Epidermal Growth Factor Receptor Inhibition Promotes Desmosome Assembly and Strengthens Intercellular Adhesion in Squamous Cell Carcinoma Cells

Abstract

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and ␤-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.

EGFR and ADAMs Cooperate to Regulate Shedding and Endocytic Trafficking of the Desmosomal Cadherin Desmoglein 2

“…Both cadherins were detected as early as 5 min and accumulated over time, the levels decreasing beginning at 90 min presumably due to degradation of the internalized pool and/or recycling back to the surface (Figure 2, B and C). Both full-length and a 100-kDa cell-retained Dsg2 fragment, previously shown to result from MMP-dependent processing of Dsg2 (Lorch et al, 2004), appeared in the internalized pool over a similar time course. Importantly, internalization occurred at calcium concentrations ranging from 0.09 to 1 mM, Figure 2.…”

“…We have previously demonstrated that Dsg2 shedding occurs in an MMP-dependent manner (Lorch et al, 2004). Subsequently, ADAM10 was shown to shed N-and E-cadherin from the surface and reduce adhesion of neuronal and epithelial cells Reiss et al, 2005), and Dsg2 was recently identified by a proteomics approach as a substrate for both ADAM10 and 17 (Bech-Serra et al, 2006).…”

“…We previously showed that MMP-dependent Dsg2 processing generates a cell-associated 100-kDa band that is reduced by EGFR inhibition and an ectodomain fragment shed into the medium (Lorch et al, 2004). Immunoprecipitation of endogenous Dsg2 with antibodies recognizing specific extra-and intracellular domains of Dsg2, and subsequent immunoblotting with a mAb directed against the terminal portion of the cytoplasmic domain indicated that the 100-kDa cleavage product comprises the entire Dsg2 cytoplasmic tail, transmembrane domain, and an extracellular juxtamembrane domain (Supplemental Figure S2).…”

“…Importantly, however, as discussed below, similar results were seen at physiological calcium concentrations (Supplemental Figure S1). Like PKI and the mAb inhibitor C225 (Lorch et al, 2004), TAPI treatment led to a dramatic redistribution of Dsg2 to intercellular borders (Figure 1, A and B). In contrast, there was little impact on the extent to which cell-cell interfaces were occupied by E-cadherin, although both inhibitors resulted in a more organized appearance of this classic cadherin (Figure 1, A and B).…”

“…Our previous work suggested that epidermal growth factor receptor (EGFR), which is overexpressed in many tumor types (Rogers et al, 2005;Kalyankrishna and Grandis, 2006), regulates desmosome assembly and function in squamous cell carcinoma (SCC) cells, by decreasing the level and cell surface localization of desmosomal cadherins (Lorch et al, 2004). The resulting weakened intercellular adhesive strength was accompanied by phosphorylation of desmoglein 2 (Dsg2) and associated proteins (which occurred preferentially on desmosomal and not classic cadherins), and MMP-dependent shedding of the Dsg2 ectodomain.…”