title: MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma.
abstract: BACKGROUND
  Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.
  
  
  RESULTS
  In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133(+)/CD15(+) tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1(+/-) p53(-/-)), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.
  
  
  CONCLUSIONS
  Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.
authors.affiliation: Centro di Ingegneria Genetica e Biotecnologia Avanzate, CEINGE, Naples, Italy.
authors.forename: Pasqualino
authors.initials: P
authors.lastname: de Antonellis
authors.forename: Chiara
authors.initials: C
authors.lastname: Medaglia
authors.forename: Emilio
authors.initials: E
authors.lastname: Cusanelli
authors.forename: Immacolata
authors.initials: I
authors.lastname: Andolfo
authors.forename: Lucia
authors.initials: L
authors.lastname: Liguori
authors.forename: Gennaro
authors.initials: G
authors.lastname: De Vita
authors.forename: Marianeve
authors.initials: M
authors.lastname: Carotenuto
authors.forename: Annamaria
authors.initials: A
authors.lastname: Bello
authors.forename: Fabio
authors.initials: F
authors.lastname: Formiggini
authors.forename: Aldo
authors.initials: A
authors.lastname: Galeone
authors.forename: Giuseppe
authors.initials: G
authors.lastname: De Rosa
authors.forename: Antonella
authors.initials: A
authors.lastname: Virgilio
authors.forename: Immacolata
authors.initials: I
authors.lastname: Scognamiglio
authors.forename: Manuela
authors.initials: M
authors.lastname: Sciro
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Basso
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Schulte
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cinalli
authors.forename: Achille
authors.initials: A
authors.lastname: Iolascon
authors.forename: Massimo
authors.initials: M
authors.lastname: Zollo
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis*; D017209:Apoptosis; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006023:Glycoproteins / Q000096:biosynthesis*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D016256:Lewis X Antigen / Q000096:biosynthesis*; D008527:Medulloblastoma / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D009474:Neurons / Q000166:cytology*; D010455:Peptides; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D002135:Calcium-Binding Proteins; C512941:DLK1 protein, human; C512925:Dlk1 protein, mouse; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D016256:Lewis X Antigen; C522425:MIRN34 microRNA, human; C551848:MIRN34a microRNA, mouse; D008565:Membrane Proteins; D035683:MicroRNAs; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53