Original Article | Published:

Epidermal growth factor receptor variant III mediates head and neck cancer cell invasion via STAT3 activation

Oncogene volume 29, pages 51355145 (16 September 2010) | Download Citation

Abstract

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to antitumor drugs including the EGFR-targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. This study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased signal transducer and activator of transcription 3 (STAT3) activation, which was not abrogated by cetuximab treatment. Further investigation showed that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion by increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors.

Introduction

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that regulates crucial cellular signaling pathways contributing to tumor progression. EGFR is frequently amplified and overexpressed in several human solid tumors including in a high percentage of head and neck squamous cell carcinomas (HNSCCs). EGFR overexpression in HNSCC has been correlated with tumor progression, resistance to conventional therapy and poor prognosis (Grandis and Tweardy, 1993). Preclinical studies showed the antitumor effects of EGFR targeting and the FDA approved the EGFR monoclonal antibody cetuximab for clinical use in HNSCC based on the results of a phase III trial (Bonner et al., 2006). However, although combining EGFR targeting with radiation prolonged overall survival, it did not reduce the incidence of metastasis. Despite the nearly ubiquitous expression of EGFR in HNSCC, there is only a 13% response rate when cetuximab is administered as a single agent (Vermorken et al., 2007). The tumor features that contribute to resistance to EGFR targeting are incompletely understood.

Receptor alterations that influence ligand and antibody binding may have a role in therapeutic resistance. The most common EGFR alteration in several cancers, including HNSCC, consists of a truncation in the extracellular domain known as EGFR variant III (EGFRvIII). This mutation eliminates exons 2–7 resulting in a distorted ligand-binding region (Bigner et al., 1990; Sugawa et al., 1990). EGFRvIII does not bind ligand but is constitutively activated in a ligand-independent manner. The presence of EGFRvIII in human tumors has been associated with tumor growth, metastasis and survival in several malignancies, including glioma, carcinomas of the breast, lung and HNSCC (Pedersen et al., 2001). Furthermore, EGFRvIII has been reported to increase resistance to antitumor agents including EGFR inhibitors (Sok et al., 2006).

We previously reported the expression of EGFRvIII in up to 42% of HNSCC where coexpression of EGFRvIII with wild-type EGFR increased HNSCC cell proliferation in vitro and tumor volume in vivo. Moreover, EGFRvIII decreased HNSCC cell apoptosis in response to cisplatin and decreased growth inhibition after treatment with cetuximab (Sok et al., 2006). Although these results support the role of EGFRvIII in mediating tumor growth in response to EGFR targeting, the contribution of EGFRvIII to invasion and the precise downstream pathways that are induced by EGFRvIII are incompletely understood. EGFRvIII expression in glioma has been reported to correlate with expression of phosphotyrosine signal transducer and activator of transcription 3 (STAT3; Mizoguchi et al., 2006). The lethality of HNSCC is associated with the tendency of these cancers to invade surrounding structures and metastasize. This study was undertaken to test the hypothesis that EGFRvIII induces HNSCC invasion and subsequently, metastasis through activation of STAT3 signaling.

Results

Expression of EGFRvIII in engineered HNSCC cells

We previously reported that EGFRvIII is expressed in approximately 40% of HNSCC tumors (Sok et al., 2006). For reasons that are incompletely understood, expression of EGFRvIII in human tumors is routinely lost in tissue culture (Bigner et al., 1990). Therefore, to study the consequences of EGFRvIII in HNSCC, we transfected EGFRvIII into a representative HNSCC cell line (686LN) as described in the Materials and Methods section. Due to the high level of wild-type EGFR in HNSCC cell lines and tissues, commercially available EGFR antibodies that are reported to detect both EGFRvIII and wild-type EGFR in other tumor systems are unable to identify EGFRvIII expression in HNSCC. Expression of the EGFRvIII transcript was therefore determined by reverse transcription (RT)–PCR where the HNSCC cell line Cal33 stably expressing EGFRvIII was used as the control for EGFRvIII. EGFRvIII gene expression was detected in transient transfectants through 6 days (the duration of the experiments used) after transfection (Figure 1a). For the animal experiments, the HNSCC cell line Cal33 was stably transfected with EGFRvIII or vector control as described previously (Sok et al., 2006). Flow cytometry was performed to measure the degree of expression of EGFRvIII in the stably transfected Cal33 clone. FACS analysis revealed no EGFRvIII expression in the vector-transfected control cells and approximately 5 × 103 EGFRvIII receptors per cell in the EGFRvIII-transfected Cal33 cells (Figure 1b). Quantitative PCR was also performed to determine the relative expression levels of wild-type EGFR and EGFRvIII in these cells and showed a sevenfold higher level of wild-type EGFR compared with EGFRvIII (data not shown). These results are consistent with findings in human HNSCC where all HNSCC tumors that express EGFRvIII also express wild-type EGFR, with a higher level of wild-type EGFR compared to EGFRvIII (Sok et al., 2006).

