title: Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells.
abstract: BACKGROUND
  The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear.
  
  
  METHODS
  The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity.
  
  
  RESULTS
  Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent.
  
  
  CONCLUSIONS
  RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.
authors.affiliation: Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Horio
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Sato
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Takeyama
authors.forename: Momen
authors.initials: M
authors.lastname: Elshazley
authors.forename: Ryo
authors.initials: R
authors.lastname: Yamashita
authors.forename: Tetsunari
authors.initials: T
authors.lastname: Hase
authors.forename: Kenya
authors.initials: K
authors.lastname: Yoshida
authors.forename: Noriyasu
authors.initials: N
authors.lastname: Usami
authors.forename: Kohei
authors.initials: K
authors.lastname: Yokoi
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Sekido
authors.forename: Masashi
authors.initials: M
authors.lastname: Kondo
authors.forename: Shinya
authors.initials: S
authors.lastname: Toyokuni
authors.forename: Adi F
authors.initials: AF
authors.lastname: Gazdar
authors.forename: John D
authors.initials: JD
authors.lastname: Minna
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hasegawa
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015536:Down-Regulation; D000071858:Epithelial Cell Adhesion Molecule; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008654:Mesothelioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010997:Pleural Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation; D014746:Vimentin / Q000378:metabolism; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D000951:Antigens, Neoplasm; D015820:Cadherins; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D018398:Homeodomain Proteins; D014157:Transcription Factors; D014746:Vimentin; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1
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