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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Int J Oncol. 2009 Oct;35(4):845–850. doi: 10.3892/ijo_00000398

Biological significance of EPHA2 expression in neuroblastoma

Bing Kung 1,4, Huaqing Zhao 2,5, Sakeenah L Hicks 3, Xao X Tang 3, Naohiko Ikegaki 3
PMCID: PMC2827331  NIHMSID: NIHMS177748  PMID: 19724921

Abstract

Neuroblastoma is a pediatric solid tumor that exhibits striking clinical bipolarity. Despite extensive efforts to treat unfavorable neuroblastoma, survival rate of children with the disease is among the lowest. Previous studies suggest that EPHA2, a member of the EPH family receptor kinases, can either promote or suppress cancer cell growth depending on cellular contexts. In this study, we investigated the biological significance of EPHA2 in neuroblastoma. It was found that tumorigenic N-type neuroblastoma cell lines expressed low levels of EPHA2, whereas hypo-tumorigenic S-type neuroblastoma cell lines expressed high levels of EPHA2 (p<0.005). Notably, inhibitors of DNA methylation and histone deacetylase enhanced EPHA2 expression in N-type cells, suggesting that EPHA2 is epigenetically silenced in unfavorable neuroblastoma cells. Furthermore, ectopic high-level expression of EPHA2 in N-type neuroblastoma cell lines resulted in significant growth suppression. However, Kaplan-Meier survival analysis showed that high EPHA2 expression was not associated with a good disease outcome of neuroblastoma, indicating that EPHA2 is not a favorable neuroblastoma gene, but a growth suppressive gene for neuroblastoma. Accordingly, EPHA2 expression was markedly augmented in vitro in neuroblastoma cells treated with doxorubicin, which is commonly used for treating unfavorable neuroblastoma. Taken together, EPHA2 is one of the effectors of chemotherapeutic agents (e.g., gene silencing inhibitors and DNA damaging agents). EPHA2 expression may thus serve as a biomarker of drug responsiveness for neuroblastoma during the course of chemotherapy. In addition, pharmaceutical enhancement of EPHA2 by non-cytotoxic agents may offer an effective therapeutic approach in the treatment of children with unfavorable neuroblastoma.

Keywords: neuroblastoma, favorable neuroblastoma genes, drug responsiveness, biomarkers

Introduction

Neuroblastoma is a neural crest-derived tumor and is the most common extracranial pediatric malignancy. The tumor accounts for 7–10% of all childhood cancers and is the cause for ~15% of fatalities in children with cancer. Neuroblastoma is unique because of its propensity to exhibit either a favorable or an unfavorable phenotype. Favorable neuroblastomas undergo spontaneous regression or maturation or are curable by surgical removal with or without adjuvant chemotherapy. In contrast, unfavorable neuroblastomas exhibit unrestrained growth despite the most intensive treatment (1).

EPHA2 is a member of the EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases. Like most EPH family receptor tyrosine kinases, EPHA2 is expressed during early development (2), but its expression is also detected in adult epithelial cells (3). Although specific cellular functions of EPHA2 in normal epithelia are not well understood, several studies suggest potential roles for EPHA2 in the control of cell growth, survival, migrations, and angiogenesis (4–8). EPHA2 is overexpressed and functionally altered in a variety of solid tumors, including colon cancer, glioma/glioblastoma, mammary cancer, melanoma and prostate cancer (5,9–17). EPHA2 expression is thus linked to the regulation of cellular behavior that confer a metastatic phenotype. In contrast, EPHA2 promotes apoptosis and suppresses growth of lung cancer, breast cancer, and melanoma cells (18–20). EPHA2 also acts as a tumor suppressor in a chemically-induced skin cancer model in mouse (21). In addition, EPHA2 transcription is regulated by p53 (18,19). These observations suggest that EPHA2 can exhibit opposite biological effects: promotion or suppression of cell growth on cancer cells depending upon their cellular context. In this study, we investigated the biological significance of EPHA2 in neuroblastoma. Our results show that EPHA2 is a neuroblastoma growth-suppressive gene and that EPHA2 expression has potential therapeutic and clinical applications in neuroblastoma.

Materials and methods

Neuroblastoma cell lines

All neuroblastoma cell lines were grown in RPMI-1640 supplemented with 5% fetal bovine serum and 1% OPI (Gibco, Grand Island, NY). These cell lines were tested negative for mycoplasma, and their identity was validated by the original source or by microsatellite analysis. NBL-S was obtained from Dr Susan L. Cohn (University of Chicago). OAN, SKNAS, LHN, KAN, SAN, LAN5, KPN, LA1-55N, LA1-5S, KCN, and KCNR were from Dr C. Patrick Reynolds (Children's Hospital Los Angeles, CA). Nb69, IMR5 (a clone of IMR32), and CHP134 were from Dr Roger H. Kennett (Department of Biology, Wheaton College, Wheaton, IL; a former faculty member of Department of Human Genetics, The University of Pennsylvania School of Medicine). SY5Y and SHEP were from Dr Robert Ross (Fordham University, Bronx, NY). NGP, NMB, and NLF were from Dr Garrett M. Brodeur (The Children's Hospital of Philadelphia). CHP902 was established by Dr Hiro Kuroda (The Children's Hospital of Philadelphia). CHP901 and CHP902R were established by Dr Naohiko Ikegaki.

Primary neuroblastoma tumor samples

Fifty neuroblastoma tumor specimens were obtained from the Tumor Bank of the former Pediatric Oncology Group, the Tumor Bank of the Children's Hospital of Philadelphia, and Memorial Sloan-Kettering Cancer Center. The neuroblastoma cohort included 10 of stage 1, 8 of stage 2, 5 of stage 4S, 12 of stage 3, and 15 of stage 4. Among these, 9 are MYCN-amplified (18%). Of 50 neuroblastoma specimens, 49 had survival data. The neuroblastoma cohort was verified by the results of Kaplan-Meier analyses, which demonstrated that the established prognostic markers of neuroblastoma (age, stage, MYCN amplification, EPHB6) predict disease outcome in this cohort. The clinical correlative studies were performed at the Children's Hospital of Philadelphia, and the use of human tumor samples for this study was reviewed and approved by its institutional review board.

Quantitative reverse transcription-PCR (RT-PCR)

RNAs were isolated from neuroblastoma cell lines or primary neuroblastoma tumors using the Qiagen RNeasy kit. Experimental procedures for the quantitative RT-PCR were previously described elsewhere (22,23). Primer sequences for EPHA2 are 5'-TGCAGCAGTATACGGAGCAC-3', and 5'-TTCACCTGGTCCTTGAGTCC-3'.

Preparation of 5AdC, 4PB, and doxorubicin

5-Aza-2'-deoxycytidine or 5AdC (Fluka) and sodium 4-phenylbutyrate or 4PB (Aldrich) were prepared as previously described (23). Doxorubicin (Sigma) was prepared by dissolving in acidic H2O at the concentration of 2.5 mg/ml as a stock.

Western blot analysis

Western blot was performed according to the method previously described (24) except SuperSignal West Dura Extended Duration Substrate (Pierce) was used. Light emission signals were captured by either a Versadoc 5000 (Bio-Rad) or a LAS-3000 (Fuji) digital image analyzer. Cell extracts were made in the 2D gel sample buffer (9 M urea, 2% Nonidet-P40, 2% 2-mercaptoethanol, and 0.32% pH 3–10 2D Pharmalyte) and the protein content of the samples was determined by the Bio-Rad protein assay kit using bovine serum albumin as a standard and the sample buffer as the blank. The anti-EPHA2 mouse monoclonal antibody D7 was purchased from Upstate USA, Inc. The monoclonal antibody specific for p53, PAB1801, was purchased from Santa Cruz Biotechnology. The monoclonal antibody specific for p21waf1, EA10, was purchased from Calbiochem.

Transient transfection of neuroblastoma cells with EPHA2

A cDNA clone of human EPHA2 (3) was subcloned into pCI-neo mammalian expression vector (Promega). Neuroblastoma cell lines were transfected with pCI-neo or pCI/EPHA2 by electroporation using a Gene Pulser Xcell electroporator (Bio-Rad) (120 V, 25 msec, a single square wave).

MTT assay

One and a half million SY5Y or IMR5 cells were transfected by electroporation with either pCI-neo eukaryotic expression vector (Invitrogen) alone or the vector containing a human EPHA2 cDNA. The resulting transfectants were plated into 6 wells of a 24-well plate and selected for 5 days with 500 µg/ml neomycin. After selection, the cells were treated with 0.5 µg/ml MTT for 4 h to stain viable cells and to examine the effect of EPHA2 on growth of neuroblastoma cells.

Statistical analysis

A χ2 test with Yates' correction was employed to examine statistical significance of EPHA2 expression in neuroblastoma cell lines. Survival probabilities in neuroblastoma subgroups were estimated according to the methods of Kaplan and Meier (25). Survival distributions were compared using log-rank tests (26). P<0.05 was considered statistically significant.

Results

Neuroblastoma cell lines derived from unfavorable neuroblastoma express low levels of EPHA2

We first examined EPHA2 expression in 23 neuroblastoma cell lines and determined its expression pattern. As shown in Fig. 1, among the cell lines, N-type neuroblastoma cells expressed low levels of EPHA2. In contrast, S-type neuroblastoma cell lines (SHEP and LA1-5S) expressed high levels of EPHA2, and this differential expression was statistically significant (p<0.005). The above observation was intriguing because S-type neuroblastoma cells are known to be hypo-tumorigenic in mouse xenograft models and are considered more benign than N-type cells (27,28).

Figure 1.

Figure 1

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EPHA2 expression in neuroblastoma cell lines. Quantitative RT-PCR was used to assess levels of EPHA2 expression in neuroblastoma cell lines. Neuroblastoma cell lines derived from unfavorable neuroblastoma express low levels of EPHA2. However S-type neuroblastoma cells (SHEP and LA1-5S), which are more benign as they seldom form tumors in nude mice, express high levels of EPHA2. Differential expression of EPHA2 was examined by a χ2 test with Yates' correction.

EPHA2 expression is silenced in N-type neuroblastoma cell lines

Our gene profiling study has shown that treatment of the neuroblastoma cell line IMR5 with inhibitors of DNA methylation and HDAC resulted in an increased EPHA2 expression (Tang and Ikegaki, unpublished data). In this report, we confirmed the previous observation by quantitative RT-PCR. As shown in Fig. 2, EPHA2 expression was significantly increased in neuroblastoma cell lines IMR5, CHP134 and SY5Y upon treatment with inhibitors of DNA methylation (5-Aza-2'-deoxycytidine or 5AdC) and HDAC (sodium 4-phenylbutyrate or 4PB).

Figure 2.

Figure 2

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Inhibitors of DNA methylation and HDAC enhance EPHA2 expression in neuroblastoma cells. EPHA2 expression is enhanced by 5AdC and 4PB in neuroblastoma cell lines. EPHA2 expression in neuroblastoma cell lines was examined using quantitative RT-PCR. SY5Y is a MYCN non-amplified cell line. IMR5 and CHP134 are a MYCN-amplified cell lines. These cells were treated with 5AdC (2.5 µM) and/or 4PB (2.5 mM) for 4 days and subjected to the gene expression studies. Levels of EPHA2 expression were presented as fold increase over control (no drug treated cells).

Forced expression of EPHA2 in unfavorable neuroblastoma cells results in growth suppression

The facts that EPHA2 expression is low in N-type neuroblastoma cells, and that gene silencing inhibitors suppress growth of these neuroblastoma cells (23) and can increase EPHA2 expression suggest that high EPHA2 expression confers a growth suppressive effect on tumorigenic N-type neuroblastoma cell lines. We therefore examined the effect of forced EPHA2 expression on two N-type neuroblastoma cell lines (SY5Y and IMR5). SY5Y is a MYCN-non-amplified neuroblastoma cell line and IMR5 is a MYCN-amplified line, which express low levels of endogenous EPHA2 (Fig. 1). As shown in Fig. 3A, transfection of SY5Y and IMR5 with an EPHA2 cDNA, in fact, significantly inhibited the clonogenicity in vitro. The expression of EPHA2 protein in the transfectants was also confirmed by Western blot analysis (Fig. 3B). These EPHA2 transfectants expressed EPHA2 protein at similar levels to that in SHEP cells.

Figure 3.

Figure 3

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Forced expression of EPHA2 in neuroblastoma cell lines results in growth suppression. (A) A full-length cDNA of EPHA2 was cloned into a eukaryotic expression vector, pCI-neo (Promega). IMR5 and SY5Y cells were transfected with either the vector control or the pCI/EPHA2 cDNA construct by electroporation. The resulting transfectants were selected by neomycin (500 µg/ml) for 4 days. MTT assay was used to assess viable cells in each culture on the seventh day. (B) Expression of EPHA2 protein in the EPHA2 transfectant neuroblastoma cells. SHEP was used as a positive control.

EPHA2 expression is not associated with neuroblastoma disease outcome

The above results indicate that EPHA2 shares several characteristics with favorable neuroblastoma genes (EPHB6, EFNB2, EFNB3, TrkA, CD44 and MIZ-1) (22,23,29). Their expression is low in neuroblastoma cell lines and can be enhanced by gene silencing inhibitors. In addition, forced expression of these genes in neuroblastoma cell lines results in growth suppression. We therefore investigated whether or not EPHA2 expression would be associated with disease outcome of neuroblastoma in a cohort of 50 primary tumors. This cohort was representative of the general neuroblastoma population as the expression of known favorable neuroblastoma genes such as EPHB6 and TrkA was associated with disease outcome (data not shown). As shown in Fig. 4, the Kaplan-Meier analysis demonstrated that high EPHA2 expression was not associated with a good disease outcome of neuroblastoma. Hence, EPHA2 is not a favorable neuroblastoma gene (see Discussion), but a growth suppressive gene for neuroblastoma.

Figure 4.

Figure 4

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EPHA2 expression is not associated with outcome of neuroblastoma. Survival probabilities of two groups of neuroblastomas with low- or high-level EPHA2 expression were estimated by the method of Kaplan-Meier. The median expression value of EPHA2 based on the entire cohort (n=50) was used as a cut-off to define high- and low-expression subgroups. Of 50 neuroblastoma specimens, 49 had survival data. The same analysis was performed for TrkA and EPHB6 as a comparison. Five-year survival was calculated for each group, and the log-rank test was used to compare survival probabilities of the two groups.

Doxorubicin augments the expression of EPHA2 in neuroblastoma cells

The growth suppressive effect of EPHA2 on tumorigenic N-type neuroblastoma cells suggests that EPHA2 is potentially involved in the drug responsiveness of unfavorable neuroblastoma. We therefore examined EPHA2 expression in neuroblastoma cell lines treated with doxorubicin, a commonly used chemotherapeutics for patients with neuroblastoma. As shown in Fig. 5A, treatment of SY5Y and IMR5 neuroblastoma cells with doxorubicin resulted in a significant increase in EPHA2 transcript expression. Thus, EPHA2 is a doxorubicin-inducible gene. Furthermore, Western blot analysis showed that EPHA2 expression was up-regulated in the doxorubicin-treated IMR5 cells in a time-dependent fashion. The increase in EPHA2 expression was accompanied by a similar time-dependent increase in p53 as well as p21WAF1, a p53 target in these cells (Fig. 5B). As EPHA2 is a known target of p53 (18,19), this result suggests that the effect of doxorubicin on the increase in EPHA2 expression is mediated by p53. To address this question, IMR5 cells were treated with CoCl2, which is known to mimic hypoxia and increases p53 expression (30). The expression of EPHA2, p53 and p21waf1 was then examined in the treated cells. As shown in Fig. 5B, the CoCl2 treatment caused a marked increase in p53 and p21waf1 expression in a time dependent fashion but resulted only in a slight increase in EPHA2 expression.

Figure 5.

Figure 5

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Chemotherapeutics enhances EPHA2 expression in neuroblastoma cells. (A) Doxorubicin enhances the expression of EPHA2 in SY5Y and IMR5 neuroblastoma cells. SY5Y and IMR5 cells were treated with 0.5 µg/ml doxorubicin for eight hours, and then subjected to RT-PCR analysis for EPHA2 expression. (B) Treatment of the neuroblastoma cell line IMR5 with doxorubicin or CoCl2 results in concomitant up-regulation of EPHA2, p53 and p21WAF1. IMR5 cells were treated with either 0.5 µg/ml doxorubicin or 250 µM CoCl2 for the time indicated and are subjected to Western blot analysis using antibodies specific for the proteins indicated.

