title: The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis.
abstract: Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.
authors.affiliation: Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu and Fundació Sant Joan de Déu,  Barcelona, Spain.
authors.forename: Carla
authors.initials: C
authors.lastname: Casalà
authors.forename: Estel
authors.initials: E
authors.lastname: Gil-Guiñón
authors.forename: José Luis
authors.initials: JL
authors.lastname: Ordóñez
authors.forename: Solange
authors.initials: S
authors.lastname: Miguel-Queralt
authors.forename: Eva
authors.initials: E
authors.lastname: Rodríguez
authors.forename: Patricia
authors.initials: P
authors.lastname: Galván
authors.forename: Cinzia
authors.initials: C
authors.lastname: Lavarino
authors.forename: Francina
authors.initials: F
authors.lastname: Munell
authors.forename: Enrique
authors.initials: E
authors.lastname: de Alava
authors.forename: Jaume
authors.initials: J
authors.lastname: Mora
authors.forename: Carmen
authors.initials: C
authors.lastname: de Torres
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism / Q000473:pathology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D017404:In Situ Hybridization, Fluorescence; D007223:Infant; D051379:Mice; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009006:Monosomy; D000071447:N-Myc Proto-Oncogene Protein; D009367:Neoplasm Staging; D009447:Neuroblastoma / Q000235:genetics / Q000401:mortality / Q000473:pathology*; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D044169:Receptors, Calcium-Sensing / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: C095550:CASR protein, human; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3