Integrin α6ß4 Confers Doxorubicin Resistance in Cancer Cells by Suppressing Caspase-3-Mediated Apoptosis: Involvement of N-Glycans on ß4 Integrin Subunit.

Drug resistance is a major obstacle to successful cancer treatment. Therefore, it is essential to understand the molecular mechanisms underlying drug resistance to develop successful therapeutic strategies. α6ß4 integrin confers resistance to apoptosis and regulates the survival of cancer cells; however, it remains unclear whether α6ß4 integrin is directly involved in chemoresistance. Here, we show that α6ß4 integrin promotes doxorubicin resistance by decreasing caspase-3-mediated apoptosis. We found that the overexpression of α6ß4 integrin by the ß4 integrin gene rendered MDA-MB435S and Panc-1 cells more resistant to doxorubicin than control cells. The acquired resistance to doxorubicin by α6ß4 integrin expression was abolished by the deletion of the cytoplasmic signal domain in ß4 integrin. Similar results were found in MDA-MB435S and Panc-1 cells when N-glycan-defective ß4 integrin mutants were overexpressed or bisecting GlcNAc residues were increased on ß4 integrin by the co-expression of N-acetylglucosaminyltransferase III with ß4 integrin. The abrogation of α6ß4 integrin-mediated resistance to doxorubicin was accompanied by reduced cell viability and an increased caspase-3 activation. Taken together, our results clearly suggest that α6ß4 integrin signaling plays a key role in the doxorubicin resistance of cancer cells, and N-glycans on ß4 integrin are involved in the regulation of cancer cells.

Tissue-Specific Regulation of HNK-1 Biosynthesis by Bisecting GlcNAc.

Human natural killer-1 (HNK-1) is a sulfated glyco-epitope regulating cell adhesion and synaptic functions. HNK-1 and its non-sulfated forms, which are specifically expressed in the brain and the kidney, respectively, are distinctly biosynthesized by two homologous glycosyltransferases: GlcAT-P in the brain and GlcAT-S in the kidney. However, it is largely unclear how the activity of these isozymes is regulated in vivo. We recently found that bisecting GlcNAc, a branching sugar in N-glycan, suppresses both GlcAT-P activity and HNK-1 expression in the brain. Here, we observed that the expression of non-sulfated HNK-1 in the kidney is unexpectedly unaltered in mutant mice lacking bisecting GlcNAc. This suggests that the biosynthesis of HNK-1 in the brain and the kidney are differentially regulated by bisecting GlcNAc. Mechanistically, in vitro activity assays demonstrated that bisecting GlcNAc inhibits the activity of GlcAT-P but not that of GlcAT-S. Furthermore, molecular dynamics simulation showed that GlcAT-P binds poorly to bisected N-glycan substrates, whereas GlcAT-S binds similarly to bisected and non-bisected N-glycans. These findings revealed the difference of the highly homologous isozymes for HNK-1 synthesis, highlighting the novel mechanism of the tissue-specific regulation of HNK-1 synthesis by bisecting GlcNAc.

Expression of N-Acetylglucosaminyltransferase III Promotes Trophoblast Invasion and Migration in Early Human Placenta.

INTRODUCTION: Trophoblast migration and invasion at the maternal-fetal interface are crucial events for normal placentation and successful pregnancy. This progress is well controlled by many placenta-specific factors. Inadequate trophoblast invasion results in poor placenta plantation or even complications such as preeclampsia. It has been shown that N-acetylglucosaminyltransferase III (GnT-III) participates in tumor invasion and metastasis as a suppressor; however, the expression of GnT-III and its role in normal pregnancy is unclear. Our objective was to characterize GnT-III expression and function during placental development and identify the underlying mechanisms. METHODS: The expression of GnT-III in human placental tissue from the first trimester was determined by immunohistochemistry. The HTR8/SVneo cell line was used to investigate the effects of GnT-III on proliferation, apoptosis, migration/invasion, matrix metalloproteinase (MMP) 2/9 activity, and the expression of the tissue inhibitor of metalloproteinase (TIMP) 1/2 using cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays, flow cytometric analysis, transwell migration/invasion assays, gelatin zymography, and Western blot, respectively. Moreover, a placental villous explant model was employed to determine its functions in placentation. RESULTS: In the first-trimester placental tissue, GnT-III was localized within the cytotrophoblast, the syncytiotrophoblast and the trophoblast columns of human placental villi, decidual cells, and some extravillous cells in the maternal decidua. GnT-III silencing significantly inhibited HTR8/SVneo cell invasion and migration as well as extravillous explant outgrowth. The application of GnT-III siRNA significantly attenuated MMP2/9 activity and increased TIMP1/2 expression. DISCUSSION AND CONCLUSION: GnT-III is expressed in trophoblasts during normal human pregnancy and is involved in regulating trophoblast function.