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• W4211087729 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. 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. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors in the world. It is characterized by a poor prognosis and a failure to respond to therapy (1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2019.CA Cancer J. Clin. 2019; 69: 7-34Google Scholar). Genomic analysis of PDAC revealed that the most frequent genetic alterations include the activation of oncogene KRAS and inactivation of the tumor suppressors tumor protein p53 (TP53), SMA-related and MAD-related protein 4 (SMAD4), and cyclin-dependent kinase inhibitor 2A (CDKN2A) (2Vincent A. Herman J. Schulick R. Hruban R.H. Goggins M. Pancreatic cancer.Lancet. 2011; 378: 607-620Google Scholar, 3Jones S. Zhang X. Parsons D.W. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Kamiyama H. Jimeno A. Hong S.M. Fu B. Lin M.T. Calhoun E.S. Kamiyama M. et al.Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.Science. 2008; 321: 1801-1806Google Scholar, 4Pan S. Brentnall T.A. Chen R. 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The transforming growth factor-β (TGF-β) signaling pathway is involved in many cellular processes, such as cell proliferation, apoptosis, migration, invasion, and immune evasion, contributing to various diseases, including cancer (18Colak S. Ten Dijke P. Targeting TGF-β signaling in cancer.Trends Cancer. 2017; 3: 56-71Google Scholar, 19Batlle E. Massague J. Transforming growth factor-β signaling in immunity and cancer.Immunity. 2019; 50: 924-940Google Scholar). TGF-β is a secreted cytokine for which cellular responses are initiated by binding to specific cell surface TGF-β type I and type II receptors, that is, TβRI and TβRII. Upon TGF-β interaction with TβRII, TβRI is recruited, and a heteromeric complex is formed (20Massague J. How cells read TGF-β signals.Nat. Rev. Mol. Cell Biol. 2000; 1: 169-178Google Scholar, 21Massague J. TGFβ signalling in context.Nat. Rev. Mol. Cell Biol. 2012; 13: 616-630Google Scholar). Thereafter, the TβRII kinase phosphorylates the serine and threonine residues of TβRI, and thereby the extracellular signal is transduced across the plasma membrane (22Heldin C.H. Miyazono K. ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Google Scholar). Subsequently, intracellular signaling by TβRI proceeds via the phosphorylation of SMAD proteins, that is, SMAD2 and SMAD3. Then, phosphorylated SMAD2/3 can form heteromeric complexes with the common SMAD mediator, that is, SMAD4, which translocates into the nucleus to regulate the transcription of target genes (23Budi E.H. Duan D. Derynck R. Transforming growth factor-β receptors and smads: Regulatory complexity and functional versatility.Trends Cell Biol. 2017; 27: 658-672Google Scholar). SMAD4 is a critical mediator of TGF-β-induced growth arrest (24Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Partnership between DPC4 and SMAD proteins in TGF-β signalling pathways.Nature. 1996; 383: 832-836Google Scholar, 25Zhang Y. Feng X. We R. Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-β response.Nature. 1996; 383: 168-172Google Scholar) and apoptosis (26Schuster N. Krieglstein K. Mechanisms of TGF-β-mediated apoptosis.Cell Tissue Res. 2002; 307: 1-14Google Scholar), which results in its role as a tumor suppressor at the early stages of cancer progression. However, the tumor-suppressive action of TGF-β–SMAD4 signaling is lost in nearly half of PDACs because of the inactivation of SMAD4 (27Dardare J. Witz A. Merlin J.L. Gilson P. Harle A. SMAD4 and the TGFβ pathway in patients with pancreatic ductal adenocarcinoma.Int. J. Mol. Sci. 2020; 21: 3534Google Scholar). The SMAD4 gene deletion, frameshift mutation, or single point mutation can lead to the deficiency of functional SMAD4 protein, which further prevents or disrupts transduction of the canonical TGF-β–SMAD4 signaling pathway. Phosphorylated TGF-β receptors can activate SMAD2/3-dependent and SMAD4-independent pathways; in that case, receptor-regulated SMADs can make complexes with sex-determining region Y-related high-mobility group box 4 (SOX4) or thyroid transcription factor-1 (also known as NKX2-1), which compete with SMAD4 for interaction with SMAD3 (28Vervoort S.J. Lourenco A.R. Tufegdzic Vidakovic A. Mocholi E. Sandoval J.L. Rueda O.M. Frederiks C. Pals C. Peeters J.G.C. Caldas C. Bruna A. Coffer P.J. SOX4 can redirect TGF-β-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis.Nucleic Acids Res. 2018; 46: 9578-9590Google Scholar, 29Isogaya K. Koinuma D. Tsutsumi S. Saito R.A. Miyazawa K. Aburatani H. Miyazono K. A Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression.Cell Res. 2014; 24: 994-1008Google Scholar). The SMAD3–thyroid transcription factor-1 and SMAD3–SOX4 complexes accumulate in the nucleus, regulate the expression of pro-oncogenic TGF-β target genes, and induce tumorigenesis (28Vervoort S.J. Lourenco A.R. Tufegdzic Vidakovic A. Mocholi E. Sandoval J.L. Rueda O.M. Frederiks C. Pals C. Peeters J.G.C. Caldas C. Bruna A. Coffer P.J. SOX4 can redirect TGF-β-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis.Nucleic Acids Res. 2018; 46: 9578-9590Google Scholar, 29Isogaya K. Koinuma D. Tsutsumi S. Saito R.A. Miyazawa K. Aburatani H. Miyazono K. A Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression.Cell Res. 2014; 24: 994-1008Google Scholar, 30David C.J. Huang Y.H. Chen M. Su J. Zou Y. Bardeesy N. Iacobuzio-Donahue C.A. Massague J. 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In this study, the PaTu-8988S (PaTu-S) cell line, a human epithelial-like PDAC cell line exhibiting KRAS activation and inactivation of SMAD4 and CDKN2A, was employed to investigate TGF-β responses and resulting glycosylation changes. Upon TGF-β treatment, PaTu-S cells were first analyzed for any effects on gene expression, morphological changes, loss of epithelial traits, and gain of mesenchymal markers. Next, by combining transcriptomic analysis of glycosylation-associated genes with mass spectrometry glycomics, we systematically assessed the TGF-β-induced alterations in the three major classes of cell surface glycans of PaTu-S, namely, N-glycans, O-glycans, and GSL-linked glycans. Furthermore, we investigated the critical role of SOX4 in TGF-β signaling and TGF-β-induced glycosylation in PaTu-S cells. This provides a stepping-stone for further studies on how glycosylation alterations contribute to TGF-β-mediated tumorigenesis of PDAC. To investigate whether the PaTu-S cell line, which lacks SMAD4, is responsive to TGF-β treatment (Fig. S1A), we performed Western blot analysis of phosphorylated SMAD2 levels of PaTu-S cells without and with TGF-β challenge. In PaTu-S cells, SMAD2 phosphorylation was significantly upregulated upon TGF-β stimulation for 1 h, which was blocked by treatment with TβRI kinase inhibitor SB431542 (Fig. 1A). The expression of TGF-β target genes, including cellular communication network factor 2 (CCN2) (66Meyer C. Godoy P. Bachmann A. Liu Y. Barzan D. Ilkavets I. Maier P. Herskind C. Hengstler J.G. Dooley S. Distinct role of endocytosis for Smad and non-Smad TGF-β signaling regulation in hepatocytes.J. Hepatol. 2011; 55: 369-378Google Scholar, 67Samarakoon R. Dobberfuhl A.D. Cooley C. Overstreet J.M. Patel S. Goldschmeding R. Meldrum K.K. Higgins P.J. Induction of renal fibrotic genes by TGF-β1 requires EGFR activation, p53 and reactive oxygen species.Cell Signal. 2013; 25: 2198-2209Google Scholar), serpin family E member 1 (SERPINE1) (68Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. Direct binding of Smad3 and Smad4 to critical TGFβ-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.EMBO J. 1998; 17: 3091-3100Google Scholar, 69Samarakoon R. Higgins P.J. Integration of non-SMAD and SMAD signaling in TGF-β1-induced plasminogen activator inhibitor type-1 gene expression in vascular smooth muscle cells.Thromb. Haemost. 2008; 100: 976-983Google Scholar), parathyroid hormone–like hormone (PTHLH), (70Kakonen S.M. Selander K.S. Chirgwin J.M. Yin J.J. Burns S. Rankin W.A. Grubbs B.G. Dallas M. Cui Y. Guise T.A. Transforming growth factor-β stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways.J. Biol. 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In response to TGF-β stimulation for 2 days, the PaTu-S cells showed an upregulation in the expression of both the epithelial marker gene cadherin 1 (CDH1, encoding the protein E-cadherin) and the mesenchymal marker genes CDH2 (encoding the protein N-cadherin), SNAIL family transcriptional repressor 2 (SNAI2, encoding the protein SLUG), and VIM (encoding the protein VIM) (Fig. 1C). At the protein level, the mesenchymal marker N-cadherin was increased after TGF-β stimulation, whereas E-cadherin and SLUG expression levels were not significantly affected (Fig. 1D). In addition, no morphological changes of PaTu-S cells were observed after 2 days of TGF-β treatment (Fig. S1B) or even longer treatment times (data not shown). The response of PaTu-S cells to TGF-β treatment was further analyzed by immunofluorescence (IF) staining of E-cadherin and filamentous (F)-actin. We observed that TGF-β induced an increase in the formation of lamellipodia and broadened and increased flat membrane protrusions at the leading edge of cells (Fig. S1C). However, in response to TGF-β, no significant changes in E-cadherin expression and localization were observed. As an increase of lamellipodia formation has been linked to an increase in cell migration (73Krause M. Gautreau A. Steering cell migration: Lamellipodium dynamics and the regulation of directional persistence.Nat. Rev. Mol. Cell Biol. 2014; 15: 577-590Google Scholar), we next examined the TGF-β response of PaTu-S cells using an embryonic zebrafish xenograft extravasati" @default.
• W4211087729 created "2022-02-13" @default.
• W4211087729 creator A5009750348 @default.
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• W4211087729 date "2022-03-01" @default.
• W4211087729 modified "2023-10-01" @default.
• W4211087729 title "Transforming growth factor-β challenge alters the N-, O-, and glycosphingolipid glycomes in PaTu-S pancreatic adenocarcinoma cells" @default.
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