Discussion

We previously mapped the human EPHA2 gene (or ECK) to 1p36.1 (31), a region where putative neuroblastoma tumor suppressor genes have been suggested (32). Extensive efforts from several laboratories have focused on mutation analysis on the genes localized to the 1p36 region to identify putative tumor suppressor genes of neuroblastoma. However, no consistent gene mutation in any gene in this region has been found so far. One exception to this is KIF1B, where mutations in this gene were found in a small number of familial neuroblastoma cases (33). Nevertheless, because of its chromosomal localization, mutation analysis was performed for the EPHA2 gene in a dozen neuroblastoma cell lines. We found no mutation in EPHA2 in the cell lines examined (Kung and Ikegaki, unpublished data), indicating that EPHA2 is not a classic tumor suppressor gene. Rather, results of this study suggest that the low expression of EPHA2 in neuroblastoma cell lines is due to an epigenetic mechanism. In fact, similar observations were previously made in genes residing in 1p36. These include ZBTB17 (MIZ-1) (29) and CHD5 (34,35).

As mentioned earlier, EPHA2 is not a favorable neuroblastoma gene, as its expression does not predict neuroblastoma disease outcome. This is an unexpected finding because 1p deletion is associated with a poor disease outcome of neuroblastoma (36,37) and EPHA2 resides in this region of chromosome (31) and is subjected to epigenetic silencing (this study). This inconsistency may be explained by the fact that S-type cells express EPHA2 at very high levels in comparison to N-type cells. As has been reported, primary neuroblastomas are composed of a mixture of neuroblastic and Schwannian stroma cells of a common tumor progenitor cell origin, and their proportional representation may vary in given tumors (38). We speculate that the progressive neuroblastomas expressing high EPHA2 at diagnosis may have included a greater number of Schwannian stroma cells, an equivalent to S-type cells in vitro, but neuroblastic and perhaps neuroblastoma stem cells, in vivo equivalents of N-type and/or ‘I-type’ cells (28), respectively (39), may eventually dominate, leading to a poor outcome.

The effect of ectopic overexpression of EPHA2 on growth of neuroblastoma cell lines (Fig. 4) and the augmentation on EPHA2 expression by doxorubicin in neuroblastoma cells (Fig. 5) have their own biological and clinical implications. Firstly, although EPHA2 expression was augmented along with a p53 up-regulation by doxorubicin, p53 expression induced by CoCl2 had a small effect on the expression of EPHA2 in neuroblastoma. These observations suggest that EPHA2 expression can be augmented by both p53-dependent and p53-independent mechanisms in neuroblastoma cells. Secondly, our results suggest that high-level EPHA2 expression is incompatible with the aggressive growth of unfavorable neuroblastoma cells. Moreover, the fact that doxorubicin effectively induces EPHA2 expression in unfavorable neuroblastoma cells could explain why cytotoxic drugs are effective during the initial course of treatment in patients with neuroblastoma (Fig. 5). However, the cytotoxic drugs may also cause genetic changes that would ultimately lead to the drug resistance phenotype of unfavorable neuroblastoma cells (40,41). If so, pharmaceutical augmentation of EPHA2 by non-cytotoxic agents would be among the attractive therapeutic approaches in treatment for children with unfavorable neuroblastoma.

Our previous study has demonstrated that epigenetic silencing inhibitors suppress neuroblastoma growth in vitro and in vivo (23). In this study, we have shown that chemotherapeutic agents such as gene silencing inhibitors (Fig. 2) and doxorubicin (Fig. 5) markedly increase EPHA2 expression in neuroblastoma cells. In addition, we have observed that 13-cis-retinoic acid (a current maintenance therapy agent for neuroblastoma) alone or its combination with an HDAC inhibitor MS-275 enhances EPHA2 expression in neuroblastoma cells (Ikegaki and Tang, unpublished data). Together these observations suggest that EPHA2 expression can be considered a biomarker of drug responsiveness during the course of chemotherapy of unfavorable neuroblastomas.

Acknowledgements

The tumor specimens used in this study were obtained from the tumor banks of the former POG, Children's Hospital of Philadelphia, and Memorial Sloan-Kettering Cancer Center. This work was supported by NIH grants CA97255 (X.X.T), CA70958, CA85519 and CA127571 (N.I.).

References

  • 1.Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369:2106–2120. doi: 10.1016/S0140-6736(07)60983-0. [DOI] [PubMed] [Google Scholar]
  • 2.Ruiz JC, Robertson EJ. The expression of the receptor-protein tyrosine kinase gene, eck, is highly restricted during early mouse development. Mech Dev. 1994;46:87–100. doi: 10.1016/0925-4773(94)90078-7. [DOI] [PubMed] [Google Scholar]
  • 3.Lindberg RA, Hunter T. cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases. Mol Cell Biol. 1990;10:6316–6324. doi: 10.1128/mcb.10.12.6316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Andres AC, Reid HH, Zurcher G, Blaschke RJ, Albrecht D, Ziemiecki A. Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and arcinogenesis. Oncogene. 1994;9:1461–1467. [PubMed] [Google Scholar]
  • 5.Rosenberg IM, Goke M, Kanai M, Reinecker HC, Podolsky DK. Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function. Am J Physiol. 1997;273:G824–G832. doi: 10.1152/ajpgi.1997.273.4.G824. [DOI] [PubMed] [Google Scholar]
  • 6.Pandey A, Shao H, Marks RM, Polverini PJ, Dixit VM. Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF alpha-induced angiogenesis. Science. 1995;268:567–569. doi: 10.1126/science.7536959. [DOI] [PubMed] [Google Scholar]
  • 7.Pandey A, Lazar DF, Saltiel AR, Dixit VM. Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity. J Biol Chem. 1994;269:30154–30157. [PubMed] [Google Scholar]
  • 8.Ganju P, Shigemoto K, Brennan J, Entwistle A, Reith AD. The Eck receptor tyrosine kinase is implicated in pattern formation during gastrulation, hindbrain segmentation and limb development. Oncogene. 1994;9:1613–1624. [PubMed] [Google Scholar]
  • 9.Easty DJ, Bennett DC. Protein tyrosine kinases in malignant melanoma. Melanoma Res. 2000;10:401–411. doi: 10.1097/00008390-200010000-00001. [DOI] [PubMed] [Google Scholar]
  • 10.Hess AR, Seftor EA, Gardner LM, Carles-Kinch K, Schneider GB, Seftor RE, Kinch MS, Hendrix MJ. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2) Cancer Res. 2001;61:3250–3255. [PubMed] [Google Scholar]
  • 11.Walker-Daniels J, Coffman K, Azimi M, Rhim JS, Bostwick DG, Snyder P, Kerns BJ, Waters DJ, Kinch MS. Overexpression of the EphA2 tyrosine kinase in prostate cancer. Prostate. 1999;41:275–280. doi: 10.1002/(sici)1097-0045(19991201)41:4<275::aid-pros8>3.0.co;2-t. [DOI] [PubMed] [Google Scholar]
  • 12.Zantek ND, Azimi M, Fedor-Chaiken M, Wang B, Brackenbury R, Kinch MS. E-cadherin regulates the function of the EphA2 receptor tyrosine kinase. Cell Growth Differ. 1999;10:629–638. [PubMed] [Google Scholar]
  • 13.Zelinski DP, Zantek ND, Stewart JC, Irizarry AR, Kinch MS. EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res. 2001;61:2301–2306. [PubMed] [Google Scholar]
  • 14.Zantek ND, Walker-Daniels J, Stewart J, Hansen RK, Robinson D, Miao H, Wang B, Kung HJ, Bissell MJ, Kinch MS. MCF-10A-NeoST: a new cell system for studying cell-ECM and cell-cell interactions in breast cancer. Clin Cancer Res. 2001;7:3640–3648. [PubMed] [Google Scholar]
  • 15.Zhou Z, Yuan X, Li Z, Tu H, Li D, Qing J, Wang H, Zhang L. RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells. Surg Neurol. 2008;70:562–569. doi: 10.1016/j.surneu.2008.04.031. [DOI] [PubMed] [Google Scholar]
  • 16.Wang LF, Fokas E, Bieker M, Rose F, Rexin P, Zhu Y, Pagenstecher A, Engenhart-Cabillic R, An HX. Increased expression of EphA2 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients. Oncol Rep. 2008;19:151–156. [PubMed] [Google Scholar]
  • 17.Wykosky J, Gibo DM, Stanton C, Debinski W. EphA2 as a novel molecular marker and target in glioblastoma multiforme. Mol Cancer Res. 2005;3:541–551. doi: 10.1158/1541-7786.MCR-05-0056. [DOI] [PubMed] [Google Scholar]
  • 18.Dohn M, Jiang J, Chen X. Receptor tyrosine kinase EphA2 is regulated by p53-family proteins and induces apoptosis. Oncogene. 2001;20:6503–6515. doi: 10.1038/sj.onc.1204816. [DOI] [PubMed] [Google Scholar]
  • 19.Jin YJ, Wang J, Qiao C, Hei TK, Brandt-Rauf PW, Yin Y. A novel mechanism for p53 to regulate its target gene ECK in signaling apoptosis. Mol Cancer Res. 2006;4:769–778. doi: 10.1158/1541-7786.MCR-06-0178. [DOI] [PubMed] [Google Scholar]
  • 20.Zhang G, Njauw CN, Park JM, Naruse C, Asano M, Tsao H. EphA2 is an essential mediator of UV radiation-induced apoptosis. Cancer Res. 2008;68:1691–1696. doi: 10.1158/0008-5472.CAN-07-2372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Guo H, Miao H, Gerber L, Singh J, Denning MF, Gilliam AC, Wang B. Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin. Cancer Res. 2006;66:7050–7058. doi: 10.1158/0008-5472.CAN-06-0004. [DOI] [PubMed] [Google Scholar]
  • 22.Tang XX, Zhao H, Robinson ME, Cohen B, Cnaan A, London W, Cohn SL, Cheung NK, Brodeur GM, Evans AE, Ikegaki N. Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma. Proc Natl Acad Sci USA. 2000;97:10936–10941. doi: 10.1073/pnas.190123297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tang XX, Robinson ME, Riceberg JS, Kim DY, Kung B, Titus TB, Hayashi S, Flake AW, Carpentieri D, Ikegaki N. Favorable neuroblastoma genes and molecular therapeutics of neuroblastoma. Clin Cancer Res. 2004;10:5837–5844. doi: 10.1158/1078-0432.CCR-04-0395. [DOI] [PubMed] [Google Scholar]
  • 24.Ikegaki N, Kennett RH. Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay. J Immunol Methods. 1989;124:205–210. doi: 10.1016/0022-1759(89)90354-2. [DOI] [PubMed] [Google Scholar]
  • 25.Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53:457–481. [Google Scholar]
  • 26.Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep. 1966;50:163–170. [PubMed] [Google Scholar]
  • 27.Piacentini M, Piredda L, Starace DT, Annicchiarico-Petruzzelli M, Mattei M, Oliverio S, Farrace MG, Melino G. Differential growth of N- and S-type human neuroblastoma cells xenografted into scid mice. Correlation with apoptosis. J Pathol. 1996;180:415–422. doi: 10.1002/(SICI)1096-9896(199612)180:4<415::AID-PATH684>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  • 28.Ross RA, Spengler BA. Human neuroblastoma stem cells. Semin Cancer Biol. 2007;17:241–247. doi: 10.1016/j.semcancer.2006.04.006. [DOI] [PubMed] [Google Scholar]
  • 29.Ikegaki N, Gotoh T, Kung B, Riceberg JS, Kim DY, Zhao H, Rappaport EF, Hicks SL, Seeger RC, Tang XX. De novo identification of MIZ-1 (ZBTB17) encoding a MYC-interacting zinc-finger protein as a new favorable neuroblastoma gene. Clin Cancer Res. 2007;13:6001–6009. doi: 10.1158/1078-0432.CCR-07-0071. [DOI] [PubMed] [Google Scholar]
  • 30.An WG, Kanekal M, Simon MC, Maltepe E, Blagosklonny MV, Neckers LM. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature. 1998;392:405–408. doi: 10.1038/32925. [DOI] [PubMed] [Google Scholar]
  • 31.Sulman EP, Tang XX, Allen C, Biegel JA, Pleasure DE, Brodeur GM, Ikegaki N. ECK, a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers. Genomics. 1997;40:371–374. doi: 10.1006/geno.1996.4569. [DOI] [PubMed] [Google Scholar]
  • 32.Maris JM, Matthay KK. Molecular biology of neuroblastoma. J Clin Oncol. 1999;17:2264–2279. doi: 10.1200/JCO.1999.17.7.2264. [DOI] [PubMed] [Google Scholar]
  • 33.Schlisio S, Kenchappa RS, Vredeveld LC, George RE, Stewart R, Greulich H, Shahriari K, Nguyen NV, Pigny P, Dahia PL, Pomeroy SL, Maris JM, Look AT, Meyerson M, Peeper DS, Carter BD, Kaelin WG., Jr The kinesin KIF1Bbeta acts downstream from EglN3 to induce apoptosis and is a potential 1p36 tumor suppressor. Genes Dev. 2008;22:884–893. doi: 10.1101/gad.1648608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Okawa ER, Gotoh T, Manne J, Igarashi J, Fujita T, Silverman KA, Xhao H, Mosse YP, White PS, Brodeur GM. Expression and sequence analysis of candidates for the 1p36.31 tumor suppressor gene deleted in neuroblastomas. Oncogene. 2008;27:803–810. doi: 10.1038/sj.onc.1210675. [DOI] [PubMed] [Google Scholar]
  • 35.Fujita T, Igarashi J, Okawa ER, Gotoh T, Manne J, Kolla V, Kim J, Zhao H, Pawel BR, London WB, Maris JM, White PS, Brodeur GM. CHD5, a tumor suppressor gene deleted from 1p36.31 in neuroblastomas. J Natl Cancer Inst. 2008;100:940–949. doi: 10.1093/jnci/djn176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Caron H, van Sluis P, de Kraker J, Bokkerink J, Egeler M, Laureys G, Slater R, Westerveld A, Voute PA, Versteeg R. Allelic loss of chromosome 1p as a predictor of unfavorable outcome in patients with neuroblastoma. N Engl J Med. 1996;334:225–230. doi: 10.1056/NEJM199601253340404. [DOI] [PubMed] [Google Scholar]
  • 37.Maris JM, Weiss MJ, Guo C, Gerbing RB, Stram DO, White PS, Hogarty MD, Sulman EP, Thompson PM, Lukens JN, Matthay KK, Seeger RC, Brodeur GM. Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study. J Clin Oncol. 2000;18:1888–1899. doi: 10.1200/JCO.2000.18.9.1888. [DOI] [PubMed] [Google Scholar]
  • 38.Mora J, Cheung NK, Juan G, Illei P, Cheung I, Akram M, Chi S, Ladanyi M, Cordon-Cardo C, Gerald WL. Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell. Cancer Res. 2001;61:6892–6898. [PubMed] [Google Scholar]
  • 39.Walton JD, Kattan DR, Thomas SK, Spengler BA, Guo HF, Biedler JL, Cheung NK, Ross RA. Characteristics of stem cells from human neuroblastoma cell lines and in tumors. Neoplasia. 2004;6:838–845. doi: 10.1593/neo.04310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Keshelava N, Zuo JJ, Chen P, Waidyaratne SN, Luna MC, Gomer CJ, Triche TJ, Reynolds CP. Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines. Cancer Res. 2001;61:6185–6193. [PubMed] [Google Scholar]
  • 41.Xue C, Haber M, Flemming C, Marshall GM, Lock RB, Mackenzie KL, Gurova KV, Norris MD, Gudkov AV. p53 determines multidrug sensitivity of childhood neuroblastoma. Cancer Res. 2007;67:10351–10360. doi: 10.1158/0008-5472.CAN-06-4345. [DOI] [PubMed] [Google Scholar]

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