title: The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/βIII-Tubulin Axis.
abstract: MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintains normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma; however, the molecular mechanisms underlying miR-34a activity in osteosarcoma are not well-defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in osteosarcoma. Levels of miR-34a were attenuated in human osteosarcoma cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pull-down assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3'-untranslated region. Overexpression of miR-34a in osteosarcoma cells suppressed STMN1 expression and reduced cell growthin vitroRestoration of miR-34a led to microtubule destabilization and increased βIII-tubulin expression, with corresponding G1-G2phase cell-cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated βIII-tubulin expression in osteosarcoma cells, suggesting that miR-34a indirectly affects Sp1. Validating the coordinating role of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in osteosarcoma cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates βIII-tubulin, leading to disruption of the microtubule network and cell death.Implications:The miR-34a/STMN1/βIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy.Mol Cancer Res; 15(7); 953-64. ©2017 AACR.
authors.affiliation: Nemours Center for Cancer and Blood Disorders, Nemours/Alfred I. duPont Hospital for Children, Wilmington, Delaware.
authors.forename: Nancy S
authors.initials: NS
authors.lastname: Vetter
authors.affiliation: Nemours Center for Cancer and Blood Disorders, Nemours/Alfred I. duPont Hospital for Children, Wilmington, Delaware.
authors.forename: E A
authors.initials: EA
authors.lastname: Kolb
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Mills
authors.affiliation: Nemours Center for Cancer and Blood Disorders, Nemours/Alfred I. duPont Hospital for Children, Wilmington, Delaware. vsampson@nemours.org.
authors.forename: Valerie B
authors.initials: VB
authors.lastname: Sampson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D059447:Cell Cycle Checkpoints / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D003599:Cytoskeleton / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D008870:Microtubules / Q000235:genetics; D012516:Osteosarcoma / Q000235:genetics* / Q000473:pathology; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000235:genetics; D050777:Stathmin / Q000235:genetics*; D014404:Tubulin / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: C522425:MIRN34 microRNA, human; D035683:MicroRNAs; C493995:STMN1 protein, human; D016329:Sp1 Transcription Factor; C108209:SP1 protein, human; D050777:Stathmin; C498585:TUBB3 protein, human; D014404:Tubulin