Naohiko Ikegaki

Resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumor cells correlates with a loss of caspase-8 expression.

TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in adult malignant glioma and various other human solid tumor models but not in normal tissues. To characterize the TRAIL death pathway in childhood primitive neuroectodermal brain tumor (PNET), 8 human PNET cell lines were tested for TRAIL-induced apoptosis. TRAIL-sensitivity of the PNET cell lines was correlated with mRNA expression levels of TRAIL, its agonistic (TRAIL-R1, TRAIL-R2) and antagonistic (TRAIL-R3, TRAIL-R4) receptors, cellular FLICE-like inhibitory protein (cFLIP), caspase-3 and caspase-8. Three of 8 PNET cell lines tested were susceptible to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cFLIP. However, all TRAIL-sensitive PNET cell lines expressed caspase-8 mRNA and protein, while none of the five TRAIL-resistant PNET cell lines expressed caspase-8 protein. Treatment with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored mRNA expression of caspase-8 and TRAIL-sensitivity in formerly TRAIL-resistant PNET cells, suggesting that gene methylation inhibits caspase-8 transcription in these cells. We conclude, that loss of caspase-8 mRNA is an important mechanism of TRAIL-resistance in PNET cells. Treatment with recombinant soluble TRAIL, possibly in combination with methyltransferase inhibitors, represents a promising therapeutic approach for PNET that deserves further investigation.

Expression of TrkA, TrkB and TrkC in human neuroblastomas.

There is considerable interest in the role of the TRK family of neurotrophin receptors in regulating the survival, growth and differentiation of normal and neoplastic nerve cells. Indeed, there is increasing evidence that TRK genes play an important role in the biology and clinical behavior of neuroblastomas, tumors of the peripheral nervous system. Evidence from several independent studies suggests that high expression of TrkA is an indicator of favorable outcome, and there is an inverse correlation between TrkA expression and N-myc amplification. In addition, some primary neuroblastomas differentiate in vitro in the presence of NGF but die in its absence. We have evidence that coexpression of full-length TrkB and BDNF is associated with N-myc amplification and may represent an autocrine survival pathway. Conversely, truncated TrkB is expressed predominantly in differentiated tumors. Finally, TrkC is expressed in favorable neuroblastomas, essentially all of which also express TrkA. In summary, the study of neurotrophin receptor expression and function in neuroblastomas may provide important insights into the role that these pathways play in the pathogenesis and clinical behavior of this tumor. Ultimately, these pathways may provide attractive targets for the development of therapy aimed at inducing differentiation or programmed cell death in these tumors.

Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.

The MYCN Enigma: Significance of MYCN Expression in Neuroblastoma

MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified.

Cloning and chromosomal localization of the human TRK-B tyrosine kinase receptor gene (NTRK2)

There is increasing evidence that neutrophins and their receptors play an important role in regulating development of both the central and the peripheral nervous systems. Human TRK-A (NTRK1) and TRK-C (NTRK3) have been cloned and sequenced, but only a truncated form of human TRK-B has been published. Therefore, we isolated complementary DNAs spanning the entire coding region of both human full-length and truncated forms of TRK-B from human brain cDNA libraries. Human full-length TRK-B codes for a protein of 822 amino acid residues. The putative mature peptide sequence is 49% homologous to human TRK-A and 55% to full-length human TRK-C, with 40% amino acid identify among TRK-A, -B, and -C. Nine of 13 cysteine residues, 4 of 12N-glycosylation sites in the extracellular domain, and 10 of 13 tyrosine residues in the intracellular domain are conserved among human TRK-A, -B, and -C. There is a cluster of 10 serine residues in the juxtamembrane region of TRK-B that is absent in TRK-A. Two major sizes of TRK-B transcripts were expressed in human brain. Northern blot analysis using probes specific for the extracellular or the tyrosine kinase domain revealed that the 9.5-kb band encodes the full-length TRK-B mRNA and the 8.0-kb band encodes the truncated form of TRK-B mRNA. By fluorescence in situ hybridization and somatic cell hybrid mapping, the human TRK-B gene was localized to chromosome 9q22.1.

EXPRESSION OF N-METHYL-D-ASPARTATE (NMDA) AND NON-NMDA GLUTAMATE RECEPTOR GENES IN NEUROBLASTOMA, MEDULLOBLASTOMA, AND OTHER CELL LINES

We evaluated expression of N‐methyl‐D‐aspartate (NMDA) and non‐NMDA glutamate receptor (GluR) genes by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and Southern blotting in nine established cell lines: rat CG‐4 (oligodendroglial lineage) and RINm5F insulinoma cells; human CHP134, SMS‐KCNR, SKNSH, and Nb69 neuroblastoma cells; and human D384Med, D425Med, and D458Med medulloblastoma cells. CG‐4 expressed mRNAs encoding GluR2–7, KA‐1, and KA‐2 non‐NMDA GluR (Yoshioka et al.: J Neurochem 64:2442–2448, 1995) and NR1 (NMDAR1) and NR2D NMDA GluR. After differentiation to oligodendrocyte‐like cells, CG‐4 also expressed NR2B mRNA. Rat insulinoma cells expressed GluR5, and KA‐2 non‐NMDA and NR1 and NR2D NMDA GluR mRNAs. The four human neuroblastoma lines all expressed mRNAs encoding GluR2–4, 6, 7 and KA‐1 non‐NMDA and NR1 NMDA GluR, and the three human medulloblastoma cell lines all expressed mRNAs encoding GluR1, 6 and KA‐1, but none of the NMDA GluRs. Whereas CG‐4 is susceptible to kainate excitotoxicity, treatment of insulinoma, neuroblastoma, and medulloblastoma lines with L‐glutamate, kainate, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), or NMDA failed to cause cell damage or to augment 45Ca2+ influx. Thus, despite expressing a variety of non‐NMDA and NMDA GluR genes, the human neuroblastoma and medulloblastoma and rat insulinoma lines failed to assemble Ca2+‐permeable NMDA or non‐NMDA GluR channels. This failure confers protection against excitotoxicity and may contribute to progression of tumors of these types.

Favorable Neuroblastoma Genes and Molecular Therapeutics of Neuroblastoma

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.

Effect of CEP‐751 (KT‐6587) on neuroblastoma xenografts expressing TrkB

BACKGROUND The compound CEP-751 (KT-6587), a potent and selective inhibitor of the Trk family of tyrosine kinases, has been shown to inhibit the growth of human neuroblastoma (NB) xenografts in nude mice [1]. PROCEDURE To address its mechanism of action, we studied SY5Y, a human NB cell line with no detectable Trk expression, and two subclones transfected with TrkB. The transfected clones, SY5Y (G8) and SY5Y (G12), expressed moderate and high levels, respectively, of TrkB mRNA and protein. These TrkB-expressing subclones and the parental line were then grown as xenografts in nude mice, and CEP-751 was used to inhibit TrkB tyrosine kinase activity in these xenografts. Animals were treated twice a day with CEP-751 (21 mg/kg), or with the carrier vehicle as a control. TrkB expression in the resultant tumors was examined by quantitative RT-PCR. The effect of CEP-751 on TrkB activation by BDNF was examined in G12 cells in culture by immunoprecipitation with antipan Trk antiserum, followed by Western blot analysis using antiphosphotyrosine antibodies. To determine if CEP-751 was causing apoptosis, the TUNEL assay was used. RESULTS CEP-751 had little effect on the growth of SY5Y tumors, but did slow the growth rate of the C8 and G12 tumors. The daily growth rate of the treated tumors was 0.16, 0.13, and 0.10 cm3, respectively, for the SY5Y, G8, and G12 tumors. RT PCR analysis confirmed the expression of TrkB in G8 and G12, but not in SY5Y tumors. Activation of TrkB by BDNF in G12 cells was inhibited by CEP-751 in a dose dependent fashion. The treated tumors showed marked evidence of apoptosis. CONCLUSIONS These data suggest that the effect of CEP-751 is due, at least in part, to its inhibition of TrkB kinase, and that CEP-751 may become a useful therapeutic tool for the treatment of aggressive neuroblastomas, which often express TrkB.

Expression and function of trk-C in favourable human neuroblastomas

Human neuroblastomas express the neurotrophin receptors trk-A and trk-B. Favourable outcome is associated with expression of trk-A, while unfavourable, MYCN amplified tumours express trk-B. In this study we examined the expression of trk-C in primary neuroblastoma tumour-derived cell lines. We found by Northern blot analysis that trk-C mRNA is expressed in 14 of 55 (25%) primary tumours. Trk-C was expressed in significantly more lower stage tumours (stage 1, 2, 4S) than higher stage tumours (stage 3, 4, P < 0.04). The expression of trk-C was correlated positively with survival and negatively correlated with MYCN amplification. We also studied the function of trk-C in transfected cell lines and found that NT-3 promotes both cell survival and differentiation. Our results suggest that trk-C is involved in the biology of favourable neuroblastomas.

Modulation of N-myc expression alters the invasiveness of neuroblastoma.

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.