title: p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.
abstract: Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.
authors.affiliation: Division of Neuroblastoma Genomics.
authors.forename: Daniel
authors.initials: D
authors.lastname: Dreidax
authors.affiliation: Division of Neuroblastoma Genomics.
authors.forename: Steffen
authors.initials: S
authors.lastname: Bannert
authors.affiliation: Division of Neuroblastoma Genomics.
authors.forename: Kai-Oliver
authors.initials: KO
authors.lastname: Henrich
authors.affiliation: Division of Neuroblastoma Genomics.
authors.forename: Christina
authors.initials: C
authors.lastname: Schröder
authors.affiliation: Division of Pediatric Neurooncology.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bender
authors.affiliation: Division of Epigenomics and Cancer Risk Factors.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Oakes
authors.affiliation: Department of Pediatric Oncology and Hematology, University Children's Hospital, Essen, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Lindner
authors.affiliation: Department of Pediatric Oncology and Hematology, University Children's Hospital, Essen, Germany.
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Schulte
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin, Ireland.
authors.forename: David
authors.initials: D
authors.lastname: Duffy
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin, Ireland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarzl
authors.affiliation: Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Maral
authors.initials: M
authors.lastname: Saadati
authors.affiliation: Department of Pathology, University of Heidelberg, Heidelberg, Germany and.
authors.forename: Volker
authors.initials: V
authors.lastname: Ehemann
authors.affiliation: Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Benner
authors.affiliation: Division of Pediatric Neurooncology.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pfister
authors.affiliation: Department of Pediatric Oncology and Center for Molecular Medicine Cologne (CMMC), University Children's Hospital, Cologne, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Fischer
authors.affiliation: Division of Neuroblastoma Genomics f.westermann@dkfz.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Westermann
mesh_terms: D000293:Adolescent; D000328:Adult; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D000071447:N-Myc Proto-Oncogene Protein; D009367:Neoplasm Staging; D009423:Nervous System Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D016019:Survival Analysis; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; C493974:CDKN2D protein, human; D050764:Cyclin-Dependent Kinase Inhibitor p19; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D014212:Tretinoin; D000077209:Decitabine; D001374:Azacitidine