title: Expression of insulin-like growth factor-binding protein 5 in neuroblastoma cells is regulated at the transcriptional level by c-Myb and B-Myb via direct and indirect mechanisms.
abstract: Neuroblastoma (NB), a malignant childhood tumor deriving from the embryonic neural crest, is sensitive to the growth-stimulating effects of insulin-like growth factors (IGFs). Aggressive cases of this disease often acquire autocrine loops of IGF production, but the mechanisms through which the different components of the IGF axis are regulated in tumor cells remain unclear. Upon conditional expression of c-Myb in a NB cell line, we detected up-regulation of IGF1, IGF1 receptor, and insulin-like growth factor-binding protein 5 (IGFBP-5) expression. Analysis of the IGFBP-5 promoter revealed two potential Myb binding sites at position -59 to -54 (M1) and -429 to -424 (M2) from the transcription start site; both sites were bound by c-Myb and B-Myb in vitro and in vivo. Reporter assays carried out using the proximal region of the human IGFBP-5 promoter demonstrated that c-Myb and B-Myb enhanced transcription. However, site-directed mutagenesis and deletion of the Myb binding sites coupled with reporter assays revealed that M2 but not M1 was important for Myb-dependent transactivation of the IGFBP-5 promoter. The double mutant M1/M2 was still transactivated by c-Myb, suggesting the existence of Myb binding-independent mechanisms of IGFBP-5 promoter regulation. A constitutively active AKT transactivated the IGFBP-5 promoter, whereas the phosphatidylinositol 3-kinase inhibitor LY294002 suppressed it. Moreover, the kinase dead dominant negative K179M AKT mutant was able to inhibit transcription from the M2 and M1/M2 IGFBP-5 mutant promoters. Deletion analysis of the IGFBP-5 promoter revealed that the AKT-responsive region lies between nucleotides -334 and -83. Together, these data suggest that the Myb binding-independent transactivation of the IGFBP-5 promoter was due to the activation of the phosphatidylinositol 3-kinase/AKT pathway likely mediated by IGF1 receptor-dependent signals. Finally, IGFBP-5 was able to modulate proliferation of NB cells in a manner dependent on its concentration and on the presence of IGFs.
authors.affiliation: Ente Nuove Tecnologie Energia Ambiente (ENEA), Section of Toxicology and Biomedical Sciences, Via Anguillarese 301, 00060 S. Maria di Galeria, Rome, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Tanno
authors.forename: Anna
authors.initials: A
authors.lastname: Negroni
authors.forename: Roberta
authors.initials: R
authors.lastname: Vitali
authors.forename: Maria Celeste
authors.initials: MC
authors.lastname: Pirozzoli
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cesi
authors.forename: Camillo
authors.initials: C
authors.lastname: Mancini
authors.forename: Bruno
authors.initials: B
authors.lastname: Calabretta
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Raschellà
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D020570:Genes, myb / Q000502:physiology*; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics*; D007335:Insulin-Like Growth Factor II / Q000502:physiology; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018975:Insulin-Like Growth Factor Binding Protein 5; C064352:MYBL2 protein, human; C499306:Mybl2 protein, mouse; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D007335:Insulin-Like Growth Factor II; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt