title: Targets of miR-200c mediate suppression of cell motility and anoikis resistance.
abstract: BACKGROUND
  miR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.
  
  
  METHODS
  Putative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.
  
  
  RESULTS
  We found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.
  
  
  CONCLUSIONS
  miR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.
authors.affiliation: Program in Cancer Biology, Department of Pathology, University of Colorado, Anschutz Medical Campus, Mail Stop 8104, PO Box 6511, Aurora, CO, USA.
authors.forename: Erin N
authors.initials: EN
authors.lastname: Howe
authors.forename: Dawn R
authors.initials: DR
authors.lastname: Cochrane
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Richer
mesh_terms: D023102:Anoikis; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D016889:Endometrial Neoplasms / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D005353:Fibronectins / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020813:Receptor, trkB / Q000235:genetics; D054411:Receptors, Leptin / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: C470196:ARHGAP32 protein, human; D015820:Cadherins; C067625:FN1 protein, human; D005353:Fibronectins; D020690:GTPase-Activating Proteins; D018398:Homeodomain Proteins; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D008840:Microfilament Proteins; D012333:RNA, Messenger; D054411:Receptors, Leptin; D012097:Repressor Proteins; D014157:Transcription Factors; C486307:ZEB1 protein, human; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; C071058:moesin; D020813:Receptor, trkB