Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase.
Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked beta(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.
Guo
Hua-Bei
HB
Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Randolph
Matthew
M
Pierce
Michael
M
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors*; D005260:Female; D005434:Flow Cytometry; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000517:prevention & control*; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D014945:Wound Healing
D009838:Oligodeoxyribonucleotides; D034741:RNA, Small Interfering; D004815:Epidermal Growth Factor; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D051416:Focal Adhesion Protein-Tyrosine Kinases