Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P < 0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P < 0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a ''favorable NB gene,'' we examined the effect of EPHB6 on NB cell lines. Transfection of EPHB6 cDNA into IMR5 and SY5Y expressing little endogenous EPHB6 resulted in inhibition of their clonogenicity in culture. Furthermore, transfection of EPHB6 suppressed the tumorigenicity of SY5Y in a mouse xenograft model, demonstrating that high-level expressions of favorable NB genes, such as EPHB6, can in fact suppress malignant phenotype of unfavorable NB. N euroblastoma (NB) is a common pediatric solid tumor of neural crest origin. The tumor occurs frequently in infants and young children and originates in the adrenal glands or the sympathetic chain. NB exhibits a wide range of clinical heterogeneity, ranging from cases that are curable without treatment to those that progress relentlessly despite the most aggressive treatment. Several markers have been described that can predict disease outcome of NB, including patient age at diagnosis, tumor stage, Shimada histology, DNA ploidy, serum ferritin or lactate dehydrogenase levels, and MYCN amplification (1-7). Others, such as deletion or allelic loss of chromosome 1p (8, 9), allelic gain of 17q (10), TrkA expression (11-13), and CD44 expression (14), are also significant prognostic markers of NB. Although these factors have been known for some time, the molecular mechanism as to why these factors are predictive of NB outcome has remained elusive.EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ephrin ligands are involved in fundamental developmental processes in the nervous system (15-19). Their participation in angiogenesis during cardiovascular development also has been demonstrated (20-23). In addition, their involvement in human cancers through autocrine and͞or juxtac...

Purpose and Experimental Design: Neuroblastoma (NB) is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. Favorable NB genes (EPHB6, EFNB2, EFNB3, NTRK1, and CD44) are genes whose high-level expression predicts favorable NB outcome, and forced expression of these genes inhibits growth of unfavorable NB cells. In this study, we investigated whether favorable NB gene expression could be augmented in unfavorable NB cells by chemical compounds and whether an increased expression of these genes was associated with suppression of NB growth and metastasis.Results: We found that inhibitors of DNA methylation [5-aza-2-deoxycytidine (5AdC)], histone deacetylase (HDAC) [4-phenylbutyrate (4PB)], and proteasome (MG262) enhanced the expression of favorable NB genes in NB cell lines and inhibited the growth of these cells in vitro (P < 0.0005). The growth-inhibitory effects of 5AdC and 4PB in vitro were in part due to caspase-dependent cell death and inhibition of DNA synthesis. Administration of 5AdC and/or 4PB also suppressed growth of subcutaneous NB xenografts in nude mice (P < 0.001), which was accompanied by enhanced favorable NB gene expression and an increase in apoptosis. Moreover, 4PB suppressed bone marrow and liver metastases of NB cells in severe combined immunodeficient/Beige mice (P ‫؍‬ 0.007 and P ‫؍‬ 0.008, respectively). The growth-suppressive activity of HDAC inhibitors on NB was further confirmed by the efficacy of trichostatin A, a potent and specific HDAC inhibitor.Conclusions: Collectively, these observations further emphasize the link between the elevated favorable NB gene expression and a benign phenotype of NB.

EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups.