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. 2016 Dec;12(6):4598-4604.
doi: 10.3892/ol.2016.5287. Epub 2016 Oct 18.

Epidermal growth factor receptor mutation enhances expression of vascular endothelial growth factor in lung cancer

Ming-Szu Hung  1 , I-Chuan Chen  2 , Paul-Yann Lin  3 , Jr-Hau Lung  4 , Ya-Chin Li  4 , Yu-Ching Lin  1 , Cheng-Ta Yang  5 , Ying-Huang Tsai  6
Affiliations

Epidermal growth factor receptor mutation enhances expression of vascular endothelial growth factor in lung cancer

Ming-Szu Hung et al. Oncol Lett. 2016 Dec.

Abstract

Epidermal growth factor receptor (EGFR) activation has been demonstrated to have a critical role in tumor angiogenesis. In the present study, the correlation between EGFR mutations and vascular endothelial growth factor (VEGF) was investigated in lung cancer cell lines and non-small-cell lung cancer (NSCLC) tumor tissues. VEGF levels were significantly increased in culture medium of lung cancer cells and NSCLC tissues with EGFR mutations (H1650 vs. A549, P=0.0399; H1975 vs. A549, P<0.0001). Stable lung cancer cell lines expressing mutant (exon 19 deletion, E746-A750; exon 21 missense mutation, L858R) and wild-type EGFR genes were established. Significantly increased expression of VEGF and stronger inhibitory effects of gefitinib to VEGF expression were observed in exon 19 deletion stable lung cancer cells (exon 19 deletion vs. wild-type EGFR, P=0.0005). The results of the present study may provide an insight into the association of mutant EGFR and VEGF expression in lung cancer, and may assist with further development of targeted therapy for NSCLC in the future.

Keywords: erpidermal growth factor receptor; lung cancer; mutation; vascular endothelial growth factor.

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Figures

Figure 1.
Figure 1.
(A) VEGF levels secreted from cell lines with EGFR mutations (H1975 and H1650) and wild-type EGFR (A549 and H460). VEGF was measured by enzyme-linked immunosorbent assay as described previously. VEGF values are normalized to total cell numbers. Values are expressed as the means ± SD of triplicate experiments. (B) VEGF expression was determined by immunohistochemical staining in mutant and wild-type non-small cell lung cancer tissues. The expression of VEGF was quantified by IRS and values are expressed as the mean ± SD. Representative pictures for VEGF expression in EGFR mutant and wild-type non-small cell lung cancer tissues are also shown. EGFR M-, wild-type EGFR; EGFR M+, mutant EGFR. Magnification, ×200. VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor; SD, standard deviation; URS, immunoreactive score.
Figure 2.
Figure 2.
(A) Western blot analysis of EGFR and Akt expression/phosphorylation in EGFR transfected A549 stable cell lines. EGFR expression was normalized to the EV group using γ-tubulin as the internal control. (B) Western blot analysis of EGFR and Akt expression/phosphorylation in pooled EGFR transfected A549 stable cell lines with and without 100 ng/ml of EGF for 10 min following serum starvation for 48 h. L5 and L7 clones were pooled as L858R. D6 and D7 clones were pooled as DEL. EGFR, epidermal growth factor receptor; EV, empty vector; WT, wild-type EGFR; L, L858R; D, DEL E746-A750; p, phophorylated.
Figure 3.
Figure 3.
(A) Expression of VEGF mRNA in EGFR transfected A549 stable cells. (B) Western blot analysis of VEGF in EGFR transfected A549 stable cells. VEGF expression was normalized to the EV group using β-actin as the internal control. (C) IHC analysis of VEGF in EGFR transfected A549 stable cells. Magnification, ×200. (D) Intensities of IHC staining for VEGF were quantified. VEGF intensity values were normalized to the EV group. Values are expressed as the mean ± standard deviation of six experiments. VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor; EV, empty vector; IHC, immunohistochemical; WT: wild-type EGFR.
Figure 4.
Figure 4.
(A) Expression of VEGF in culture medium of EGFR transfected A549 stable lung cancer cell lines with or without EGF (100 ng/ml) stimulation for 24 h was measured by enzyme-linked immunosorbent assay. Groups with gefitinib (20 µM) and EGF stimulation were also measured. Values are expressed as the mean ± SD of triplicate experiments normalized to cell numbers and subsequently normalized to the group of empty vector transfected cells without EGF stimulation. (B) HUVECs were co-cultured with EGFR transfected A549 stable lung cancer cell lines in a Transwell system for 5 days. Cells in each group were counted. Values are expressed as the mean ± SD of triplicate experiments. (C) Inhibition effects of VEGF in culture medium with EGF (100 ng/ml) stimulation for 24 h were measured following gefitinib treatment. Values are expressed as the mean ± SD of triplicate experiments normalized to each group of EGFR transfected A549 stable lung cancer cell lines without gefitinib treatment. EV, Empty vector; WT, wild-type EGFR; VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor; SD, standard deviation; HUVECs, human umbilical vein endothelial cells; IRESSA, gefitinib.
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