Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells

Abstract

Elevated focal adhesion kinase (FAK) expression in human tumor cells has been correlated with an increased cell invasion potential. In cell culture, studies with FAK-null fibroblasts have shown that FAK function is required for cell migration. To determine the role of elevated FAK expression in facilitating epidermal growth factor (EGF)-stimulated human adenocarcinoma (A549) cell motility, antisense oligonucleotides were used to reduce FAK protein expression >75%. Treatment of A549 cells with FAK antisense (ISIS 15421) but not a mismatched control (ISIS 17636) oligonucleotide resulted in reduced EGF-stimulated p130Cas-Src complex formation, c-Jun NH2-terminal kinase (JNK) activation, directed cell motility, and serum-stimulated cell invasion through Matrigel. Because residual FAK protein in ISIS 15421-treated A549 cells was highly phosphorylated at the Tyr-397/Src homology (SH)2 binding site, expression of the FAK COOH-terminal domain (FRNK) was also used as an inhibitor of FAK function. Adenoviral-mediated infection and expression of FRNK promoted FAK dephosphorylation at Tyr-397, resulted in reduced EGF-stimulated JNK as well as extracellular-regulated kinase 2 (ERK2) kinase activation, inhibited matrix metalloproteinase-9 (MMP-9) secretion, and potently blocked both random and EGF-stimulated A549 cell motility. Equivalent expression of a FRNK (S-1034) point-mutant that did not promote FAK dephosphorylation also did not affect EGF-stimulated signaling or cell motility. Dose-dependent reduction in EGF-stimulated A549 motility was observed with the PD98059 MEK1 inhibitor and the batimastat (BB-94) inhibitor of MMP activity, but not with the SB203580 inhibitor of p38 kinase. Finally, comparisons between normal, FAK-null, and FAK-reconstituted fibroblasts revealed that FAK enhanced EGF-stimulated JNK and ERK2 kinase activation that was required for cell motility. These data indicate that FAK functions as an important signaling platform to coordinate EGF-stimulated cell migration in human tumor cells and support a role for inhibitors of FAK expression or activity in the control of neoplastic cell invasion.