Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of β1-integrin at threonine 788/789

Abstract

Increased extracellular pressure stimulates β1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in β1-integrin binding affinity or avidity and whether these changes are phosphorylation dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab′)2multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of β1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-β1-integrin-null murine fibroblasts stably transfected with either wild-type β1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of β1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab′)2multimeric complexes, but not soluble RGD-Fc ligands, was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting that pressure modulates β1-integrin affinity. Pressure stimulated both β1-integrin S785 and T788/9 phosphorylation. GD25-β1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in β1-integrin-null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by β1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity.