Lung Cancer Cells That Survive Ionizing Radiation Show Increased Integrin α2β1- and EGFR-dependent Invasiveness

Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin α2 and β1 subunits were significantly elevated in IR cells. Knockdown of α2 expression or functional blockade of integrin α2β1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule's essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin α2β1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy.