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[Cancer Research 65, 9868-9875, November 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Proliferation of Human Neuroblastomas Mediated by the Epidermal Growth Factor Receptor

Ruth Ho, Jane E. Minturn, Tomoro Hishiki, Huaqing Zhao, Qun Wang, Avital Cnaan, John Maris, Audrey E. Evans and Garrett M. Brodeur

Division of Oncology, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: Garrett M. Brodeur, Division of Oncology, Children's Hospital of Philadelphia, Abramson Research Center, Room 902-D, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318. Phone: 215-590-2817; Fax: 215-590-3770; E-mail: brodeur{at}email.chop.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma is a common solid tumor of childhood that is derived from the neural crest. Expression of epidermal growth factor (EGF) receptors (EGFRs) has been associated with enhanced cell growth and aggressive behavior in other tumors. Here, we examined the expression profile of EGFRs in neuroblastoma cell lines and primary tumors. We found that all 13 neuroblastoma cell lines examined expressed EGFR1 (HER1), most at readily detectable levels. Low levels of other human EGFR family receptors were also detected in almost all cell lines. All primary tumors examined expressed readily detectable levels of HER1 and HER3 and lower levels of HER2 and HER4. EGF had a significant effect on the proliferation of neuroblastoma cell lines in vitro. EGF treatment (100 ng/mL) of the cell lines SY5Y and NLF significantly increased cell number (P < 0.01). EGF stimulated more cells to enter S and G2-M phase, as suggested by flow cytometry, indicating that EGF increases cell number by increasing proliferation, with no appreciable change in apoptosis. EGF exposure resulted in receptor autophosphorylation and activation of both the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Exposure to 0.5 µmol/L ZD1839, a HER1-specific inhibitor, caused a 40% to 50% reduction in the number of SY5Y and NLF cells grown in medium containing 10% fetal bovine serum (P < 0.01). Even at 0.01 µmol/L, ZD1839 inhibited autophosphorylation of HER1 by EGF. At 0.1 µmol/L, it also blocked phosphorylation of AKT, but not MAPK, in NLF cells. Additional studies showed that the PI3K/AKT–specific inhibitor LY294002 had a more profound effect than the MAPK-specific inhibitor U0126 in blocking EGF-induced cell proliferation. This suggests that the PI3K/AKT pathway is the main signaling pathway responsible for the proliferation effects of EGF in neuroblastomas. Our results also indicate that ZD1839 is a potent inhibitor of neuroblastoma cell proliferation; therefore, it may be a useful, biologically based therapeutic agent for these tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma is the most common extracranial solid tumor in children. It arises from neural crest cells, and the tumors are most commonly found in the adrenal medulla or along the sympathetic chain (1). Neuroblastomas are heterogeneous both clinically and biologically. Tumors in infants may regress spontaneously, whereas tumors in older patients may mature into benign ganglioneuromas. Unfortunately, the majority of tumors occur between the ages of 1 and 5 years, and these tumors are generally unresectable or metastatic at the time of diagnosis and have a poor prognosis. Although intensive multimodality therapy has produced some improvements in the overall cure rate of these patients, this therapy has considerable short- and long-term toxicities. Therefore, a better understanding of the molecular pathogenesis of neuroblastomas may lead to biologically based therapy that is more effective and less toxic.

Tyrosine kinase receptors play an important role in survival, growth, and differentiation of many normal and malignant cells. For example, TrkA, the receptor for nerve growth factor, is commonly expressed in the most favorable neuroblastomas (2–4). Conversely, TrkB, along with its ligand, brain-derived neurotrophic factor (BDNF), is expressed in the more aggressive tumors, particularly those with MYCN amplification (5). TrkC, the receptor for neurotrophin-3, is expressed in a subset of TrkA-expressing tumors and is similarly associated with favorable clinical features and outcome (6, 7). Evidence suggests that the Trk family of tyrosine kinase receptors plays an important role in the behavior of both favorable and unfavorable neuroblastomas. However, the ultimate behavior of these tumors is probably dependent on additional pathways that also affect growth, survival, or differentiation.

