Transforming growth factor-β challenge alters the N-, O-, and glycosphingolipid glycomes in PaTu-S pancreatic adenocarcinoma cells

Jing Zhang,Zejian Zhang,Stephanie Holst,Constantin Blöchl,Katarina Madunic,Manfred Wuhrer,Peter Ten Dijke,Tao Zhang

doi:10.1016/j.jbc.2022.101717

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-β (TGF-β) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-β and the role of glycosylation therein is not well known. Here, we investigated the TGF-β-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-β signaling. PaTu-S cells responded to TGF-β by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-β induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-β stimulation. TGF-β treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-β-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-β treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-β stimulation, and its depletion blocked TGF-β-induced N-glycomic changes. Thus, TGF-β-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4–dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-β signaling in SMAD4-inactivated PDAC.

Highlights

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors in the world
KRAS mutations occur in the early stage of PDAC [5], whereas mutations of tumor protein p53 (TP53), SMAD4, and cyclin-dependent kinase inhibitor 2AJournal Pre-proof (CDKN2A) arise in advanced pancreatic intraepithelial neoplasias and invasive pancreatic adenocarcinomas [6,7,8]
Our study demonstrated the significant upregulation of branching, sialylation, and core fucosylation of N-glycans in Transforming growth factor-β (TGF-β)-treated PaTu-S cells in a SMAD4-independent manner

Summary

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors in the world. Taken together, these results indicate that the PaTu-S cells responded to TGF-β with an upregulation of SMAD2 phosphorylation and target gene expression. Journal Pre-proof Differences of O-glycosylation in PaTu-S cells with and without TGF-β-treatment Following the N-glycan analysis, we analyzed 22 O-glycan species spanning 16 glycan compositions with relative quantification (Figure S3A). TGF-β-induced increases in complex structures, as well as Journal Pre-proof the gene expression levels including MGAT4A, MGAT4B, were attenuated by CRISPRimediated SOX4 depletion (Figure S6C, D). SOX4 depletion significantly decreased the relative abundance of sialylation and expression levels of associated genes, i.e., ST3GAL2, ST3GAL4, and ST6GAL1 (Figure S6C, D) These results indicated that SOX4 is a critical mediator for TGF-β signaling in the PaTu-S cell line. SOX4 plays a role in TGF-β-mediated increase of N-glycan-associated genes in multiple SMAD4-deficient PDAC lines

Discussion

Findings

Experimental procedures