title: Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells.
abstract: BACKGROUND
  Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells.
  
  
  METHODS
  MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05).
  
  
  RESULTS
  Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity.
  
  
  CONCLUSIONS
  This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.
authors.forename: Honor J
authors.initials: HJ
authors.lastname: Hugo
authors.forename: Lloyd
authors.initials: L
authors.lastname: Pereira
authors.forename: Randy
authors.initials: R
authors.lastname: Suryadinata
authors.forename: Yvette
authors.initials: Y
authors.lastname: Drabsch
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gonda
authors.forename: N P A Devika
authors.initials: NP
authors.lastname: Gunasinghe
authors.forename: Cletus
authors.initials: C
authors.lastname: Pinto
authors.forename: Eliza T  L
authors.initials: ET
authors.lastname: Soo
authors.forename: Bryce J W
authors.initials: BJ
authors.lastname: van Denderen
authors.forename: Prue
authors.initials: P
authors.lastname: Hill
authors.forename: Robert G
authors.initials: RG
authors.lastname: Ramsay
authors.forename: Boris
authors.initials: B
authors.lastname: Sarcevic
authors.forename: Donald F
authors.initials: DF
authors.lastname: Newgreen
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D049109:Cell Proliferation / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D018398:Homeodomain Proteins; D020598:Proto-Oncogene Proteins c-myb; D034741:RNA, Small Interfering; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1