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. Author manuscript; available in PMC: 2013 Sep 6.
Published in final edited form as: Oncogene. 2011 Dec 5;31(36):4054–4066. doi: 10.1038/onc.2011.563

EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway

R Bonavia 1,2,9, MM Inda 1,9, S Vandenberg 2,3, S-Y Cheng 4,5, M Nagane 6, P Hadwiger 7, P Tan 7, DWY Sah 8, WK Cavenee 1,3, FB Furnari 1,3
PMCID: PMC3537826  NIHMSID: NIHMS426422  PMID: 22139077

Abstract

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wildtype (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Keywords: glioblastoma, EGFR, ΔEGFR, angiogenesis, NF-κB, IL-8

Introduction

Amplification of the epidermal growth factor (EGFR) is present in almost 50% of glioblastomas (GBMs) (Hurtt et al., 1992; Jaros et al., 1992; Schlegel et al., 1994), and is often associated with activating mutations such as the deletion of exons 2–7 (EGFRvIII, EGFRde2-7, hereafter referred to as ΔEGFR) that generates a truncated receptor that is unable to bind its ligands but is constitutively active (Huang et al., 1997; Narita et al., 2002) and correlates with poor prognosis (Shinojima et al., 2003; Heimberger et al., 2005). The ability of ΔEGFR to enhance the tumorigenic properties of human gliomas has been clearly demonstrated in animal models: human glioma cells, or primary mouse astrocytes, engineered to overexpress ΔEGFR, when injected either intracranially or subcutaneously, form tumors much faster than their respective parental cell lines (Huang et al., 1997; Holland et al., 1998; Bachoo et al., 2002). Although it has been previously shown that ΔEGFR is constitutively phosphorylated and bound to Shc-Grb2-Ras and phosphoinositide-3-kinase (PI-3K) (Prigent et al., 1996; Huang et al., 1997), many aspects of ΔEGFR signaling have not yet been completely defined. Although there is evidence that ΔEGFR induces transcriptional activation of selected genes (Inda et al., 2010), the downstream effectors responsible for ΔEGFR-mediated enhanced tumor growth have not been fully elucidated, nor have specific transcription factors driving the ΔEGFR phenotype been identified.

The wild-type EGFR (wtEGFR) is known to activate a variety of transcription factors, including signal transducers and activators of transcriptions (Zhong et al., 1994), AP-1 (Malliri et al., 1998) and nuclear factor (NF)-κB (Biswas et al., 2000). The NF-κB family of transcription factors includes seven proteins sharing a DNA-binding domain known as the REL homology domain (Li and Verma, 2002) that, in their active form, associate to form homo- or heterodimers that bind to a common sequence motif known as the κB site. In the absence of stimulation, NF-κB factors are held in the cytoplasm by inhibitory subunits called IκB (Li and Verma, 2002). The stimulation produced by some inflammatory cytokines activates the IκB kinase complex, which phosphorylates IκB promoting its subsequent targeting to the proteasome, and thus the release of the active NF-κB.

NF-κB has been implicated primarily in hematological malignancies, but also appears to have a role in solid tumors such as breast, ovarian, colorectal, gastric cancers and others (Lee et al., 2007). Its role in tumor development likely results from its ability to suppress apoptosis through the activation of anti-apoptotic genes such as cellular inhibitors of apoptosis, cellular FLICElike inhibitory protein (c-FLIP), B-cell lymphoma 2 (BCL-2) and BCL-XL (Karin and Lin, 2002). Additional evidence that NF-κB has a role in tumorigenesis derives from its ability to induce the expression of many cytokines and genes involved in immune responses and in cell proliferation (interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor, CD40L and G1 cyclins), angiogenesis (IL-8 and vascular endothelial growth factor (VEGF)) and metastasis (matrix metalloproteinases) (Karin et al., 2002). Although it was suggested that EGFR induces NF-κB activation, and this pathway is important in the maintenance of tumors bearing amplification/activation of EGFR or others members of the ErbB family (Biswas et al., 2000), the molecular mechanisms linking EGFR with NF-κB are not completely understood.

The AP-1 family of transcription factors is composed of a wide variety of dimeric basic region-leucine zipper proteins belonging to the Jun, Fos, Maf and ATF subfamilies, which are regulated at the gene expression level, at the protein level through modification by direct phosphorylation from mitogen-activated protein (MAP) kinases, or by interaction with other transcription factors (Whitmarsh and Davis, 1996). Several AP-1 proteins can transform cells in vitro simply by overexpression (Jochum et al., 2001) and have been implicated in the development of various tumors, in particular those of epithelial origin (Wang et al., 1991; Young et al., 1999). The AP-1 proteins are the main activators downstream of MAP kinases, thus their activity is typically increased by receptor tyrosine kinase stimulation. However, the signaling events causing activation of AP-1 are not always identical and may depend on several cell-specific factors including differential expression/activation of MAP kinases (Bancroft et al., 2002; Sparmann and Bar-Sagi, 2004) and the composition of AP-1 dimers themselves (van Dam and Castellazzi, 2001).

We recently reported that the secretion of IL-6 is markedly elevated in glioma cell lines and glioma stem cells that overexpress ΔEGFR and its expression is also increased in human glioma samples where the ΔEGFR variant is present (Inda et al., 2010). Here, we demonstrate that ΔEGFR also promotes the expression of high levels of IL-8 in glioma clinical samples and cell lines. This overexpression is mediated at the transcriptional level by NF-κB, AP-1 and C/EBP, which show constitutive activation dependent on the kinase activity of ΔEGFR. We further show that blocking this pathway by RNA interference knock-down of either NF-κB or IL-8 in ΔEGFR-positive cells abrogated tumor angiogenesis with a concomitant reduction in tumor formation, while overexpression of IL-8 in ΔEGFR-negative cells significantly enhanced tumorigenicity and associated blood vessel formation. Our results also show NF-κB to be an important mediator of ΔEGFR enhancement of tumorigenicity in glioma, as direct short hairpin RNA (shRNA) knock-down of NF-κB also inhibited the in vivo tumor growth of cells overexpressing ΔEGFR.

Results

GBM cell lines overexpressing ΔEGFR form highly vascularized tumors

The presence of an abundant and disorganized vasculature is believed to be an important determinant of the aggressive phenotype of high-grade gliomas (Maher et al., 2001). To identify a potential link between angiogenesis and the amplification/mutation of EGFR commonly observed in gliomas, we analyzed the vasculature in mouse intracranial xenografts derived from U87MG (U87Par) glioma cells and compared with tumors derived from the same cells engineered to express tumor-associated levels of ΔEGFR (U87Δ), wild-type EGFR (U87wt) and control kinase-dead ΔEGFR (U87DK). Immunohistochemical staining for the endothelial cell and pericyte marker CD31 in xenografts derived from ΔEGFR cells demonstrated the highest vessel density throughout the tumor mass, compared with tumors derived from all other U87 lines as determined by quantification of the area occupied by vessels (Figure 1a).

Figure 1.

Figure 1

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ΔEGFR upregulates IL-8 and induces angiogenesis in glioma cells. (a) IHC analysis by CD31 staining of intracranial xenografts: representative fields of U87Par (I), U87wt (II), U87Δ (III) or U87DK (IV) xenografts (left) and relative quantification of vessel density expressed as percentage of stained area. (b) Quantification by ELISA of VEGF secretion in U87Par and U87Δ after 1, 2, 3 and 4 days. (c) IL-8 concentration in CM generated from U87MG, U178, U373 and LNZ308 GBM cell lines engineered to overexpress wtEGFR (wt), ΔEGFR (Δ) or a dead kinase version of ΔEGFR was determined by ELISA. (d) IL-8 expression in U87MG and U178 cell lines and derivatives was determined by real-time PCR. (e) Comparison of IL-8 relative expression in ΔEGFR-positive and -negative GBM clinical samples (left) and GBM tumorspheres (right) determined by real-time PCR. *P<0.05; **P<0.01.

IL-8 is upregulated in GBM cell lines overexpressing ΔEGFR

It is known that gliomas respond to hypoxic conditions by upregulating VEGF and thus stimulate the formation of blood vessels. However, no differences in VEGF secretion were observed between U87Δ and U87Par cells in vitro (Figure 1b) suggesting that there might be other factors specifically induced in the former responsible for the markedly different angiogenic phenotype observed in tumors derived from those cells compared with the other U87 derivatives. To identify such secreted factors we analyzed the conditioned media (CM) of U87 glioma cell line derivatives using a cytokine array that qualitatively detects 79 human cytokines and growth factors in the supernatants of these cultured cells. Among these, IL-8 (also known as CXCL8) was the most upregulated molecule in U87Δ CM compared with CM from U87wt, control kinase-dead ΔEGFR-expressing (U87DK) or parental cell lines (data not shown). Quantification of IL-8 by enzyme-linked immunosorbent assay (ELISA) showed that its expression varied among cell lines (from 218.42 to 4921.7 pg/ml), however, all ΔEGFR-engineered cells had a significant increase in IL-8 secretion and expression when compared with their isogenic partners that did not express the receptor. This varied from approximately 2-fold for LNZ-308 to up to 60-fold more for U87MG (Figure 1c). In no cell line did the overexpression of wtEGFR or DK alone induce an increase in IL-8 secretion (Figure 1c). Analysis by realtime quantitative PCR of IL-8 RNA expression in U87MG and U178MG cell line derivatives confirmed the results obtained by ELISA (Figure 1d), thus indicating that the increased IL-8 production is the result of a transcriptional activation of the IL-8 gene. Furthermore, real-time PCR demonstrated significantly higher levels of IL-8 expression in ΔEGFR-positive human GBM clinical samples, as well as tumor spheres derived from GBM specimens than in those without ΔEGFR expression (P<0.05) (Figure 1e).

IL-8 contributes to ΔEGFR-promoted angiogenesis and tumor growth

Ectopic expression of ΔEGFR in GBM cell lines dramatically increases their ability to grow as xenografts (Huang et al., 1997). To test the contribution of IL-8 to this ΔEGFR-mediated enhancement of tumorigenesis, we inhibited its expression in U87Δ cells by small interfering RNA (siRNA) transfection (Figure 2a). There was no significant effect on cell proliferation in vitro (Supplementary Figure 1). In contrast, subcutaneous injection into nude mice of U87Δ cells transfected with IL-8 siRNA significantly decreased tumor growth compared with cells transfected with an irrelevant siRNA against the green fluorescent protein (GFP) gene (Figure 2b). Additionally, U87wt and U87 Par engineered to overexpress IL-8 at levels comparable to U87Δ cells (Supplementary Figure 2) formed tumors more efficiently than the original cells (Figures 2c and d), thereby phenocopying the effects of ΔEGFR in this respect. As IL-8 is known to be a pro-angiogenic factor (Brat et al., 2005), we analyzed the tumor vasculature by immunohistochemical staining for CD31 in the above described xenografts to determine whether the enhanced tumor growth conveyed by overexpression of IL-8 is caused by increased angiogenesis. Tumors originating from cells overexpressing IL-8 showed a more robust vascularization, indicating that the upregulation of this cytokine alone was sufficient to enhance vessel formation by glioma cells (Figures 2c and d, Supplementary Figure 3). A modest decrease in number and area of vessels was observed in tumors derived from the injection of U87Δ cells transfected with IL-8 siRNA at 17 days post-implantation (Figure 2e). As durability tests with the transfected siRNAs suggest that the effect of the siRNA-mediated knockdown would have diminished by 7 days post injection (data not shown), we interpret this modest reduction in angiogenesis as a reflection of IL-8 expression recovery in the tumors. As a complementary means to test the effect of acute IL-8 knockdown on angiogenesis, we performed an in vitro HUVEC tube formation assay using CM from U87Δ cells freshly transfected with GFP or IL-8 siRNAs. The ability of U87Δ CM to induce tube formation was strongly impaired when the IL-8 siRNA was fully effective (Figure 2f), supporting the idea that IL-8 overexpression might contribute to ΔEGFR-driven enhanced angiogenesis.

Figure 2.

Figure 2

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ΔEGFR promotes glioma angiogenesis and tumor growth through induction of IL-8 expression. (a) ELISA quantification of IL-8 secretion in U87Δ cells after transfecting with GFP or IL-8 siRNAs. Cells not transfected were included as negative control (−). (b) Tumor growth curve after subcutaneous injection of U87Δ cells transfected with 25 nM of GFP or IL-8 siRNAs. (c) Top: tumor growth curve after subcutaneous injection of U87wt cells infected with empty vector (pBABE) or engineered to overexpress IL-8 (IL-8). Bottom: representative fields of CD31 immunohistochemistry on sections of U87wt-pBABE (left) or U87wt-IL-8 (right) xenografts. (d) Top: tumor growth curve after subcutaneous injection of U87MG cells infected with empty vector (pBABE) or engineered to overexpress IL-8 (IL-8). Bottom: CD31 immunohistochemistry on corresponding xenografts, U87Par-pBABE (left) and U87Par-IL-8 (right). (e) Quantification of vessel relative area (left) and CD31 immunohistochemistry (right) of xenografts obtained in (b). (f) Representative images of HUVEC cells treated with CM from U87Δ cells transfected with GFP or IL-8 siRNA showing in vitro tube formation. *P<0.05; **P<0.01.

IL-8 gene expression is regulated by NF-κB, AP-1 and C/EBP-binding sequences

To analyze the transcriptional regulation of IL-8, we isolated its promoter and inserted it into a reporter plasmid to drive the expression of the luciferase gene. Point mutations in the NF-κB, AP-1 and C/EBP-binding sites, previously reported as important regulators of IL-8 transcription (Matsusaka et al., 1993; Holtmann et al., 1999) were also introduced (Figure 3a), and the activity of each construct was evaluated by luciferase assays in U87Δ cells and compared with U87Par. As expected, we found that the activity of the wild-type IL-8 promoter was significantly increased in U87Δ (Figure 3b; P<0.01). Mutation of either the AP-1 or the C/EBP sites caused a significant reduction in reporter gene expression (P<0.001), while mutation of the NF-κB site completely abolished the promoter activity (Figure 3b; P<0.001).

Figure 3.

Figure 3

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ΔEGFR activates regulatory elements in IL-8 promoter. (a) Scheme of IL-8 proximal promoter (from −400 bp) showing the responsive elements that were mutated in the following experiments. (b) U87Par (Parental) and U87Δ (Δ) cells were transfected with a reporter construct consisting of the luciferase gene under the control of wild-type IL-8 promoter (WT) or the IL-8 promoter mutated at binding sites for individual transcription factors indicated; transcriptional activity was measured by luciferase assay of cellular extracts after 24 h of serum starvation. Data represent mean±s.e.m. Luciferase activity assay is expressed in relative light units (RLU) normalized to corresponding readings in U87Par. **P<0.01.

NF-κB, AP-1, C/EBP are activated in ΔEGFR cells

As full activation of NF-κB transcriptional activity requires phosphorylation of its p65 subunit at serine 536 (Jiang et al., 2003), we interrogated the phosphorylation of this residue and found that it was increased in ΔEGFR cells regardless of the presence of serum in the culture medium, suggesting it is constitutive lyactivatedin these cells (Figure 4a). No increase in phosphorylation of p65 was observed in cells overexpressing wtEGFR even in the presence of serum. To confirm that p65 phosphorylation corresponds to increased transcriptional activity of NF-κB in ΔEGFR cells, we performed a reporter assay using a construct containing the firefly luciferase gene under the control of four copies of the NF-κB consensus sequence. U87Δ cells showed significantly increased luciferase activity (P<0.001), indicating that NF-κB target genes could be upregulated in these cells (Figure 4b). These results were further validated by analyzing the binding of nuclear NF-κB in U87 cell derivatives to a radiolabeled oligonucleotide containing the NF-κB consensus sequence: the increased binding observed in U87Δ was ablated for an IκB construct mutated in all activating serines and threonines to alanines that prevents NF-κB from shuttling to the nucleus (known as IκB superrepressor, IκBsr) (Van Antwerp et al., 1996) (Figure 4c). Similar results were obtained by using AP-1 or C/EBP reporter plasmids: the activity measured by luciferase assay was increased in ΔEGFR cells (P<0.05), but not in wtEGFR or DK cells (Figures 4d and f). We then analyzed the AP-1 pathway both at the level of upstream kinases and transcription factor expression in these isogenic cell lines. Western blots for the phosphorylated forms of the different mitogen-activated protein kinase (MAPK) classes revealed increased c-Jun N-terminal kinase (JNK) activity in unstimulated ΔEGFR cells (Figure 4e), but not ERK or p38 (data not shown). Moreover, AP-1 was also induced in ΔEGFR cells at the expression level, as demonstrated by the upregulation of c-Jun, the substrate of JNK (Figure 4e). A similar mechanism of ΔEGFR-dependent regulation of the C/EBPβ transcription factor was also observed in U87 cells (Figure 4g).

Figure 4.

Figure 4

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NF-κB, AP-1 and C/EBP are activated in ΔEGFR-expressing glioma cells. (a) Phosphorylation of Ser 536 of p65 (ph-p65), indicative of NF-κB activation, was analyzed by western blot in U87MG cell line derivatives grown in media supplemented with 10% FBS or serum-starved for 24 h or 48 h. (b) U87MG cells and their derivatives were transfected with the NF-κB reporter and transcriptional activity was measured by luciferase activity assay. (c) Activation of NF-κB in U87MG cells and their derivatives serum-starved for 48 h and then treated where indicated with 50 ng/ml TNF-α or 50 ng/ml EGF for 15 min. Activation of NF-κB was analyzed by binding of nuclear extracts to a radiolabeled NF-κB consensus oligonucleotide. (d) Promoter assay using U87MG cells and their derivatives transfected with an AP-1 reporter plasmid followed by quantification of luciferase activity. (e) Phosphorylation of JNK (ph-JNK) and c-Jun expression were analyzed by western blot in U87MG cell line derivatives, showing increased JNK activation and high levels of c-Jun in ΔEGFR-expressing cells. (f) Promoter assay using U87MG cells and their derivatives transfected with a C/EBP reporter plasmid followed by quantification of luciferase activity. (g) Analysis of C/EBPβ expression by western blot analysis in U87MG cell line derivatives. Data represent mean±s.e.m. Luciferase activity is expressed in RLU after normalizing by Renilla luciferase reporter activity. *P<0.05; **P<0.01.

The secretion of IL-8 is reduced by inhibitors of NF-κB and JNK

To verify whether NF-κB and AP-1 pathways are required by ΔEGFR cells to produce high levels of IL-8, we analyzed its secretion in serum-starved cells treated with pharmacological inhibitors that target the two pathways at different levels. Treatment of U87Δ cells with BAY11-7082, an inhibitor of IκB phosphorylation, significantly (P<0.001) reduced IL-8 secretion (Figure 5a). As AP-1 activity is regulated post-transcriptionally by MAPK-mediated phosphorylation, we next assessed whether ΔEGFR activates AP-1 through MAPKs and to identify which MAPK was involved. We treated serum-starved U87Δ cells with selective inhibitors for each MAPK-class for 24 h and then analyzed the secretion of IL-8 by ELISA. No effect on IL-8 secretion was observed after treatment with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 (10 μM) or the p38 inhibitor SB203580 (10 μM) (Figure 5b). In contrast, a significant reduction of IL-8 secretion was achieved when U87Δ cells were treated with the JNK inhibitor SP600125 (10 μM) (Figure 5b; P<0.05), confirming the results obtained by western blotting (Figure 4e).

Figure 5.

Figure 5

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Inhibition of NF-κB and JNK/cJun reduces IL-8 promoter activity and secretion in ΔEGFR-expressing glioma cells. (a) Quantification of IL-8 secretion by ELISA in supernatants of U87Δ cells serum-starved for 48 h and treated for 24 h with 10 or 20 μM of BAY11-7082, a pharmacological NF-κB inhibitor. Cells not treated (−) were used as control. (b) IL-8 quantification by ELISA in supernatants of U87Δ (Δ) cells treated with inhibitors of the different MAP kinases: PD=PD98059, MEK/ERK inhibitor (10 μM); SB=SB203580, p38 inhibitor (10 μM); SP=SP600125, JNK inhibitor (10 μM). (c) IL-8 promoter reporter and a NF-κB reporter construct (pNFκB-Luc, Clontech, containing multiple copies of the NF-κB consensus sequence) were transfected into U87Par (Par), U87Δ cells stably infected with empty pCLBabePuro (Δ-pCLBP) or U87Δ stably infected with pCLBabe-Puro containing IκB super-repressor (Δ-IκBsr) and transcriptional activity was measured by luciferase assay. (d) IL-8 promoter reporter or AP-1 reporter (pAP1-Luc, Clontech) were co-transfected into U87Δ with c-Jun dominant-negative (cJundn) or the same amount of empty vector (−) and transcriptional activity was measured by luciferase activity assay. Data represent mean±s.e.m. Luciferase activity assays are expressed in RLU. *P<0.05; **P<0.01.

IL-8 promoter activity is reduced by blocking NF-κB and JNK/c-Jun pathways

We next sought to modulate IL-8 promoter activity by transducing negative regulators of NF-kB and JNK/AP-1 into ΔEGFR-expressing cells. The transcription of the luciferase gene driven by this promoter was reduced to parental levels by co-expression of IκBsr (Figure 5c). Similarly, secretion of IL-8 was significantly reduced in ΔEGFR-expressing cells stably infected with IκBsr (P<0.01) (Supplementary Figure 4). The activity of IL-8 promoter was also reduced by the co-expression of a dominant-negative c-Jun construct in which the amino acids 3–122 of the transactivation domain were deleted (TAM67, c-Jundn) (Brown et al., 1993) (Figure 5d). Assays performed with NF-κB and AP-1 reporter constructs to corroborate the effective blockade of these pathways, mirrored the results obtained with the IL-8 construct (Figures 5c and d).

IL-8 transcription is regulated in part by Ras but not by PI-3K

It has been proposed that Ras is the common upstream regulator of NF-κB and AP-1, through the activation of PI-3K and Raf or Rac (Sparmann and Bar-Sagi, 2004). Moreover, Ras is activated by ΔEGFR (Prigent et al., 1996), thus a single signaling pathway emanating from ΔEGFR might explain the activation of both transcription factors. To test this hypothesis, we analyzed the activity of the IL-8 promoter in ΔEGFR-expressing cells transduced with a retrovirus-expressing dominant-negative Ras (Ras17N). Similar experiments were also performed using a dominant-negative PI-3K p85 subunit lacking its SH2 domain (p85ΔSH2), as well as IκBsr. IL-8 promoter activity was reduced by all constructs, except for p85ΔSH2 (Figure 6a). Analysis of the phosphorylation of Akt revealed that p85ΔSH2 was only partially effective in blocking the PI-3K pathway (Figure 6b). Based on this result, we further assessed the involvement of PI-3K in the regulation of IL-8 by using the PI-3K inhibitor, LY294002. Although the treatment of U87Δ with this inhibitor efficiently blocked Akt activation for up to 24 h (Figure 6d), no effect on IL-8 secretion was observed (Figure 6c), suggesting that the induction of this cytokine is independent of PI-3K activity. Furthermore, in reporter assays designed to elucidate interconnections between Ras and the other pathways, we found that Ras17N was able to reduce AP-1, but not NF-κB activity inU87Δ (Figure 6e), and, similarly, the JNK inhibitor SP600125 blocked the activity of the AP-1 but not of the NF-κB or C/EBP reporters (Figure 6f). These data indicate that Ras regulates IL-8 transcription solely through the activation of the JNK/AP-1 pathway, while it does not affect the other transcription factors identified.

Figure 6.

Figure 6

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Ras and PI-3K involvement in the activation of IL-8 promoter. (a) Transcriptional activity of IL-8 promoter in U87Δ cells infected with different retroviruses: pBabe-Puro empty vector (pBP) or pBabe-Puro-expressing p85ΔSH2, Ras17N or IκBsr. (b) Western blot analysis of Akt phosphorylation from lysates of U87Δ cells infected with pBabe-Puro empty vector (pBP) or containing p85ΔSH2, and Ras17N. (c) IL-8 quantification by ELISA in supernatants of U87Par (Par) and U87Δ (Δ) cells serum-starved for 24 h, replenished with fresh serum-free media and vehicle treated (dimethylsulphoxide (DMSO)) or treated with LY294002 10 μM (LY) for 4 h or 24 h. (d) Western blot analysis of Akt phosphorylation of lysates fromU87Δ treated for different times with LY294002 10 μM. (e) Transcriptional activity of IL-8 promoter, and AP-1 and NF-κB reporter constructs in U87Δ cells infected with pBabe-Puro empty vector (pBP) or pBabe-Puro-expressing Ras17N. (f) Transcriptional activity of AP-1, NF-κB and C/EBP reporter constructs in U87Δ cells treated 24 h with vehicle (DMSO) or the JNK inhibitor SP600125 (10 μM). Data represent mean±s.e.m. Luciferase activity is expressed in relative light units (RLU). *P<0.05; **P<0.01.

Blockade of NF-κB suppresses ΔEGFR-promoted glioma angiogenesis and tumor growth

As NF-κB appears to have a prominent role among the transcriptional regulators of IL-8 expression (Figure 3b), we tested the effect of NF-κB suppression on ΔEGFR tumorigenicity by shRNA-mediated knock-down. A panel of shRNAs against the p65 NF-κB subunit was tested in vitro in U87Δ cells, showing that 3 out of 4 were able to suppress p65 expression (Figure 7a). Subcutaneous injection into nude mice of U87Δ cell population stably transduced with shRNA #1 resulted in a significantly (P<0.001) reduced tumor growth when compared with U87Δ or U87Δ transduced with a shRNA against GFP (Figure 7b). As expected, the constitutive abrogation of NF-κB signaling had a profound impact on the tumor vasculature as samples from p65 shRNA cells showed a vascular density reduced by 50% compared with control tumors (Supplementary Figure 5). To confirm the effect of NF-κB on IL-8 expression, we quantified IL-8 in the same tumor lysates and observed decreased levels of this cytokine in p65 knocked-down xenografts (Figure 7c; P<0.01). Together, these results show an important role for NF-κB activation and IL-8 secretion in gliomas expressing ΔEGFR.

Figure 7.

Figure 7

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NF-κB activation is important for ΔEGFR-mediated tumorigenesis and angiogenesis. (a) Western blot analysis of p65 expression in U87Δ cells transfected with a GFP shRNA or shRNAs against the NF-κB p65 subunit. (b) Top: tumor growth curve after subcutaneous injection of U87Δ cells stably expressing a shRNA against GFP or with a shRNA against NF-κB p65 (shp65). Bottom: CD31 immunohistochemistry on corresponding xenografts, U87Δ-shGFP (right) and U87Δ-shp65 (left). (c) ELISA quantification ofIL-8 in tumor lysates generated in panel (b). Data represent mean±s.e.m. **P<0.01.

Discussion

Here, we show that a complex network of signals originating from the highly tumorigenic ΔEGFR is able to activate multiple pathways that act synergistically to potentiate the production of IL-8, a pro-inflammatory cytokine of proven importance in the progression of high-grade gliomas (Garkavtsev et al., 2004). The transcription factors activated by ΔEGFR belong to three distinct families: NF-κB, AP-1 and C/EBP.

The role of IL-8 in glioma has been under investigation for several years (Brat et al., 2005). Although in other cancers it has been demonstrated that IL-8 can directly stimulate the proliferation of tumor cells (Brew et al., 2000; Takamori et al., 2000; Yao et al., 2007), in gliomas this autocrine effect is unlikely, based on the low expression levels of its receptors in glioma cells (Zhou et al., 2002). Our data support this hypothesis, as transfection of U87Δ cells with IL-8 siRNAs did not greatly alter cell proliferation in vitro (Supplementary Figure 1) and IL-8 stimulation of U87 cells was not able to produce intracellular signaling such as ERK or Akt phosphorylation (data not shown). Rather, IL-8 may promote tumor growth by altering the microenvironment through the stimulation of angiogenesis (Garkavtsev et al., 2004). Although the uniqueness and indispensability of VEGF in regulating key events during physiological angiogenesis has been demonstrated (Ferrara et al., 1996), it is also true that some unrelated growth factors are either able to potently enhance its activity (Cao et al., 2003) or are even required for particular steps in the process (Hellstrom et al., 2007). In this context, the role of IL-8 remains controversial, because although its involvement in angiogenesis seems to be well established, the molecular mechanism and the target cells are still completely obscure. Here, we provide evidence that the ectopic overexpression of IL-8 alone in cell lines with low basal VEGF secretion can induce a switch to a more tumorigenic and more angiogenic phenotype when injected in nude mice. This suggests that in human GBMs any event that would cause IL-8 upregulation might be sufficient to enhance the pro-angiogenic phenotype, and could represent a mechanism exploited by these tumors to circumvent therapies based on anti-VEGF antibodies, even after an initial response to the treatment (Norden et al., 2008). We demonstrated a correlation between ΔEGFR expression and IL-8 upregulation in GBM clinical samples, GBM tumor spheres and, that ΔEGFR ectopic expression in GBM cell lines induces IL-8, but not VEGF, production and secretion. In other contexts, both wild-type and mutant EGFR have been found to direct VEGF expression (Gille et al., 1997; Petit et al., 1997; Magnus et al., 2010). However, although U87 cells secrete low levels of VEGF in vitro, this basal VEGF secretion could be required in vivo to obtain angiogenesis. The combined effect of constitutive EGFR signaling and hypoxic conditions on the expression of VEGF and IL-8, and the relative importance of the two cytokines, individually and together, needs to be further addressed. Our results illustrate that, in absence of other signaling inputs, signaling generated by ΔEGFR is able to produce an abundance of a pro-angiogenic factor such as IL-8, which therefore might represent in ΔEGFR-positive GBMs an additional element acting to potentiate the well-known hypoxia-VEGF axis. We showed that the inhibition of IL-8 was able to reduce the stimulatory activity of U87Δ CM on endothelial cells in vitro. In this context, the negligible amounts of VEGF secreted by U87 cells allowed us to highlight the differences between U87Δ with normal or reduced IL-8 secretion and to attribute ΔEGFR-mediated stimulation of endothelial tube formation to IL-8. However, more thorough experiments will be required to better assess the contribution of IL-8 on tumor angiogenesis in vivo and the possible existence of other factors specifically upregulated by ΔEGFR.

Prior studies have shown that EGFR-mediated activation of NF-κB required PI-3K/Akt signaling (Bancroft et al., 2002; Le Page et al., 2005), which is supported by evidence that Akt can interact with IκB kinase complex-α (Romashkova and Makarov, 1999). However, it has also been suggested that alternative mechanisms might exist for EGF-induced NF-κB activation, independent of IκB kinase complexes (Garkavtsev et al., 2004; Weissenberger et al., 2004). This model requires the activity of NF-κB inducing kinase (NIK), which has been found to be a component of all ErbB family receptors signaling complexes and to be required for NF-κB activation induced by EGF or heregulins (Chen et al., 2003). Our data support a PI-3K-independent activation of NF-κB downstream of ΔEGFR based on the inability of LY294002, a potent inhibitor of PI-3K, to reduce the secretion of IL-8, and dominant-negative PI-3K incapable of blocking the IL-8 promoter activity. Further studies are required to elucidate whether this unique signaling is a common feature of glioma cells or is specifically driven by ΔEGFR signaling.

In the presence of growth factors, Ras can activate MEKK1, either directly or indirectly through PI-3K and Rac1 or Cdc42Hs. MEKK1 in turn phosphorylates the MAPK kinases, MKK4 and MKK7, which then activate JNK (Kyriakis and Avruch, 2001). JNK has been found to be activated in cells transfected with ΔEGFR, and it was proposed that its activation is mediated by PI-3K (Antonyak et al., 1998). Our data suggest a different link between ΔEGFR and JNK, as PI-3K inhibition did not affect either the secretion of IL-8 (Figure 6) or JNK activation (data not shown). As the JNK inhibitor was quite effective in decreasing IL-8 secretion, we hypothesize that JNK activity is important for the overexpression of this cytokine, although we cannot exclude the possibility that the upregulation of c-Jun alone might be sufficient to ensure basal constitutive AP-1 activity or that in ΔEGFR-positive cells other activating mechanisms might exist, for example, through MEK/ERK. The reduction in transcriptional activity observed when introducing a dominant-negative Ras is similar to that obtained with a dominant-negative c-Jun, but was less effective than IκBsr. Moreover, Ras17N and the JNK inhibitor SP600125 reduced AP-1 reporter activity to a similar extent, while they were both ineffective on NF-kB. Collectively, these data indicate the existence of a Ras/JNK/AP-1 pathway in U87Δ cells, while NF-κB activation seems to be independent of Ras or PI-3K. It is noteworthy that VEGF can also be regulated by Rasin GBM under both normoxic and hypoxic conditions (Feldkamp et al., 1999), pointing at this pathway as a key regulator of angiogenic events in astrocytic tumors.

Our data show that ectopic expression of IL-8 in human glioma cell lines dramatically increases their ability to form tumors in nude mice. Moreover, we demonstrated that IL-8 is an important target of ΔEGFR signaling, as its knock-down was able to reduce the tumor volume by approximately 50%. In previous work, similar knock-down of IL-6 expression in ΔEGFR cells, in contrast to these results with IL-8, resulted in no difference in tumor formation when compared with non-transfected cells (Inda et al., 2010). On the contrary, in IL-6 was able to stimulate the growth of cells overexpressing wtEGFR a paracrine fashion. We hypothesize that, in the scenario of a tumor composed of different cell populations with amplified/mutated EGFR, as in most clinical samples (Biernat et al., 2004; Nishikawa et al., 2004), combined therapy targeting the two cytokines might have synergistic efficacy. As the knock-down of the NF-κB p65 subunit produced a stronger impairment in the tumor growth of ΔEGFR cells than that obtained by knocking-down IL-8, we predict that NF-κB targets are not limited to IL-8. ΔEGFR cells show a marked resistance to cytotoxic drugs in vitro, mediated through increased expression of Bcl-XL (Nagane et al., 1998). NF-κB is a well-known regulator of many anti-apoptotic genes including Bcl-XL itself (Tsukahara et al., 1999), but also Bcl-2 and cellular inhibitors of apoptosis (Karin and Lin, 2002), thus it is possible that NF-κB not only regulates growth promoting cytokines, but also induces an apoptosis-resistant phenotype.

Materials and methods

Cell lines

Glioma cell lines were engineered to overexpress wtEGFR, ΔEGFR or a dead kinase version of ΔEGFR as described previously (Nishikawa et al., 1994). U87Par-IL-8 and U87wt-IL-8 were obtained by transducing U87Par and U87wt, respectively, with a pBabePuro retrovirus expressing the full-length human IL-8 complementary DNA obtained by reverse transcriptase–PCR, and stably selected.

Plasmids

NF-κB and AP-1 reporter plasmids (pNFκB-Luc and pAP1-Luc) were purchased from Clontech (Mountain View, CA, USA). IL-8 promoter was obtained by PCR using human genomic DNA and the following primers: 5′-GCGGTACCGAATTCAGTAACCCAGGCATTATT-3′, 5′-CCTAGAAG CTTGTGTGCTCTGC-3′ (−1480 to +50). The resulting amplified fragment was sequenced and inserted into the pNFkB-Luc backbone (Clontech). The mutations in AP-1, NF-κB and C/EBP consensus sequences were inserted by PCR using the following oligonucleotides and corresponding reverse primers (point mutations in capital letters): AP-1 5′-aagtgtga tgCAGcaggtttgccctgagg-3′, C/EBP, 5′-ggccatcagAtATCaatcgt ggaatttcc-3′ and NF-κB 5′-ttgcaaatcgtAgaTCttcctctgacataa-3′.

