-β type II receptor TβRI : TGF-β type I receptor MAPK : mitogen-activated protein kinase ERK : extracellular signal-regulated kinase JNK : c-Jun amino terminal kinase IKK : IκB kinase PI3K : phosphatidylinositol-3 kinase EMT : epithelial-to-mesenchymal transition EMP : epithelial–mesenchymal plasticity SOX4 : SRY-related HMG box 4 TTF-1 : thyroid transcription factor-1 IGFBP3 : insulin-like growth factor binding protein LGALS3BP : galectin-3-binding protein TNF : tumor necrosis factor NF-κ-B : nuclear factor κ-light-chain-enhancer of activated B GALNT3 : polypeptide N-acetylgalactosaminyltransferase 3 ECM : extracelular matrix CA : cancer antigen qRT-PCR : quantitative real-time-polymerase chain reaction PGC : nano-LC-ESI-MS/MS porous graphitized carbon nano-liquid chromatography coupled to a tandem mass spectrometer TFA : trifluoroacetic acid ACN : acetonitrile RP : reverse phase PNGase F : Peptide N-glycosidase F EGCase I : Endoglycoceramidase I IB : immunoblotting IF : immunofluorescence HEK : human embryonic kidney VIM : vimentin RFP : red fluorescent protein ATCC : American Type Culture Collection DMEM : Dulbecco’s modified Eagle medium FBS : fetal bovine serum STR : short tandem repeat RIPA : radio immune precipitation assay Min : minute SDS-PAGE : sodium dodecyl sulphate-polyacrylamide gel elelectrophoresis PVDF : polyvinylidene difluoride GAPDH : glyceraldehyde 3-phosphate dehydrogenase shRNA : short hairpin RNA VSVG : vesicular stomatitis virus h : hour PES : polyethersulfone BSA : bovine serum albumin PBS : phosphate buffered saline DAPI : 4’,6-diamidino-2-phenylindole hpf : hour-post-fertilization RT : room temperature SPS : smart parameter setting CFG : Consortium of Functional Glycomics CCN2 : cellular communication network factor 2 SERPINE1 : serpin family E member 1 PTHLH : parathyroid hormone like hormone CDH : gene cadherin SNAI2 : snail family transcriptional repressor 2 F-actin : filamentous actin CHT : caudal hematopoietic tissue MAGT : N -acetylglucosaminyltransferase FUT : fucosyltransferase ST3GAL : β-galactoside alpha-2,3-sialyltransferase ST6GAL1 : β-galactoside alpha-2,6-sialyltransferase 1 CRC : colorectal cancer DCs : dendritic cells HaCaT : human immortalized keratinocytes HSCs : hepatic stellate cells siRNA : small interfering RNA EICs : extracted ion chromatograms