Project description:The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free- and overall survival of NB patients indicated that the highexpression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g. neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also that EZH2-related NTRK1 transcriptional regulation may be the key pathway for NB cell differentiation.

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.

Dataset Information

Targeting NTRK1 Enhances Immune Checkpoint Inhibitor Efficacy in NTRK1 Wild-type Non-Small Cell Lung Cancer

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets