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.