The human epidermal growth factor (EGF) receptor (EGFR/HER1, also ErbB1), a 170 kDa transmembrane tyrosine kinase receptor, is one of four members of the human EGFR (HER) family receptors. The three other members are HER2 (Neu/ErbB2), HER3 (ErbB3), and HER4 (ErbB4). HER1 becomes biologically functional by forming a homodimer with itself or a heterodimer with other members of the HER receptor family. The mechanisms that promote the formation of receptor dimers include ligand binding and high receptor density due to overexpression. Ligands that can activate HER1 include EGF, amphiregulin, and transforming growth factor-{alpha}, which bind specifically to HER1, as well as ß-cellulin, heparin-binding EGF, and epiregulin, which bind both HER1 and HER4 (8, 9).

HER1 is normally expressed in all epithelial and stromal cells but more selectively expressed in glial and smooth muscle cells. High levels of EGFRs have been observed in a variety of tumors, including head and neck, non–small cell lung, prostate, breast, gastric colorectal, and ovarian cancers. The expression of EGFRs in tumors has been correlated with tumor progression, poor survival, poor response to therapy, and resistance to cytotoxic agents. Constitutive activation of the EGFR pathway by gene amplification or other mechanisms may contribute to enhanced survival and growth of tumor cells (8, 10, 11).

HER family receptors are also important in the developing nervous system and in nervous system tumors. The HER2 and HER3 receptors and their ligand, neuregulin-1, are essential for development of the sympathetic nervous system (12). HER3 and HER4 are coexpressed in childhood medulloblastoma, and their expression is associated with high proliferative activity in ependymoma (13, 14). Overexpression of HER1 is also found in glioblastoma multiforme (15).

There are a few reports concerning the expression of functional HER1 in neuroblastoma cell lines (16–18). However, the expression profile of HER family members in neuroblastoma cell lines and primary tumors, as well as downstream signaling pathways, have not been explored in detail. Therefore, we examined the expression and function of the EGFR pathway in neuroblastomas and determined whether EGFR-specific inhibitors might have a role as novel, biologically based therapeutic agents for neuroblastoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tumor samples. All cell lines (MYCN nonamplified, n = 4; MYCN amplified, n = 9) were obtained from the Children's Hospital of Philadelphia cell line bank (19). The lines were maintained at 5% CO2 in RPMI 1640 with 10% fetal bovine serum (FBS; same lot used for all studies), 1% oxaloacete-pyruvate-insulin supplement, 1% glutamate, and 50 µg/mL gentamicin. A431, a human epidermoid carcinoma cell line that amplifies and overexpresses HER1 (20), was used as a positive control and grown under similar conditions. Cells were harvested when they reached 70-80% confluence to make RNA or protein. Eighteen neuroblastoma tumor samples were obtained from the Children's Hospital of Philadelphia tumor bank. Ten patients had biological favorable disease (ganglioneuroma, n = 1; stage I, n = 3; stage II, n = 4, stage IV-S, n = 2) and eight patients had biological unfavorable disease (stage III, n = 4; stage IV, n = 4).

Semiquantitative reverse transcription-PCR of HER family genes. Total RNA was extracted from neuroblastoma primary tumors and cell lines. Human fetal brain RNA was purchased from Stratagene (La Jolla, CA). Semiquantitative reverse transcription-PCR (RT-PCR) was carried out according to a previously described protocol (21). Primers for human HER1 are 5'-CAGCGCTACCTTGTCATTCA-3' and 3'-AGGACTCGAGAGACTCACGT-5' (PCR product size: 195 bp); for human HER2, 5'-CTGACCAGTGTGTGGCCTGT-3' and 3'-GACTGTGGATCGCCTCGCTA-5' (PCR product size: 376 bp); for human HER3, 5'-ATGTGTGCCTTTGTTTCCCATC-3' and 3'-GTGTTTCCCTTCAGCCCGTA-5' (PCR product size: 293 bp); and for human HER4, 5'-GATGGAGGTTTTGCTGAAC-3' and 3'-GGATACGCTCTGTTTGGGTTTG-5' (PCR product size: 277 bp). The primers were biotinylated at their 5' ends. PCR reactions were carried for 22 to 24 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as the internal control because it has been the most consistent compared with others, such as ß-2-microglobulin, midkine, actin, and 18S, according to the semiquantitative and quantitative RT-PCR (Taqman) and microarray analysis data from our laboratory.

Cell viability assay. To study the effect of EGF on neuroblastoma, we used one cell line with moderate HER1 expression (SY5Y) and one with high HER1 expression (NLF). Cells were seeded into 24-well plates at a density of 5 x 103 per well in RPMI with 2% FBS overnight. Cells were treated the following day with 0 or 100 ng/mL EGF in 2% FBS-RPMI. To quantitate the number of viable cells, a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay was done at days 0 (after seeding), 2, 5, and 7 as previously described (1). A multiple wavelength scanner was used to measure the absorbance at 570 to 630 nm dual wavelengths. The experiments were done in five replicates and repeated four to five times. Measurement of absorbance was used as a correlate of viable cell number.

Flow cytometry. We used flow cytometric analysis of DNA content to decide whether EGF enhances growth by increasing proliferation or decreasing apoptosis. Cells were seeded in medium containing 2% FBS and treated with EGF (100 µg/mL) overnight, with or without pretreatment of ZD 1839 for 30 minutes. Cells were collected using 0.5% trypsin and resuspended in cold PBS at a concentration of 1 x 106 per milliliter. One-tenth volume of 10x propidium iodide solution (500 µg/mL propidium iodide, 10 mg/mL sodium citrate, and 1% v/v Triton X-100) was added, and cells were acquired in a flow cytometer within half an hour. A cell fit program (CELLQuest version 5.1, Becton Dickinson Bioscience, San Jose, CA) was used to calculate the fraction of cells with 2n, 2n-4n, and 4n amounts of DNA, corresponding to G0-G1, S, and G2 + M phases of the cell cycle. Fractions of cells in different cell cycle phases under various conditions were compared.

Immunoblotting. To analyze the inhibition of HER1 autophosphorylation by ZD1839 (Iressa, AstraZeneca, Cheshire, United Kingdom), cells were grown in 10 cm2 dishes to 80% confluence in standard culture medium. Cells were then grown in serum-free medium for a minimum of 4 hours before treatment with various concentrations (0.01, 0.1, 1, and 10 µmol/L) of the EGFR inhibitor ZD1839 for 1 hour. Cells were subsequently treated with EGF (10 or 100 ng/mL) for 5 minutes. To analyze inhibition of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, cells were pretreated with LY294002 or U0126 for 30 minutes, followed by EGF exposure (100 ng/mL) for 5 minutes. The cell pellets were lysed in NP40 lysis buffer [1% NP40, 20 mmol/L Tris pH 8.0, 137 mmol/L NaCl, 0.5 mmol/L EDTA, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 0.15 units/mL aprotinin, 20 µmol/L leupeptin, 1 mmol/L sodium vanadate] on ice for 20 minutes and cleared by centrifugation for 20 minutes at 14,000 x g. Protein content was measured by the Bio-Rad protein assay. One hundred micrograms of protein were resolved by SDS-PAGE, transferred, and detected with anti–phospho-EGFR (Tyr1068), anti–phospho-AKT (Ser473), or anti–phospho-p42/p44 MAPK antibody (Cell Signaling, Beverly, MA) using an ECL chemiluminescence system (Amersham Corp., Arlington Heights, IL). Corresponding membranes were reprobed with anti-EGFR (Santa Cruz, Santa Cruz, CA), anti-AKT (Cell Signaling) or anti-MAPK (Santa Cruz) antibody to ensure equal loading of protein. HER1 expression in cell lines was analyzed by Western blotting using the anti-EGFR antibody.

Cytotoxicity assay. To study the effect of EGFR inhibition by ZD1839 in neuroblastoma, A431, SY5Y, and NLF cells were seeded into 96-well plates at a density of 3 x 104 per well in 10% FBS-RPMI medium. We started with more cells for these experiments than in the cell viability assay to obtain an accurate reading by MTT after treatment. Different concentrations (0.1, 0.5, 1, 10, and 40 µmol/L) of ZD1839 were added after the cells were attached. The cells were treated for 72 hours and a colorimetric MTT assay was then done as detailed above. To study effects of the PI3K inhibitor LY294002 and the MAPK inhibitor U0126, cells were pretreated with LY294002 (20 µmol/L) or U0126 (10 µmol/L) after seeding, and then were treated with EGF (100 ng/mL) or EGF and concurrent ZD1839 (1 µmol/L) for 72 hours. An MTT assay was then preformed. The experiments were done in five replicates and each experiment was repeated thrice. Measurement of absorbance is used to represent cell number.

Statistical analysis. The results of the MTT assay were analyzed by paired Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HER1-4 expression in neuroblastoma cells. We found readily detectable HER1 expression in 10 of 13 neuroblastoma cell lines examined by semiquantitative RT-PCR. The amount of HER1 mRNA expressed was at least half of our internal control gene (GAPDH). The expression had no apparent correlation with the presence or absence of MYCN amplification. Most of the cell lines did not express a higher level of HER1 than normal fetal brain. NLF, which had the highest expression, was comparable with the HER1 level detected in the A431 cells, which have amplification of the HER1 gene (Fig. 1A). Expression of HER1 protein in the neuroblastoma cell lines NLF, SKNSH, IMR5, SY5Y, and CHP134 was confirmed by Western blotting (Fig. 1C). NLF and SY5Y were representative of high and intermediate levels of HER1 expression, respectively, and they were used in further studies. The cell lines examined also expressed relatively low levels of HER4 mRNA. HER2 and HER3 expression were generally low in the cell lines examined except for NMB (for HER2) and LAN-5 (for HER3), which expressed an mRNA level similar to the internal control gene GAPDH (Fig. 1A).



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  		Figure 1. Expression of HER family receptors in neuroblastoma cell lines and tumors. A and B, mRNA expression of HER family receptors in neuroblastoma cell lines and primary tumors was detected by semiquantitative RT-PCR. The expression was quantified by densitometry. Ratio of the density of each target mRNA and its corresponding GAPDH was graphed. A, expression of HER family receptors in cell lines. Open white columns, cell lines without MYCN amplification. Solid black columns, MYCN-amplified cell lines. Solid gray columns, control cell line A431 and control tissue human fetal brain. B, expression of HER family receptors in primary tumors. *No data due to running out of tumor cDNA. C, protein expression of HER1 of some of the neuroblastoma cell lines. HER1 expression was detected by Western blotting.

 
HER1 mRNA was also expressed in 18 of 18 primary neuroblastoma tumors examined, and the average HER1 expression was similar to GAPDH. However, we did not find a significant correlation between expression level and tumor stage (Fig. 1B). We found readily detectable levels of HER2 expression in 9 of 15 primary tumors. The primary tumors also expressed low levels of HER4, but HER3 expression was generally higher. The expression level of the HER family genes was not significantly correlated with tumor stage (Fig. 1B). To confirm these findings, we did an analysis of HER family gene expression (by microarray analysis) in 101 additional primary tumors, representing carefully defined low, intermediate, high, and ultrahigh risk groups. There was again no correlation between HER expression and either tumor stage or risk group (data not shown).

Epidermal growth factor enhances proliferation of some neuroblastoma cells. Because HER1 is expressed in neuroblastoma cells, we studied the effect of EGF exposure to determine if the receptor was functional. SY5Y has a moderate level of HER1 expression and NLF has a high level, comparable with the HER1-overexpressing cell line A431. Treatment of SY5Y cells with EGF (100 ng/mL) caused a 2-fold increase in cell number by day 7 (P < 0.01). Similar results were obtained in NLF cells (Fig. 2A). Neurite outgrowth was used as an indication of neuronal differentiation, but no outgrowth was observed during 7 days of EGF treatment. NLF cells have a faster growth rate than SY5Y cells under normal conditions. Other neuroblastoma cell lines, such as SKNSH, IMR5, and CHP134, were also screened for their response to EGF. We found that there was a general correlation between the level of EGFR expression and the magnitude of growth enhancement by EGF (data not shown).



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  		Figure 2. EGF enhances proliferation of NLF and SY5Y cell lines. A, cells were seeded in 24-well plates in 2% FBS-RPMI medium, and treated with EGF (100 ng/mL) for 2, 5, and 7 days. An MTT assay was done at indicated time points to measure cell number. Points, means of five replicates; bars, SD. The results shown are representative of three independent experiments. B, cells were seeded in 2% FBS without or with 100 ng/mL EGF or EGF plus ZD1839 (1 µmol/L). DNA content was measured by flow cytometry. Cell fit was done by the CELLQuest (version 5.1) program.

 
To decide whether EGF increases cell number by increasing proliferation or decreasing apoptosis, we measured DNA content by flow cytometry. We found that treating cells with EGF resulted in a substantial decrease in the percentage of cells in the G0-G1 phase, with a corresponding increase in the percentage of cells in S and G2-M phase. Pretreatment of cells with ZD1839 (1 µmol/L) prevents the cell cycle progression by EGF. The apoptosis fraction was very low for cells under all conditions, and there was no appreciable change in this fraction (Fig. 2B; Table 1). Similar results were seen in SY5Y cells, although to a lesser extent. Thus, EGF increases neuroblastoma cell number primarily by increasing cell proliferation.


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  		Table 1. Percentage of NLF cells in different phases of the cell cycle under different conditions

 
ZD1839 blocks proliferation of neuroblastoma cells. ZD1839 is specific inhibitor of the EGFR tyrosine kinase at the micromole per liter range (22). Because EGF enhances the growth of some neuroblastoma cell lines, we investigated the effect of ZD1839 in neuroblastomas. A431 cells, which amplify and overexpress HER1, were sensitive to ZD1839 and used as a positive control. A431 cells, as well as the neuroblastoma cells SY5Y and NLF, were grown in 10% FBS and exposed to a range of concentrations of ZD1839 for 72 hours. Concentrations as low as 0.1 µmol/L for NLF cells and 0.5 µmol/L for SY5Y cells caused significant growth inhibition. At a concentration of 0.5 µmol/L, ZD1839 caused a 40% to 50% reduction of growth in both NLF and SY5Y cells by 72 hours. This concentration was similar to that needed for inhibition of the A431 cell line (data not shown), indicating that NLF and SY5Y were both very sensitive to ZD1839 (Fig. 3A). A similar concentration of ZD1839 was needed to inhibit EGF-stimulated growth in NLF cells to the same extent in 1% FBS (data not shown). Thus, the IC50 for ZD1839 was ~0.5 µmol/L for both NLF and SY5Y. At higher doses, ZD1839 also inhibits other kinase receptors, such as HER2, Flt-1, Raf, etc. (22). Thus, the growth inhibitory effect we observed at 10 to 40 µmol/L concentrations could be due to inhibition of other kinase receptors as well. Flow cytometry indicates that 1 µmol/L ZD1839 prevents EGF-mediated cell cycle progression from G0-G1 to the S and G2 phases. Higher doses (10 µmol/L) of ZD1839 increases apoptosis as well (data not shown), probably due to simultaneous inhibition of other important cell signaling pathways.



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  		Figure 3. ZD1839 inhibits the growth of neuroblastoma cells. A, effect of ZD1839 on survival of NLF and SY5Y cells. Cells were treated with the indicated concentrations of ZD1839 for 72 hours and cell number was measured by MTT assay. ZD1839 significantly inhibits the proliferation of NLF cells at a concentration of 0.1 µmol/L and of SY5Y cells at 0.5 µmol/L (P < 0.01). Columns, mean of five replicates; bars, SD. B, cells were pretreated with ZD1839 for 1 hour before exposure to EGF for 5 minutes. Phosphorylation of EGFR (Tyr1068), AKT (Ser473), and MAPK was examined by Western blotting. The blots were stripped and reprobed with anti-EGFR, anti-AKT, and anti-MAPK antibody, respectively, to measure total protein loading in each lane.

 
ZD1839 blocks the effect of epidermal growth factor on neuroblastoma cells primarily through the phosphoinositide 3-kinase pathway. Phosphorylation of HER1 by EGF led to activation of the MAPK and PI3K pathways. These pathways are thought to be important for cell survival and proliferation (23, 24). EGF induces phosphorylation of AKT (Ser473) and phosphorylation of both p42 and p44 MAPK (Figs. 3B and 4B, first two lanes).



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  		Figure 4. The PI3K/AKT specific inhibitor, LY294002, completely abolishes the proliferation effect of EGF. A, LY294002 completely blocks the effect of EGF. SY5Y and NLF cells were cultured in 2% serum medium in 96-well plates and pretreated with LY294002 (20 µmol/L) or U0126 (10 µmol/L) for 1 hour. EGF (100 ng/mL) or EGF plus ZD1839 (1 µmol/L) were added for 72 hours. Cell number was determined by MTT assay. LY294002 completely blocked EGF-mediated proliferation (P < 0.01), whereas 10 µmol/L U0126 only modestly reduced EGF growth enhancement; this is significant for NLF (P < 0.01) but not for SY5Y (P = 0.12). LY294002 alone significantly reduced cell number compared with control cells (P < 0.01), whereas U0126 alone did not have significant effect (P > 0.01). Columns, mean of five replicate samples; bars, SD. The results shown are representative of at least three independent experiments. B, Cells were pretreated with LY294002, U0126, or both for 30 minutes before being stimulated by EGF for 5 minutes. Phosphorylation of AKT (Ser473) and MAPK was examined by Western blotting. Blots were stripped and reprobed with antibody against total AKT and MAPK to show equal protein loading in each lane.

 
We also examined the ability of ZD1839 to block downstream signaling pathways initiated by EGF. NLF and SY5Y cells were pretreated with different concentrations of ZD1839 before EGF treatment, and phosphorylation of HER1, AKT (Ser473), and p42/p44 MAPK was examined. We found that ZD1839 greatly reduced phosphorylation of HER1 (Tyr1068) at a concentration of 0.01 µmol/L. In NLF cells at a concentration of 1 µmol/L, ZD1839 markedly reduced phosphorylation of AKT (Ser473). However, ZD1839 showed only moderate inhibition of the phosphorylation of MAPK at concentrations as high as 10 µmol/L (Fig. 3B). In SY5Y cells, inhibition of AKT phosphorylation (Ser473) was evident at a concentration of 0.01 µmol/L, whereas inhibition of p42/p44 MAPK phosphorylation was not seen until 1 µmol/L. We also examined the effect of ZD1839 on two other neuroblastoma cell lines (NB69 and SKNSH), and both showed pathway inhibition similar to SY5Y. In summary, ZD1839 reduced the phosphorylation of HER1 at very low concentrations. It has a profound effect on PI3K/AKT pathway but a more modest effect on the phosphorylation of MAPK. Thus, the PI3K/AKT pathway seems to play a more important role in EGF-meditated cell proliferation.

To further support our conclusions regarding the relative importance of the PI3K/AKT and p42/p44 MAPK pathways, we studied the effect of LY294002 and U0126, which are specific inhibitors of these respective pathways. NLF and SY5Y cells grown in medium containing 2% FBS were pretreated with LY294002 or U0126 for 1 hour before EGF treatment. Twenty micromoles per liter of LY294002 significantly decreased cell number, whereas 10 µmol/L U0126 had no significant effect on either cell line (Fig. 4A, white columns). The same dose of LY294002 completely blocked EGF-mediated growth enhancement (P < 0.01) in both cell lines, whereas 10 µmol/L U0126 only modestly reduced EGF growth enhancement (P > 0.01) in SY5Y cells as determined by MTT assay.

Western blotting was done to ensure inhibition of specific pathways by corresponding inhibitors at the appropriate concentration. Twenty micromoles per liter of LY294002 almost completely blocked phosphorylation of AKT (Ser473) induced by EGF specifically in SY5Y cells (Fig. 4B), whereas NLF needed up to 40 µmol/L LY294002 to completely block the pathway. Ten micromoles per liter U0126 completely blocked MAPK pathway in SY5Y cells, whereas as little as 1 µmol/L was effective in NLF cells. Thus, the effect of 10 µmol/L U0126 we saw in NLF (Fig. 4A) could be due to inhibition of other pathways. Given the fact that 1 µmol/L ZD1839 inhibited EGF-mediated growth enhancement and PI3K/AKT activation in NLF cells, but it has very little effect on MAPK phosphorylation (Fig. 3B), we conclude that the PI3K/AKT pathway is the primary pathway responsible for EGF-mediated proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neuroblastoma is a common pediatric cancer and a leading cause of morbidity and mortality in children. Currently, even very intensive, multimodality therapy has resulted in only modest improvement in the cure rate of the more aggressive neuroblastomas. Therefore, a better understanding of the genes, proteins, and pathways responsible for neuroblastoma tumorigenesis and progression may lead to the development of more effective, less toxic therapies.

We have shown that 10 of 13 neuroblastoma cell lines and all primary tumors tested expressed HER1 at readily detectable levels. Other members of HER family receptors (HER2, HER3, and HER4) are also expressed in neuroblastoma cell lines at lower levels. HER3 was expressed in primary neuroblastomas at levels similar to HER1. There was no correlation between tumor stage and expression of any of the HER receptors. This was further supported by data from a microarray analysis of HER expression in 101 neuroblastoma tumors (data not shown). Nevertheless, the EGF/HER1 pathway seems to be an important growth/proliferation pathway for neuroblastoma. The addition of exogenous EGF led to proliferation of SY5Y, NLF, and other neuroblastoma cells. Others have made similar observations on the NB69 neuroblastoma cell line (25). Because of the presence of other HER family receptors, HER1 may form a homodimer with itself or heterodimers with other members of this family. Other members of the HER family in neuroblastoma may be functional as well. For example, the HER3 receptor is expressed in the neuroblastoma cell line LAN-5 at readily detectable levels, and it can bind its ligand GGF-2 (neuregulin-2; ref. 26), as well as activate downstream PI3K/AKT and MAPK pathways (data not shown).

The HER1-specific tyrosine kinase inhibitor ZD1839 blocked the growth advantage induced by EGF. Furthermore, we examined downstream signaling pathways and showed that this specific inhibitor markedly reduced the EGF-induced autophosphorylation of HER1 and the phosphorylation of AKT (but not MAPK), suggesting that the EGF/HER1–mediated growth enhancement effect is primarily mediated by the PI3K/AKT pathway. Furthermore, this inhibitory effect could be mimicked using LY249002, a specific inhibitor of PI3K/AKT pathway. However, treatment of cells with U0126, a specific inhibitor of p42/p44 MAPK, had only a modest effect, further supporting our conclusion that the primary growth enhancement effect of HER1 activation is mediated by the PI3K/AKT pathway.

