Early growth response-1 induces and enhances vascular endothelial growth factor-A expression in lung cancer cells

Abstract

Vascular endothelial growth factor-A (VEGF-A) is crucial for angiogenesis, vascular permeability, and metastasis during tumor development. We demonstrate here that early growth response-1 (EGR-1), which is induced by the extracellular signal-regulated kinase (ERK) pathway activation, activates VEGF-A in lung cancer cells. Increased EGR-1 expression was found in adenocarcinoma cells carrying mutant K-RAS or EGFR genes. Hypoxic culture, siRNA experiment, luciferase assays, chromatin immunoprecipitation, electrophoretic mobility shift assays, and quantitative RT-PCR using EGR-1-inducible lung cancer cells demonstrated that EGR-1 binds to the proximal region of the VEGF-A promoter, activates VEGF-A expression, and enhances hypoxia inducible factor 1alpha (HIF-1alpha)-mediated VEGF-A expression. The EGR-1 modulator, NAB-2, was rapidly induced by increased levels of EGR-1. Pathology samples of human lung adenocarcinomas revealed correlations between EGR-1/HIF-1alpha and VEGF-A expressions and relative elevation of EGR-1 and VEGF-A expression in mutant K-RAS- or EGFR-carrying adenocarcinomas. Both EGR-1 and VEGF-A expression increased as tumors dedifferentiated, whereas HIF-1alpha expression did not. Although weak correlation was found between EGR-1 and NAB-2 expressions on the whole, NAB-2 expression decreased as tumors dedifferentiated, and inhibition of DNA methyltransferase/histone deacetylase increased NAB-2 expression in lung cancer cells despite no epigenetic alteration in the NAB-2 promoter. These findings suggest that EGR-1 plays important roles on VEGF-A expression in lung cancer cells, and epigenetic silencing of transactivator(s) associated with NAB-2 expression might also contribute to upregulate VEGF-A expression.

Figure 1

Analysis of EGR-1, HIF-1α, VEGF-A, NAB-2, and NAB-1 expression in cultivated lung cancer cells. A: Western blot analysis of whole-cell lysates from adenocarcinoma cell lines containing wild-type K-RAS and EGFR (TKB6 and TKB14), mutant K-RAS (A549, H358), or mutant EGFR (PC9 and HCC827) and from control and EGR-1-transfected TKB5 cells (TKB5pRevEmpty and TKB5pRevEGR-1) and their parental cells (TKB5). Lysates were separated by SDS-PAGE and immunoblotted using antibodies against the proteins indicated. β-Actin was analyzed as an internal control. Lung cancer cells without mutation of K-RAS/EGFR expressed minimal amounts of EGR-1, NAB-2, and VEGF-A, whereas these proteins were readily detectable in mutant K-RAS–carrying cells (A549 and H358) and mutant EGFR-carrying cells (PC9 and HCC827). EGR-1 was abundantly induced by doxycycline treatment for 24 hours in TKB5pRevEGR-1 cells, and NAB-2 and VEGF-A were also promptly induced by EGR-1 induction. NAB-1 was constitutively expressed, but HIF-1α was not detectable in all cell lines. B: Quantitative RT-PCR (qRT-PCR) analysis in TKB5pRevEmpty cells (open bars) and TKB5pRevEGR-1 cells (closed bars) after doxycycline treatment for the indicated times. HIF-1α expression weakly increased after 48 hours, irrespective of EGR-1 induction. However, EGR-1 induction by doxycycline treatment (1 μg/ml final concentration) up-regulated VEGF-A expression, and the expression levels of TKB5pRevEGR-1 cells (72 hours and 96 hours) were further increasing and much higher than those of TKB5pRevEmpty cells. NAB-2 was promptly induced by EGR-1, whereas NAB-1 expression was not altered. C: Association of VEGF-A expression with EGR-1 and HIF-1α. TKB5pRevEGR-1 cells were cultivated in the hypoxic condition (0.5% O₂ and 5% CO₂) for eight hours. Doxycycline (1 μg/ml final concentration) was added 24 hours before harvest. Lysates were separated by SDS-PAGE and immunoblotted using antibodies against the proteins indicated. β-Actin served as an internal control. Relative expression level of VEGF-A was calculated as follows: (intensity of VEGF-A/intensity of β-actin)/[intensity of VEGF-A (no EGR-1 induction and no hypoxic culture)/intensity of β-actin (no EGR-1 induction and no hypoxic culture)]. VEGF-A was not only induced by EGR-1 and HIF-1α but also enhanced by coexpression of EGR-1 and HIF-1α. D: Alteration of VEGF-A expression by forced repression of EGR-1 in H358 cells. The EGR-1–specific or scrambled control siRNA was treated on H358 cells for 24 hours. β-Actin served as an internal control. Relative expression levels of EGR-1 and VEGF-A were calculated as follows: (intensity of EGR-1 or VEGF-A/intensity of β-actin)/[intensity of EGR-1 or VEGF-A (scrambled siRNA)/intensity of β-actin (scrambled siRNA)]. The VEGF-A expression decreased by EGR-1 repression.

Figure 2

EGR-1 binds to the VEGF-A promoter and activates gene expression. A: VEGF-A promoter assays. Top: TKB5pRevEmpty or TKB5pRevEGR-1 cells were transiently cotransfected with full-length VEGF-A promoter-luciferase reporter construct and control vector for 24 hours with or without doxycycline treatment, and the cells were harvested, lysed, and subjected to luciferase assays. Data reflect means + SD from three experiments performed in triplicate. The mean luciferase activity of full-length VEGF-A promoter in TKB5-pRevEmpty without doxycycline treatment is set to 100%. Bottom: TKB5pRevEGR-1 cells were transiently transfected with wild-type VEGF-A promoter-luciferase reporter construct or the indicated VEGF-A promoter deletion or mutation construct for 24 hours in the presence of doxycycline, and the cells were harvested, lysed, and subjected to luciferase assays. Data reflect the means + SD from three experiments performed in triplicate. The mean luciferase activity of full-length VEGF-A promoter in TKB5-pRevEGR-1 is set to 100%. B: ChIP-based PCR analysis using doxycycline-treated or untreated TKB5pRevEGR-1 cells. DNA fragments conjugated with nuclear proteins were immunoprecipitated with rabbit anti–EGR-1, nonimmunized rabbit IgG, or rabbit anti–HIF-1α antibody. Immunoprecipitated DNA was PCR-amplified using primers for a 377-bp fragment containing the putative EGR-1-binding sites (Table 1) and then separated by agarose gel electrophoresis. Input indicates input DNA; water, no DNA. Weak signal was amplified from the EGR-1 immunoprecipitates even in the condition of no EGR-1 induction (top). This result was consistent with the fact that TKB5pRevEGR-1 constitutively expressed low level of EGR-1–like parental TKB5 cells (Figure 1A). The specific signal was abundantly amplified by EGR-1 induction (bottom). C: EMSA analysis using recombinant EGR-1 (left) and nuclear lysates from TKB5pRevEGR-1 with or without EGR-1 induction (right). Left: Recombinant EGR-1 was incubated with the indicated digoxigenin-labeled EGR-1-binding site probe with or without unlabeled competitor probe (100-fold molar excess) for the consensus EGR-1–binding motif 5′-GCGGGGGCG-3′. EGR-1 bound with highest affinity to sites 1 and 2-4 (arrow). Left, insets: Recombinant EGR-1 was incubated with the indicated digoxigenin-labeled EGR-1–binding site 1 (top) or site 2-4 (bottom) probe with the indicated antibody. Right: Nuclear lysates extracted from EGR-1–induced or uninduced TKB5pRevEGR-1 cells was incubated with the indicated digoxigenin-labeled EGR-1–binding site 1 (top) or site 2-4 (bottom) probe with the indicated antibody. Abundant EGR-1–specific complex was formed by EGR-1 induction and the signal supershifted by anti–EGR-1 antibody. Arrow, EGR-1–specific signal; asterisk, Supershifted EGR-1–specific signal.

Figure 3

Immunohistochemistry of VEGF-A, EGR-1, and HIF-1α expression in surgically resected lung adenocarcinomas. Representative images are shown. A: In a well-differentiated adenocarcinoma, VEGF-A expression is diffuse in the cytoplasm, and EGR-1 is highly expressed in nuclei; HIF-1α is not expressed. B: In a poorly-differentiated adenocarcinoma, VEGF-A expression is diffuse in the cytoplasm, and EGR-1 and HIF-1α are both primarily expressed in nuclei. Scale bars = 100 μm. C: Relationships between EGR-1 and VEGF-A, between HIF-1α and VEGF-A, between EGR-1 and NAB-2, and between EGR-1 and HIF-1α (Pearson’s correlation coefficient test). The expression score (ES) of EGR-1 was correlated with that of VEGF-A and NAB-2, and the ES of HIF-1α was also correlated with that of VEGF-A. However, there was no correlation between EGR-1 and HIF-1α.

Figure 4

NAB-2, EGR-1, and VEGF-A promoter CpG islands are not hypermethylated in lung cancer cells. A: Localization of CpG islands and regions subjected to bisulfite-modified DNA sequencing (BS). Nucleotide positions are relative to the transcription start site. B and C: Summary of BS analysis of NAB-2, EGR-1, and VEGF-A promoter methylation using bisulfite-modified DNA extracted from cultivated lung cancer cells (B) and in vivo adenocarcinoma cells (C). Nucleotide positions are relative to the transcription start site. Closed circle indicates methylated CpG site; open circle, unmethylated CpG site. No severe methylation was noted in NAB-2, EGR-1, or VEGF-A promoters of all cell lines and in vivo adenocarcinoma cells examined. Parenthesis indicates the expression score of each case. W1–W2, M1–M2, and P1–P6 indicated well-differentiated (W), moderately differentiated (M), and poorly differentiated (P) adenocarcinoma samples, respectively. D: Alteration of NAB-2 expression via 5-aza-2′-deoxycytidine treatment in cultivated lung cancer cells. Cells were treated with 10 nmol/ml of 5-aza-2′-deoxycytidine (AZA) for 72 hours or with 300 ng/ml of trichostatin A (TSA) for 24 hours. In addition, cells were also treated with AZA for 48 hours and then with a combination of AZA and TSA for an additional 24 hours. Apparent increase of NAB-2 expression was found in TKB6, TKB14, and H358 cells through the AZA and/or TSA treatment.