N-acetylglucosaminyltransferase V confers hepatoma cells with resistance to anoikis through EGFR/PAK1 activation.

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.