title: GSK3 inhibitors regulate MYCN mRNA levels and reduce neuroblastoma cell viability through multiple mechanisms, including p53 and Wnt signaling.
abstract: Neuroblastoma is an embryonal tumor accounting for approximately 15% of childhood cancer deaths. There exists a clinical need to identify novel therapeutic targets, particularly for treatment-resistant forms of neuroblastoma. Therefore, we investigated the role of the neuronal master regulator GSK3 in controlling neuroblastoma cell fate. We identified novel GSK3-mediated regulation of MYC (c-MYC and MYCN) mRNA levels, which may have implications for numerous MYC-driven cancers. In addition, we showed that certain GSK3 inhibitors induced large-scale cell death in neuroblastoma cells, primarily through activating apoptosis. mRNA-seq of GSK3 inhibitor-treated cells was performed and subsequent pathway analysis revealed that multiple signaling pathways contributed to the loss of neuroblastoma cell viability. The contribution of two of the signaling pathways highlighted by the mRNA-seq analysis was functionally validated. Inhibition of the p53 tumor suppressor partly rescued the cell death phenotype, whereas activation of canonical Wnt signaling contributed to the loss of viability, in a p53-independent manner. Two GSK3 inhibitors (BIO-acetoxime and LiCl) and one small-molecule Wnt agonist (Wnt Agonist 1) demonstrated therapeutic potential for neuroblastoma treatment. These inhibitors reduced the viability of numerous neuroblastoma cell lines, even those derived from high-risk MYCN-amplified metastatic tumors, for which effective therapeutics are currently lacking. Furthermore, although LiCl was lethal to neuroblastoma cells, it did not reduce the viability of differentiated neurons. Taken together our data suggest that these small molecules may hold potential as effective therapeutic agents for the treatment of neuroblastoma and other MYC-driven cancers.
authors.affiliation: Corresponding Author: David J. Duffy, University College Dublin, Belfield, Dublin 4, Ireland. david.duffy@ucd.ie.
authors.forename: David J
authors.initials: DJ
authors.lastname: Duffy
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Krstic
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarzl
authors.forename: Desmond G
authors.initials: DG
authors.lastname: Higgins
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010091:Oximes / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000187:drug effects*
chemical_list: C582966:6-bromoindirubin-3'-acetoxime; D007211:Indoles; C459801:MYCN protein, human; D008301:Maleimides; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D010091:Oximes; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C417521:SB 216763; D016159:Tumor Suppressor Protein p53; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride