Blockade of EGFR Activation Promotes TNF-Induced Lung Epithelial Cell Apoptosis and Pulmonary Injury

Abstract

Pneumonitis is the leading cause of death associated with the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) against non-small cell lung cancer (NSCLC). However, the risk factors and the mechanism underlying this toxicity have not been elucidated. Tumor necrosis factor (TNF) has been reported to transactivate EGFR in pulmonary epithelial cells. Hence, we aimed to test the hypothesis that EGFR tyrosine kinase activity regulates TNF-mediated bronchial epithelial cell survival, and that inhibition of EGFR activity increases TNF-induced lung epithelial cell apoptosis. We used surfactant protein C (SPC)-TNF transgenic (tg) mice which overexpress TNF in the lungs. In this model, gefitinib, an EGFR-TKI, enhanced lung epithelial cell apoptosis and lymphocytic inflammation, indicating that EGFR tyrosine kinase prevents TNF-induced lung injury. Furthermore, IL-17A was significantly upregulated by gefitinib in SPC-TNF tg mice and p38MAPK activation was observed, indicative of a pathway involved in lung epithelial cell apoptosis. Moreover, in lung epithelial cells, BEAS-2B, TNF stimulated EGFR transactivation via the TNF-α-converting enzyme in a manner that requires heparin binding (HB)-EGF and transforming growth factor (TGF)-α. These novel findings have significant implications in understanding the role of EGFR in maintaining human bronchial epithelial cell homeostasis and in NSCLC treatment.

Keywords: EGFR; TNF; apoptosis; lung injury; transactivation.

Figure 1

Gefitinib enhances lymphocyte infiltration and causes lung tissue inflammation. Lung tissue sections of (a) wild type (WT) mice and (b) surfactant protein C-tumor necrosis factor (SPC-TNF) transgenic (tg) mice were stained with hematoxylin and eosin. The images were obtained at a magnification of 40× and 200×, in the upper and lower rows, respectively. (c) Body weight changes are shown for WT and SPC-TNF Tg mice over 14 days of daily oral treatment with 1% tween 80 as control or with gefitinb (100 or 200 mg/kg). The WT; C57BL/6 or SPC-TNF tg mice used in these studies were 6-8 weeks old. The scale bars represent 500 μm and 100 μm for 40× and 200× magnification, respectively. Data in the figure represent mean body weights ± SEMs; * p < 0.05, Student’s t-test.

Figure 2

Gefitinib enhances lung inflammation in surfactant protein C-tumor necrosis factor (SPC-TNF) transgenic (tg) mice. Indocyanine green (ICG)-loaded liposomes carrying sialyl Lewis X were used to detect regions of inflammation in whole lungs of (a) wildtype (WT) and (b) SPC-TNF tg mice after daily gefitinib (100 mg/kg) treatment for 14 days. Images were captured using the Clairvivo OPT in vivo imaging system (Shimadzu, Kyoto, Japan).

Figure 3

Gefitinib enhanced lung epithelial cell apoptosis via p38 mitogen-activated protein kinase (MAPK) pathway in surfactant protein C-tumor necrosis factor (SPC-TNF) transgenic (tg) mice. Wildtype (WT) and SPC-TNF tg mice were administered daily with gefitinib (100 or 200 mg/kg) or 1% tween 80 by oral gavage for 14 days. Paraffin-embedded lung tissues were studied for apoptosis using in situ oligo ligation (ISOL) staining, and apoptotic nuclei were labeled with peroxidase in (a) WT and (c) SPC-TNF tg mice. Percentage of apoptosis was determined for at least 500 cells in (b) WT and (d) SPC-TNF tg mice. * p < 0.05 compared with control, Student’s t-test. The scale bar represents 200 μm and 100 μm for 100× and 200× magnification, respectively. (e) Phosphorylated (P) epithelial growth factor receptor (P-EGFR; Y1068) was detected in paraffin-embedded lung tissues by immunostaining. The scale bar represents 50 μm for 400× magnification. (f,g) The percentages of positive cells are shown in the control lung tissues of WT and SPC-TNF tg (f), and in the SPC-TNF tg mice, where both a control and gefitinib were administered (g). * p < 0.05 compared with WT (f), or control (g), Student’s t-test. (h) Western blot analysis of EGFR/P-EGFR (Y1068), AKT/P-AKT, and extracellular-signal-regulated kinase (ERK)1/2/P-ERK1/2 in lung tissues of WT and SPC-TNF tg mice. β-actin was included as a loading control. (i) Western blot analysis of p38 mitogen-activated protein kinase (MAPK)/P-p38 MAPK and MAPK kinase (MKK) 3/6/P-MKK3/6 in lung tissues of WT and SPC-TNF tg mice. β-actin was included as a loading control. Band intensity of phosphorylated EGFR (P-EGFR), AKT (P-AKT), ERK1/2 (P-ERK1/2), MKK3/6 (P-MKK3/6), and p38MAPK (P-p38MAPK) was quantified and determined using ImageJ software (NIH). The relative ratios compared to the control are shown.

Figure 3

Gefitinib enhanced lung epithelial cell apoptosis via p38 mitogen-activated protein kinase (MAPK) pathway in surfactant protein C-tumor necrosis factor (SPC-TNF) transgenic (tg) mice. Wildtype (WT) and SPC-TNF tg mice were administered daily with gefitinib (100 or 200 mg/kg) or 1% tween 80 by oral gavage for 14 days. Paraffin-embedded lung tissues were studied for apoptosis using in situ oligo ligation (ISOL) staining, and apoptotic nuclei were labeled with peroxidase in (a) WT and (c) SPC-TNF tg mice. Percentage of apoptosis was determined for at least 500 cells in (b) WT and (d) SPC-TNF tg mice. * p < 0.05 compared with control, Student’s t-test. The scale bar represents 200 μm and 100 μm for 100× and 200× magnification, respectively. (e) Phosphorylated (P) epithelial growth factor receptor (P-EGFR; Y1068) was detected in paraffin-embedded lung tissues by immunostaining. The scale bar represents 50 μm for 400× magnification. (f,g) The percentages of positive cells are shown in the control lung tissues of WT and SPC-TNF tg (f), and in the SPC-TNF tg mice, where both a control and gefitinib were administered (g). * p < 0.05 compared with WT (f), or control (g), Student’s t-test. (h) Western blot analysis of EGFR/P-EGFR (Y1068), AKT/P-AKT, and extracellular-signal-regulated kinase (ERK)1/2/P-ERK1/2 in lung tissues of WT and SPC-TNF tg mice. β-actin was included as a loading control. (i) Western blot analysis of p38 mitogen-activated protein kinase (MAPK)/P-p38 MAPK and MAPK kinase (MKK) 3/6/P-MKK3/6 in lung tissues of WT and SPC-TNF tg mice. β-actin was included as a loading control. Band intensity of phosphorylated EGFR (P-EGFR), AKT (P-AKT), ERK1/2 (P-ERK1/2), MKK3/6 (P-MKK3/6), and p38MAPK (P-p38MAPK) was quantified and determined using ImageJ software (NIH). The relative ratios compared to the control are shown.

Figure 4

Transactivation of the epithelial growth factor receptor (EGFR) protects lung epithelial cells from apoptosis induced by tumor necrosis factor (TNF). Lung epithelial cells (BEAS-2B) were cultured and serum-starved for 16 h prior to treatment unless otherwise indicated (a–e). (a–d) BEAS-2B cell lysates were immunoblotted with the indicated primary antibodies after the following treatments, and β-actin was included as a loading control. (a) BEAS-2B cells were treated with TNF (100 ng/mL) for the indicated duration. Then the cells were treated with EGF (10 ng/mL) for 15 min as positive control. (b) BEAS-2B cells were treated with the indicated concentrations of TNF for 15 min. (c) BEAS-2B cells were treated with gefitinib (1 µM) for 1 h, and then treated with either EGF (10 ng/mL) or TNF (100 ng/mL) for 15 min. Band intensity of phosphorylated EGFR (P-EGFR) was quantified and determined using the ImageJ software (NIH). The relative ratios compared to the control are shown. (d) BEAS-2B cells were cultured on collagen-coated dishes, and transfected with either non-targeting (N/T) small interfering (siRNA) or with siRNA against EGFR for 48 h; cells were serum-starved for 16 h and then treated with EGF (10 ng/mL) or TNF (100 ng/mL) for 15 min. (e) BEAS-2B cells were treated with TNF (100 ng/mL) for 8 h in the presence or absence of gefitinib (1 µM). Cells were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, with apoptotic nuclei labeled with fluorescein isothiocyanate (FITC, green) and nuclei labeled with 4′,6-diaminido-2-phenylindole (DAPI, blue). FITC- and DAPI- labeled images were taken from the same field. The scale bar represents 100 μm. (f) Percentages of apoptotic cells were determined out of at least 500 cells. * p < 0.05, Student’s t-test, compared with the control, the treatment with TNF and with gefitinib.

Figure 5

TAPI-1 enhances tumor necrosis factor (TNF)-induced lung epithelial cell apoptosis. Lung epithelial cells (BEAS-2B) were cultured and serum-starved for 16 h prior to treatment unless otherwise indicated (a–d). (a) BEAS-2B cells were treated with TAPI-1 (10 µM), a TNF-α converting enzyme (TACE) inhibitor, for 1 h, and then treated with either TNF (100 ng/mL) or epithelial growth factor (EGF, 10 ng/mL) for 15 min. Cell lysates were immunoblotted with the indicated primary antibodies. β-actin was included as a loading control. (b) BEAS-2B cells were treated with TNF (100 ng/mL) for 8 h in the presence or absence of TAPI-1 (10 µM). Cells were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, with apoptotic nuclei labeled with fluorescein isothiocyanate (FITC, green) and nuclei labeled with 4′,6-diaminido-2-phenylindole (DAPI, blue). FITC- and DAPI- labeled images were taken from the same field. The scale bar represents 50 μm. (c) Percentages of apoptotic cells were determined out of at least 500 cells. * p < 0.05, Student’s t-test, compared with the control, the treatment with TNF or TAPI-1. (d) BEAS-2B cells were treated with TNF (100 ng/mL) for 8 h in the presence or absence of a MAPK/ERK kinase (MEK) inhibitor, U0126 (10 µM), or a PI3 kinase (P13K) inhibitor, wortmannin (1 µM). Cells were fixed for TUNEL assay, with apoptotic nuclei labeled with FITC (green) and nuclei labeled with DAPI (blue). FITC- and DAPI- labeled images were taken from the same field. The scale bar represents 100 μm. (e) Percentages of apoptotic cells were determined out of at least 500 cells. * p < 0.05 compared with the control, the treatment with TNF or with wortmannin or U0126.

Figure 6

Epithelial growth factor receptor (EGFR) ligands, transforming growth factor (TGF)-α and heparin binding (HB)-EGF, promote tumor necrosis factor (TNF)-induced EGFR transactivation via the TNF-α converting enzyme (TACE). Lung epithelial cells (BEAS-2B) were cultured and serum-starved for 16 h prior to different treatments (a–e). Cell lysates were immunoblotted, and β-actin was included as a loading control (a–c). (a) BEAS-2B cells were treated with TNF (100 ng/mL) for 15 min following pretreatment with control antibody (control-IgG) or anti-EGFR antibody cetuximab (10 µg/mL) for 1 h. (b) BEAS-2B cells were treated with TNF (100 ng/mL) for 15 min with or without pretreatment (1 h) with neutralizing antibodies (10 µg/mL) for various kinds of EGFR ligands: EGF, amphiregulin (AR), TGF-β(TGF), β-cellulin (BTC), HB-EGF, and epiregulin (EPR). Band intensity of phosphorylated EGFR (P-EGFR) was quantified and determined using the ImageJ software (NIH). The relative ratios compared to the control are shown. (c) BEAS-2B cells were treated with TNF (100 ng/mL) for 15 min with or without pretreatment (1 h) with neutralizing antibodies against TGF-α (10 µg/mL) and HB-EGF (10 µg/mL). (d,e) BEAS-2B cells were treated with TNF (100 ng/mL) for the indicated duration in the presence or absence of the TACE inhibitor TAPI-1 (1 µM). (d) HB-EGF and (e) TGF-α protein levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (f,g) BEAS-2B cells were cultured on collagen-coated dishes and transfected with non-targeting (N/T) small interfering RNA (siRNA) or TACE for 48 h. The cells were serum-starved for 16 h, and then treated with TNF (100 ng/mL) for 60, 120, and 240 min. The protein levels of (f) HB-EGF and (g) TGF-α in the culture supernatants were measured by ELISA. (h) BEAS-2B cells were cultured on collagen-coated dishes and transfected with N/T siRNA or TACE for 72 h. The cell lysates were collected and protein levels of TACE were determined by Western blot analysis.