title: Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide.
abstract: BACKGROUND
  Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 μM).
  
  
  METHODS
  MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 μM. Clonogenic GSC surviving 500 μM TMZ (GSC-500 μM TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC.
  
  
  RESULTS
  The molecular signatures characterized an activation of protective stress responses in GSC-500 μM TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 μM TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 μM TMZ and GSC-parental to 35 μM TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p = 0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p = 0.0489).
  
  
  CONCLUSIONS
  These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. jtso24@ucla.edu.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Tso
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. yshuai1001@msn.cn.|Department of Neurosurgery, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China. yshuai1001@msn.cn.
authors.forename: Shuai
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. JimmyMenjivar@mednet.ucla.edu.
authors.forename: Jimmy C
authors.initials: JC
authors.lastname: Menjivar
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. mdky@xc4.so-net.ne.jp.|Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Higashiku, Fukuoka, Japan. mdky@xc4.so-net.ne.jp.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. ybzhang803@gmail.com.|Department of Orthopedics, Zhongshan Hospital, Xiamen University, Xiamen, China. ybzhang803@gmail.com.
authors.forename: Yibei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. ireneola@ucla.edu.
authors.forename: Irene
authors.initials: I
authors.lastname: Hong
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. ybui@g.ucla.edu.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Bui
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. astream@ucla.edu.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stream
authors.affiliation: Department of Radiation-Oncology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. WMcBride@mednet.ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. WMcBride@mednet.ucla.edu.
authors.forename: William H
authors.initials: WH
authors.lastname: McBride
authors.affiliation: Department of Neurosurgery, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. LLiau@mednet.ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. LLiau@mednet.ucla.edu.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Liau
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. nelsongenomics@mac.com.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. nelsongenomics@mac.com.
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.affiliation: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. tcloughe@ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. tcloughe@ucla.edu.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cloughesy
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. wyong@mednet.ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. wyong@mednet.ucla.edu.
authors.forename: William H
authors.initials: WH
authors.lastname: Yong
authors.affiliation: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. AlbertLai@mednet.ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. AlbertLai@mednet.ucla.edu.
authors.forename: Albert
authors.initials: A
authors.lastname: Lai
authors.affiliation: Department of Surgery/Surgical Oncology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA. ctso@mednet.ucla.edu.|Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, USA. ctso@mednet.ucla.edu.
authors.forename: Cho-Lea
authors.initials: CL
authors.lastname: Tso
mesh_terms: D018906:Antineoplastic Agents, Alkylating; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005909:Glioblastoma / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D014411:Neoplastic Stem Cells / Q000201:enzymology* / Q000378:metabolism; D000077204:Temozolomide; D014407:Tumor Cells, Cultured
chemical_list: D018906:Antineoplastic Agents, Alkylating; C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D006147:Guanine; D003606:Dacarbazine; C008449:O-(6)-methylguanine; D015254:DNA Modification Methylases; D000077204:Temozolomide