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.

Fig. 1.

Specificity of EGFR transactivation by PACAP analogs. A, the ability of 100 nM VIP, PACAP (PC)-38, or PC-27 to stimulate EGFR tyrosine phosphorylation in NCI-H345 cells was determined by Western blot using antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D was indicated for four determinations. C, the ability of 100 nM PC-27 or VIP to stimulate EGFR tyrosine phosphorylation was determined by using NCI-H838 cells. Ten micrometers PC(6–38) had no effect on EGF-R tyrosine phosphorylation but antagonized the effects of 100 nM PC-27. D, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p < 0.01 by ANOVA.

Fig. 2.

PACAP-27 increases EGFR and ERK tyrosine phosphorylation in a time- and dose-dependent manner. A, the ability of 100 nM PACAP-27 to increase EGFR tyrosine phosphorylation was investigated as a function of time by using NCI-H838 cells and antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for three determinations. C, the ability of 100 nM PACAP-27 to increase ERK tyrosine phosphorylation is indicated by using NCI-H838 cells. D, the mean percentage of ERK tyrosine phosphorylation ± S.D is indicated for four determinations. E, the ability of PACAP-27 to increase EGFR tyrosine phosphorylation is indicated as a function of dose by using NCI-H838 cells. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p < 0.01 by ANOVA.

Fig. 3.

Gefitinib inhibits EGFR and ERK tyrosine phosphorylation caused by PACAP. A, gefitinib in a dose-dependent manner inhibits EGFR transactivation caused by PC. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p < 0.01 relative to control by ANOVA. a, p < 0.05 relative to PC + 0.1 μg/ml gefitinib. aa, p < 0.01 relative to PC + 0.1 μg/ml gefitinib. C, gefitinib is a dose-dependent manner inhibits ERK tyrosine phosphorylation caused by 100 nM PC. D, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p < 0.01 relative to control by ANOVA. aa, p < 0.01 relative to PC + 0.1 μg/ml gefitinib. E, the EGFR tyrosine phosphorylation was determined by using antiphospho-Tyr-1068-EGFR antibody after adding 100 nM PC to NCI-H838 cells in the absence or presence anti-HB-EGF, antiamphiregulin (Amph), or anti-TGFα. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p < 0.01 relative to control. *, p < 0.05 by ANOVA. a, p < 0.05 relative to PC. aa, p < 0.01 relative to PC.

Fig. 4.

Inhibitors of EGFR transactivation. A, the ability of 10 μM GM6001 (GM) to inhibit the EGFR transactivation caused by the addition of 100 nM PC to NCI-H838 cells was investigated by using antiphospho-Tyr-1068-EGFR antibody. B, the ability of 50 μM U73122 or H89 to inhibit the EGFR transactivation caused by the addition of 100 nM PACAP-27 to NCI-H838 cells was investigated. C, the ability of 10 μM BAPTA and 1 mM EGTA as well as 10 μM GF109203X (GF) to inhibit EGFR transactivation caused by the addition of 100 nM PACAP-27 (PC) to NCI-H838 cells was investigated. D, the ability of 5 mM NAC or 5 mM tiron to inhibit the EGFR transactivation caused by 100 nM PACAP-27 to NCI-H838 cells was investigated. These experiments are representative of three others.

Fig. 5.

Signal transduction mechanisms for lung cancer PAC1. Activated PAC1 interacts with G_α-stimulating adenylyl cyclase (AC), leading to PKA activation, cAMP response element-binding protein (Creb) phosphorylation, and altered gene expression. PKC activates MMP and/or ADAM, releasing TGFα from its precursor protein in the plasma membrane causing EGFR tyrosine phosphorylation. The phosphorylated EGFR causes Ras and Raf activation, resulting in phosphorylation of mitogen-activated protein kinase kinase (MEK) and Erk1/2, leading to proliferation. The phosphorylated EGFR causes tyrosine phosphorylation of phosphatidylinositol 3-kinase (PI3K) resulting in the activation of pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK-1), Akt, and mammalian target of rapamycin (mTor) phosphorylation, leading to cancer cellular survival. Activated PAC1 interacts with G_αq, leading to phospholipase C (PLC) stimulation and PI turnover. The IP₃ and diacylglycerol (DAG) released cause elevation of cytosolic Ca²⁺ and PKC activation, respectively. The PKC or Ca²⁺ causes Src tyrosine phosphorylation, leading to phosphorylation of focal adhesion kinase (FAK), pyruvate kinase 2 (PYK2), and paxillin, affecting cellular motility, secretion and migration. ER, endoplasmic reticulum; PIP2, phosphatidylinositol bisphosphate; RTK, receptor tyrosine kinase.