title: Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.
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.
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Marianna
authors.initials: M
authors.lastname: Szemes
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Gabriella Cunha
authors.initials: GC
authors.lastname: Vieira
authors.affiliation: Department of Cellular Pathology, Southmead Hospital, Bristol, UK.
authors.forename: Zsombor
authors.initials: Z
authors.lastname: Melegh
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Sally
authors.initials: S
authors.lastname: Malik
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Heesom
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Von Wallwitz-Freitas
authors.affiliation: Colorectal Cancer Laboratory, School of Cellular & Molecular Medicine University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Greenhough
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Brown
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA.
authors.forename: Y George
authors.initials: YG
authors.lastname: Zheng
authors.affiliation: The Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Catchpoole
authors.affiliation: Cambridge Centre for Proteomics, Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Deery
authors.affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK. Electronic address: k.t.a.malik@bristol.ac.uk.
authors.forename: Karim
authors.initials: K
authors.lastname: Malik
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D006801:Humans; D008969:Molecular Sequence Data; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*
chemical_list: C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases
keywords: Arginine methylation; MYCN; Neuroblastoma; PRMT5