title: Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway.
abstract: BACKGROUND
  Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells.
  
  
  METHODS
  A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used.
  
  
  RESULTS
  Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine-phosphate-guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels.
  
  
  CONCLUSIONS
  Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling.
authors.affiliation: Corresponding Author: Chiara Riganti, MD, Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy. chiara.riganti@unito.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Riganti
authors.forename: Iris Chiara
authors.initials: IC
authors.lastname: Salaroglio
authors.forename: Valentina
authors.initials: V
authors.lastname: Caldera
authors.forename: Ivana
authors.initials: I
authors.lastname: Campia
authors.forename: Joanna
authors.initials: J
authors.lastname: Kopecka
authors.forename: Marta
authors.initials: M
authors.lastname: Mellai
authors.forename: Laura
authors.initials: L
authors.lastname: Annovazzi
authors.forename: Amalia
authors.initials: A
authors.lastname: Bosia
authors.forename: Dario
authors.initials: D
authors.lastname: Ghigo
authors.forename: Davide
authors.initials: D
authors.lastname: Schiffer
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology* / Q000627:therapeutic use; D001932:Brain Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D060509:Wnt3A Protein / Q000235:genetics* / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018906:Antineoplastic Agents, Alkylating; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D051176:beta Catenin; D003606:Dacarbazine; D004317:Doxorubicin; D038362:Glycogen Synthase Kinase 3; D000077204:Temozolomide
keywords: ABCB1/P-glycoprotein; Wnt3a; glioblastoma stem cells; temozolomide
references.citation: Stem Cells. 2009 Feb;27(2):280-9
references.pmid: 18988710
references.citation: Int J Cancer. 2009 Jul 1;125(1):15-22
references.pmid: 19291792
references.citation: J Neurosci. 1992 Nov;12(11):4565-74
references.pmid: 1432110
references.citation: Int J Radiat Oncol Biol Phys. 2009 Dec 1;75(5):1511-9
references.pmid: 19931733
references.citation: Neuropharmacology. 2010 May;58(6):903-10
references.pmid: 20045420
references.citation: Neuro Oncol. 2013 Mar;15(3):269-78
references.pmid: 23258843
references.citation: Cancer Res. 2005 Jan 15;65(2):516-25
references.pmid: 15695394
references.citation: Cancer Res. 2010 Sep 1;70(17):6870-9
references.pmid: 20736377
references.citation: DNA Repair (Amst). 2007 Aug 1;6(8):1079-99
references.pmid: 17485253
references.citation: Ann Oncol. 2009 Jan;20(1):175-81
references.pmid: 18687982
references.citation: Dev Dyn. 2007 Dec;236(12):3297-308
references.pmid: 18000980
references.citation: Cancer Cell. 2010 May 18;17(5):497-509
references.pmid: 20478531
references.citation: Mol Cancer. 2006 Dec 02;5:67
references.pmid: 17140455
references.citation: Stem Cells. 2007 Jul;25(7):1645-53
references.pmid: 17412894
references.citation: Clin Cancer Res. 2007 Jul 15;13(14):4042-5
references.pmid: 17634527
references.citation: Glia. 2006 Dec;54(8):850-60
references.pmid: 16981197
references.citation: Anticancer Drugs. 1999 Feb;10(2):179-85
references.pmid: 10211548
references.citation: Oncogene. 2008 May 1;27(20):2897-909
references.pmid: 18037961
references.citation: Mol Cell Biochem. 2006 Aug;288(1-2):17-28
references.pmid: 16583142
references.citation: Cancer. 2004 Mar 15;100(6):1199-207
references.pmid: 15022287
references.citation: Cell Death Differ. 2006 Jul;13(7):1238-41
references.pmid: 16456578
references.citation: Neurology. 2011 Mar 29;76(13):1126-34
references.pmid: 21346220
references.citation: Dev Cell. 2009 Jul;17(1):9-26
references.pmid: 19619488
references.citation: Cancer Cell. 2006 May;9(5):391-403
references.pmid: 16697959
references.citation: Cancer Cell. 2011 Oct 18;20(4):427-42
references.pmid: 22014570
references.citation: Mol Cancer Ther. 2007 Jun;6(6):1774-84
references.pmid: 17575106
references.citation: World Neurosurg. 2012 Feb;77(2):237-40
references.pmid: 22501017
references.citation: Cell Mol Life Sci. 2009 May;66(9):1580-94
references.pmid: 19288057
references.citation: Expert Opin Investig Drugs. 2009 Aug;18(8):1061-83
references.pmid: 19555299
references.citation: Physiol Rev. 2006 Oct;86(4):1179-236
references.pmid: 17015488
references.citation: Mol Pharmacol. 1996 May;49(5):832-41
references.pmid: 8622633
references.citation: Nat Rev Cancer. 2002 Jan;2(1):48-58
references.pmid: 11902585
references.citation: Neuro Oncol. 2009 Feb;11(1):69-79
references.pmid: 18772354
references.citation: Cell Cycle. 2009 Sep 15;8(18):2936-44
references.pmid: 19713741
references.citation: Trends Mol Med. 2011 Jun;17(6):301-312
references.pmid: 21411370
references.citation: J Oncol. 2011;2011:314962
references.pmid: 21869887
references.citation: Nat Rev Cancer. 2006 Jun;6(6):425-36
references.pmid: 16723989
references.citation: Cancer Invest. 2009 Nov;27(9):901-8
references.pmid: 19832037
references.citation: Oncogene. 2009 Jun 11;28(23):2245-56
references.pmid: 19421142
references.citation: Oncogene. 2002 Jan 31;21(6):878-89
references.pmid: 11840333
references.citation: Neuropathol Appl Neurobiol. 2009 Aug;35(4):380-93
references.pmid: 19508445
references.citation: J Neurooncol. 2008 Nov;90(2):157-70
references.pmid: 18612800
references.citation: Neuron. 2008 Jun 26;58(6):832-46
references.pmid: 18579075
references.citation: Mol Cancer. 2011 Oct 11;10:128
references.pmid: 21988793
references.citation: Cancer Res. 2004 Oct 1;64(19):7011-21
references.pmid: 15466194
references.citation: Cell Mol Neurobiol. 2009 Sep;29(6-7):845-58
references.pmid: 19288188