The full-length human IL-8 complementary DNA was obtained by reverse transcriptase–PCR, sequenced and cloned into the BamHI/SalI sites of pBabePuro. Dominant-negative c-Jun was obtained by PCR amplification from human fetal complementary DNA to obtain amino acids 123–331 (lacking the transactivation domain) and inserted in the EcoRI/XhoI sites of pCMV-HA (Clontech). The IκBsr construct, consisting of mouse IκB mutated at serines 32, 36, 283, 288 and 293 and threonines 291 and 296 to alanines was a kind gift of Dr Inder Verma (Salk Institute, La Jolla, CA, USA). Dominant-negative PI-3K p85 in which the SH2 domain was deleted (p85ΔSH2) was a kind gift of Dr Julian Downward. Dominant-negative Ras (Ras17N) was a kind gift of Dr Gerry Boss (UCSD, La Jolla, CA, USA).

Antibodies

IL-8 antibodies were purchased from R&D Systems (Minneapolis, MN, USA); NF-κB p65 (total and phospho-Ser536), SAPK/JNK (total and phospho-Thr183/Tyr185), c-Jun, C/EBPβ, phospho-Akt (Ser473) were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin was from Sigma (St Louis, MO, USA). The cytokine array was purchased from Ray Biotech (Norcross, GA, USA) and performed according to the manufacturer’s instructions.

Inhibitors

BAY11-7082, PD98059, SB203580, SP600125 and LY294002 were purchased from Calbiochem (Rockland, MA, USA).

Luciferase assay

Double luciferase assays were used to access the activities of reporter genes. Briefly, cells were seeded in 12-well plates at 105 cells per well and the day after were transfected with 200 ng of the indicated reporter plasmid expressing the firefly luciferase and 20 ng of the control plasmid consisting of Renilla luciferase under the control of the Herpes simplex virus thymidine kinase promoter, plus 4 μl of Lipofectamine 2000 per well. When indicated, 500 ng of an additional expression construct, or the same amount of empty vector as control, were added to the mixture. The following day the cells were washed, serum-starved for 24 h and then lysates were prepared. The luciferase activity was measured by using a dual luciferase assay kit (Promega, Madison, WI, USA) and a Tecan Genios Proluminometer (Tecan, Männedorf, Switzerland) according to the manufacturer’s instructions.

ELISA

Sandwich ELISAs were performed as described previously (Inda et al., 2010). Briefly, microplates coated overnight with capture antibody were incubated 2 h at room temperature with supernatants from 48-h starved cells, freshly collected and spun to eliminate debris, followed by a similar incubation with the detection antibody. The colorimetric reaction was measured in a microplate reader (Tecan Genios Pro) and the readings were normalized with a four-parameter logistic curve.

Electrophoretic mobility shift assay

Cells serum-starved for 48 h and treated as indicated were harvested and resuspended in buffer A (10mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl2, 1mM DTT) supplemented with protease and phosphatase inhibitor mixes (Sigma). The cells were allowed to swell on ice for 15 min, and then Nonidet P-40 was added to a final concentration of 0.1%. The suspension was then vortexed for 10 s, and centrifuged at 5000 g for 10 min. The nuclear pellet was resuspended in buffer B (20mM HEPES pH 7.9, 400mM KCl, 1mM EDTA, 20% glycerol, 1mM DTT, and protease and phosphatase inhibitors). After 30min on ice with intermittent mixings, nuclear extracts were cleared at 10 000 g for 5 min at 4 °C and supernatants were collected.

A double-stranded oligonucleotide containing the NF-κB consensus sequence (in capitals) (5′-agttgagGGGACTTTCC caggc-3′), purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), was labeled by using [γ-32P]ATP and T4 polynucleotide kinase and purified by gel filtration on S-200 spin columns (Pharmacia, Piscataway, NJ, USA). Five μg of nuclear extract protein and the labeled oligonucleotide were incubated in 10mM Tris (pH 7.4), 10mM EDTA, 0.5% bovine serum albumin, 0.5M NaCl, 10mM DTT, 50% glycerol and 1mg of poly (dI-dC) in a final volume of 10 μl for 30 min at room temperature. The protein–DNA complexes were resolved on 4% polyacrylamide gels and visualized by autoradiography.

Real-time quantitative PCR

Total RNA was extracted from 19 human GBM samples and from 9 gliomatumorspheres isolated from GBM patients as described previously (Inda et al., 2010). Real-time PCR was performed using Maxima SYBR Green qPCR Master mix (Fermentas, Glen Burnie, MD, USA) on an iCycler (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Primers used for IL-8 amplification were: 5′-TAAACATGACTTCCAAGCTGGCCG-3′ (forward) and 5′-GTGTGGTCCACTCTCAATCACTCT-3′ (reverse). Primers used for ΔEGFR and glyceraldehyde 3-phosphate dehydrogenase amplification were the same described previously (Inda et al., 2010). Glyceraldehyde 3-phosphate dehydrogenase expression was used for normalization and relative quantification using the Ct values was performed for each sample.

RNA interference

A set of 24 siRNAs targeting IL-8 was designed and synthesized on an ABI3900 DNA synthesizer using standard procedures, purified by anion exchange (AEX) high-performance liquid chromatography, and annealed in phosphate-buffered saline. SiRNAs were tested in vitro: U87Δ cells were seeded in 24-well plates at 48 000 cells per well. The following day, cells were transfected with different concentrations of siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and Opti-MEM (Gibco, Carlsbad, CA, USA). At 24 h after transfection, the medium was changed to serum-free Dulbecco’s modified Eagle’s medium media, which was collected 48 h after starvation and analyzed for IL-8 secretion by ELISA. Cells non-transfected as well as transfected with a siRNA specific for GFP (5′-GCUG ACCCUGAAGUUCAUC-3′) (Dharmacon, Lafayette, CO, USA) were included as negative control. The IL-8 siRNA sequence with chemical modifications follows (lower case letters indicate 2′-O-methyl modification at that position; s indicates phosphorothioate linkage; dT indicates deoxythymidine): antisense strand 5′-UGGCuAGcAGACuAGGGUUdTsdT-3′, sense strand 5′-AAcccuAGucuGcuAGccAdTsdT-3′.

Lentiviral vectors expressing shRNAs were purchased from the Broad Institute (Boston, MA, USA). Viral particles were produced by transient co-transfection of HEK293FT cells with 2μg of the shRNA construct, 200 ng of vesicular stomatitis virus G protein (VSV-G) and 2 μg of Δ 8.9 plasmids using Lipofectamine 2000 (Invitrogen). HEK293FT supernatants were collected 48 h after transfection, filtered and applied to U87Δ cells. Infected cells were selected in medium containing 400 ng/ml of Puromycin.

Tumor engraftment

For intracranial injection, 5×105 cells in 5 μl of phosphate-buffered saline were injected into 4–5 weeks old athymic nude mice using a guide-screw system according to the protocol described by Lal et al. (2000). For subcutaneous injection, the different U87MG cell lines were harvested, suspended in phosphate-buffered saline and injected subcutaneously into the right flank of 4 to 5 weeks old female athymic nude mice (106 cells per mouse in 100 μl for U87Par and U87wt, and 5×105 cells per mouse in 100 μl for U87Δ). Tumors were measured periodically with a vernier caliper and tumor volumes were calculated using the formula V=a2×b/2 where a≤b. Mice were euthanized when tumor volumes reached 1500mm3 (subcutaneous) or at the first neurological symptoms (intracranial). For in vivo experiments with IL-8 siRNA, the cells were transfected with 25 nM siRNA as described above, propagated in vitro and injected subcutaneously 3 to 4 days after transfection.

In vitro HUVEC tube formation assay

We used an in vitro angiogenesis assay kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. HUVECs were exposed to CM taken from U87Δ cells 3 days after transfection with GFP or IL-8 siRNA. The tube formation was monitored and captured using an Olympus IX51 fluorescence microscope (Center Valley, PA, USA).

Supplementary Material

Acknowledgments

We thank Donna Harclerode and Donald Pizzo for technical assistance with immunostaining and Dr Cameron Brennan for providing the GBM tumorspheres. This work was supported by an award from the Goldhirsh Foundation (to FBF), and NIH Grant P01-CA95616 (to WKC, FBF), as well as NIH RO1CA130966, a grant with Pennsylvania Department of Health and an Innovative Research Scholar Award of the Hillman Foundation (to SYC). WKC is a fellow of the National Foundation for Cancer Research. MM Inda thanks the Gobierno de Navarra, Spain, and the American Brain Tumor Association in Honor of Walter Terlik for the fellowships received. R Bonavia was supported by a fellowship from Federazione Italiana Ricerca Cancro.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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

References

  1. Antonyak MA, Moscatello DK, Wong AJ. Constitutive activation of c-Jun N-terminal kinase by a mutant epidermal growth factor receptor. J Biol Chem. 1998;273:2817–2822. doi: 10.1074/jbc.273.5.2817. [DOI] [PubMed] [Google Scholar]
  2. Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, et al. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell. 2002;1:269–277. doi: 10.1016/s1535-6108(02)00046-6. [DOI] [PubMed] [Google Scholar]
  3. Bancroft CC, Chen Z, Yeh J, Sunwoo JB, Yeh NT, Jackson S, et al. Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. Int J Cancer. 2002;99:538–548. doi: 10.1002/ijc.10398. [DOI] [PubMed] [Google Scholar]
  4. Biernat W, Huang H, Yokoo H, Kleihues P, Ohgaki H. Predominant expression of mutant EGFR (EGFRvIII) is rare in primary glioblastomas. Brain Pathol. 2004;14:131–136. doi: 10.1111/j.1750-3639.2004.tb00045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biswas DK, Cruz AP, Gansberger E, Pardee AB. Epidermal growth factor-induced nuclear factor kappa B activation: a major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells. Proc Natl Acad Sci USA. 2000;97:8542–8547. doi: 10.1073/pnas.97.15.8542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol. 2005;7:122–133. doi: 10.1215/S1152851704001061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brew R, Erikson JS, West DC, Kinsella AR, Slavin J, Christmas SE. Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine. 2000;12:78–85. doi: 10.1006/cyto.1999.0518. [DOI] [PubMed] [Google Scholar]
  8. Brown PH, Alani R, Preis LH, Szabo E, Birrer MJ. Suppression of oncogene-induced transformation by a deletion mutant of c-jun. Oncogene. 1993;8:877–886. [PubMed] [Google Scholar]
  9. Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, Wahlberg E, et al. Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med. 2003;9:604–613. doi: 10.1038/nm848. [DOI] [PubMed] [Google Scholar]
  10. Chen D, Xu LG, Chen L, Li L, Zhai Z, Shu HB. NIK is a component of the EGF/heregulin receptor signaling complexes. Oncogene. 2003;22:4348–4355. doi: 10.1038/sj.onc.1206532. [DOI] [PubMed] [Google Scholar]
  11. Feldkamp MM, Lau N, Rak J, Kerbel RS, Guha A. Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras. Int J Cancer. 1999;81:118–124. doi: 10.1002/(sici)1097-0215(19990331)81:1<118::aid-ijc20>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  12. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–442. doi: 10.1038/380439a0. [DOI] [PubMed] [Google Scholar]
  13. Garkavtsev I, Kozin SV, Chernova O, Xu L, Winkler F, Brown E, et al. The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis. Nature. 2004;428:328–332. doi: 10.1038/nature02329. [DOI] [PubMed] [Google Scholar]
  14. Gille J, Swerlick RA, Caughman SW. Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO J. 1997;16:750–759. doi: 10.1093/emboj/16.4.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heimberger AB, Hlatky R, Suki D, Yang D, Weinberg J, Gilbert M, et al. Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients. Clin Cancer Res. 2005;11:1462–1466. doi: 10.1158/1078-0432.CCR-04-1737. [DOI] [PubMed] [Google Scholar]
  16. Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Lindblom P, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445:776–780. doi: 10.1038/nature05571. [DOI] [PubMed] [Google Scholar]
  17. Holland EC, Hively WP, DePinho RA, Varmus HE. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev. 1998;12:3675–3685. doi: 10.1101/gad.12.23.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holtmann H, Winzen R, Holland P, Eickemeier S, Hoffmann E, Wallach D, et al. Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways. Mol Cell Biol. 1999;19:6742–6753. doi: 10.1128/mcb.19.10.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huang HS, Nagane M, Klingbeil CK, Lin H, Nishikawa R, Ji XD, et al. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J Biol Chem. 1997;272:2927–2935. doi: 10.1074/jbc.272.5.2927. [DOI] [PubMed] [Google Scholar]
  20. Hurtt MR, Moossy J, Donovan-Peluso M, Locker J. Amplification of epidermal growth factor receptor gene in gliomas: histopathology and prognosis. J Neuropathol Exp Neurol. 1992;51:84–90. doi: 10.1097/00005072-199201000-00010. [DOI] [PubMed] [Google Scholar]
  21. Inda MM, Bonavia R, Mukasa A, Narita Y, Sah DW, Vandenberg S, et al. Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev. 2010;24:1731–1745. doi: 10.1101/gad.1890510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jaros E, Perry RH, Adam L, Kelly PJ, Crawford PJ, Kalbag RM, et al. Prognostic implications of p53 protein, epidermal growth factor receptor, and Ki-67 labelling in brain tumours. Br J Cancer. 1992;66:373–385. doi: 10.1038/bjc.1992.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jiang X, Takahashi N, Matsui N, Tetsuka T, Okamoto T. The NF-kappa B activation in lymphotoxin beta receptor signaling depends on the phosphorylation of p65 at serine 536. J Biological Chem. 2003;278:919–926. doi: 10.1074/jbc.M208696200. [DOI] [PubMed] [Google Scholar]
  24. Jochum W, Passegue E, Wagner EF. AP-1 in mouse development and tumorigenesis. Oncogene. 2001;20:2401–2412. doi: 10.1038/sj.onc.1204389. [DOI] [PubMed] [Google Scholar]
  25. Karin M, Cao Y, Greten FR, Li ZW. NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer. 2002;2:301–310. doi: 10.1038/nrc780. [DOI] [PubMed] [Google Scholar]
  26. Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nat Immunol. 2002;3:221–227. doi: 10.1038/ni0302-221. [DOI] [PubMed] [Google Scholar]
  27. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001;81:807–869. doi: 10.1152/physrev.2001.81.2.807. [DOI] [PubMed] [Google Scholar]
  28. Lal S, Lacroix M, Tofilon P, Fuller GN, Sawaya R, Lang FF. An implantable guide-screw system for brain tumor studies in small animals. J Neurosurg. 2000;92:326–333. doi: 10.3171/jns.2000.92.2.0326. [DOI] [PubMed] [Google Scholar]
  29. Lee CH, Jeon YT, Kim SH, Song YS. NF-kappaB as a potential molecular target for cancer therapy. Biofactors. 2007;29:19–35. doi: 10.1002/biof.5520290103. [DOI] [PubMed] [Google Scholar]
  30. Le Page C, Koumakpayi IH, Lessard L, Saad F, Mes-Masson AM. Independent role of phosphoinositol-3-kinase (PI3K) and casein kinase II (CK-2) in EGFR and Her-2-mediated constitutive NF-kappaB activation in prostate cancer cells. Prostate. 2005;65:306–315. doi: 10.1002/pros.20291. [DOI] [PubMed] [Google Scholar]
  31. Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol. 2002;2:725–734. doi: 10.1038/nri910. [DOI] [PubMed] [Google Scholar]
  32. Magnus N, Garnier D, Rak J. Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells. Blood. 2010;116:815–818. doi: 10.1182/blood-2009-10-250639. [DOI] [PubMed] [Google Scholar]
  33. Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev. 2001;15:1311–1333. doi: 10.1101/gad.891601. [DOI] [PubMed] [Google Scholar]
  34. Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, et al. The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells. J Cell Biol. 1998;143:1087–1099. doi: 10.1083/jcb.143.4.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, et al. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA. 1993;90:10193–10197. doi: 10.1073/pnas.90.21.10193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nagane M, Levitzki A, Gazit A, Cavenee WK, Huang HJ. Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc Natl Acad Sci USA. 1998;95:5724–5729. doi: 10.1073/pnas.95.10.5724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Narita Y, Nagane M, Mishima K, Huang HJ, Furnari FB, Cavenee WK. Mutant epidermal growth factor receptor signaling down-regulates p27 through activation of the phosphatidylinositol 3-kinase/Akt pathway in glioblastomas. Cancer Res. 2002;62:6764–6769. [PubMed] [Google Scholar]
  38. Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA. 1994;91:7727–7731. doi: 10.1073/pnas.91.16.7727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nishikawa R, Sugiyama T, Narita Y, Furnari F, Cavenee WK, Matsutani M. Immunohistochemical analysis of the mutant epidermal growth factor, deltaEGFR, in glioblastoma. Brain Tumor Pathol. 2004;21:53–56. doi: 10.1007/BF02484510. [DOI] [PubMed] [Google Scholar]
  40. Norden AD, Drappatz J, Wen PY. Novel anti-angiogenic therapies for malignant gliomas. Lancet Neurol. 2008;7:1152–1160. doi: 10.1016/S1474-4422(08)70260-6. [DOI] [PubMed] [Google Scholar]
  41. Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B, et al. Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol. 1997;151:1523–1530. [PMC free article] [PubMed] [Google Scholar]
  42. Prigent SA, Nagane M, Lin H, Huvar I, Boss GR, Feramisco JR, et al. Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway. J Biol Chem. 1996;271:25639–25645. doi: 10.1074/jbc.271.41.25639. [DOI] [PubMed] [Google Scholar]
  43. Romashkova JA, Makarov SS. NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature. 1999;401:86–90. doi: 10.1038/43474. [DOI] [PubMed] [Google Scholar]
  44. Schlegel J, Merdes A, Stumm G, Albert FK, Forsting M, Hynes N, et al. Amplification of the epidermal-growth-factor-receptor gene correlates with different growth behaviour in human glioblastoma. Int J Cancer. 1994;56:72–77. doi: 10.1002/ijc.2910560114. [DOI] [PubMed] [Google Scholar]
  45. Shinojima N, Tada K, Shiraishi S, Kamiryo T, Kochi M, Nakamura H, et al. Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res. 2003;63:6962–6970. [PubMed] [Google Scholar]
  46. Sparmann A, Bar-Sagi D. Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis. Cancer Cell. 2004;6:447–458. doi: 10.1016/j.ccr.2004.09.028. [DOI] [PubMed] [Google Scholar]
  47. Takamori H, Oades ZG, Hoch OC, Burger M, Schraufstatter IU. Autocrine growth effect of IL-8 and GROalpha on a human pancreatic cancer cell line, Capan-1. Pancreas. 2000;21:52–56. doi: 10.1097/00006676-200007000-00051. [DOI] [PubMed] [Google Scholar]
  48. Tsukahara T, Kannagi M, Ohashi T, Kato H, Arai M, Nunez G, et al. Induction of Bcl-x(L) expression by human T-cell leukemia virus type 1 Tax through NF-kappaB in apoptosis-resistant T-cell transfectants with Tax. J Virol. 1999;73:7981–7987. doi: 10.1128/jvi.73.10.7981-7987.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science. 1996;274:787–789. doi: 10.1126/science.274.5288.787. [DOI] [PubMed] [Google Scholar]
  50. van Dam H, Castellazzi M. Distinct roles of Jun: Fos and Jun: ATF dimers in oncogenesis. Oncogene. 2001;20:2453–2464. doi: 10.1038/sj.onc.1204239. [DOI] [PubMed] [Google Scholar]
  51. Wang ZQ, Grigoriadis AE, Mohle-Steinlein U, Wagner EF. A novel target cell for c-fos-induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras. EMBO J. 1991;10:2437–2450. doi: 10.1002/j.1460-2075.1991.tb07783.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weissenberger J, Loeffler S, Kappeler A, Kopf M, Lukes A, Afanasieva TA, et al. IL-6 is required for glioma development in a mouse model. Oncogene. 2004;23:3308–3316. doi: 10.1038/sj.onc.1207455. [DOI] [PubMed] [Google Scholar]
  53. Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med. 1996;74:589–607. doi: 10.1007/s001090050063. [DOI] [PubMed] [Google Scholar]
  54. Yao C, Lin Y, Chua MS, Ye CS, Bi J, Li W, et al. Interleukin-8 modulates growth and invasiveness of estrogen receptor-negative breast cancer cells. Int J Cancer. 2007;121:1949–1957. doi: 10.1002/ijc.22930. [DOI] [PubMed] [Google Scholar]
  55. Young MR, Li JJ, Rincon M, Flavell RA, Sathyanarayana BK, Hunziker R, et al. Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion. Proc Natl Acad Sci USA. 1999;96:9827–9832. doi: 10.1073/pnas.96.17.9827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhong Z, Wen Z, Darnell JE., Jr Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 1994;264:95–98. doi: 10.1126/science.8140422. [DOI] [PubMed] [Google Scholar]
  57. Zhou Y, Larsen PH, Hao C, Yong VW. CXCR4 is a major chemokine receptor on glioma cells and mediates their survival. J Biol Chem. 2002;277:49481–49487. doi: 10.1074/jbc.M206222200. [DOI] [PubMed] [Google Scholar]

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