Aberrant amplification of the crosstalk between canonical Wnt signaling and N-glycosylation gene DPAGT1 promotes oral cancer.

Oral cancer is one of the most aggressive epithelial malignancies, whose incidence is on the rise. Previous studies have shown that in a subset of human oral squamous cell carcinoma (OSCC) tumor specimens, overexpression of the DPAGT1 gene, encoding the dolichol-P-dependent N-acetylglucoseamine-1-phosphate transferase, a key regulator of the metabolic pathway of protein N-glycosylation, drives tumor cell discohesion by inhibiting E-cadherin adhesive function. Recently, we reported that DPAGT1 was a target of the canonical Wnt signaling pathway. Here, we link overexpression of DPAGT1 in human OSCC tumor specimens to aberrant activation of canonical Wnt signaling. We report dramatic increases in ß- and γ-catenins at the DPAGT1 promoter and correlate them with reduced expression of a Wnt inhibitor, Dickkopf-1 (Dkk-1). Using human squamous carcinoma cell lines of the head and neck, we show that partial inhibition of DPAGT1 reduces canonical Wnt signaling, indicating that DPAGT1 and canonical Wnt signaling function in a positive feedback loop. We provide evidence that E-cadherin inhibits DPAGT1, canonical Wnt signaling and the OSCC cancer phenotype by depleting nuclear ß- and γ-catenins, with hypoglycosylated E-cadherin being the most effective. This suggests that in human OSCC, extensive N-glycosylation of E-cadherin compromises its ability to inhibit canonical Wnt signaling and DPAGT1 expression. Our studies reveal a novel interplay between DPAGT1/N-glycosylation and canonical Wnt signaling and suggest that dysregulation of this crosstalk is a key mechanism underlying OSCC. They also suggest that partial inhibition of DPAGT1 may represent an effective way to restore normal interactions among these essential pathways in oral cancer.

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct ß-catenin- and γ-catenin-containing AJs.

N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/ß-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct ß-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.