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. Author manuscript; available in PMC: 2015 Aug 14.
Published in final edited form as: Oncogene. 2013 Sep 30;33(33):4253–4264. doi: 10.1038/onc.2013.400

An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

Li Li 1,, Sharmistha Chakraborty 1,, Chin-Rang Yang 2, Kimmo J Hatanpaa 3, Daisha J Cipher 6, Vineshkumar Thidil Puliyappadamba 1, Alizeh Rehman 1, Ameena J Jiwani 1, Bruce Mickey 4, Christopher Madden 4, Jack Raisanen 3, Sandeep Burma 5, Debabrata Saha 5, Zhixiang Wang 7, Sandeep C Pingle 9, Santosh Kesari 9, David A Boothman 2, Amyn A Habib 1,2,8,*
PMCID: PMC3969863  NIHMSID: NIHMS549270  PMID: 24077285

Abstract

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention.

Keywords: EGFRvIII, glioblastoma, EGFR wild type, activation, model, dimerization, HB-EGF, ligands

Introduction

Aberrant receptor tyrosine kinase signaling plays a key role in glioblastoma in glioblastoma (GBM) (1–2). Although increased expression of other RTKs such as PDGFRA is detected in GBMs (3), EGFR gene amplification and mutation are the most striking abnormality detected in about 40–50% of patients with GBM and usually found in the classical subtype of GBM (2). In about 50% of tumors with EGFR amplification, a specific EGFR mutant (EGFR Type III, EGFRvIII, de2–7, ΔEGFR) can be detected (4–5). EGFRvIII is generated from a deletion of exons 2–7 of the EGFR gene, which results in an in frame deletion of 267 amino acids. EGFRvIII is unable to bind ligand and signals constitutively. Since EGFRvIII is a tumor specific EGFR mutant, it has attracted intense interest both for its potential role in the biology of GBM, as well as a target for treatment (5–6). Anti-EGFR treatment with EGFR specific kinase inhibitors appears to be less successful in GBM compared to lung cancer, possibly secondary to altered kinetics of inhibitor binding or sensitivity of EGFRvIII compared to EGFR mutants in lung cancer (7–8).

A number of studies have demonstrated that the EGFRvIII is more tumorigenic than the EGFR wild type (EGFRwt) (9–12). EGFRvIII does not bind ligand, becomes tyrosine phosphorylated and appears to signal constitutively when expressed in cells (13). Dimerization of EGFRvIII plays an important role in its activation (14–17). EGFRvIII may homodimerize or heterodimerize with the EGFR. We and others have reported biologically significant interactions between EGFRwt and EGFRvIII in glioma, suggesting that key effector signals from EGFRvIII require participation of EGFRwt (18–19). These studies have provided evidence of EGFRvIII induced autocrine or paracrine loops in GBM mediated by RTK ligands such as HB-EGF (18) or cytokines such as IL6 (19), or heterodimerization between the receptors (16). Such interactions have the potential to profoundly alter the biological outcome of EGFRvIII signaling.

In this study, we show that EGFRwt activates EGFRvIII and is required for EGFRvIII to exert its oncogenic effect in glioma cells. Thus, when EGFRwt is silenced, tyrosine phosphorylation of EGFRvIII is highly attenuated. Conversely, increasing the EGFRwt level results in amplified EGFRvIII activation. Examination of the transcriptional program of EGFRvIII in the presence or absence of EGFRwt led to the identification of heparin binding epidermal growth factor like growth factor (HB-EGF) which is induced by EGFRvIII only in the presence of EGFRwt. HB-EGF is a ligand for EGFRwt and, remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII tyrosine phosphorylation/activation and oncogenicity. Importantly, we find that the EGFRwt is an important partner for EGFRvIII in promoting the malignant phenotype. Thus, silencing EGFRwt or HB-EGF in EGFRvIII expressing glioma cells severely inhibits tumor formation, while increased expression of EGFRwt or HB-EGF in EGFRvIII expressing cells accelerates tumor formation. Thus, we propose that EGFRvIII is activated by co-expressed EGFRwt, which is activated in turn, by HB-EGF induced by EGFRvIII in a feed forward loop.

Results

EGFRwt influences activation of EGFRvIII

EGFRwt and EGFRvIII are usually co-expressed in GBMs. To investigate the role of EGFRwt in EGFRvIII signaling, we used U251MG cells to conditionally express EGFRvIII in response to tetracycline (U251vIII cells, Figure 1A) as described previously (18). Next, we selectively and stably silenced endogenous EGFRwt but not EGFRvIII in U251vIII cells by using two different shRNA sequences directed against exon 3. We generated two clones with selective silencing of EGFRwt designated U251vIII-5 and U251vIII-2 (also referred to as clone 5 or 2), along with a control with scrambled shRNA (Figures 1B and 1I). U251vIII-5 has more efficient silencing of EGFRwt compared to U251vIII-2. In addition, we generated U251vIII clones constitutively overexpressing a HA-tagged EGFRwt designated as U251vIII-15 and U251vIII-18 (clone 15 and 18) as shown in Figures 1C, D, E and I. Thus, we generated U251EGFRvIII expressing cell lines that conditionally express EGFRvIII in response to tetracycline on a background of overexpressed EGFRwt (15 and 18), endogenous EGFRwt (control) and silenced EGFRwt (2 and 5). EGFRvIII expression induced by tetracycline is similar in all lines.

Figure 1.

Figure 1

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A. U251MG cells expressing EGFRvIII or EGFRwt in response to tetracycline. Endogenous EGFRwt can be detected in cells not exposed to tetracycline. B. Silencing of endogenous EGFRwt in U251vIII cells using shRNA sequences specific to EGFRwt or control (scrambled shRNA). Two clones designated U251vIII-5 and U251vIII-2 show silencing of EGFRwt in varying degrees. C. EGFRwt tagged with an HA epitope was stably transfected into U251vIII cells and two clones (U251vIII-15 and U251vIII-18) with constitutive overexpresson of EGFRwt-HA are shown. D. EGFRwt overexpression in U251vIII-15 is compared with endogenous EGFRwt expression in (C) control cells. Both lanes are exposed to tetracycline and the lower doublet represents EGFRvIII. E. A comparison of EGFRwt levels in control (C), U251vIII-5 and U251vIII-15 cells in the absence (upper panel) or presence of tetracycline (middle panel) which induces expression of EGFRvIII. F. Tyrosine phosphorylation of EGFRvIII in U251vIII-15, U251vIII-5, U251vIII-control (C) cells. Phospho-specific antibodies directed against Tyr 1068, Tyr 1173, and Tyr 845 were tested. G. U87MG cells expressing EGFRvIII in a tet-on inducible system were transiently transfected with EGFRwt-HA in lanes 1 and 4 or empty vector in lanes 2 and 3. Lane 1 shows basal tyrosine phosphorylation of the EGFRwt in EGFRvIII-15 cells while lanes 3–4 are treated with tetracycline to induce expression of EGFRvIII. There is increased phosphorylation of EGFRvIII when EGFRwt-HA is overexpressed. H. Level of EGFRvIII in U87-vIII and U251-vIII cells in response to tetracycline is compared to EGFRvIII levels in lysates made directly from frozen GBM tumor tissue from 4 tumors (394, 908, 331 and 369). The levels are comparable. I. Table of various U251EGFRvIII clones used in the study.

We find that the level of EGFRwt has a profound effect on tyrosine phosphorylation of EGFRvIII. As can be seen in Figure 1F, there is a striking increase in tyrosine phosphorylation of EGFRvIII in U251vIII-15 cells while tyrosine phosphorylation of EGFRvIII is significantly attenuated in U251vIII-5 cells and intermediate in control cells (Figure 1F). A similar result was found in a 2nd clone overexpressing EGFRwt or with EGFRwt silenced (SFigure 1A). Constitutive overexpression of EGFRwt results in a low level of activation/tyrosine phosphorylation of EGFRwt (Figure 1F and SFigure 1A–B), consistent with previous observations (20–21). The phospho-EGFR antibody used first in this experiment is directed against tyrosine 1068 of the EGFR. We confirmed a similar increase in phosphorylation of EGFRvIII in EGFRvIII-15 cells using phospho-EGFR antibodies directed against Tyrosine 1173 and Tyrosine 845 (Figure 1F), but not in U251vIII-5 or U251vIII-control cells. This may reflect the sensitivity of the antibodies used and/or the need for a high level of EGFRwt for EGFRvIII to become phosphorylated at these residues. Furthermore, transient overexpression of EGFRwt-HA in U87-vIII cells also results in increased phosphorylation of EGFRvIII (Figure 1G). The level of EGFRvIII expression in U87-vIII and U251-vIII cells is similar to GBM tumors (Figure 1H). A table describing the various U251MG-EGFRvIII clones used is presented in Figure 1I. Next, we examined a panel of primary GBM neurosphere cultures and found two tumors, GBM9 and GBM748, that retain EGFRvIII expression (Figure 2A). GBM748 with EGFRwt overexpression, has increased EGFRvIII phosphorylation compared to GBM9 expressing a low level of EGFRwt (Figure 2B). Transient overexpression of EGFRwt-Myc results in increased EGFRvIII tyrosine phosphorylation in GBM9 cells (Fig. 2C). Furthermore, selective silencing of EGFRwt results in decreased tyrosine phosphorylation of EGFRvIII compared to control siRNA (Fig. 2D). Thus, EGFRwt regulates EGFRvIII activation in established GBM lines as well as primary GBM neurosphere cultures.

Figure 2.

Figure 2

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A. A panel of primary GBM neurosphere cultures was tested for expression of EGFRvIII and EGFRwt expression by Western blot. B. A comparison of pEGFR levels in neurosphere cultures from GBM9 and GBM748. C. GBM9 neurosphere cultures were transfected with EGFRwt-Myc or empty vector followed by Western blot. D. GBM9 neurosphere cultures were transfected with siRNA sequences specific to EGFRwt or control scrambled siRNA followed by Western blot. E. EGFRwt-Myc and EGFRvIII-HA or empty vector were co-transfected in COS-7 cells. Cell lysates were denatured followed by immunoprecipitation with HA antibodies. F. The endogenous EGFRwt in COS-7 cells was silenced using siRNA sequences specific for EGFRwt (E) or control (C) siRNA. Silencing of EGFRwt is detected in the short exposure in the middle lane. Expression of EGFRvIII in lanes 2 and 3 are seen in the longer exposure WB and in the HA WB. The pEGFR antibody used in this figure is pEGFR Tyr 1068. G. EGFRvIII-HA was cotransfected with EGFRwt or EGFRwt mutants in COS-7 cells. Cell lysates were denatured followed by immumoprecipitation with HA antibodies and WB with pEGFR antibodies. H. EGFRwt-Myc was co-transfected with EGFRvIII with an intact kinase domain, or an EGFRvIII kinase inactive mutant, both tagged with HA. Cell lysates were denatured followed by IP with HA antibodies and WB with pEGFR. I. The dimerization of EGFR was examined in U251vIII-15 and U251vIII-5, using BS3 as a cross-linker with or without tetracycline. In U251vIII-5 cells, EGFRwt is silenced and there is no signal in the no tetracycline lane. In U251vIII-15 cells there is constitutive overexpression of EGFRwt and EGFRwt can be detected in the absence of tetracycline. In the presence of tetracycline, EGFRvIII is expressed in both lines. J. Coimmunoprecipitation of EGFRwt and EGFRvIII is demonstrated. Transient transfection of COS-7 cells with EGFRwt-Myc and EGFRvIII-HA was followed by IP with Myc and WB with HA. A phospho-EGFR (1068) antibody was used.

Next, we transiently transfected epitope tagged EGFRwt-Myc and EGFRvIII-HA in COS-7 cells. EGFRvIII phosphorylation is substantially increased by co-expression of EGFRwt-Myc in whole cell lysates (SFigure 1B) and by immunoprecipitation of EGFRvIII using HA antibodies. In order to eliminate any contribution of co-immunoprecipitating EGFRwt, we denatured lysates prior to immunoprecipitation. As shown in Figure 2E, co-expression of EGFRwt induces a substantial increase in EGFRvIII tyrosine phosphorylation. Surprisingly, exposure to exogenous EGF does not increase EGFRvIII phosphorylation, and seems to decrease it in COS cells suggesting that a low level of EGFRwt activation is optimal for EGFRvIII activation. Furthermore, transient silencing of endogenous EGFRwt in COS-7 cells results in a significant decrease in phosphorylation of transfected EGFRvIII (Figure 2F). It should be noted that the EGF induced dephosphorylation of EGFRvIII is not detected in glioma cells (Puliyappadamba et al., in press), so there may be cell type specific differences.

EGFRwt induced activation of EGFRvIII requires both the kinase activity and the dimerization loop of EGFRwt

Binding of ligand to the extracellular domain of EGFRwt leads to its dimerization and activation of the tyrosine kinase activity of the receptor (22–23). Dimerization appears to play a key role in EGFR activation and internalization. The CR1 region in the extracellular domain of the EGFRwt is known to play an essential role in dimerization of the EGFR (24–25). We transiently transfected EGFRwt, EGFR kinase mutant, or an EGFR dimerization loop mutant (CR1) mutant along with EGFRvIII-HA into COS cells and find that both the kinase activity of EGFRwt and its dimerization loop are required for inducing EGFRvIII phosphorylation as shown in Figure 2G.

EGFRwt facilitates dimerization of EGFRvIII but does not transphosphorylate EGFRvIII

Transfection of an EGFRvIII kinase inactive mutant-HA into COS cells along with EGFRwt-Myc was followed by immunoprecipitation with HA antibody and Western blot with pEGFR antibodies. No phosphorylation of EGFRvIII-kinase mutant can be detected while there is robust phosphorylation of the kinase intact EGFRvIII mutant (Figure 2H). This result argues against EGFRwt transphosphorylating EGFRvIII. Next, we examined the influence of EGFRwt on EGFRvIII dimerization by using a cross linker, BS3. U251vIII-15 and U251vIII-5 cells were exposed to tetracycline to induce EGFRvIII expression. Dimerization of EGFRvIII is significantly amplified in U251vIII-15 compared to U251vIII-5 cells (Fig. 2I). In addition, by co-transfecting EGFRvIII-HA and EGFRwt-Myc into COS-7 cells we find that EGFRvIII and EGFRwt form a physical complex (Figure 2J). Thus, our data suggest that EGFRwt promotes EGFRvIII phosphorylation by facilitating dimerization of EGFRvIII.

Increased EGFRvIII activaton leads to induction of EGFRwt ligands

Next, we undertook an analysis of the EGFRvIII induced transcriptional program in U251vIII-15, U251vIII-5 and U251vIII-C cells. The transcriptional program of EGFRvIII was profoundly influenced by the level of EGFRwt in cells (Figure 3A, SFigure 2 and STable 1). Ingenuity pathway analysis revealed that the top canonical pathway induced by EGFRvIII expression in control and U251vIII-15 cells but not U251vIII-5 cells, is the “role of tissue factor in cancer” a network of oncogenic ligands such as HB-EGF and IL-8 (26). Remarkably, under conditions of maximal EGFRvIII activation in U251vIII-15 cells, EGFRvIII induced the activation of three EGFRwt ligands (HB-EGF, TGFA and BTC), while two ligands are induced in control cells and none in U251vIII-5 cells (Figure 3A). Figure 3B shows upregulation of HB-EGF by EGFRvIII in in U251vIII-15 cells and U251vIII-control cells but not in U251vIII-5 cells. An ELISA was performed to examine protein levels of HB-EGF and the results are similar to the quantitative PCR results (SFigure 3A). HB-EGF was also induced in U87MG cells by EGFRvIII (SFigure 3B). Confirmation of EGFRvIII-mediated induction of TGF-alpha and IL-8 by qPCR is shown in SFigures 3C and 3D.

Figure 3.

Figure 3

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A. Heatmap of microarray gene expression in response to EGFRvIII expression in cells with varying levels of EGFRwt expression. B. HBEGF expression in response to tetracycline induced EGFRvIII expression in U25vIII-15 and control cells by quantitative real time PCR (1way ANOVA, p=0.005). HB-EGF is not induced in U251vIII-5 cells. C. Stable silencing of HB-EGF in U251vIII expressing cells using HB-EGF shRNA in clones 50 and 8 as detected with real time qPCR. GAPDH was used as an internal control (1way ANOVA, p<0.0001). D. Generation of U251vIII lines overexpressing Myc-tagged HB-EGF or empty vector (C). Clones 34 and 1 show the highest levels of HB-EGF-Myc expression. E. Increased expression of HB-EGF-Myc (clone 34) results in increased tyrosine phosphorylation of EGFRvIII while silencing HB-EGF (clone 50) inhibits tyrosine phosphorylation of EGFRvIII. F. Transient transfection of HB-EGF-Myc in primary GBM neurosphere cultures (GBM9) results in increased EGFRvIII phosphorylation compared to empty vector. A part of this figure is also shown in Figure 2C. G. GBM9 cells were transfected with HB-EGF siRNA followed by preparation of lysates after 72h and Western blot. As a control (C) we used scrambled (non-sequence siRNA). A part of this figure is also shown in Figure 2D. H. HB-EGF silencing was confirmed with quantitative real time PCR. A two tailed t test was statistically significant (p<0.0001). The pEGFR antibody used in experiments in this figure was Tyr-1068. Gene expression analysis was performed with two replicates and a change of 2 fold or greater was considered significant.

HB-EGF induces EGFRvIII activation

Next, we stably silenced HB-EGF in U251EGFRvIII cells using lentiviral shRNA. Two clones with HB-EGF stably silenced were identified (Figure 3C). We confirmed HB-EGF silencing at a protein level by conducting an ELISA (SFigure 3E). We also generated lines stably overexpressing Myc-tagged-HB-EGF in U251vIII cells (Figure 3D). Importantly, increased expression of HB-EGF results in a striking increase in tyrosine phosphorylation of EGFRvIII while silencing HB-EGF inhibits tyrosine phosphorylation of EGFRvIII (Figure 3E and SFigure 4A). Thus, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Importantly, tyrosine phosphorylation of the endogenous EGFRwt can be detected in cells overexpressing HB-EGF (Figure 3E), demonstrating that HB-EGF activates EGFRwt. Also, stable overexpression of HB-EGF in U87vIII cells or in GBM 9 neurosphere cells results in increased EGFRvIII activation (SFigure 4B and Figure 3F). GBM9 cells express a high level of endogenous HB-EGF compared to U251MG and U87MG cells (SFigure 4C) and when HB-EGF is silenced there is decreased EGFRvIII activation (Figure 3G). HB-EGF overexpression was confirmed by Western blot with Myc (Figure 3F) while HB-EGF silencing was confirmed with qPCR and ELISA (Figure 3H and SFigure 3E). Furthermore, increased HB-EGF levels lead to substantially increased dimerization of EGFRvIII compared to cells with silenced HB-EGF (Figure 4A).

Figure 4.

Figure 4

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A. The dimerization of EGFRvIII was examined in HB-EGF overexpressing U251vIII cells (clone 34) and HB-EGF silenced U251EGFRvIII cells (clone 50) using BS3 as a cross-linker. B. Expression of EGFRvIII leads to increased activation of EGFRwt. U251vIII-15 cells were exposed to tetracycline with or without EGF followed by immunoprecipitation with HA antibodies. The tyrosine phosphorylated band aligns perfectly with the tyrosine phosphorylated band in the EGF treated lane indicating that it is EGFRwt. C. EGFRvIII mediated activation of EGFRwt is demonstrated by co-transfecting COS-7 cells with EGFRwt-Myc and EGFRvIII-HA followed by denaturing lysates and IP with Myc antibody. The tyrosine phosphorylation of EGFRwt is amplified when EGFRvIII-HA is co-transfected. D. Activation of EGFRvIII is attenuated by using an EGFR antibody (528) that inhibits ligand binding to EGFRwt. In the right lane cells were exposed to EGF in the absence of tetracycline (no EGFRvIII) and show the position of the slower migrating pEGFRwt band. E. COS-7 cells were transfected with EGFRvIII-HA+EGFRwt-Myc or empty vector (v) followed by incubation with EGFR neutralizing antibody (528) or isotype matched control antibody. A phospho-EGFR (1068) antibody was used. F. HB-EGF neutralizing antibodies result in attenuation of both EGFRwt and EGFRvIII phosphorylation in U251vIII-15 cells while normal goat antibody has no effect. G. Analysis of EGFRvIII induced signal transduction in U251vIII-15, U251vIII-5 and U251vIII-control cells shows that ERK activation is equal is all clones, while Met activation is amplified in U251vIII-15 cells and attenuated in U251vIII-5 cells. Note that In the absence of tetracycline constitutive EGFRwt overexpression in EGFRvIII-15 cells results in increased Met phosphorylation. H. Phospho-specific antibody microarrays comparing the effects of tetracycline induced expression of EGFRvIII in U251vIII-15 cells to U251vIII-5 cells. There is substantial increase in phosphorylation of multiple RTKs (including EGFRvIII) in response to EGFRvIII expression in U251vIII-15+tet compared to U251vIII-5+tet cells. Two independent experiments were performed for H.

Next, we investigated whether EGFRvIII leads to EGFRwt activation. We examined tyrosine phosphorylation of EGFRwt-HA in U251vIII-15 cells with or without tetracycline, by immunoprecipitating EGFRwt using HA antibodies followed by immunoblotting with pEGFR antibodies. Figure 4B shows an increased phosphorylation of EGFRwt when EGFRvIII is expressed. A similar result is detected in COS-7 cells (Figure 4C). Importantly, a neutralizing EGFR antibody (528) that inhibits ligand binding to the EGFRwt attenuates activation of EGFRvIII (Figure 4D) in U251vIII-15 cells and in COS cells (Figure 4E). Similarly, a neutralizing antibody to HB-EGF inhibits EGFRvIII and EGFRwt tyrosine phosphorylation in U251vIII-15 cells (Figure 4F). These findings strongly suggest that that an EGFRwt-EGFRvIII-HB-EGF feed forward loop regulates activation of EGFRvIII.

EGFR activation levels influence MET and other RTK phosphorylation

There is no difference in ERK activation in U251vIII-15 compared to U251vIII-5, suggesting that a low level of EGFRvIII activation is sufficient to activate ERK (Fig. 4G). EGFRvIII can transactivate the tyrosine kinase Met and other RTKs (27–29). When EGFRvIII is expressed using tetracycline there are significantly larger increases in Met phosphorylation in EGFRvIII-15 cells compared to EGFRvIII-5 cells (Figure 4G). Furthermore, there is increased transactivation of other RTKs such as IR, IGF1R, AXL, RET and EPHA2 in U251vIII-15 cells compared to U251vIII-5 cells as detected by a phospho-specific antibody microarray (Figure 4H).

EGFRwt and HB-EGF influence EGFRvIII mediated oncogenicity in vivo

It has been reported that EGFRvIII does not have a significant effect on proliferation of glioma cells in culture, but profoundly influences oncogenicity in vivo (9). We also did not find a significant influence of EGFRwt levels on the proliferation of EGFRvIII expressing cells in monolayer culture (SFigure 5A). Next, we tested the cell lines for growth in an orthotopic xenograft model of GBM in nude mice. First, we confirmed that doxycycline penetrated into brain tumors by examining expression of EGFRvIII in lysates from mouse brain tumors implanted with U251-vIII cells (Figure 5A). Subsequently, we implanted U251vIII-5, U251vIII-15 and U251vIII-control shRNA cells intracranially in nude mice and provided doxycycline in food and water. Kaplan-Meier survival analyses revealed that the U251vIII-5 mice remained symptom-free for a significantly longer period of time than the U251vIII-15 mice, while the U251vIII-15 mice formed tumors faster compared to the control mice (Figure 5B). We repeated the experiment with EGFRvIII-5 cells in a second group of mice with a very similar result (SFigure 6A). H&E sections from representative brains are shown in Figure 5D. EGFRwt and EGFRvIII levels in representative tumors are shown in Figure 5C. Three out of eight mice in the EGFRvIII-5 group formed tumors after long latencies while 5 mice did not form tumors, whereas all mice in control and EGFRvIII-15 groups formed tumors. In the three EGFRvIII-5 tumors EGFRvIII and EGFRwt levels are very high, presumably due to multiple rounds of selection for high EGFR expressing cells (Figure 5C). A second clone with less complete EGFRwt silencing, U251vIII-2 also forms tumors after a longer latency compared to control. Thus, increasing the EGFRwt level accelerates the tumorigenicity of EGFRvIII while silencing EGFRwt inhibits tumor formation by EGFRvIII. The oncogenic effect of EGFRwt alone (30) is weaker and resulted in tumor formation in 102 days compared to 64 days for EGFRvIII (control group) (SFigure 6B).

Figure 5.

Figure 5

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A. Analysis of EGFRvIII expression in brain tumors in response to doxycycline in food and water. Brain tumor lysates from mice injected intracranially with U251vIII cells exposed to doxycycline (252 and 253) or not exposed to doxycycline (241 and 242) were examined by Western blot with EGFR antibodies. EGFRvIII was detected only in tumors of doxycycline treated animals. B. Kaplan-Meier survival analysis of U251EGFRvIII-15, U251EGFRvIII-5, U251EGFRvIII-2, and U251EGFRvIII-C (control) cells (n=8) implanted intracranially in nude mice. All mice were exposed to doxycycline in food and water. The log rank test was significant (χ2(4) =77.9, p <.001). The median number of days until the appearance of neurological symptoms for the U251vIII-5 group was 240 days, as opposed to 26 days for the U251vIII-15 group (Fig. 5B). Post-hoc Kaplan-Meier pairwise analyses revealed significant differences between each pair of conditions (p < .01), U251vIII-5 compared to U251vIII-control (median survival = 240 days vs. 64 days respectively), U251vIII-control compared to U251vIII-15 (median survival = 64 days vs. 26 days respectively), and U251vIII-2 mice (with EGFRwt silenced less efficiently) compared to U251vIII=control (median survival = 112 days vs. 64 days respectively). C. Western blot from tumor lysates from intracranial tumors from three mice each from U251vIII-15 group and U251vIII-5 group. D. H&E sections from a representative brain section from U251vIII-15 and U251vIII-control mice showing presence of large brain tumors and from U251vIII-5 showing absence of tumor. The bottom panel shows immunohistochemistry in tumor sections with HA antibody showing positive staining in U251vIII-15 (EGFRwt-HA).

Next, we examined if altering HB-EGF has an effect on EGFRvIII mediated tumorigenicity. While there was no difference in the proliferation of HB-EGF altered cells in culture (SFigure 5B), we did note a significant difference in tumor growth in the orthotopic model. Kaplan-Meier survival analyses revealed that the Clone 50 mice (HB-EGF silenced) remained symptom-free for a significantly longer period of time than the clone 34 mice (HB-EGF overexpressed) as shown in Figure 6A. Four out of eight mice in the Clone 50 group formed tumors after a long latency whereas all mice formed tumors in the Clone 34 group faster than the control group. Figure 6B shows H&E sections of representative tumors or normal brain (clone 50). The Myc tagged HB-EGF can be detected in lysates from mouse tumors as shown in Figure 6C. Thus, the results of altering HB-EGF on EGFRvIII induced tumorigenicity are similar to the effect of altering EGFRwt.

Figure 6.

Figure 6

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A. Kaplan-Meier survival analysis of U251vIII-34 cells (clone 34) with HB-EGF overexpression and U251vIII-50 (clone50) cells with HB-EGF silenced implanted intracranially in mice (n=8). All mice were exposed to doxycycline in food and water. The log rank test was significant (χ2(2) =32.5, p <.00001). The median number of days until the appearance of neurological symptoms for the Clone 50 group was 166 days, as opposed to 47 days for the Clone 34 group (Fig. 6A). Post-hoc Kaplan-Meier pairwise analyses revealed significant differences between each pair of conditions (p < .01), the Clone 50 compared to control group (median survival = 166 days vs. 64 days respectively), and control compared to Clone 34 (median survival = 64 days vs. 47 days respectively). Four out of eight mice in the Clone 50 group formed tumors after a long latency whereas all mice formed tumors in the Clone 34 group faster than the control group. B. H&E sections from a representative brain section from clone 34 and control showing presence of a large brain tumor and from clone 50 showing absence of tumor. C. Western blot from tumor lysates from intracranial tumors from two mice from clone 34 and clone 50 groups showing Myc-HBEGF expression in clone 34 tumors.

EGFR and HB-EGF expression in glioblastoma

Next, we assessed the presence of EGFRwt and EGFRvIII by Western blots in tumor lysates made directly from GBM tissue (Fig. 7A). EGFRvIII mRNA levels were also detected by quantitative real time PCR (SFigure 7B). In this series of GBMs, 36% of tumors express both EGFRwt and EGFRvIII. While EGFRwt is expressed in tumors without EGFRvIII, EGFRvIII was detected only in GBMs that have co-expression of EGFRwt. The presence of HB-EGF was detected by qPCR and a two fold increase in HB-EGF levels was deemed positive (Figure 7B–D). Thus, a statistically significant correlation between the presence of EGFRvIII and HB-EGF expression in GBMs was noted (Figure 7E and STable 2). HB-EGF protein levels were also determined by ELISA in 8 tumors (SFigure 7A) and the results are consistent with the qPCR data. These data show that the components of the EGFRvIII-HBEGF-EGFRwt-EGFRvIII feed forward loop can be detected in GBM.

Figure 7.

Figure 7

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EGFRvIII expression correlates with the presence of HB-EGF in glioblastoma samples. A. Expression of EGFRwt and EGFRvIII in a panel of GBMs. EGFRvIII migrates faster than EGFRwt in electrophoresis gels and is expressed in about a third of the tumors. B-D show expression of HB-EGF determined by quantitative real time PCR in GBMs. E. HB-EGF expression is considered upregulated if there is a 2 fold increase. A Mann-Whitney U test was computed to determine the difference in HBEGF values by presence/absence of EGFRvIII. HBEGF values were significantly higher in tumors positive for EGFRvIII (z = 3.56, p < .0001). HBEGF values were recoded into a binary variable where a 1 represented 2.0 or higher, and a 0 represented values less than 2.0. This new HB-EGF variable was significantly associated with EGFRvIII (OR = 5.50, 95% CI: 1.57 – 19.3). The Fisher’s exact test was also significant (p < .0001). Two independent experiments were performed.

Discussion

EGFRvIII is a prime oncogene in GBM. EGFRvIII does not bind any EGFR ligand and it has thus been assumed that EGFRvIII signals constitutively. Here, we propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed EGFRwt activated by ligands such as HB-EGF. Previous studies and our data show that increased expression of EGFRwt is sufficient to induce a low level tyrosine phosphorylation of EGFRwt. We propose that the activated EGFRwt then promotes EGFRvIII activation and tyrosine phosphorylation. Furthermore, EGFRvIII contributes to its own activation by inducing expression of HB-EGF, a ligand for EGFRwt. Thus EGFRvIII activation is amplified by a positive feedback loop involving EGFRwt→EGFRvIII→ HBEGF→ EGFRwt.

We present several lines of evidence demonstrating that EGFRwt plays a critical role in EGFRvIII activation. Silencing EGFRwt in cells expressing EGFRvIII inhibits tyrosine phosphorylation of EGFRvIII and confers a loss of tumorigenicity in vivo, while overexpression of the EGFRwt results in a striking increase in EGFRvIII activation/tyrosine phosphorylation and rapid tumor growth in vivo in an orthotopic model. Furthermore, EGFRvIII induces expression of HB-EGF and other EGFRwt ligands. Remarkably, altering HB-EGF recapitulates the EGFRwt phenotype. Thus, increased expression of HB-EGF also leads to increased EGFRvIII tyrosine phosphorylation and tumorigenicity while silencing HB-EGF markedly inhibits EGFRvIII/activation tyrosine phosphorylation and tumorigenicity. Furthermore, EGFRvIII activation is attenuated by neutralizing antibodies to EGFR or HB-EGF. Consistent with our feed forward model, EGFRvIII expression also leads to activation of EGFRwt and this activation can also be blocked by a neutralizing antibody to EGFRwt or HB-EGF. Our model is also supported by a previous study showing that an EGFR antibody (528) that inhibits binding of ligand to EGFRwt inhibits EGFRvIII mediated tumor growth in xenograft studies (31). EGFRwt ligands including HB-EGF and TGF-alpha are expressed in GBM (18, 32–33). We also find that EGFRwt, EGFRvIII and HB-EGF are commonly co-expressed and that EGFRvIII expression correlates with a high expression of HB-EGF.

EGFRvIII is expressed primarily in GBMs with EGFR gene amplification and overexpression of EGFRwt making it likely that the genetic abnormalities underlying overexpression of EGFRwt and EGFRvIII co-exist in individual tumor cells (34–35) (Figure 7). Immunohistochemical studies comparing EGFRwt and EGFRvIII expression by IHC using antibodies that are specific to EGFRwt and EGFRvIII suggest that EGFRwt and EGFRvIII expression commonly overlap (34, 36). Nishikawa et al., found that in GBMs in which EGFRwt was detected almost all tumors cells expressed EGFRwt while the distribution of EGFRvIII was scattered in 65% of the tumors. These studies suggest that while in some GBMs the distribution of EGFRvIII may be more restricted compared to EGFRwt (36), EGFRvIII is almost always expressed in EGFRwt expressing cells. Another study recently reported similar findings (37). Thus, all of the available data demonstrates that EGFRvIII is usually co-expressed with EGFRwt in the same GBM cells.

We have previously identified HB-EGF as a gene induced by EGFRvIII and hypothesized that an autocrine loop extends from EGFRvIII to EGFRwt (38). In the current study, we show that the primary function of this loop is to activate EGFRvIII. Thus, we show by both loss of function and gain of function studies, that HB-EGF and EGFRwt critically influence EGFRvIII activation and tumorigenicity. The EGFRvIII-EGFRwt interplay in GBM may involve multiple mechanisms in GBM including EGFRvIII induced paracrine activation of EGFRwt (19). Our results further suggest that the best experimental model for studying EGFRvIII signaling is one in which both receptors are co-overexpressed, as they are in GBM, since EGFRvIII in a background of high EGFRwt is highly activated.

Is EGFRwt absolutely required for EGFRvIII activation? Previous studies have shown that EGFRvIII becomes tyrosine phosphorylated in immortalized fibroblasts, CHO cells and hematopoietic cells that do not express EGFRwt and can even transform immortalized fibroblasts (14, 17, 39–40), arguing against an absolute requirement of EGFRwt for activation of EGFRvIII. Our data demonstrate that in glioma cells if the EGFRwt level is low (we have not been able to completely silence EGFRwt), EGFRvIII activation is low and EGFRvIII loses it oncogenicity, whereas EGFRvIII activation is amplified and oncogenicity enhanced if EGFRwt is overexpressed. Thus, we conclude that EGFRwt plays an important role in oncogenic activation of EGFRvIII in glioma cells.

Forced dimerization of EGFRvIII results in stronger activation and enhanced oncogenicity of EGFRvIII (17). We show that increased EGFRwt or HB-EGF promotes EGFRvIII dimerization. The resultant increased activation of EGFRvIII leads to increased transactivation of multiple receptor tyrosine kinase families such as Met and EphA2 that may mediate the increased oncogenicity detected in vivo. Structurally, the EGFRvIII has a deletion of the extracellular amino acids 6–273 that constitute the dimerization arm and a part of the ligand-binding domain. Despite the lack of the dimerization arm, it is known that EGFRvIII is able to dimerize with EGFRwt (16). Our data indicate that the dimerization arm of EGFRwt plays a key role in EGFRvIII activation (Figure 2G).

Previous studies have shown that EGFRwt and EGFRvIII can heterodimerize, and we also detect the formation of a physical complex between EGFRwt and EGFRvIII. Also, it appears that a low level of EGFRwt activation is optimal for EGFRvIII activation. Surprisingly, addition of exogenous EGF did not result in increased EGFRvIII activation, while overexpression of HB-EGF facilitates EGFRvIII activation. An important role for endogenous HB-EGF is suggested by experiments showing attenuated EGFRvIII phosphorylation with HB-EGF neutralizing antibodies or if HB-EGF is silenced.

We propose that the EGFRwt-EGFRvIII-HBEGF feed forward loop is a key regulatory network in GBM that maintains continuous activation and oncogenicity of EGFRvIII. These findings have implications for our understanding of EGFRvIII function and for therapeutic approaches to target the EGFR signaling system in GBM. The likely relevance of this model to human GBM is supported by data demonstrating a significant correlation between the expression of EGFRwt, EGFRvIII and HB-EGF components in human GBM samples. While we have focused on HB-EGF in this study, EGFRvIII appears to induce additional EGFRwt ligands such as TGF-alpha, and betacellulin which may also participate in this feed forward loop. This feed forward loop may be an attractive target for GBM therapy.

Materials and Methods

Plasmids, transfection and generation of Cell lines

U251MG and U87MG were used to generate cell lines conditionally expressing either EGFRwt or EGFRvIII using the T-Rex Tet-on system from Invitrogen as described previously (18). EGFRwt-HA in PcDNA 3.1 (Neo) was transfected into U251EGFRvIII expressing cells followed by selection of stable clones with G418. Lipofectamine 2000 (Invitrogen) was used for all transfections. Primary GBM cultures were generated directly from human GBM tumor specimens. Cells were cultured in Neurobasal medium supplemented with B27 without Vitamin A, and with EGF (10ng/ml) and bFGF (10ng/ml). GBM9 cells were a kind gift of Dr. James Van Brocklyn (41). COS-7 cells were transfected with equal amounts of DNA with empty vector or 5ug of EGFRwt-Myc and/or 5ug of EGFRvIII-HA for 48h. Three independent experiments were performed unless specified otherwise.

RNA interference

We used a pool of siRNA sequences directed against exons 3–6 of the EGFRwt obtained from Qiagen (catalog no S100300104) according to the manufacterer’s protocol. For stable silencing of EGFRwt two different shRNA sequences targeting sequences within exons 2–7 cloned into pSilencer 3.1-H1 neo vector (Ambion) were obtained from Dr. Stephen Lee (University of Ottawa, Canada) (42) or control shRNA was used. HB-EGF was silenced using lentiviral shRNA particles from Santa Cruz Biotechnology (sc-39421-V) and puromycin selection.

Antibodies and phospho-specific Antibody microarray

A phospho-specific Proteome-Profiler RTK antibody microarray from R&D systems was used. EGFR antibody was purchased from Millipore. β-actin, and EGFR neutralizing antibodies (528) were from Santa Cruz Biotechnology. Phospho-EGFR antibodies (Tyr-1068, Tyr-845, Tyr-1173), pERK, ERK, HA, Myc, pMet, Met antibodies were obtained from Cell Signaling Technology. HB-EGF neutralizing antibody (AF-259-NA) was obtained from R&D systems.

Dimerization Assay

BS3 (bis[sulfosuccinimidyl] suberate) was obtained from Thermo Scientific and used according to the manufacterer’s instructions. Cells were incubated with BS3 (2mM) at room temperature for 30 minutes followed by quenching and Western blot.

Microarray Analysis

Cells were incubated with or without tetracycline followed by RNA extraction. The Illumina humanHT-12 v4 Expression BeadChip was used using the manufacterer’s protocol. Significantly altered genes (fold change > 2 or < −2 with p value < 0.1) were further analyzed using the Ingenuity Pathway Analysis (IPA, Ingenuity Systems, inc.) for enrichment analyses.

cDNA Synthesis and Real Time PCR

Were performed as we have described previously (18). Primer sequences are available upon request.

Primary Tumors

Frozen tissue specimens of human glioblastomas (GBMs) were obtained according to IRB approved protocols. Resected tumors were initially frozen at −80°C.

Cell proliferation Assays

Cells were plated at 20,000 per well in a 12-well tissue culture plate in the presence and absence of tetracycline (1μg/μl). The cells were trypsinized and counted using an automated cell counter after 72 h.

Orthotopic implants

2 × 106 U251MG derived cells were suspended in PBS and matrigel (5 μL) and injected into the right corpus striatum of the brains of 6–8 week old nude mice using a stereotactic frame. Animals were monitored and sacrificed when neurological signs appeared or after 240 days. All animal studies were done under IACUC approved protocols.

HBEGF ELISA

HBEGF ELISA was performed using an HB-EGF ELISA kit from R&D Systems (DY259) according to the manufacturer’s protocol.

Statistical Anaysis

Error bars represent the means ± standard deviations of three independent experiments. All data were analyzed for significance using GraphPad Prism 5.0 software, where P < 0.05 was considered statistically significant. One-way ANOVA and two-tail t-test were used to compare groups. Survival analysis for mice is described in the results and figure legends.

Supplementary Material

Li et al Oncogene Supplemental Data.pdf

Acknowledgments

We thank Dr. Mien-Chie Hung (MD Anderson Cancer Center) for the EGFRwt-Myc plasmid and Dr. James Van Brocklyn (Ohio State University) for GBM9 cells. This work was supported in part by NIH grant RO1NS062080 to AH and by RO1 CA139217 to DAB. SB is supported by grants from the National Institutes of Health (RO1 CA149461), National Aeronautics and Space Administration (NNX13AI13G) and the Cancer Prevention and Research Institute of Texas (RP100644).

Footnotes

The authors declare that there are no financial conflicts of interest.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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