Figure 1
Figure 1

EGFRvIII expression in HNSCC-transfected cells. (a) EGFRvIII mRNA levels were examined in HNSCC transiently transfected cells (686LN) by RT–PCR. Representative ethidium bromide-stained gel showing EGFRvIII and GAPDH PCR products from two vector control transfections (1 and 6 days after transfection) and EGFRvIII-transfected cells (1–6 days after transfection). The EGFR-transfected Cal33vIII cell line was used as positive control for EGFRvIII-transfected cells. Experiments were repeated three times with similar results. (b) Flow cytometry results for the Cal33 clone stably transfected with EGFRvIII and its respective vector control clone. Surface EGFRvIII on Cal33vIII was detected by flow cytometry. A vector-transfected control clone (control-1) was used as negative control. Data are represented as mean fluorescent intensity. Experiments were repeated three times with similar results.

EGFRvIII increases HNSCC motility and invasion

We previously reported that EGFRvIII induces HNSCC cell proliferation in vitro and tumor growth in vivo (Sok et al., 2006). EGFRvIII has been shown to induce motility in murine fibroblasts (Pedersen et al., 2004). To determine the consequences of EGFRvIII on directional HNSCC cell motility, we performed cell migration assays using a transwell assay. As shown in Figure 2a, HNSCC cell migration was increased in EGFRvIII-expressing cell compared to a vector-transfected control (P=0.03). To validate these findings in other HNSCC cell lines, we transiently transfected 1483 and PCI-37A cells with an EGFRvIII expression plasmid and tested the migration of the cells. EGFRvIII-expressing cells showed increased migration (data not shown). We next assessed the consequences of EGFRvIII on HNSCC cell invasion through Matrigel, controlling for proliferation. As shown in Figure 2a, EGFRvIII-expressing HNSCC cells were significantly more invasive than vector-transfected controls (P=0.03).

Figure 2
Figure 2

EGFRvIII increases HNSCC migration and invasion. (a) The migration of EGFRvIII-transfected 686LN cells (vIII) compared with a vector-transfected control was determined using a Boyden chamber assay. The fold increase in the EGFRvIII-transfected cells was compared with vector-transfected controls. Cumulative results are shown from four independent experiments (*P=0.03). The invasive capacity of EGFRvIII-expressing 686LN cells (vIII) compared with a vector-transfected control (control-1) was determined using a Boyden chamber Matrigel assay. The fold increase in invasion relative to vector-transfected control is shown. Cumulative results are shown from four independent experiments (*P=0.03). (b) EGFRvIII-transfected 686LN cells (vIII) and vector-transfected control cells (control) were subjected to a migration assay in the presence of EGF (10 ng/ml) and/or C225 (7 μg/ml) for 24 h. The fold increase in migration relative to untreated vector-transfected control (control) is shown. The migration of EGFRvIII-expressing HNSCC cells was not abrogated by C225 treatment compared with vector-transfected control cells (*P=0.03). (c) The experiment from b was repeated using an invasion assay. The invasion of EGFRvIII-expressing HNSCC (686LN) cells was not abrogated by C225 treatment compared to vector-transfected control cells (*P=0.03).

Cetuximab was FDA-approved for the treatment of HNSCC in 2006. We previously reported that HNSCC cells expressing EGFRvIII are relatively resistant to the growth inhibitory effects of cetuximab in vitro and in vivo (Sok et al., 2006). Because the addition of cetuximab to radiation did not prevent metastasis in HNSCC patients, we next determined the effects of cetuximab on EGFRvIII-mediated migration and invasion (Bonner et al., 2006). As shown in Figures 2b and c, although cetuximab abrogated EGF-induced migration and invasion of vector control-transfected HNSCC cells, treatment of EGFRvIII-expressing HNSCC cells with cetuximab failed to decrease migration or invasion. Although EGF induced migration and invasion of vector-transfected control cells, EGF treatment had no significant effect on the motility or invasive capacity of EGFRvIII cells (which are more motile and invasive than controls in the absence of growth factor stimulation). These results suggest that EGFRvIII induces HNSCC cell motility and invasion in vitro, which are not abrogated by treatment with the only clinically approved EGFR-targeting strategy in HNSCC. These results were also validated in a transitional cell carcinoma model (T-24 cells) stably expressing EGFRvIII (data not shown).

EGFRvIII increases STAT3 activation

The precise signaling pathways induced by EGFRvIII in the setting of cancer cells that also express wild-type EGFR are incompletely understood. STAT3 is activated downstream of several receptor and nonreceptor tyrosine kinases including EGFR. A correlation between EGFRvIII expression and expression of phosphotyrosine STAT3 has been noted in glioblastomas (Mizoguchi et al., 2006). We previously reported that STAT3 is activated downstream of wild-type EGFR in HNSCC (Grandis et al., 1998). To determine whether STAT3 is differentially activated by EGFRvIII, we analyzed expression of tyrosine phosphorylated STAT3 by immunoblotting in addition to STAT3 transcriptional activity in the EGFRvIII-expressing HNSCC cells compared with vector controls. As shown in Figure 3a, phosphotyrosine STAT3 was expressed at higher levels in HNSCC cells that contain EGFRvIII compared to vector-transfected controls. Other HNSCC cells (1483 and PCI-37A) transiently transfected with the EGFRvIII cDNA construct and the EGFRvIII stably expressing urothelial line T-24 also showed higher levels of phosphorylated STAT3 (data not shown). In addition, EGFRvIII-expressing HNSCC cells showed increased STAT3 transcriptional activity using an hSIE-luciferase reporter assay compared with controls (P<0.001) (Figure 3b). To determine the relative expression levels of phosphorylated and total STAT3 in EGFRvIII-expressing HNSCC tumors, we analyzed xenografts derived from EGFRvIII-expressing cells. As shown in Figure 3c, EGFRvIII-expressing tumors contained higher levels of phosphorylated STAT3 compared to tumors derived from vector-transfected control cells (P=0.04). Further, EGFRvIII expression was associated with increased tumor growth in a xenograft model, thus confirming our previous findings (Sok et al., 2006) (data not shown). To determine the effects of cetuximab on EGFRvIII-mediated STAT3 activation, we treated HNSCC cells expressing EGFRvIII with cetuximab followed by STAT3 promoter assays. Although cetuximab decreased STAT3 promoter activity in vector-transfected control cells, cetuximab was unable to abrogate STAT3 activation in HNSCC cells expressing EGFRvIII (Figure 3d). Similarly, EGF increased STAT3 promoter activity in vector-transfected controls, whereas EGF was unable to augment the already elevated levels of STAT3 promoter activation in HNSCC cells expressing EGFRvIII. These results indicate that EGFRvIII enhances STAT3 transcription and phosphorylation in HNSCC, effects that are resistant to treatment with cetuximab.

Figure 3
Figure 3

EGFRvIII increases STAT3 activation. (a) After serum starvation for 24 h, cell extracts from vector-transfected control 686LN cells (control) and EGFRvIII-transfected cells (vIII) were analyzed by western blot analysis. The blot was incubated with phosphotyrosine-STAT3 antibody, stripped and probed for total STAT3 and β-tubulin to ensure equivalent loading. The experiment was performed three times with similar results. (b) Vector-transfected control 686LN cells (control) and EGFRvIII cells (vIII) were transiently co-transfected with a luciferase construct under the control of STAT3 responsive promoter and a Renilla luciferase construct 24 h after transfection with vector control or EGFRvIII, incubated in complete media for 24 h and assayed for luciferase activity. The firefly luciferase activity units (relative light unit, RLU) were normalized to Renilla luciferase RLU and micrograms of total protein and expressed as a fold of the activity of the control in each experiment. Cumulative results from three independent experiments indicate a significant increase in STAT3 promoter activity in 686LN HNSCC cells expressing EGFRvIII (*P<0.001). (c) HNSCC xenografts derived from EGFRvIII-expressing Cal33 cells and xenografts derived from vector-transfected control cells were analyzed for phosphorylated and total STAT3 expression by immunoblotting. A representative immunoblot of pSTAT3 and STAT3 levels in control and vIII xenograft tumors are depicted. Densitometry analysis was performed on immunoblots from all tumors (n=17) and phosphorylated STAT3 expression levels relative to total STAT3 are shown as mean±s.e. EGFRvIII-expressing Cal33 HNSCC cell xenograft tumor (n=10) expressed 1.5-fold higher levels of phosphorylated STAT3 compared to control Cal33 HNSCC xenografts (n=7) (*P=0.043). (d) EGFRvIII-transfected 686LN cells (vIII) and vector-transfected control cells (control) were transiently co-transfected with an hSIE-luciferase construct and Renilla luciferase construct for 4 h. Cells were incubated in serum-free media with or without EGF (10 ng/ml) and/or cetuximab (C225) (0.7 μg/ml) for 24 h and assayed for luciferase activity. The luciferase RLU were normalized to Renilla luciferase RLU and to micrograms of total protein and expressed as a fold of the activity of the vector control (control) in each experiment. Cumulative results are shown from two independent experiments. The hSIE promoter activity of EGFRvIII-expressing 686LN cells was not stimulated by EGF or abrogated by cetuximab treatment. In contrast, cetuximab effectively abrogated EGF induced STAT3 promoter activity in HNSCC cells (*P=0.03).

STAT3 is required for EGFRvIII-mediated motility and invasion

STAT3 has been implicated in several oncogenic processes, including proliferation, survival and invasion and may represent a therapeutic target for cancer (Germain and Frank, 2007). To determine whether STAT3 is required for EGFRvIII-mediated cell motility and invasion, we performed migration and invasion assays in the presence or absence of small-interfering RNA (siRNA) targeting STAT3, under conditions where siRNA did not modulate proliferation. To examine the phenotypic effects of EGFRvIII signaling through STAT3, we assessed cell invasion and migration in the absence of EGFR ligand. STAT3 siRNA effectively abrogated STAT3 levels in vector control and EGFRvIII-expressing cells (Figure 4a). As shown in Figures 4b and c, knockdown of STAT3 (and phosphotyrosine STAT3) reduced the motility and invasion of EGFRvIII-expressing HNSCC cells, under conditions controlling for proliferation (migration: P=0.03; invasion: P=0.02). In fact, on STAT3 knockdown, the degree of migration and invasion in the EGFRvIII cells were comparable to levels in vector-transfected controls. In addition to downmodulation of STAT3 expression using siRNA, we also blocked STAT3 in the cells with a transcription factor decoy directed against STAT3 as described previously (Leong et al., 2003). The STAT3 decoy interferes with STAT3-mediated DNA binding and abrogates STAT3 target gene expression. As shown in Figures 4d and e, treatment with the STAT3 decoy resulted in reduction of both migration and invasion in EGFRvIII-expressing cells compared to treatment with a mutant control decoy, under conditions where the decoy did not affect cell growth. In the absence of EGFR ligand, there was a significant reduction in the migration or invasion of control cells treated with the STAT3 decoy (migration: P=0.05; invasion: P=0.03). However, the STAT3 decoy abrogated the migration and invasion of EGFRvIII-expressing HNSCC cells to a significantly greater degree than the vector-transfected control cells (P=0.03 and 0.05, respectively).

Figure 4
Figure 4

STAT3 has a critical role in EGFRvIII-mediated migration and invasion. (a) EGFRvIII-transfected 686LN cells (vIII) and vector-transfected control cells (control) were subsequently transfected with nontargeting siRNA or STAT3 siRNA for 6 h. Cells were collected at 48 h for the analysis of total STAT3 protein levels by immunoblotting. β-Tubulin levels show equal loading of protein in all lanes. The experiment was repeated four times with similar results. (b) STAT3 siRNA decreases HNSCC migration and invasion. At 48 h after nontargeting or STAT3 siRNA transfection, EGFRvIII-transfected 686LN HNSCC cells (vIII) and vector-transfected controls (control) were subjected to migration (left panel) and invasion assays (right panel) with the same methods described in Materials and Methods section. Both control cells and EGFRvIII-transfected cells decreased their migration and invasion when cells are treated with STAT3 siRNA compared to nontargeting siRNA treated condition. (c) Percent reduction of migration and invasion with STAT3 siRNA in both control and vIII was calculated from the results shown in panel b. The degree of both migration (left panel) and invasion (right panel) with STAT3 siRNA was greater in EGFRvIII-expressing 686LN cells than in control cells (*P=0.03 and 0.016, respectively). (d) STAT3 decoy decreases HNSCC migration and invasion. After mutant or STAT3 decoy treatment, EGFRvIII-expressing HNSCC 686LN cells (vIII) and vector-transfected controls (control) were subjected to migration (left panel) and invasion assays (right panel). Both control cells and EGFRvIII-expressing cells showed decreased migration and invasion when treated with STAT3 decoy compared to mutant control decoy treatment. (e) Percent reduction of migration and invasion with STAT3 decoy in both control and vIII was calculated from the results shown in Figure 4d. HNSCC cells expressing EGFRvIII had significantly reduced migration (*P=0.05) and invasion (*P=0.05) compared to control cells in the presence of STAT3 decoy.

Others have reported that phosphatidylinositol 3-kinase (PI3K)/AKT is activated downstream of EGFRvIII in glioma (Antonyak et al., 1998; Li et al., 2004). To determine whether motility and invasion are also mediated by this pathway, in addition to STAT3, we examined the expression of AKT phosphorylation in EGFRvIII and vector-transfected control HNSCC cells and found that similar to results in previous reports, EGFRvIII-expressing HNSCC cells expressed modestly increased levels of pAKT (Figure 5a). However, in contrast to our observations with STAT3 targeting, blockade of PI3K/AKT using the pharmacological inhibitor NVP-BEZ235-AN-4 (BEZ) abrogated cell growth but not invasion or metastasis, in HNSCC cells expressing EGFRvIII (Figures 5b and c). Similar results were also obtained using siRNA directed against the p85 subunit of PI3K in a urothelial cancer model expressing EGFRvIII (data not shown). These results suggest that STAT3 is specifically required for the EGFRvIII-mediated enhancement of HNSCC cell motility and invasion.

Figure 5
Figure 5

Increased PI3K/AKT signaling in EGFRvIII-expressing HNSCC cells mediates proliferation, but not invasion. (a) EGFRvIII-expressing HNSCC 686LN cells express modestly increased levels of phosphorylated AKT. After serum starvation for 24 h, cell extracts from vector-transfected control cells (control) and EGFRvIII-transfected cells (vIII) were analyzed by western blot analysis. The blot was incubated with phosphoserine-AKT antibody (Ser473), stripped and immunoblotted for total AKT and β-tubulin to ensure equivalent loading. The experiment was performed three times with similar results. (b) Blockade of PI3K/AKT abrogates cell growth in HNSCC 686LN cells expressing EGFRvIII. HNSCC cells expressing EGFRvIII and vector-transfected control cells were plated onto six-well plates with presence (♦, vIII), (, control) or absence (, vIII), (▪, control) of 100nM of NVP-BEZ235-AN-4. Growth curve of the transfected cell lines were obtained by MTT at several time points for 6 days. HNSCC cells expressing EGFRvIII showed increased growth rates compared with the vector-transfected control cells where cell growth was abrogated by PI3K/AKT blockade in both cell lines. Experiment was repeated three times with similar results. (c) Cell migration (left panel) and invasion (right panel) was not abrogated by PI3K/AKT blockade. Cell invasion assay was performed with HNSCC 686LN cells transfected with EGFRvIII or vector-transfected control with or without PI3K/AKT inhibition using 100 nM of NVP-BEZ235-AN-4. HNSCC cells expressing EGFRvIII show increased cell invasion compared with the vector-transfected control cells but cell invasive capacity was not decreased by PI3K/AKT blockade in either cell line.

