Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal di erentiation in vitro and might play an important role in the di erentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their e ects on di erentiation in NB, we stably expressed wild-type TrkA and ®ve di erent 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 di erentiation, activation of mitogenactivated 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 NGFtreatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced di erentiation 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 di erentiation of NB cells. Our results also show that the signaling e ector 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 di erences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB. Oncogene (2000) 19, 2043 ± 2051.Keywords: neuroblastoma; tyrosine kinase receptors; neurotrophins; signal transduction; di erentiation IntroductionNeuroblastoma is one of the most common pediatric neoplasms and is derived from the neural crest. Neurotrophic factors and their tyrosine kinase receptors (Trks) play an important role in the pathogenesis, biology and clinical behavior of NB (Brodeur, 1993). Observations from several independent studies suggest that high expression of the neurotrophin receptor TrkA is present in NB with favorable biological features and highly correlated with patient survival (Nakagawara et al., 1993;Kogner et al., 1993). Activation of TrkA by its ligand NGF leads to survival and di erentiation of TrkA expressing cells in vitro (Klein et al., 1991) and might play an important role in the regression or di erentiation of NB in vivo.Signal transduction pathways used by TrkA have been studied mainly in the PC12 rat pheochromocytoma cell line. Following NGF binding, TrkA receptors rapidly become phosphorylated on tyrosine residues, and their tyrosine kinase domain is activated (Klein et al., 1991). Phosphorylated tyrosine residues in the TrkA cytoplasmic domain serve as anchors for binding downstream signaling molecules (Schlessinger and Ullrich, 1992). Proteins known to become phosphorylated and activated in response to NGF include phosph...

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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, Trk-C 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.

In comparison to parental SY5Y cells, mRNA and protein levels of angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium (CM) of parental SY5Y and SY5Y-TrkB cells induced endothelial cell proliferation, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model, and was associated with reduced angiogenic factor expression and less vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay.