title: Wnt/β-Catenin Signaling Activation beyond Robust Nuclear β-Catenin Accumulation in Nondysplastic Barrett's Esophagus: Regulation via Dickkopf-1.
abstract: BACKGROUND
  Wnt/β-catenin signaling activation has been reported only during the late steps of Barrett's esophagus (BE) neoplastic progression, but not in BE metaplasia, based on the absence of nuclear β-catenin. However, β-catenin transcriptional activity has been recorded in absence of robust nuclear accumulation. Thus, we aimed to investigate the Wnt/β-catenin signaling in nondysplastic BE.
  
  
  METHODS
  Esophageal tissues from healthy and BE patients without dysplasia were analyzed for Wnt target gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Esophageal squamous (EPC1-& EPC2-hTERT), BE metaplastic (CP-A), and adenocarcinoma (OE33) cell lines were characterized for Wnt activation by qRT-PCR, Western blot, and luciferase assay. Wnt activity regulation was examined by using recombinant Wnt3a and Dickkopf-1 (Dkk1) as well as Dkk1 short interfering RNA.
  
  
  RESULTS
  Wnt target genes (AXIN2, c-MYC, Cyclin D1, Dkk1) and Wnt3a were significantly upregulated in nondysplastic BE compared with squamous mucosa. Elevated levels of dephosphorylated β-catenin were detected in nondysplastic BE. Nuclear active β-catenin and TOPflash activity were increased in CP-A and OE33 cells compared with squamous cells. Wnt3a-mediated β-catenin signaling activation was abolished by Dkk1 in CP-A cells. TOPFlash activity was elevated following Dkk1 silencing in CP-A but not in OE33 cells. Dysplastic and esophageal adenocarcinoma tissues demonstrated further Dkk1 and AXIN2 overexpression.
  
  
  CONCLUSIONS
  Despite the absence of robust nuclear accumulation, β-catenin is transcriptionally active in nondysplastic BE. Dkk1 overexpression regulates β-catenin signaling in BE metaplastic but not in adenocarcinoma cells, suggesting that early perturbation of Dkk1-mediated signaling suppression may contribute to BE malignant transformation.
authors: Division of Gastroenterology and Hepatology of Wisconsin, Milwaukee, USA; Department of Visceral, Transplant, Thoracic and Vascular Surgery, University Hospital, Leipzig, Germany.
authors: Orestis
authors: O
authors: Lyros
authors: Department of Surgery of Medical College of Wisconsin, Milwaukee, USA.
authors: Parvaneh
authors: P
authors: Rafiee
authors: Department of Surgery of Medical College of Wisconsin, Milwaukee, USA.
authors: Linghui
authors: L
authors: Nie
authors: Department of Surgery of Medical College of Wisconsin, Milwaukee, USA.
authors: Rituparna
authors: R
authors: Medda
authors: Division of Gastroenterology and Hepatology of Wisconsin, Milwaukee, USA.
authors: Nebojsa
authors: N
authors: Jovanovic
authors: Department of Surgery of Medical College of Wisconsin, Milwaukee, USA.
authors: Mary F
authors: MF
authors: Otterson
authors: Department of Pathology of Medical College of Wisconsin, Milwaukee, USA.
authors: Behnaz
authors: B
authors: Behmaram
authors: Department of Visceral, Transplant, Thoracic and Vascular Surgery, University Hospital, Leipzig, Germany.
authors: Ines
authors: I
authors: Gockel
authors: Department of Pathology of Medical College of Wisconsin, Milwaukee, USA.
authors: Alexander
authors: A
authors: Mackinnon
authors: Division of Gastroenterology and Hepatology of Wisconsin, Milwaukee, USA. Electronic address: rshaker@mcw.edu.
authors: Reza
authors: R
authors: Shaker
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology*; D060466:Axin Protein / Q000096:biosynthesis; D001471:Barrett Esophagus / Q000473:pathology*; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000096:biosynthesis; D004789:Enzyme Activation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering; D060449:Wnt Signaling Pathway / Q000502:physiology*; D060509:Wnt3A Protein / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: C491761:AXIN2 protein, human; D060466:Axin Protein; C530104:CCND1 protein, human; C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D051176:beta Catenin; D019938:Cyclin D1