The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the alpha v beta 3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the alpha v beta 3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different alpha v beta 3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of alpha v beta 3 integrins (alpha v beta 3(high) or alpha v beta 3(low) cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. alpha v beta 3(high) cells showed a threefold increased cell invasion compared to alpha v beta 3(low) cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the beta 3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for alpha v beta 3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in alpha v beta 3(high) cells but not in alpha v beta 3(low) cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in alpha v beta 3(low) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of alpha v beta 3(high) cells, whereas the invasiveness of beta 3 specific knock-down cells and alpha v beta 3(low) cells was not altered. In summary, these results suggest that the alpha v beta 3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.