NTRK1/TrkA Signaling in Neuroblastoma Cells Induces Nuclear Reorganization and Intra-Nuclear Aggregation of Lamin A/C.

Abstract

Simple SummaryNeuroblastoma (NB) accounts for 15% of all cancer-related deaths of children. While the amplification of the Myc-N proto-oncogene (MYCN) is a major driver of aggressive NB, the expression of the neurotrophin receptor, NTRK1/TrkA, has been shown to be associated with an excellent outcome. MYCN downregulates NTRK1 expression, but it is unknown if the molecular effects of NTRK1 signaling also affect MYCN-induced networks. The aim of this study was to decipher NTRK1 signaling using an unbiased proteome and phosphoproteome approach. To this end, we realized inducible ectopic NTRK1 expression in a NB cell line with MYCN amplification and analyzed the proteomic changes upon NTRK1 activation in a time-dependent manner. In line with the phenotypes observed, NTRK1 activation induced markers of neuronal differentiation and cell cycle arrest. Most prominently, NTRK1 upregulated the expression and phosphorylation of the nuclear lamina component Lamin A/C. Moreover, NTRK1 signaling also induced the aggregation of LMNA within nucleic foci, which accompanies differentiation in other cell types.(1) Background: Neuroblastomas (NBs) are the most common extracranial solid tumors of children. The amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. (2) Methods: Inducible NTRK1 expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1 activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. (3) Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1 targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed the upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. (4) Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. The phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.