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FUT8 promotes breast cancer cell invasiveness by remodeling TGF-? receptor core fucosylation.

ABSTRACT: Core fucosylation (addition of fucose in ?-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-? (TGF-?)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-? signaling during breast cancer progression remains largely unknown.In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-?-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-?1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model.Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-?-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-? receptor complexes might be core fucosylated by FUT8 to facilitate TGF-? binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis.Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-? signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.

SUBMITTER: Tu CF

PROVIDER: S-EPMC5629780 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation.

Tu Cheng-Fen CF Wu Meng-Ying MY Lin Yuh-Charn YC Kannagi Reiji R Yang Ruey-Bing RB

Breast cancer research : BCR 20171005 1

<h4>Background</h4>Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF ...[more]

PMID: 28982386

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Project description:FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Dataset Information

FUT8 promotes breast cancer cell invasiveness by remodeling TGF-? receptor core fucosylation.

Publications

FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation.

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