Cetuximab does not abrogate EGFRvIII-induced HIF-1α expression under hypoxic conditions

Solid tumors, including HNSCC, contain large regions of low oxygen concentrations (hypoxic) regions, which contribute to resistance to treatment with standard approaches including chemotherapy and radiation. Hypoxia potently induces expression of hypoxia-inducible factor (HIF-1α), which has been shown to be a STAT3 target gene (Niu et al., 2008). EGFRvIII has been reported to contribute to hypoxia-mediated tumor growth in conjunction with radiation therapy but has not been previously linked to HIF-1α expression (Weppler et al., 2007). We therefore examined the expression of HIF-1α after treatment of HNSCC cells expressing EGFRvIII (or vector-transfected controls) with EGF and/or cetuximab. As shown in Figure 6, hypoxia-induced expression of HIF-1α was reduced by cetuximab in vector-transfected control cells but not in HNSCC cells expressing EGFRvIII. This experiment was also validated in urothelial cancer T-24 cells stably transfected with EGFRvIII (data not shown). These results suggest that STAT3 signaling through HIF-1α may contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors under hypoxia.

Figure 6
Figure 6

EGFRvIII induces HIF-1α expression under hypoxic conditions, which is not abrogated by cetuximab. EGFRvIII-expressing 686LN cells (vIII) and vector-transfected control cells (control) were treated with (or without) EGF (10 ng/ml) and/or cetuximab (C225, 7 μg/ml) in hypoxic conditions for 24 h. Under hypoxic conditions and EGF treatment, EGFRvIII-transfected cells maintained expression of HIF-1α with cetuximab treatment whereas the expression of HIF-1α in vector-transfected control cells was significantly decreased by cetuximab. The experiment was performed four times with similar results.

Discussion

We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where expression of this altered receptor mediates growth and resistance to chemotherapy or EGFR targeting using cetuximab (Sok et al., 2006). Patients with HNSCC died because of invasion into surrounding tissues and regional and distant metastasis. Although the addition of cetuximab to radiation was shown to improve survival, it did not decrease metastasis (Bonner et al., 2006). This study was undertaken to determine the effects of EGFRvIII on the migration and invasion of HNSCC cells and the signaling pathways that mediate these properties. Our results suggest that EGFRvIII increases HNSCC motility and invasion, at least in part, through activation of STAT3.

EGFRvIII is the most common EGFR alteration in human cancers. Deletion of exons 2–7 gives rise to a receptor that lacks a ligand-binding site and is constitutively activated in a ligand-independent manner. EGFRvIII has not been observed in normal tissue, but it has been detected in carcinomas of the brain, breast, ovary (Moscatello et al., 1995), lung (Garcia de Palazzo et al., 1993), prostate (Olapade-Olaopa et al., 2000), and head and neck (Sok et al., 2006). Expression of EGFRvIII has been correlated with poor prognosis in brain tumors (Diedrich et al., 1995). Furthermore, EGFRvIII can to transform fibroblasts in vitro (Pedersen et al., 2004) and enhance the tumorigenicity of cancer cells in vivo, supporting its oncogenic function (Tang et al., 2000).

The effect of EGFRvIII on tumor cell behavior is incompletely understood. Nagane et al. (1998) reported that EGFRvIII-expressing cells showed less apoptosis in response to cisplatin treatment. Others have reported that EGFRvIII induced glioma cell migration and invasion through induction of metalloproteases and extracellular matrix components (Lal et al., 2002; (Cai et al., 2005). The precise signaling events that mediate EGFRvIII-induced migration and invasion need further investigation. Moscatello et al. (1998) reported that EGFRvIII activates PI3K pathway instead of the Ras/Raf/MEK pathway, which is preferentially activated by wild-type EGFR. Further investigation suggested that constitutive PI3K/AKT activation by EGFRvIII may contribute to chemoresistance and radioresistance in these cells (Narita et al., 2002; Li et al., 2004). Antonyak et al. (1998) showed that c-Jun N-terminal kinase was constitutively activated by EGFRvIII and was downregulated by PI3K inhibition. To date, signaling through PI3K/AKT has not been correlated with tumor cell migration or invasion mediated by EGFRvIII. We found that although EGFRvIII-expressing HNSCC cells expressed increased levels of phosphorylated AKT, abrogation of PI3K/AKT using either NVP-BEZ235-AN-4 or p85 siRNA did not abrogate invasion in EGFRvIII-expressing HNSCC cells. NVP-BEZ235-AN-4 treatment decreased the proliferation of EGFRvIII-expressing HNSCC cells, suggesting that activated PI3K/AKT by EGFRvIII contributes to EGFRvIII-induced HNSCC cell proliferation, but not migration or invasion (Figure 5).

There are few, but conflicting, reports linking EGFRvIII to STAT3 signaling. Although Mizoguchi et al. (2006) reported a correlation of expression levels of EGFRvIII and phosphotyrosine STAT3 in glioblastoma, Andersen et al. (2008) recently reported that glioma cells that express EGFRvIII fail to induce IRF-1 through STAT3 phosphorylation. Cumulative evidence has implicated STAT3 as an critical oncogene where elevated expression levels of tyrosine phosphorylated STAT3 are detected in numerous human cancers (Bowman et al., 2000). Studies in HNSCC show that STAT3 is activated downstream of receptor and nonreceptor tyrosine kinases including EGFR and Src family kinases as well as IL-6/gp130 (Kijima et al., 2002; Xi et al., 2003; Sriuranpong et al., 2003). Targeting STAT3 in HNSCC preclinical HNSCC models inhibited tumor growth but not the growth of normal epithelial cells (Leong et al., 2003). Expression of tyrosine phosphorylated STAT3 in the primary HNSCC tumor has been correlated with nodal metastasis, advanced tumor stage and decreased survival (Masuda et al., 2002). The results of this study suggest that activation of STAT3 downstream of EGFRvIII in HNSCC contributes to the increased migration and invasion.

STAT3 target genes include cell-cycle regulators (Sinibaldi et al., 2000), anti-apoptotic genes (Oritani et al., 1999) and pro-angiogenic factors (Huang et al., 2002), each of which has been implicated in tumorigenic processes including invasion and metastasis. STAT3 has been shown to contribute to cancer migration and invasion through regulation of genes that stimulate these processes including matrix metalloproteinases (for example, MMP-2 and MMP-9), VEGF and/or basic fibroblast growth factor (Dechow et al., 2004; Qiu et al., 2007). In addition to the transcriptionally mediated effects of STAT3 on cell migration and invasion, transcription-independent pathways have also been described for the effects of STAT3 on cell motility. Specifically, STAT3 has been found to directly interact with cell motility components such as focal adhesion components, FAK, paxillin (Silver et al., 2004), p130CAS (Kira et al., 2002) or cytoskeletal microtubles (Ng et al., 2006). We did not detect increased expression of MMP-2, MMP-9 or VEGF in association with the EGFRvIII-mediated migration and invasion observed in these cells (data not shown), suggesting that other STAT3 target genes or STAT3 interacting proteins may be having a role. In addition, the lack of HIF-1α decrease in EGFRvIII cells treated with cetuximab under hypoxic conditions suggests a more complex regulatory balance between oxygen-dependent and oxygen-independent factors that influence HIF-1α.

Aberrant activation of STAT3 has been shown to contribute to tumor progression making STAT3 an attractive therapeutic target. To date, no STAT3-targeting strategies have undergone clinical evaluation. We have developed a highly specific transcription factor decoy approach to block STAT3 signaling and showed that it inhibits tumor growth in vitro and in vivo in HNSCC preclinical models (Leong et al., 2003). Using the same STAT3 decoy, others have reported antitumor effects in a murine model of cutaneous squamous cell carcinoma (Sano et al., 2005). Toxicology studies in nonhuman primates were recently completed and showed no evidence of toxicity (Sen et al., 2008). In this study, treatment of EGFRvIII-expressing HNSCC cells with the STAT3 decoy abrogated cell motility and invasion. These results suggest that selective activation by EGFRvIII in HNSCC contributes to the invasive phenotype, which can potentially be targeted with therapeutic strategies that inhibit STAT3. In addition to the decoy, others have reported the use of siRNA, peptidomimetic strategies and G-quartet oligonucleotides to inhibit STAT3 in cancer models (Leeman et al., 2006).

These cumulative results suggest that STAT3 activation is critical for cancer progression mediated by both wild-type EGFR and EGFRvIII, which are often coexpressed in HNSCC tumors (Sok et al., 2006). Knockdown or blockade of STAT3 preferentially abrogated the migration and invasion of HNSCC cells that expressed EGFRvIII implicating STAT3 as a critical pathway in mediating HNSCC invasion in tumors that express this altered receptor. We have previously shown that both PLCγ-1 and c-Src mediate HNSCC invasion downstream of EGFR (Thomas et al., 2003; Zhang et al., 2004). Thus, EGF stimulation in cells expressing both EGFR and EGFRvIII likely results in invasion through multiple downstream signaling molecules including STAT3.

Therapeutic agents with selective activity against EGFRvIII are presently under clinical investigation including the immunotoxin MR1-1, the chimeric antibody 806 (Li et al., 2007) and irreversible HER1/HER2 inhibitors (Ji et al., 2006) that appear to have selective activity against EGFRvIII. The EGFR monoclonal antibody cetuximab is the only FDA-approved EGFR-targeting strategy for HNSCC. We previously reported that HNSCC xenografts expressing EGFRvIII were resistant to the growth inhibitory effects of cetuximab (Sok et al., 2006). Here we show that EGFRvIII cells are resistant to anti-invasive effects of cetuximab in HNSCC. Furthermore, EGFRvIII expression results in an increase in phosphorylation of STAT3 in HNSCC cells. These results suggest that HNSCC tumors that express EGFRvIII may be best treated with strategies that selectively block EGFRvIII, or its downstream signaling pathways, in addition to targeting wild-type EGFR.

Materials and methods

Cell lines, reagents and cell culture

C225/cetuximab was purchased from the research pharmacy at the University of Pittsburgh Cancer Institute. For in vitro cell stimulation, recombinant human EGF (Sigma-Aldrich Corp., St Louis, MO, USA) was used. PI3K inhibitor NVP-BEZ235-AN-4 was obtained from Novartis (East Hanover, NJ, USA). EGFRvIII-transfected HNSCC cells (Cal33vIII) and vector control-transfected HNSCC cells (Cal33control) were generated as described previously (Sok et al., 2006). Cal33 and 686LN cells were a kind gift from Dr Gerard Milano (Centre Antoine-Lacassagne, Nice, France) and Dr Georgia Chen (Emory University, Atlanta, GA, USA), respectively. Cal33 cells were maintained in Dulbecco's modified Eagle's medium (Mediatech Inc., Herndon, VA, USA) with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). 686LN cells were maintained in Dulbecco's modified Eagle's medium/F12 (1:1) (Invitrogen) and 10% heat-inactivated FBS (Invitrogen). All cells were incubated at 37 °C in the presence of 5% CO2. To establish hypoxic conditions (1% O2), we placed cells in an InVivo 300 hypoxia workstation (Ruskinn Lifesciences Ltd, Bridgend, UK).

