ABSTRACT
Neuroblastomas (NBs) have heterogeneous clinical behavior, from spontaneous regression or differentiation to relentless progression. Evidence from our laboratory and others suggests that neurotrophin receptors contribute to these disparate behaviors. Previously, the role of TRK receptors in NB pathogenesis was investigated. In the present study, the expression of RET and its coreceptors in a panel of NB cell lines was investigated and responses to cognate ligands GDNF, NRTN, and ARTN with GFRα13 coreceptor expression, respectively were found to be correlated. RET expression was high in NBLS, moderate in SY5Y, low/absent in NBEBc1 and NLF cells. All cell lines expressed at least one of GFRα coreceptors. In addition, NBLS, SY5Y, NBEBc1 and NLF cells showed different morphological changes in response to ligands. As expected, activation of RET/GFRα3 by ARTN resulted in RET phosphorylation. Interestingly, activation of TrkA by its cognate ligand NGF resulted in RET phosphorylation at Y905, Y1015, and Y1062, and this was inhibited in a dosedependent manner by the TRK inhibitor (CEP701). Conversely, RET activation by ARTN in NBLS cells led to phosphorylation of TrkA. This suggests a physical association between RET and TRK proteins, and crosstalk between these two receptor pathways. Finally, RET, GFR and TRK expression in primary tumors was investigated and a significant association between RET, its coreceptors and TRK expression was demonstrated. Thus, the present data support a complex model of interacting neurotrophin receptor pathways in the regulation of cell growth and differentiation in NBs.
Subject(s)
Neuroblastoma/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Receptor, trkA/metabolism , Up-Regulation , Carbazoles/pharmacology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Furans , Gene Expression Regulation, Neoplastic , Guanine Nucleotide Exchange Factors/pharmacology , Humans , Neuroblastoma/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction , ras Guanine Nucleotide Exchange FactorsABSTRACT
Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors.
