title: The T-box transcription factor Brachyury regulates epithelial-mesenchymal transition in association with cancer stem-like cells in adenoid cystic carcinoma cells.
abstract: BACKGROUND
  The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) emphasize the need to better understand the biological factors associated with these outcomes. To analyze the mechanisms of AdCC metastasis, we established the green fluorescence protein (GFP)-transfected subline ACCS-GFP from the AdCC parental cell line and the metastatic ACCS-M GFP line from an in vivo metastasis model.
  
  
  METHODS
  Using these cell lines, we investigated the involvement of the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSCs) in AdCC metastasis by real-time RT-PCR for EMT related genes and stem cell markers. Characteristics of CSCs were also analyzed by sphere-forming ability and tumorigenicity. Short hairpin RNA (shRNA) silencing of target gene was also performed.
  
  
  RESULTS
  ACCS-M GFP demonstrated characteristics of EMT and additionally displayed sphere-forming ability and high expression of EMT-related genes (Snail, Twist1, Twist2, Slug, zinc finger E-box binding homeobox 1 and 2 [Zeb1 and Zeb2], glycogen synthase kinase 3 beta [Gsk3β and transforming growth factor beta 2 [Tgf-β2]), stem cell markers (Nodal, Lefty, Oct-4, Pax6, Rex1, and Nanog), and differentiation markers (sex determining region Y [Sox2], Brachyury, and alpha fetoprotein [Afp]). These observations suggest that ACCS-M GFP shows the characteristics of CSCs and CSCs may be involved in the EMT of AdCC. Surprisingly, shRNA silencing of the T-box transcription factor Brachyury (also a differentiation marker) resulted in downregulation of the EMT and stem cell markers. In addition, sphere-forming ability, EMT characteristics, and tumorigenicity were simultaneously lost. Brachyury expression in clinical samples of AdCC was extremely high and closely related to EMT. This finding suggests that regulation of EMT by Brachyury in clinical AdCC may parallel that observed in vitro in this study.
  
  
  CONCLUSIONS
  The use of a single cell line is a limitation of this study. However, parallel data from in vitro and clinical samples suggest the possibility that EMT is directly linked to CSCs and that Brachyury is a regulator of EMT and CSCs.
authors.affiliation: Division of Maxillofacial Diagnostic and Surgical Sciences, Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Shimoda
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Sugiura
authors.forename: Ikumi
authors.initials: I
authors.lastname: Imajyo
authors.forename: Kotaro
authors.initials: K
authors.lastname: Ishii
authors.forename: Satomi
authors.initials: S
authors.lastname: Chigita
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Seki
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Kanemitsu
authors.initials: K
authors.lastname: Shirasuna
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D003528:Carcinoma, Adenoid Cystic / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D005326:Fetal Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009362:Neoplasm Metastasis; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology*; D010641:Phenotype; D018874:Spheroids, Cellular; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism*; D000098908:Brachyury Protein
chemical_list: D014408:Biomarkers, Tumor; D005326:Fetal Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein