Overexpression of DPAGT1 leads to aberrant N-glycosylation of E-cadherin and cellular discohesion in oral cancer

Abstract

Cancer cells are frequently characterized by aberrant increases in protein N-glycosylation and by disruption of E-cadherin-mediated adherens junctions. The relationship between altered N-glycosylation and loss of E-cadherin adhesion in cancer, however, remains unclear. Previously, we reported that complex N-glycans on the extracellular domains of E-cadherin inhibited the formation of mature adherens junctions. Here, we examined whether dysregulated N-glycosylation was one of the underlying causes for cellular discohesion in oral cancer. We show that dense cultures of human salivary epidermoid carcinoma A253 cells exhibited elevated expression of DPAGT1, the gene that initiates protein N-glycosylation. Overexpression of DPAGT1 correlated with the production of E-cadherin-bearing complex N-glycans in nascent adherens junctions. Partial inhibition of DPAGT1 with small interfering RNA reduced the complex N-glycans of E-cadherin and increased the abundance of alpha-catenin and stabilizing proteins in adherens junctions. This was associated with the assembly of functional tight junctions. The inverse relationship between DPAGT1 expression and intercellular adhesion was a feature of oral squamous cell carcinoma. Oral squamous cell carcinomas displayed overexpression of DPAGT1 that correlated with diminished localization of E-cadherin and alpha-catenin at the sites of adherens junctions. Our studies show for the first time that DPAGT1 is an upstream regulator of E-cadherin N-glycosylation status and adherens junction composition and suggest that dysregulation of DPAGT1 causes disturbances in intercellular adhesion in oral cancer.

Fig. 1

Salivary epidermoid carcinoma A253 cells produce extensively N-glycosylated E-cadherin and diminished intercellular adhesion. (A) Schematic representation of an inverse relationship between DPAGT1 expression and cell-cell adhesion was reflected in immunofluorescence localization of E-cad in dense HOK and A253 cultures (Size bars: 10 µm) and Western blot of DPAGT1, E-cadherin (E-cad) and ZO-1 expression. Bargraph, Fold changes in expression levels in A253 cells in comparison to HOK cells after normalization to actin. (B) Western blot of EndoH- or PNGaseF-treated E-cad from A253 and HOK cells. (C) Western blot of selected proteins in E-cad immunoprecipitates. Bargraph, Fold changes in expression levels in A253 cells in comparison to HOK cells after normalization to E-cad. (D) Western blot of selected proteins in ZO-1 immunoprecipitates. Bargraph, Fold change of occludin, claudin-1 and E-cadherin levels in A253 cells in comparison to HOK cells after normalization to ZO-1. All studies represent one of three independent experiments (*P<0.01; **P<0.005; ***P<0.001).

Fig. 2

Partial silencing of DPAGT1 enhances intercellular adhesion in A253 cells. (A). Effects of DPAGT1 siRNA (S) on DPAGT1 expression in NS and S cells. DPAGT1 transcript levels were measured by real-time PCR, and protein levels were determined by Western blot. Bargraph, Fold change of DPAGT1 abundance in S cells in comparison with NS cells after normalization to actin. (B) Western blot of associated proteins in E-cad immunoprecipitates from NS and S cells. Bargraph, Fold changes in expression levels in S cells in comparison to NS cells after normalization to E-cad. (C) Immunofluorescence localization of E-cad in NS and S cells. Cells were counterstained for F-actin with rhodamine-phalloidin to assess cytoarchitecture. Size bars: 20 µm. All studies represent results from three different experiments (*P<0.01; **P<0.005; ***P<0.001).

Fig. 3

Overexpression of DPAGT1 and compromised intercellular adhesion are signatures of OSCC. (A) Comparison of H&E staining of AE and OSCC. Distinct basal cell layer (filled arrow) and characteristic stratification (unfilled arrow) were features of AE, while OSCC was marked by invasive epithelial islands displaying dyskeratosis and keratin pearls (unfilled arrow) with mild cytologic atypia and nuclear pleomorphism. Size bar: 50 µm. Immunofluorescence localization of Ki67, cytokeratin and DPAGT1. In AE, Ki67 was prominent in the basal cell layer (arrow), while in OSCC it was detected throughout the invasive epithelium (arrow). Cytokeratin was insignificant in the basal layer of AE (filled arrow) and increased in intensity with cellular maturation (unfilled arrow); in OSCC, cytokeratin staining was markedly reduced (arrow). In AE, DPAGT1 staining was most intense in the basal layer (filled arrow) being diminished in stratified regions (unfilled arrow); in OSCC, DPAGT1 expression was extensive throughout the invasive tumor islands (arrow). Merged images of cytokeratin (unfilled arrow) and DPAGT1 (filled arrow) in AE and in OSCC highlighted their inverse relationship. Size bars: 20 µm. (B) Immunofluorescence localization of E-cad and associated catenins. Sections were either doubly immunostained for E-cad and α-catenin or for β-catenin only. In AE, E-cad and α-catenin were at cell-cell borders (arrows, insets) while in OSCC, E-cad displayed more punctate staining and α-catenin was diffuse (arrows, insets). Immunostaining of β-catenin did not appear greatly altered between AE and OSCC. Size bar: 20 µm. (C) Immunolocalization of ZO-1. ZO-1 was detected at cell-cell borders in AE (arrow), but was significantly diminished at these sites in OSCC. Size bars: 5 µm. Results represent one of three independent experiments.

Fig. 4

OSCC exhibits overexpression of DPAGT1 and aberrantly N-glycosylated E-cadherin in nascent AJs. (A) Western blot of DPAGT1, E-cad and ZO-1 expression and immunofluorescence localization of DPAGT1 in AE and well-differentiated OSCC. Bargraph, Fold changes in DPAGT1, E-cad and ZO-1 in OSCC levels in comparison to AE after normalization to actin. Overexpression of DPAGT1 in OSCC correlated with a loss of differentiation-dependent immunofluorescence localization. Size bar: 25 µm. (B) Western blot of EndoH- or PNGaseF-treated E-cad from AE and OSCC. (C) Western blot of proteins in E-cad immunoprecipitates and their immunofluorescence localization in AE and OSCC. Bargraph, Fold changes of γ-catenin, α-catenin, vinculin, IQGAP1 and PP2A levels in OSCC in comparison to AE after normalization to E-cad. Immunofluorescence localization of E-cad, α-catenin and IQGAP1 confirmed that while in AE, E-cad and α-catenin were organized at cell-cell borders (arrows, insets), but in OSCC both exhibited diffuse staining (arrows, insets). IQGAP1 was better organized at cell-cell contacts in OSCC than in AE (unfilled arrows). Size bar: 20 µm. (D) Western blot of PP2A in ZO-1 immunoprecipitates and immunofluorescence localization of ZO-1 in AE and OSCC. Bargraph, Fold change of PP2A OSCC level in comparison to AE after normalization to ZO-1. Increased interaction between ZO-1 and PP2A in OSCC correlated with its diminished organization at cell-cell contact sties. Size bar: 10 µm. Results represent one of three independent experiments (*P<0.01; ***P<0.001).

Fig. 5

Schematic representation of how partial silencing of DPAGT1 enhances intercellular adhesion in cancer cells. Overexpression of DPAGT1 in cancer cells produces E-cad extensively modified with complex N-glycans that is unable to form mature AJs and prevents the assembly of TJs. Attenuation of DPAGT1 expression reduces N-glycosylation of E-cad with complex N-glycans and leads to the remodeling and stabilization of AJs and to the formation of functional TJs. Diminished DPAGT1 expression and enhanced intracellular adhesion revert the cancer phenotype to a more organized epithelial morphology.