Background: Whereas histone deacetylase inhibitors are known to modulate chromatin structure, the precise mechanisms by which these novel agents induce apoptosis in cancer cells remain unknown. Previously we reported that depsipeptide FK228 depletes epidermal growth factor receptor (EGFR), erbB2, and Raf-1 kinases in non-small cell lung cancer cells. In the present study we sought to further define the mechanisms by which FK228 modulates oncoprotein signaling and to ascertain whether altered signal transduction contributes to FK228-mediated apoptosis in lung cancer cells.
Methods: Cultured non-small cell lung cancer cells were treated with FK228 alone or FK228 with a variety of kinase inhibitors. Proliferation and apoptosis mediated by drug exposure were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, and Apo-BrdU techniques. Western blot and kinase assays were used to evaluate EGFR-related signal transduction pathways. Lung cancer cells were transduced with adenoviral vectors expressing activated AKT or mitogen-activated protein kinase kinase (MEK) 1 or beta-galactosidase to determine whether constitutive activation of mitogen-activated protein kinase signaling could abrogate FK228-mediated apoptosis.
Results: FK228 treatment induced time-dependent apoptosis in lung cancer cells expressing wild-type or mutant EGFR. FK228 inhibited a variety of EGFR-related pathways including Src, RAF-MEK-extracellular signal-regulated kinase (ERK) 1/2 and phosphatidyl inositol-3 kinase (PI3K)/AKT, resulting in down-regulation of Bcl-2 and Bcl-xL and up-regulation of Bax. The kinase inhibitors AG1478, AG825, PD98059, and LY294002 markedly enhanced FK228-induced apoptosis in lung cancer cells. Coincident with inhibition of ERK1/2 and PI3K/AKT survival pathways, FK228 enhanced p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase stress signaling. Constitutive expression of MEK1 but not AKT markedly reduced FK228-mediated apoptosis in lung cancer cells.
Conclusions: FK228 inhibits EGFR expression and modulates a variety of downstream mediators regulating proliferation and stress responses in lung cancer cells. These data highlight the significance of MEK signaling with respect to FK228-mediated apoptosis and support evaluation of histone deacetylase inhibitors in conjunction with agents specifically targeting mitogen-activated protein kinases in patients with lung cancer.