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

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

Abstract

© 2014 The Authors. 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.

• Journal Article Type: Article
• Acceptance Date: Oct 30, 2014
• Online Publication Date: Nov 15, 2014
• Publication Date: Jan 1, 2015
• Deposit Date: Feb 12, 2019
• Publicly Available Date: Feb 14, 2019
• Journal: Molecular Oncology
• Print ISSN: 1574-7891
• Electronic ISSN: 1878-0261
• Publisher: Wiley Open Access
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Issue: 3
• Pages: 617-627
• DOI: https://doi.org/10.1016/j.molonc.2014.10.015
• Public URL: https://uwe-repository.worktribe.com/output/838105
• Publisher URL: https://doi.org/10.1016/j.molonc.2014.10.015
• Contract Date: Feb 12, 2019