Project description:Simple Summary: Neuroblastoma (NB) accounts for 15% of all cancer related deaths of children. While amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB, expression of the neurotrophin receptor, NTRK1/TrkA, has been shown to associate with an excellent outcome. MYCN down-regulates 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 phenotypes observed, NTRK1-activation induced markers of neuronal differentiation and cell cycle arrest. Most prominently, NTRK1 upregulated expression and phosphorylation of the nuclear lamina component Lamin A/C. Moreover, NTRK1 signaling also induced aggregation of LMNA within nucleic foci, which accompanies differentiation in other cell types. Abstract: Background: Neuroblastomas (NB) are the most common extracranial solid tumors of childhood. 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. 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. 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 upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. Phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.

Project description:Neuroblastoma (NB) is an embryonal tumor with various clinical presentations and behaviors. Several genomic alterations has been well-studied in NB, among which genomic amplification of MYCN oncogene, is a strong prognostic biomarker with worsens outcome. Long noncoding RNAs (lncRNAs), constitute major proportion of the cellular transcripts with no coding capacity. One of their function is to guide transcription factors to the target genes and facilitate gene expression. However, relative contribution of lncRNA and MYCN to the advanced NB has remained unclear. Herein, by applying a network-based integrative analysis on MYCN amplified and MYCN nonamplified lncRNA expression profile from both RNA-seq and microarray platform, we identified lncRNA, SNHG1 to be differentially expressed and strongly correlated with MYCN in MYCN-amplified NB. The expression of SNHG1 was validated by RT-qPCR in NB cell lines. Survival analysis revealed that higher expression of SNHG1 significantly associates with poor patient survival. Moreover, knockdown of MYCN in MYCN-amplified NB cell lines inhibited SNHG1 expression. Furthermore, to unravel the role of SNHG1 in NB, we extracted SNHG1-interacting proteins by RNA-protein pull down assay coupled with doi:10.6342/NTU201701980 ! ! VI liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 27 SNHG1-interacting proteins in common from three NB cell lines. However, only three SNHG1-interacting proteins, MATR3, YBX1 and HHRNPL have binding site detected by DeepBind motif analysis. Western blot confirms interaction of MATR3 with SNHG1. Additionally, we further validated the direct interaction between MATR3 and SNHG1 by RNA-immunoprecipation (IP). MATR3 is known to be involved in RNA transport and stabilization. Therefore, we proposed that MATR3 after interacting with SNHG1 might help in SNHG1 transcription and stabilization. In conclusion, our study unveils that SNHG1 could be a prognostic marker for high-risk NB and possibly stabilized by MATR3. Our results might provide future directions for the development of therapeutic strategies against high-risk NB.

Dataset Information

A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma

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A MYCN-independent mechanism mediating secretome reprogramming and metastasis in <i>MYCN</i>-amplified neuroblastoma.

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