Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4

Cell Cycle. 2014;13(24):3857-66. doi: 10.4161/15384101.2014.972875.

Abstract

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

Keywords: BE, Barrett's esophagus; CDX1, caudal type homeobox 1; dnMAML, dominant-negative Mastermind-like; DSC1, desmocollin 1; DSC3, desmocollin 3; EAC, esophageal adenocarcinoma; EGJ, esophago-gastric junction; ESC, embryonic stem cell; ESCC, esophageal squamous; Barrett's esophagus; KLF4; Notch signaling; metaplasia; transdifferentiation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Barrett Esophagus / metabolism
  • Barrett Esophagus / pathology
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / metabolism
  • Cell Culture Techniques
  • Cell Line
  • Cell Transdifferentiation
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Esophagus / cytology
  • Esophagus / pathology*
  • Gene Expression Regulation
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Intercellular Signaling Peptides and Proteins / chemistry
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Jagged-1 Protein
  • Keratins / metabolism
  • Kruppel-Like Transcription Factors / antagonists & inhibitors
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism*
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Metaplasia
  • Mucins / metabolism
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA Interference
  • Receptors, Notch / antagonists & inhibitors
  • Receptors, Notch / metabolism*
  • Serrate-Jagged Proteins
  • Signal Transduction
  • Tissue Array Analysis
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • CDX1 protein, human
  • Calcium-Binding Proteins
  • DNA-Binding Proteins
  • GKLF protein
  • Homeodomain Proteins
  • Intercellular Signaling Peptides and Proteins
  • JAG1 protein, human
  • Jagged-1 Protein
  • Kruppel-Like Transcription Factors
  • MAML1 protein, human
  • MYC protein, human
  • Membrane Proteins
  • Mucins
  • Proto-Oncogene Proteins c-myc
  • Receptors, Notch
  • Serrate-Jagged Proteins
  • Transcription Factors
  • Keratins