Abstract
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