The stratified squamous epithelium of the esophagus is regulated at an exquisite level. Exiting from the cell cycle, basal keratinocytes migrate towards the luminal surface. They undergo terminal differentiation in the suprabasal layer, expressing Involucrin (IVL) and cytokeratins such as CK13, and eventually desquamated into the lumen to complete epithelial renewal. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas (SCCs)(1
) and is a paradigm for the investigation for all types of SCCs. Squamous differentiation contributes to tumor heterogeneity in SCCs. An individual tumor often consists of both well-differentiated cell nests with central keratinization (i.e. keratin pearl) and poorly-differentiated cell nests.
The Notch pathway regulates cell fate and differentiation through cell-cell communication. Ligand binding triggers a series of enzymatic cleavages of one of four Notch receptor paralogs (NOTCH1-4), resulting in nuclear translocation of the intracellular domain of Notch (ICN). ICN forms a transcriptional activation complex containing a common transcription factor CSL [C
ag-1] and the coactivator Mastermind-like (MAML)(2
). Notch target genes include the HES/HEY family of transcription factors. CSL-dependent canonical Notch signaling regulates squamous differentiation and epidermal barrier functions (3
). Loss of Notch receptors or γ-secretase, a key Notch processing enzyme, or inhibition of the CSL-dependent activity by dominant negative MAML1 (DNMAML1) impairs squamous differentiation and causes epidermal barrier defects, promoting SCC (4
). We have demonstrated recently that NOTCH1 regulates esophageal epithelial homeostasis through NOTCH3, which is activated in a CSL-dependent manner at the onset of squamous differentiation and required for induction of IVL and CK13 (10
). However, it remains to be elucidated as to how Notch signaling may contribute to esophageal cancer initiation and progression.
Epithelial-to-mesenchymal transition (EMT) is marked by loss of epithelial characteristics (e.g. cell polarity and cell-cell junctions) and gain of mesenchymal characteristics (e.g. fibroblastic spindle-shaped morphology and an increased motility). EMT occurs during cancer cell invasion and metastasis (11
). In a mouse xenograft model using viral-oncogene transformed human esophageal cells, we have documented EMT in vivo
). In primary ESCC, EMT markers are upregulated at the invasive front (15
). TGF-β is a potent EMT inducer in the tumor microenvironment (20
) and expressed by both tumor and stromal cells in ESCC (21
). EMT occurs also during the early stages of carcinogenesis to bypass oncogene-induced senescence (19
). We have found recently that malignant transformation of human esophageal cells by EGFR oncogene causes enrichment of EMT-competent cells negating oncogene-induced senescence through transcriptional repression of the INK4 locus by zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 (23
), transcription factors essential in TGF-β-mediated EMT, senescence and maintenance of cancer stem cells (24
). ZEBs are subjected to negative regulation by the microRNA (miR)-200 family members (26
). However, neither the status of ZEBs nor their regulation in ESCC is known to date.
Herein, we demonstrate that ZEB1 is induced in ESCC at the invasive front undergoing EMT-like dedifferentiation. Loss of the NOTCH3-mediated CSL-dependent transcriptional activity allows expansion of EMT-competent cells expressing ZEBs, providing a novel mechanistic link between the Notch pathway and cell fate regulatory transcription factors during cancer progression.