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

Abstract

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...

Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas

“…Interestingly, upregulation of IGF-II expression has previously been reported as a feature of TrkA, but not TrkB, activation in SY5Y cells (Kim et al, 1999). Despite this finding, high TrkA expression results in growth inhibition and decreased tumorigenesis of SY5Y cells (Eggert et al, 2000b. Here, recombinant IGFBP-3 inhibited proliferation of neuroblastoma cells in the absence and presence of IGF.…”

“…SH-SY5Y is a subclone of the human SK-N-SH neuroblastoma cell line described previously (Biedler et al, 1973). Full-length TrkA or TrkB cDNA were cloned into the retroviral expression vector pLNCX or pLNCX2 (Clontech, Palo Alto, CA, USA), respectively, as described (Eggert et al, 2000b). For confirmation of target gene expression in a second cell line, TrkA was also stably expressed in IMR5 human neuroblastoma cells.…”

“…Activation of TrkA or TrkB signaling pathways and the resulting phenotypes have been well studied in stable Trkexpressing SY5Y cells (Eggert et al, 2000b. Briefly described, activation of TrkA by NGF resulted in neuronal differentiation, growth inhibition, inhibition of angiogenesis and sensitization to drug-induced apoptosis (Eggert et al, 2000b. Whereas, activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) mainly resulted in enhanced proliferation and chemotherapy resistance of the cells (Ho et al, 2002).…”

“…The human SH-SY5Y neuroblastoma cell line, lacking endogenous expression of Trk receptors, served as a model system. Activation of TrkA or TrkB signaling pathways and the resulting phenotypes have been well studied in stable Trkexpressing SY5Y cells (Eggert et al, 2000b. Briefly described, activation of TrkA by NGF resulted in neuronal differentiation, growth inhibition, inhibition of angiogenesis and sensitization to drug-induced apoptosis (Eggert et al, 2000b.…”

Galanin and galanin receptor expression in neuroblastic tumours: correlation with their differentiation status

“…In the sympathetic ganglion, injury-induced galanin overexpression was triggered by deprivation of NGF (Zigmond and Sun, 1997). Similar mechanisms promoting tumoral cell survival or counteracting neuronal differentiation may be also involved in NB where NGF/TrkA interaction induced differentiation of tumour cells (Eggert et al, 2000).…”