title: Osteopontin up-regulates critical epithelial-mesenchymal transition transcription factors to induce an aggressive breast cancer phenotype.
abstract: BACKGROUND
  Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPN's role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPN's role in Twist activation is examined through immunoprecipitation and Western blot.
  
  
  METHODS
  MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation.
  
  
  RESULTS
  Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN+APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression.
  
  
  CONCLUSIONS
  This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis.
authors.affiliation: Department of Surgery, Loyola University, Maywood, IL 60153, USA.
authors.forename: Neill Y
authors.initials: NY
authors.lastname: Li
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Weber
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Mi
authors.forename: Philip Y
authors.initials: PY
authors.lastname: Wai
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Cuevas
authors.forename: Paul C
authors.initials: PC
authors.lastname: Kuo
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D061986:MCF-7 Cells; D009361:Neoplasm Invasiveness; D053495:Osteopontin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010641:Phenotype; D063150:Polycomb Repressive Complex 1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D051879:Twist-Related Protein 1 / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C069077:BMI1 protein, human; D014408:Biomarkers, Tumor; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D053495:Osteopontin; D063150:Polycomb Repressive Complex 1