A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors

Cancer Res. 2011 Nov 1;71(21):6836-47. doi: 10.1158/0008-5472.CAN-11-0846. Epub 2011 Sep 2.

Abstract

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.

Publication types

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

MeSH terms

  • Animals
  • Antigens, CD
  • Cadherins / biosynthesis
  • Cadherins / genetics
  • Carcinoma, Squamous Cell / genetics
  • Carcinoma, Squamous Cell / pathology*
  • Cell Division
  • Cell Line, Tumor
  • DNA-Binding Proteins / physiology
  • Epithelial-Mesenchymal Transition / genetics
  • Epithelial-Mesenchymal Transition / physiology*
  • Esophageal Neoplasms / genetics
  • Esophageal Neoplasms / pathology*
  • Gene Expression Regulation, Neoplastic
  • Homeodomain Proteins / physiology*
  • Humans
  • Mice
  • Mice, Nude
  • MicroRNAs / biosynthesis
  • MicroRNAs / genetics
  • Neoplasm Proteins / biosynthesis
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / physiology*
  • Neoplasm Transplantation
  • RNA Interference
  • RNA, Neoplasm / biosynthesis
  • RNA, Neoplasm / genetics
  • RNA, Small Interfering / pharmacology
  • Receptor, Notch3
  • Receptors, Notch / physiology*
  • Recombinant Fusion Proteins / physiology
  • Repressor Proteins / physiology*
  • Signal Transduction
  • Transcription Factors / physiology*
  • Tumor Stem Cell Assay
  • Zinc Finger E-box Binding Homeobox 2
  • Zinc Finger E-box-Binding Homeobox 1

Substances

  • Antigens, CD
  • CDH1 protein, human
  • Cadherins
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MAML1 protein, human
  • MIRN200 microRNA, human
  • MIRN205 microRNA, human
  • MicroRNAs
  • NOTCH3 protein, human
  • Neoplasm Proteins
  • RNA, Neoplasm
  • RNA, Small Interfering
  • Receptor, Notch3
  • Receptors, Notch
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Transcription Factors
  • ZEB1 protein, human
  • ZEB2 protein, human
  • Zinc Finger E-box Binding Homeobox 2
  • Zinc Finger E-box-Binding Homeobox 1

Grants and funding