Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Ji Hyun Park, Marianna Szemes, Gabriella Cunha C Vieira, Zsombor B Melegh, Sally Malik, Kate J Heesom, Laura Von Wallwitz-Freitas, Alexander Greenhough, Keith W Brown, Y George Zheng, Daniel Catchpoole, Michael J Deery, Karim T A Malik

Research output: Contribution to journalArticle (Academic Journal)peer-review

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Abstract

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

Original languageEnglish
Pages (from-to)617-27
Number of pages11
JournalMolecular Oncology
Volume9
Issue number3
Early online date15 Nov 2014
DOIs
Publication statusPublished - 1 Mar 2015

Keywords

  • Amino Acid Sequence
  • Apoptosis
  • Brain Neoplasms
  • Cell Cycle
  • Cell Line, Tumor
  • Gene Knockdown Techniques
  • Humans
  • Molecular Sequence Data
  • Neuroblastoma
  • Nuclear Proteins
  • Oncogene Proteins
  • Protein Binding
  • Protein Stability
  • Protein-Arginine N-Methyltransferases

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