Pituitary Adenylate Cyclase-Activating Polypeptide Causes Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an autocrine growth factor for some lung cancer cells. The activated PACAP receptor (PAC1) causes phosphatidylinositol turnover, elevates cAMP, and increases the proliferation of lung cancer cells. PAC1 and epidermal growth factor receptor (EGFR) are present in non-small-cell lung cancer (NSCLC) cells, and the growth of NSCLC cells is inhibited by the PAC1 antagonist PACAP(6-38) and the EGFR tyrosine kinase inhibitor gefitinib. Here, the ability of PACAP to transactivate the EGFR was investigated. Western blot analysis indicated that the addition of PACAP but not the structurally related vasoactive intestinal peptide increased EGFR tyrosine phosphorylation in NCI-H838 or H345 cells. PACAP-27, in a concentration-dependent manner, increased EGFR transactivation 4-fold 2 min after addition to NCI-H838 cells. The ability of 100 nM PACAP-27 to increase EGFR or extracellular signal-regulated kinase tyrosine phosphorylation in NCI-H838 cells was inhibited by PACAP(6-38), gefitinib, 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2; Src inhibitor), (R)-N4-hydroxy-N1-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide (GM6001; matrix metalloprotease inhibitor), or antibody to transforming growth factor α (TGFα). By enzyme-linked immunosorbent assay, PACAP addition to NCI-H838 cells increased TGFα secretion into conditioned media. EGFR transactivation caused by the addition of PACAP to NCI-H838 cells was inhibited by N-acetyl-cysteine (antioxidant), tiron (superoxide scavenger), diphenylene iodonium (NADPH oxidase inhibitor), or 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; phospholipase C inhibitor), but not N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide (H89; protein kinase A inhibitor). PACAP addition to NCI-H838 cells significantly increased reactive oxygen species, and the increase was inhibited by tiron. The results indicate that PACAP causes transactivation of the EGFR in NSCLC cells in an oxygen-dependent manner that involves phospholipase C but not protein kinase A.