Abstract
Background: DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied. Materials and Methods: A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene. Results: Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not. Conclusion: Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.
Epidermal growth factor receptor (EGFR) is frequently overexpressed in various solid tumors (1, 2) and is regarded as a definitive oncogene. Accumulating data on EGFR and its signal pathway in cancer cells suggests that EGFR is a promising therapeutic target molecule; indeed, benefits from treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) and anti-EGFR antibody have been confirmed in clinical settings (3, 4). Common EGFR mutations of DelE746_A750 and L858R, characterized by 15-base in-frame deletions or substitutions clustered around the ATP-binding site in exons 19 and 21 of EGFR, have been identified in patients with non-small cell lung cancer (NSCLC); these mutations are major determinants of sensitivity to EGFR-TKIs (5-8). Such mutations confer a constitutively active EGFR signal pathway to cancer cells (9).
The activated EGFR signal pathway has been intensively investigated, including studies on alterations in downstream signaling, the underlying mechanism responsible for sensitivity to EGFR-TKIs, the involvement in carcinogenesis, oncogene addiction, and clinico-pathological analyses. It has been previously reported that a lung cancer cell line, PC-9, with a deletional mutant of EGFR (delE746_A750) was hypersensitive to EGFR-TKIs and that this mutant EGFR was constitutively active and activated the ERK and AKT pathways (10-13). However, the changes in gene expression that occur in mutant EGFR-harboring cells have not been fully studied.
To identify changes in the gene expressions of downstream molecules that arise as a result of EGFR mutation and activated EGFR signaling, a microarray analysis of cells, in which the DelE746_A750-type of EGFR mutation had been stably introduced, was performed.
Materials and Methods
Reagents. The purified recombinant human EGF was purchased from R&D systems (Minneapolis, MN, USA). LY294002 2-(4-Morpholinyl)-8-phenyl-4H-benzopyran-4-one was purchased from Calbiochem (San Diego, CA, USA), U0126 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadine was purchased from Cell Signaling Technology (Beverly, MA, USA).
Expression constructs and viral production. Full-length cDNA of wild-type EGFR was amplified by RT-PCR from a human embryonal kidney cell line (HEK293), and mutant EGFR (delE746_A750) was amplified from an NSCLC cell line (PC-9) (10, 14). Wild-type and mutant EGFR cDNA in a pcDNA3.1 vector (Clontech, Palo Alt, CA, USA) was cut out and introduced into a pQCLIN retroviral vector (BD Biosciences Clontech, San Diego, CA, USA) together with EGFP, followed by the internal ribosome entry sequence (IRES) to monitor the expression of the inserts indirectly. A pVSV-G vector (Clontech, Palo Alt, CA, USA) for the constitution of the viral envelope and pQCXIX constructs were co-transfected into the GP2-293 cells using FuGENE6 transfection reagent. Briefly, 80% confluent cells cultured on a 10-cm dish were transfected with 2 μg of pVSV-G plus 6 μg of pQCXIX vectors. Forty-eight hours after transfection, the culture medium was collected and the viral particles were concentrated by centrifugation at 15,000 g for 3 h at 4°C. The viral pellet was then resuspended in fresh RPMI1640 medium. The titer of the viral vector was calculated by counting the EGFP-positive cells that were infected by serial dilutions of virus-containing medium, and the multiplicity of infection (MOI) was then determined.
Cell culture and transfection. The HEK293 cell line was cultured in DMEM (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin (Sigma) in a humidified atmosphere of 5% CO2 at 37°C. The HEK293 cells were retrovirally transfected with the mock, wild-type and mutant EGFR, and the stable established cell lines were designated as HEK293-Mock, HEK293-Wild and HEK293-Del.
Real-time RT-PCR. One microgram of total RNA from a cultured cell line was converted to cDNA using a GeneAmp® RNA-PCR kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was carried out using the Applied Biosystems 7900HT Fast Real-time PCR System (Applied Biosystems) under the following conditions: 95°C for 6 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. GAPD was used to normalize the expression levels in the subsequent quantitative analyses. To amplify the target genes, the following primers were purchased from TaKaRa (Yotsukaichi, Japan): EGR1-FW, GTA CAG TGT CTG TGC CAT GGA TTT C; EGR1-RW, GAG GAT CAC CAT TGG TTT GCT TG; GAPD-FW, GCA CCG TCA AGG CTG AGA AC; and GAPD-RW, ATG GTG GTG AAG ACG CCA GT. The results of three independent experiments were analyzed.
In vitro growth-inhibition assay. The growth-inhibitory effects of AG1478 (Biomol International, Plymouth Meeting, PA, USA) on the HEK293-Mock, -Wild and -Del cells were examined using an MTT assay. A 180-μL volume of an exponentially growing cell suspension (2×103 cells/well) was seeded into 96-well microtiter plates and 20 μL of various drug concentrations were added. After incubation for 72 h at 37°C, 20 μL of MTT solution (5 mg/mL in PBS) were added to each well and the plates were incubated for an additional 3 h at 37°C. After centrifuging the plates at 400 g for 5 min, the medium was aspirated from each well and 200 μL of DMSO was added to each well to dissolve the formazan. The optical density was measured at 570 nm. The results of three independent experiments were analyzed.
Immunoblotting. The antibodies used for immunoblotting were as follows: anti-EGFR (Upstate Biotechnology), anti-phospho-EGFR (Tyr1068), anti-p44/42 MAP kinase, anti-phospho-p44/42 MAP kinase, anti-Akt (Cell Signaling), anti-EGR1, anti-βactin (Santa Cruz), and anti-phospho-Akt (Ser473) (BD Bioscience, SanJose, CA, USA). Sub-confluent cells were washed with cold PBS and harvested with Lysis A buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.0), 5 mM EDTA, 50 mM sodium chloride, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, and a protease inhibitor mix, complete™ (Roche Diagnostics). Whole-cell lysates and the culture medium were separated using a 2-15% gradient SDS-PAGE and blotted onto a polyvinylidene fluoride membrane. After blocking with 3% bovine serum albumin in a TBS buffer (pH 8.0) with 0.1% Tween-20, the membrane was probed with primary antibody. After rinsing twice with TBS buffer, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling) and washed, followed by visualization using an ECL detection system (Amersham) and LAS-3000 (Fujifilm, Tokyo, Japan). The immunoblotting was performed in two independent experiments.
Microarray analysis. The microarray procedure was performed according to the Affymetrix protocols (Santa Clara, CA, USA). In brief, the total RNA extracted from the cell lines was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) as a quality check, and cRNA was synthesized using the GeneChip® 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix). The labeled cRNAs were then purified and used to construct the probes. Hybridization was performed using the Affymetrix GeneChip HG-U133 Plus2.0 array for 16 h at 45°C. The signal intensities were measured using a GeneChip®Scanner3000 (Affymetrix) and converted to numerical data using the GeneChip Operating Software, Ver.1 (Affymetrix).
Statistical analysis. The microarray analysis was performed using the BRB Array Tools software ver. 3.3.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html), developed by Dr. Richard Simon and Dr. Amy Peng. The microarray analysis was performed as described previously (15). Additional statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, WA, USA) to calculate the standard deviation (SD) and statistically significant differences between each sample using the Student t-test. P-values of <0.05 were considered statistically significant.
Results
Early growth response 1 (EGR1) expression in mutant EGFR. The DelE746_A750-type EGFR mutation mediates a constitutively active EGFR signal and induces cellular hypersensitivity to EGFR-TKIs (11, 13). Mock, wild and mutant EGFR was introduced and stable cell lines was established as HEK293-Mock, -Wild and -Del cells. HEK293-Del cells showed increased phosphorylation levels of EGFR and ERK1/2 and were significantly hypersensitive to the EGFR-tyrosine kinase inhibitor AG1478, compared with the other cell lines (Figure 1A and 1B). To identify which gene expressions were changed by the EGFR mutation, a microarray analysis was performed for these stable cell lines. Twenty-three genes were identified as differentially expressed genes, the expressions of which differed by more than three-fold between HEK293-Wild and HEK293-Del cells (Table I). These genes included several cancer-related genes such as EGR1, GALNT3, TACSTD1 (EpCAM), MAFF, NLK, FOXN4, RUNX3 and CD70. Among them, EGR1 was the most up-regulated gene in the HEK293-Del cells (>10-fold higher than in HEK293-Mock and -Wild cells, Figure 1C, 1D). The ratios of the signal intensity relative to that in the HEK293-Mock cells were 3.0-fold in the HEK293-Wild cells and 34.5-fold in the HEK293-Del cells. Thus, the role of the EGR1 transcription factor in EGFR signal activation was the focus of subsequent studies.
EGF stimulates EGR1 expression. The mRNA and protein levels of EGR1 up-regulation were confirmed using real-time RT-PCR and western blotting for these stable cell lines. Real-time RT-PCR revealed that EGR1 mRNA expression in the HEK293-Wild cells was slightly (~3-fold) higher than that in the HEK293-Mock cells. On the other hand, EGR1 mRNA expression was remarkably increased in the HEK293-Del cells (133-fold, compared with the HEK293-Mock cells). Similar results were obtained for the protein levels (Figure 2A, 2B). These results indicate that EGR1 expression was constitutively up-regulated in the EGFR mutation-harboring cells.
To examine whether the up-regulation of EGR1 expression is regulated by EGFR signaling, the change in expression induced by EGF stimulation was evaluated. EGF increased EGR1 mRNA expression in HEK293-Mock, -Wild and -Del cells (Figure. 2C). EGR1 up-regulation by EGF was also confirmed by immunoblotting (Figure 2D). HEK293-Wild cells stimulated with EGF expressed EGR1 to the same extent as in HEK293-Del cells, possibly reflecting the constitutively active function of EGFR in the HEK293-Del cells. In addition, EGR1 expression was closely correlated with the phospho-ERK1/2 expression levels. These findings suggest that EGR1 expression is involved in the ERK1/2 pathway.
EGFR-TKI down-regulates EGR1 expression. To elucidate the further relationship between EGR1 up-regulation and EGFR signaling activity, the three cell lines were treated with EGFR-TKI. An EGFR-TKI, AG1478, inhibited the expression of both EGR1 mRNA (Figure 3A) and protein (Figure 3B). EGR1 expression was also correlated with the phospho-ERK1/2 expression levels detected by immunoblotting. These results support the concept that EGR1 up-regulation by mutant EGFR is regulated by EGFR signaling.
EGR1 expression is regulated through the ERK1/2 pathway. EGR1 is thought to be a downstream molecule in the ERK1/2 pathway (16). To elucidate whether EGR1 up-regulation in mutant EGFR cells is regulated via the ERK1/2 pathway, ERK1/2 and AKT, two major downstream pathways of EGFR was evaluated. LY294002, a phosphatidylinositol 3-kinase inhibitor, inhibited the phosphorylation levels of AKT but did not modify the expression of EGR1 (Figure 4A). However, the MEK inhibitor U0126 clearly down-regulated EGR1 expression in HEK293-Del cells (Figure 4B). EGR1 expression was consistent with the phospho-ERK1/2 expression levels. These results strongly suggest that EGR1 up-regulation by mutant EGFR is regulated through the ERK pathway. Based on these findings, a model was propossed to explain the up-regulation of EGR1 by mutant EGFR (Figure 4C). In this model, mutant EGFR activates the ERK pathway and induces EGR1 transcription.
Discussion
EGR1 transcription factor is induced by various stimuli, including growth factors, hypoxia, UV and cytokines, and mediates multiple cellular responses such as mitogenesis, differentiation, cellular survival, anti-apoptosis, angiogenesis and apoptosis (17). In cancer biology, EGR1 is basically regarded as a tumor suppressor gene because it directly regulates p53, PTEN and TGFβ1. Deletion of the EGR1-containing 5q31 region has been associated with a certain type of lymphoma and small cell lung carcinoma. Low EGR1 expression in tumor tissue is frequently observed in breast cancer, glioblastoma and other solid tumors (18). In contrast, the oncogenic property of EGR1 is observed in prostate cancer (19).
An increased expression of EGR1 was observed in mutant EGFR cells. Previous reports have demonstrated that mutant EGFR is oncogenic in non-small cell lung cancer (20). However, Ferraro et al. have demonstrated that EGR1 expression is strongly correlated with PTEN expression and that patients with high levels of EGR1 had better overall and disease-free survival periods than patients with low levels of EGR1 in patients with NSCLC (21). It was speculated that the overexpression of EGR1 in mutant EGFR cells may play some role in the biological behaviors of mutant EGFR in cancer.
In general, ERK and JNK kinases phosphorylate ternary complex factors (TCF), which cooperate with serum response factor (SRF) to induce EGR1 transcription in vascular biology (22). EGR1 can displace Sp1 and other transcription factors, and EGR1 transactivation leads to the transcription of many EGR1-target genes. To date, several putative EGR1-target genes related to cancer have been identified, including cyclin D, EGFR, FGF, IGF-I, thymidine kinase, PDGF-A, Bcl2, CD44, p53, PTEN, TNF-α and VEGF. Further investigation of the biological role of EGR1 overexpression in mutant EGFR may lead to a better understanding of the roles of mutant EGFR in cancer cells.
In conclusion, it was found that mutant EGFR induced EGR1 overexpression and that this overexpression was correlated with EGFR signal activation through ERK1/2. These results provide a novel insight into the oncogenic properties of EGFR in cancer cells.
Acknowledgements
This work was supported by funds for the Third-Term Comprehensive 10-Year Strategy for Cancer Control and the program for the promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NiBio) and the Japan Health Sciences Foundation.
We thank Dr. Richard Simon and Dr. Amy Peng for providing the BRB ArrayTools software.
- Received October 20, 2008.
- Revision received January 19, 2009.
- Accepted February 13, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved