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Title: NOTCH3 knockdown in non-transformed human esophageal cells      
dateReleased:
12-01-2011
description:
To determine the role of NOTCH3 in human esophageal epitheila homeostasis/squamous cell differentiation Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial to mesenchymal transition (EMT), senescence and cancer stem cell maintenance through mutual negative regulation of the microRNA (miR)-200 family members. However, little is known as to how ZEB expressing tumor cells may emerge during invasive growth. We find that canonical Notch signaling prevents expansion of a unique subset of cells expressing ZEBs through NOTCH3 (N3). In primary esophageal squamous cell carcinoma (ESCC), ZEB1 is induced in tumor cells displaying EMT-like dedifferentiation at the invasive front of tumor nests with reciprocal downregulation of the miR-200. ZEB expression was associated with the lack of cellular capability of undergoing squamous differentiation through dysfunction of N3, implicated at the onset of normal esophageal squamous differentiation. Dominant-negative Mastermind-like1 (DNMAML1), a genetic pan-notch inhibitor, prevented CSL-dependent transcription, resulting in suppression of N3 expression and squamous differentiation while enriching EMT competent cells with robust upregulation of ZEBs and downregulation of the miR-200. Such a cell population demonstrated enhanced anchorage independent growth as well as tumor formation in nude mice. RNA interference (RNAi) experiments documented the requirement of ZEBs in TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition in organotypic 3D culture, a form of human tissue engineering. Finally, RNAi experiments revealed N3 as a key factor limiting the expansion of the ZEB expressing cells, providing novel mechanistic insights into the role of Notch signaling in ESCC cell fate regulation and disease progression. NOTCH3 was knockdown stably in immortalized human esophageal keratinocytes EPC2-hTERTstably by lentivirus-mediated gene transfer with shRNA directed against NOTCH3 (Open BiosystemsV2LHS_229748). A scrambled shRNA (Open Biosystems RHS4346) served as acontrol. Cells were stimulated with 0.6 mM calcium chloride to induce squamous cell differentiation for 72 hrs (0.09 mM Calcium Chloride as a unstimulated control) as described in Gastroenterology. 2010 Dec;139(6):2113-23 by Ohashi et al.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-27424
refinement:
raw
alternateIdentifiers:
27424
keywords:
functional genomics
dateModified:
06-26-2012
availability:
available
types:
gene expression
name:
Homo sapiens
ID:
A-AFFY-44
name:
Affymetrix GeneChip Human Genome U133 Plus 2.0 [HG-U133_Plus_2]
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-27424/E-GEOD-27424.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-27424/E-GEOD-27424.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27424
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress

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