Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of beta1-integrin at threonine 788/789

Abstract

Increased extracellular pressure stimulates beta1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in beta1-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')2 multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of beta1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-beta1-integrin-null murine fibroblasts stably transfected with either wild-type beta1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of beta1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab')2 multimeric 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 beta1-integrin affinity. Pressure stimulated both beta1-integrin S785 and T788/9 phosphorylation. GD25-beta1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in beta1-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 beta1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity.

Fig. 1.

Effect of increased extracellular pressure on integrin affinity and avidity. SW620 cell adhesion to fibronectin or binding of soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab′)₂ multimeric complexes was assessed under ambient and 15-mmHg increased pressures for 30 min in the absence or presence of 1 mM MnCl₂ and 1 μM cytochalasin D (Ctyo D). A: effect of 1-min vs. 30-min exposure to increased pressure on cell adhesion to 12 μg/ml fibronectin (n = 6). Data from individual experiments were normalized to their respective ambient pressure controls. MFI, mean fluorescence intensity. B: effect of fibronectin substrate concentration (0.78–25 μg/ml) on pressure-stimulated cell adhesion (n = 3). C: effect of increased pressure, MnCl₂, and cytochalasin D on cell adhesion to 12 μg/ml fibronectin (n = 6). Data from individual experiments were normalized to their respective DMSO-treated ambient pressure controls. D: dose response of RGD-Fc soluble ligand binding under ambient and increased pressure (n = 4). E: determination of affinity modulation by assessment of the effects of increased pressure, MnCl₂, cytochalasin D, or MnCl₂ and cytochalasin D in combination on soluble RGD-Fc ligand binding (n = 9). F: determination of affinity/avidity modulation by assessment of the effects of increased pressure, MnCl₂, cytochalasin D, or MnCl₂ and cytochalasin D in combination on RGD-Fc-F(ab′)₂ multimeric complex binding (n = 9). Data from individual experiments are graphically expressed as means ± SE. *P < 0.05 compared with respective ambient pressure control. #P < 0.05 compared with DMSO-treated ambient pressure control.

Fig. 2.

Effect of increased pressure on exposure of the β₁-integrin 9EG7 epitope. A: effect of functional blockade of β₁- and β₃-integrin on pressure-stimulated SW620 cell adhesion to 12 μg/ml fibronectin (n = 6). B: induction of the 9EG7 epitope on SW620 cells was assessed following exposure to ambient or 15-mmHg increased pressure for 30 min in the presence of DMSO, 1 mM MnCl₂, or 1 μM cytochalasin D (n = 4). Representative histograms are shown (right) of typical 9EG7 staining following exposure to increased (red) or ambient (blue) pressure conditions. Solid gray peaks represent the unstained cell populations. C: effect of increasing pressures on 9EG7 induction (n = 3). MFI values of 9EG7-positive cells were determined by flow cytometric analysis. Data from individual experiments were normalized to respective IgG- or DMSO-treated ambient pressure controls and are graphically expressed as means ± SE. *P < 0.05 compared with ambient pressure control.

Fig. 3.

Effect of increased pressure on β₁-integrin phosphorylation at serine 785 and threonines 788/789. Protein lysates from suspended SW620 cells (n = 6) and surgically resected human primary colon cancer cells (n = 7) exposed to either ambient or increased pressure conditions were assessed for β₁-integrin phosphorylation at S785 and T788/9 by Western blot. A: effect of increased pressure on β₁-integrin S785 phosphorylation (pS785) in SW620 cells and primary colon cancer cells. B: effect of elevated pressure on β₁-integrin phosphorylation at T788/9 in SW620 cells and primary colon cancer cells. Data from individual experiments were normalized to respective ambient pressure controls and are graphically expressed as means ± SE. *P < 0.05 compared with respective ambient pressure controls.

Fig. 4.

Effect of β₁-integrin phosphorylation site-specific mutations on pressure-stimulated cell adhesion and soluble ligand binding. GD25-β₁-null murine fibroblast cells and stably transfected derivatives GD25-β_1A [wild-type (WT)], GD25-β_1A,S785A, GD25-β_1A,TT788/9AA, and GD25-β_1A,T788D were exposed to either ambient [control (C)] or 15-mmHg increased pressure [pressure (P)] conditions for 30 min and assessed for adhesion to fibronectin and RGD-Fc soluble ligand binding. A: effect of β₁-integrin site-specific mutations on pressure-stimulated adhesion to 12.5 μg/ml fibronectin (left; n = 6) and 0.78–25 μg/ml fibronectin (right; n = 3). B: effect of β₁-integrin site-specific mutations on RGD-Fc soluble ligand binding under ambient and increased pressure conditions (n = 9). Data from individual experiments were normalized to respective GD25-β_1A ambient pressure controls and are graphically expressed as means ± SE. *P < 0.05 compared with respective GD25-β_1A ambient pressure controls.

Fig. 5.

Effect of increased pressure on 9EG7 induction and β₁-integrin surface expression on GD25-β_1A phosphorylation mutants. A: induction of the 9EG7 epitope on GD25, GD25-β_1A, GD25-β_1A,S785A, GD25-β_1A,TT788/9AA, and GD25-β_1A,T788D cells was assessed following exposure to ambient or 15-mmHg increased pressure for 30 min (n = 4). Individual MFI values for 9EG7 staining were divided by respective MFI values for total β₁-integrin expression and were normalized to the GD25-β_1A ambient pressure controls. Data from individual experiments are graphically expressed as means ± SE. Representative histograms are shown (right) of typical 9EG7 staining following exposure to increased (red) or ambient (blue) pressure conditions. Solid gray peaks represent the unstained cell populations. B: flow cytometric analysis of β₁-integrin surface expression on GD25 cell transfectants following 30-min exposure to ambient or increased pressure. Data are representative of MFI values from 3 individual experiments and are expressed as means ± SE. *P < 0.05 compared with respective GD25-β_1A ambient pressure controls.

Fig. 6.

Temporal effects of 1-min exposure to increased extracellular pressure on tumor cell adhesion and β₁-integrin T788/9 phosphorylation. A: effect of 1-min exposure to 15-mmHg increased extracellular pressure on SW620 cell adhesion to 12 μg/ml fibronectin at 30, 45, 60, 90, and 180 min (n = 6). B: protein lysates from parallel nonadherent cell populations were collected and assessed for pressure-induced β₁-integrin T788/9 phosphorylation at 1, 30, 45, and 60 min (n = 5). Data from individual experiments were normalized to respective (1 min) ambient pressure controls and are graphically expressed as means ± SE. *P < 0.05 compared with respective ambient pressure controls at individual time points.

Fig. 7.

Effect of α-actinin-1, focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase, and ERK 1/2 inhibition on pressure-stimulated cell adhesion and β₁-integrin T788/9 phosphorylation. A: typical reduction of total α-actinin-1 and FAK protein in SW620 cells transfected with small interfering RNA (siRNA) targeted to either α-actinin-1 (siACTN1) or FAK (siFAK) as measured by Western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and α-actinin-1 and FAK protein expression was normalized against that of nontarget siRNA (siNT) transfectant controls (n = 7). B: SW620 cells were treated with either DMSO (n = 9), 20 μM PP2 (n = 9), 20 μM LY-294002 (n = 9), 20 μM U–0126 (n = 5), siNT (n = 7), siACTN1 (n = 7), or siFAK (n = 7) and assessed for ability to adhere to 12.5 μg/ml fibronectin under ambient or increased pressure conditions. C: protein lysates from parallel nonadherent cell populations were collected and assessed for pressure-induced β₁-integrin T788/9 phosphorylation by Western blot. Data from individual experiments were normalized to respective ambient pressure DMSO or siNT controls and are graphically expressed as means ± SE. *P < 0.05 compared with respective ambient pressure DMSO or siNT controls.