title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200.
abstract: BACKGROUND
  Glioblastoma multiforme is the most common form of primary brain tumor, often characterized by poor survival. Glioblastoma initiating cells (GICs) regulate self-renewal, differentiation, and tumor initiation properties and are involved in tumor growth, recurrence, and resistance to conventional treatments. The sonic hedgehog (SHH) signaling pathway is essential for normal development and embryonic morphogenesis. The objectives of this study were to examine the molecular mechanisms by which GIC characteristics are regulated by NPV-LDE-225 (Smoothened inhibitor; (2,2'-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine).
  
  
  METHODS
  Cell viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively.
  
  
  CONCLUSIONS
  NPV-LDE-225 inhibited cell viability, neurosphere formation, and Gli transcriptional activity and induced apoptosis by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. NPV-LDE-225 increased the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, TRAIL-R2/DR5, and Fas and decreased the expression of platelet derived growth factor receptor-α and Bcl2, and these effects were abrogated by Gli1 plus Gli2 short hairpin RNAs. NPV-LDE-225 enhanced the therapeutic potential of FasL and TRAIL by upregulating Fas and DR4/5, respectively. Interestingly, NPV-LDE-225 induced expression of programmed cell death 4 and apoptosis and inhibited cell viability by suppressing micro RNA (miR)-21. Furthermore, NPV-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. The inhibition of Bmi1 by NPV-LDE-225 was regulated by induction of miR-128. Finally, NPV-LDE-225 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1 through modulating the miR-200 family. Our data highlight the importance of the SHH pathway for self-renewal and early metastasis of GICs.
authors.affiliation: Department of Pathology and Laboratory Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
authors.forename: Junsheng
authors.initials: J
authors.lastname: Fu
authors.forename: Mariana
authors.initials: M
authors.lastname: Rodova
authors.forename: Rajesh
authors.initials: R
authors.lastname: Nanta
authors.forename: Daniel
authors.initials: D
authors.lastname: Meeker
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Van Veldhuizen
authors.forename: Rakesh K
authors.initials: RK
authors.lastname: Srivastava
authors.forename: Sharmila
authors.initials: S
authors.lastname: Shankar
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001713:Biphenyl Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D024202:Electrophoretic Mobility Shift Assay; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D035683:MicroRNAs / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D018874:Spheroids, Cellular / Q000187:drug effects; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000071676:Zinc Finger Protein GLI1
chemical_list: C069077:BMI1 protein, human; D001713:Biphenyl Compounds; D015820:Cadherins; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; C545189:MIRN128 microRNA, human; C533896:MIRN200 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D011725:Pyridines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; C561435:sonidegib; D063150:Polycomb Repressive Complex 1; D053148:Caspase 3
keywords: Gli transcription factors; NPV-LDE-225; brain cancer stem cells; cell survival; glioblastoma initiating cells; sonic hedgehog signaling