title: Proliferation and Survival of Embryonic Sympathetic Neuroblasts by MYCN and Activated ALK Signaling.
abstract: Neuroblastoma (NB) is a childhood tumor that arises from the sympathoadrenal lineage. MYCN amplification is the most reliable marker for poor prognosis and MYCN overexpression in embryonic mouse sympathetic ganglia results in NB-like tumors. MYCN cooperates with mutational activation of anaplastic lymphoma kinase (ALK), which promotes progression to NB, but the role of MYCN and ALK in tumorigenesis is still poorly understood. Here, we use chick sympathetic neuroblasts to examine the normal function of MYCN and MYC in the control of neuroblast proliferation, as well as effects of overexpression of MYCN, MYC, and activated ALK, alone and in combination. We demonstrate that MYC is more strongly expressed than MYCN during neurogenesis and is important for in vitro neuroblast proliferation. MYC and MYCN overexpression elicits increased proliferation but does not sustain neuroblast survival. Unexpectedly, long-term expression of activated ALKF1174Lleads to cell-cycle arrest and promotes differentiation and survival of postmitotic neurons. ALKF1174Linduces NEFM, RET, and VACHT and results in decreased expression of proapototic (BMF, BIM), adrenergic (TH), and cell-cycle genes (e.g., CDC25A, CDK1). In contrast, neuroblast proliferation is maintained when MYCN and ALKF1174Lare coexpressed. Proliferating MYCN/ALKF1174Lneuroblasts display a differentiated phenotype but differ from ALK-expressing neurons by the upregulation of SKP2, CCNA2, E2F8, and DKC1 Inhibition of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets the CDK inhibitor p27 for degradation, reduces neuroblast proliferation, implicating SKP2 in the maintained proliferation of MYCN/ALKF1174Lneuroblasts. Together, our results characterize MYCN/ALK cooperation leading to neuroblast proliferation and survival that may represent initial steps toward NB development.
  
  
  
  MYCN overexpression combined with activated anaplastic lymphoma kinase (ALK) is sufficient to induce neuroblastoma (NB) in mouse sympathoadrenal cells. To address cellular and molecular effects elicited by MYCN/ALK cooperation, we used cultures of chick sympathetic neuroblasts. We demonstrate that MYCN increases proliferation but not survival, whereas long-term expression of ALKF1174Lelicits cell-cycle exit, differentiation, and survival of postmitotic neurons. Combined MYCN/ALKF1174Lexpression allows long-term proliferation and survival of neuroblasts with differentiated characteristics. In the presence of ALKF1174Lsignaling, MYCN induces the expression of the ubiquitin ligase SKP2 (S-phase kinase-associated protein 2), which targets p27 for degradation and is also upregulated in high-risk NB. SKP2 inhibition supports a function for SKP2 in the maintained neuroblast proliferation downstream of MYCN/ALK, which may represent an early step toward tumorigenesis.
authors.affiliation: Max-Planck-Institute for Brain Research, Developmental Neurobiology, 60438 Frankfurt/Main, Germany, Institute of Clinical Neuroanatomy, Goethe University Frankfurt/Main, 60590 Frankfurt/Main, Germany, Ernst-Strüngmann-Institute, 60528 Frankfurt/Main, Germany, and.
authors.forename: Marco
authors.initials: M
authors.lastname: Kramer
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Diogo
authors.identifier: 0000-0003-4400-8752
authors.initials: D
authors.lastname: Ribeiro
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-17177 Stockholm, Sweden.
authors.forename: Marie
authors.initials: M
authors.lastname: Arsenian-Henriksson
authors.affiliation: Institute of Clinical Neuroanatomy, Goethe University Frankfurt/Main, 60590 Frankfurt/Main, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Deller
authors.affiliation: Max-Planck-Institute for Brain Research, Developmental Neurobiology, 60438 Frankfurt/Main, Germany, Institute of Clinical Neuroanatomy, Goethe University Frankfurt/Main, 60590 Frankfurt/Main, Germany, Ernst-Strüngmann-Institute, 60528 Frankfurt/Main, Germany, and hermann.rohrer@brain.mpg.de rohrer@med.uni-frankfurt.de.
authors.forename: Hermann
authors.initials: H
authors.lastname: Rohrer
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D002642:Chick Embryo; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D000071447:N-Myc Proto-Oncogene Protein / Q000235:genetics*; D058953:Neural Stem Cells; D009447:Neuroblastoma / Q000473:pathology*; D009474:Neurons / Q000473:pathology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
chemical_list: C489427:MYC protein, human; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D016271:Proto-Oncogene Proteins c-myc; C000626173:ALK protein, human; C000626174:Alk protein, mouse; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: ALK; MYCN; neuroblast; neuroblastoma; neurogenesis; sympathetic