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.

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