Core 1 β1,3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation and is overexpressed in various human malignancies. However, its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here we demonstrate that C1GALT1 expression is upregulated in HNSCC tumors and is associated with adverse clinicopathologic features. Moreover, high C1GALT1 expression predicts poor disease-free and overall survivals. C1GALT1 overexpression enhances HNSCC cell viability, migration, and invasion, which can be reversed by erlotinib. Silencing of C1GALT1 suppresses the malignant behavior both in vitro and in vivo. Mass spectrometry and lectin pull-down assays demonstrate that C1GALT1 modifies O-glycans on EGFR. Blocking O-glycan elongation on EGFR by C1GALT1 knockdown decreases EGF-EGFR binding affinity and inhibits EGFR signaling, thereby suppressing malignant phenotypes. Using molecular docking simulations, we identify itraconazole as a C1GALT1 inhibitor that directly binds C1GALT1 and promotes its proteasomal degradation, leading to significant blockade of C1GALT1-mediated effects in HNSCC cells in vitro and in vivo. Collectively, our findings demonstrate a critical role of O-glycosylation in HNSCC progression and highlight the therapeutic potential of targeting C1GALT1 in HNSCC treatment.
C1GALT1 controls the crucial step of GalNAc-type O-glycosylation and is associated with both physiologic and pathologic conditions, including cancers. EPH receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate a diverse range of developmental processes and human diseases. However, the role of C1GALT1 in the signaling of EPH receptors remains largely overlooked. Here, we showed that C1GALT1 high expression in gastric adenocarcinomas correlated with adverse clinicopathologic features and is an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth and metastasis, as well as increased apoptosis and cytotoxicity of 5-fluorouracil in AGS and MKN45 cells. Phospho-RTK array and western blot analysis showed that C1GALT1 depletion suppressed tyrosine phosphorylation of EPHA2 induced by soluble Ephrin A1-Fc. O-glycans on EPHA2 were modified by C1GALT1 and both S277A and T429A mutants, which are O-glycosites on EPHA2, dramatically enhanced phosphorylation of Y588, suggesting that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased Ephrin A1-Fc induced migration and reduced Ephrin A1 binding to cell surfaces. The effects of C1GALT1 knockdown or knockout on cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown in gastric cancer cells. These results suggest that C1GALT1 promotes phosphorylation of EPHA2 and enhances soluble Ephrin A1-mediated migration primarily by modifying EPHA2 O-glycosylation. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.
Ovarian cancer is the most lethal of the gynecologic malignancies. N-acetylgalactosaminyltransferase 6 (GALNT6), an enzyme that mediates the initial step of mucin type-O glycosylation, has been reported to regulate mammary carcinogenesis. However, the expression and role of GALNT6 in ovarian cancer are still unclear. Here we showed that high GALNT6 expression correlates with increased recurrence, lymph node metastasis, and chemoresistance in ovarian endometrioid and clear cell carcinomas; and higher GALNT6 levels are significantly associated with poorer patient survivals. GALNT6 knockdown with two independent siRNAs significantly suppressed viability, migration, and invasion of ovarian cancer cells. Using phospho-RTK array and Western blot analyses, we identified EGFR as a critical target of GALNT6. GALNT6 knockdown decreased phosphorylation of EGFR, whereas GALNT6 overexpression increased the phosphorylation. Lectin pull-down assays with Vicia villosa agglutinin (VVA) indicated that GALNT6 was able to modify O-glycans on EGFR. Moreover, the GALNT6-enhanced invasive behavior was significantly reversed by erlotinib, an EGFR inhibitor. Our results suggest that GALNT6 expression is associated with poor prognosis of ovarian cancer and enhances the aggressive behavior of ovarian cancer cells by regulating EGFR activity.
Mucins are heavily glycosylated proteins that play critical roles in the pathogenesis of tumour malignancies. Pancreatic ductal adenocarcinoma (PDAC) is characterised by the aberrant expression of mucins. However, the role of mucin (MUC) 20 in PDAC remains unclear. PDAC is usually surrounded by a dense fibrotic stroma consisting of an extracellular matrix and pancreatic stellate cells (PSCs). The stroma creates a nutrient-deprived, hypoxic, and acidic microenvironment, and promotes the malignant behaviours of PDAC cells. In this study, immunohistochemical staining demonstrated that high MUC20 expression correlated with poor progression-free survival and high local recurrence rate of PDAC patients (n = 61). The expression of MUC20 was induced by serum deprivation, hypoxia, and acidic pH in PDAC cells. MUC20 knockdown with siRNA decreased cell viability, as well as migration and invasion induced by PSCs in HPAC and HPAF-II cells. In intraperitoneal, subcutaneous, and orthotopic injection models, MUC20 knockdown decreased tumour growth in immunodeficient mice. Phospho-RTK array and western blot analysis indicated that MUC20 knockdown decreased HGF-mediated phosphorylation of MET in PDAC cells. Moreover, HGF-induced malignant phenotypes could be suppressed by MUC20 knockdown. Co-immunoprecipitation revealed the physical association of MUC20 and MET. These findings suggest that MUC20 knockdown suppresses the malignant phenotypes of PDAC cells at least partially through the inhibition of the HGF/MET pathway and that MUC20 could act as a potential therapeutic target.
Pancreatic adenocarcinoma (PDAC) is a leading cause of cancer-related death. Altered glycosylation contributes to tumor progression and chemoresistance in many cancers. C1GALT1 is the key enzyme controlling the elongation of GalNAc-type O-glycosylation. Here we showed that C1GALT1 was overexpressed in 85% (107/126) of PDAC tumors compared with adjacent non-tumor tissues. High expression of C1GALT1 was associated with poor disease-free and overall survival (n = 99). C1GALT1 knockdown using siRNA suppressed cell viability, migration, and invasion as well as increased gemcitabine sensitivity in PDAC cells. In contrast, C1GALT1 overexpression enhanced cell migration and invasion. In subcutaneous and pancreatic orthotopic injection models, C1GALT1 knockdown decreased tumor growth and metastasis of PDAC cells in NOD/SCID mice. Mechanistically, C1GALT1 knockdown dramatically suppressed cell-extracellular matrix (ECM) adhesion, which was associated with decreased phosphorylation of FAK at Y397/Y925 and changes in O-glycans on integrins including the β1, αv, and α5 subunits. Using functional blocking antibodies, we identified integrin αv as a critical factor in C1GALT1-mediated invasiveness of PDAC cells. In conclusion, this study not only reveals that C1GALT1 could be a potential therapeutic target for PDAC but also provides novel insights into the role of O-glycosylation in the α subunits of integrins.
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