Inhibition of tyrosine kinase receptors is an attractive approach for treating cancers for which a specific tyrosine kinase receptor pathway can be identified as important. ZD1839 (Iressa, gefitinib) has proven to be useful preclinically and clinically for treating EGFR-expressing tumors, such as non–small cell lung cancer, head and neck cancer, breast cancer, prostate cancer, and others; the general doses used clinically are 250 to 500 mg/d (27–30). Other EGFR inhibitors, such as Herceptin (trastuzumab), an anti-HER2 monoclonal antibody, have shown activity in a subset of breast cancers (31).

We have shown previously that members of the Trk family are expressed in both favorable and unfavorable neuroblastomas (2, 5, 7). TrkB and its ligand BDNF are expressed in the most aggressive, MYCN-amplified neuroblastomas, and this seems to represent an autocrine survival pathway for these tumors. The TrkB/BDNF pathway mediates survival and leads to activation of the PI3K/AKT pathway as well (1). CEP-751 is a Trk-specific tyrosine kinase inhibitor that is also under investigation in our laboratory. We have shown that CEP-751 inhibits growth of TrkB-expressing neuroblastoma cells in vitro and in vivo in mouse xenografts (32).

Here, we have shown that the EGF/HER1 pathway may also be an important growth/proliferation pathway for neuroblastomas, and low concentrations of ZD1839 inhibit growth of neuroblastoma cells. The IC50 of ZD1839 for NLF is 0.5 µmol/L in 10% FBS serum (and a similar dose under low serum conditions with EGF stimulation). This dose is comparable with that needed to inhibit the HER1-expressing A431 cell line in our study. Clinically, the geometric mean of plasma concentration is 0.3 to 1 mmol/L when patients were treated with a dose of 225 to 400 mg/d of ZD1839 (33, 34), which is more than serum-stimulated ZD1839 IC50 needed for NLF and SY5Y. Thus, ZD1839 seems promising as a novel agent for treating human neuroblastomas.

Other tyrosine kinase receptors may also facilitate the activity of EGFR or have an independent effect on growth or survival. Previous reports have shown that other receptors, such as insulin-like growth factor-IR (IGF-IR) and EPHR family receptors, are expressed in neuroblastomas (35, 36). IGF-IR activates AKT and MAPK pathways in SY5Y cells and leads to survival and differentiation (35). Interestingly, ligand binding of all of these tyrosine kinase receptors results in activation of similar intracellular signaling pathways, suggesting that activation of any of these growth factor/receptor pathways could lead to enhanced growth or survival. Deregulated tumor cell growth, especially for highly resistant tumor cells, could result from the overexpression of one or several tyrosine receptors.

A combination of currently available reagents inhibiting the HER family or Trk family receptors is an attractive, biologically based approach for the treatment of highly resistant neuroblastomas. In fact, the cooperative inhibitory effect of ZD1839 and trastuzumab has been shown on human breast cancer cell growth (37). We are currently investigating whether EGFR inhibition alone, or in combination with either Trk inhibition or conventional chemotherapeutic agents, may be a more effective and less toxic approach for treating human neuroblastoma xenografts or other mouse models of neuroblastoma.


   Acknowledgments
 
Grant support: NIH grants CA-94194 and CA97323 (G.M. Brodeur, R. Ho, J.E. Minturn, and A.E. Evans), Young Investigator Award and Career Development Award from the American Society of Clinical Oncology (J.E. Minturn), Richard and Nancy Wolfson Young Investigator Award (J.E. Minturn and T. Hishiki), and Audrey E. Evans Endowed Chair (G.M. Brodeur).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank AstraZeneca for providing ZD1839 and Wing M. So for performing some confirmatory semiquantitative RT-PCR experiments.

Received 7/ 7/04. Revised 6/13/05. Accepted 8/17/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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