title: Tumour suppressor p16(INK4a)  - anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model.
abstract: Tumour suppressor p16(INK4a)  is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with α2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for α2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (β1,4-galactosyltransferase-IV, α2,3-sialyltransferase-I) and detected p16(INK4a) -dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16(INK4a) /GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering α2,6-sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.
authors.affiliation: Field of Drug Discovery Research, Graduate School of Life Sciences, Hokkaido University, Sapporo, Japan. shin@sci.hokudai.ac.jp
authors.forename: Maho
authors.initials: M
authors.lastname: Amano
authors.forename: Hanna
authors.initials: H
authors.lastname: Eriksson
authors.forename: Joachim C
authors.initials: JC
authors.lastname: Manning
authors.forename: Katharina M
authors.initials: KM
authors.lastname: Detjen
authors.forename: Sabine
authors.initials: S
authors.lastname: André
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Gabius
mesh_terms: D023102:Anoikis; D002238:Carbohydrate Epimerases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D037483:Galectin 1 / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D016147:Genes, Tumor Suppressor; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D037483:Galectin 1; D037102:Lectins; D011134:Polysaccharides; D020543:Proteome; D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; D019158:N-Acetylneuraminic Acid