Transfection with vector control or EGFRvIII

Transfection with vector control or EGFRvIII was performed as previously described (Sok et al., 2006). HNSCC cells were plated at a density of 1 × 106 cells in a 100 mm tissue culture dish. After 16 h incubation in complete media, cells were transfected using 9 μg of the expression vector pLERNL containing the EGFRvIII cDNA or vector control and Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Plasmid DNAs were a kind gift from Dr Frank Furnari, Ludwig Institute for Cancer Research, La Jolla, CA, USA. Transfection media was replaced with complete media after 6 h and cells were incubated for 24 h for further transfection or 48 h for use in assays.

RT–PCR analysis and cDNA sequencing of EGFRvIII

Total RNA was isolated from HNSCC cell lines (5 × 106 cells) using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Total RNA (1 μg) was reverse transcribed with the first-strand cDNA synthesis using SuperScript First-Strand Synthesis System for RT–PCR (Invitrogen). To detect the deleted region of EGFRvIII, we performed standard RT–PCR as described previously (Sok et al., 2006).

Flow cytometry

Indirect analytic flow cytometry was carried out on a Becton Dickinson FACS calibur equipped with CellQuest Pro software (Becton Dickinson, San Jose, CA, USA). Assays were performed at 4 °C; all washes were carried out with iced medium to facilitate the detection of cell-surface receptors without allowing internalization to occur. All profiles were obtained with cells maintained in ice-cold 1% bovine serum albumin/phosphate-buffered saline (PBS). The percentage of a population designated as positive was arbitrarily defined as that region in which only the highest fluorescing 10% of the isotype-control stained cells graphed, corrected for background; this is a conservative estimate of the total positive staining population. To examine the cell-surface expression of EGFRvIII proteins, we stained target cultured cells with anti-EGFRvIII monoclonal antibody L8A4 under nonpermeabilized conditions. Subconfluent cells were detached from culture flasks by incubation with 0.02% EDTA/PBS; 106 cells were maintained in 0.5% paraformaldehyde/PBS for 10 min at 4 °C, washed, resuspended in 150 ml PBS containing 10% FBS and blocked for 20 min at 4 °C. After two washes, the samples were reacted with L8A4 monoclonal antibody (10 mg/ml, black line) and irrelevant mouse IgG1k (10 mg/ml, solid gray) in PBS for 60 min. After two additional washes, cells were incubated with FITC-labeled secondary antibody for 30 min at 4 °C and analyzed on a Becton Dickinson FACS calibur instrument (Becton Dickinson).

Western blotting

Cell lines were lysed in detergent containing 1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin and 1 mg/ml aprotinin, and protein levels were determined using the Bio-Rad protein assay method (Bio-Rad Laboratories, Hercules, CA, USA). Total protein (40 μg) were separated on 8% SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes using the semidry transfer machine (Bio-Rad Laboratories). Membranes were blocked with 5% skim milk/Tris-buffered saline with Tween 20 (TBS-T) solution for 2 h at room temperature, and incubated with primary antibodies in 5% skim milk in TBS-T overnight at 4 °C. After washing with TBS-T three times, membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories) 1:3000 diluted in 5% skim milk in TBS-T. The filters were rinsed with TBS-T three times, and the blot was developed using Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) by autoradiography. Antibodies used for blotting included β-tubulin (Abcam, Cambridge, UK), HIF-1α (BD Transduction Laboratories, San Jose, CA, USA), phospho-AKT (Ser473), AKT, phospho-STAT3 (Tyr705) and STAT3 (Cell Signaling Technology, Beverly, MA, USA).

Luciferase reporter assay

HNSCC cells (4 × 105 per ml) were plated onto six-well tissue culture plates. After cells were transiently transfected with vector control or EGFRvIII and incubated in complete medium for 24 h, cells were then co-transfected with pSTAT3TALuc, a generous gift from Dr Jacqueline Bromberg (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) and pRL-TK (Promega, Madison, WI, USA) a Renilla luciferase construct, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions (Besser et al., 1999). The transfection media was replaced to complete Dulbecco's modified Eagle's medium after 4 h of transfection. Cells were lysed and luciferase assays were performed 24 h after the transfection using the Dual-Luciferase Reporter Assay System kit (Promega). Cell lysates were subjected to protein estimation using the Bio-Rad protein assay kit (Bio-Rad Laboratories). Relative light units from luciferase were normalized to relative light units from Renilla luciferase (to account for differences in transfection efficiency) and to micrograms of protein (to account for differences in protein concentrations between samples).

Matrigel invasion assay and cell migration assay

Cell invasion was evaluated in vitro using Matrigel-coated semipermeable modified Boyden inserts with a pore size of 8 μm (Becton Dickinson/Biocoat, Bedford, MA, USA). Cell migration was evaluated in vitro using semipermeable modified Boyden inserts with a pore size of 8 μm (Becton Dickinson/Biocoat). For both assays, cells were plated in duplicate at a density of 1.3 × 104 cells per well in serum-free media in the insert. At the same time, cells were plated in 24-well plates to serve as loading controls. Both the insert and the holding well were subjected to the same medium composition with the exception of serum. The insert contained no serum, whereas the lower well contained 10% FBS that served as a chemoattractant. After 24 h of treatment at 37 °C in a 5% CO2 incubator, the cells in the insert were removed by wiping gently with a cotton swab. Cells on the reverse side of the insert were fixed and stained with Hema 3 (Fisher Scientific, Hampton, NH, USA) according to the manufacturer's instructions. Cells plated in 24-well plates were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and the cell numbers across the groups were normalized. The number of invading or migrating cells was adjusted accordingly.

STAT3 siRNA and STAT3 decoy transfection

The STAT3 decoy and the mutant control decoy sequences (double-stranded deoxyribonucleotides with phosphorothioate modifications in the first three bases and last three bases of the sequences) were generated as described previously (Leong et al., 2003). The mutant control decoy, carrying a single base mutation, that does not abrogate STAT3 DNA binding activity, was used as a control as in previous studies (Leong et al., 2003; Xi et al., 2005). The siRNA sequences targeting STAT3 human mRNA (D-003544-01, sense 5′-CCAACGACCUGCAGCAAUAUU-3′ and antisense 5′-PUAUUGCUGCAGGUCGUUGGUU-3′; Dharmacon, Lafayette, CO, USA) were transfected into HNSCC cells for STAT3 silencing. The nontargeting siRNA (D-001210-01, sense 5′-UAGCGACUAAACACAUCAAUU-3′ and antisense 5-UUGAUGUGUUUAGUCGCUAUU-3′; Dharmacon) was used as a control. The siRNA or decoy transfections were performed using the Lipofectamine 2000 (Invitrogen). In brief, HNSCC cells were transfected with vector control or EGFRvIII and after 24 h incubation in complete media cells were transfected with 800 pmol of STAT3 siRNA or nontargeting control siRNA, or 50.4 pmol of STAT3 decoy or mutant control decoy. The transfection medium was replaced with complete media after 4 h of transfection.

In vitro growth assay

To determine if the sensitivity of HNSCC cells to PI3K/AKT inhibition was affected by EGFRvIII expression, we seeded vector-and EGFRvIII-transfected HNSCC cells (1.5 × 104) onto 24-well plates 24 h after transfection and treated with a PI3K inhibitor NVP-BEZ235-AN-4 (100 nM) or dimethyl sulfoxide (1 μl/ml of media). Each cell population was then assayed every other day for 6 days in triplicate using MTT. MTT was combined with warm PBS (5 mg/ml) and placed on cells for 1 h at 37 °C in a 5% CO2 incubator. The MTT was removed and replaced with an equal volume of dimethyl sulfoxide to solubilize the cells. The absorbance was read on a μQuant spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Percent cell growth was determined by normalizing each cell population to the average day 2 absorbance value for that population.

In vivo growth of HNSCC cell expressing EGFRvIII and analysis of HNSCC xenografts

HNSCC cells (Cal33 expressing EGFRvIII (Cal33vIII) or empty vector-transfected parental cells (control-1)) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. Cells were trypsinized and cell number and viability were determined using Trypan blue dye exclusion. A suspension of 7.5 × 105 HNSCC cells in 50 μl serum-free media was injected into the flanks of nu/nu athymic nude mice (n=20; Harlan Sprague-Dawley, Indianapolis, IN, USA) subcutaneously. Cal33vIII was injected into the right flank and control cells were injected into the left flank. Tumor volumes were measured in two dimensions with vernier calipers and calculated using the formula: (length × width2) × 0.52. At the end of the study, mice were killed by cervical dislocation under anesthesia, the tumors surgically excised and snap frozen in dry ice. To evaluate the expression of phosphorylated and total STAT3 in HNSCC xenografts, we homogenized tumors, then sonicated them in detergent containing 1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin and 1 mg/ml aprotinin. Total protein (40 μg) were separated on 8% SDS–polyacrylamide gel electrophoresis gels and immunoblotted for phosphorylated and total STAT3 and β-tubulin. Animal use and care was in strict compliance with institutional guidelines established by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Statistical analysis

For migration and invasion studies, the statistical significance of differences in the number of invading cells or migrated area was assessed using Wilcoxon–Mann–Whitney two-tailed exact test.

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Acknowledgements

This work was supported by Grants RO1 CA77308, RO1 CA101840 and P50 CA097190, and an American Cancer Society Clinical Research professorship CRP-08-229-01 (to JRG) and the NIH core Grant EY08098.

Author information

Author notes

    • S E Wheeler
    •  & S Suzuki

    These authors contributed equally to this work.

    • S Y Lai

    Current address: Department of Head and Neck Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

Affiliations

  1. Department of Otolaryngology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    • S E Wheeler
    • , S Suzuki
    • , S M Thomas
    • , M Sen
    • , S Y Lai
    •  & J R Grandis
  2. Department Pathology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    • R J Leeman-Neill
    •  & S I Chiosea
  3. Department of Pathology, Duke University Medical Center, Durham, NC, USA

    • C-T Kuan
    •  & D D Bigner
  4. Department Biostatistics, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    • W E Gooding
  5. Department Pharmacology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

    • J R Grandis

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The authors declare no conflict of interest.

Corresponding author

Correspondence to J R Grandis.

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https://doi.org/10.1038/onc.2009.279

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