Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

Mitsuteru Natsuizaka,Jonathan P Katz,Shingo Kagawa,Jianwen Que,E Paul Wileyto,Adam J Bass,Yizeng Yang,Anil K Rustgi,Andres J Klein‐Szanto ,Shoji Natsugoe,Prasanna M Chandramouleeswaran,J Alan Diehl,Kwok-Kin Wong,Douglas S Darling,Véronique Giroux ,Devraj Basu,Varun Sahu,Koji Tanaka,Yoshiaki Kita,Kelly A Whelan,Seiji Naganuma,Apple Long,Hiroshi Nakagawa

doi:10.1038/s41467-017-01500-9

Abstract

Notch1 transactivates Notch3 to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial–mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-β present in the tumor microenvironment. We find that TGFβ activates the transcription factor ZEB1 to repress Notch3, thereby limiting terminal differentiation. Concurrently, TGFβ drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC.

Highlights

Notch[1] transactivates Notch[3] to drive terminal differentiation in stratified squamous epithelia
We demonstrate that Notch[1] activation and epithelial–mesenchymal transition (EMT) are coupled to promote tumor initiation and intratumoral cancer cell heterogeneity in squamous cell carcinomas (SCCs)
Flow cytometry revealed the presence of cells displaying both negative and positive expression of EpCAM (EpCAMneg and EpCAMpos), an epithelial cell surface marker, within the tdTomato-positive fractions of 4-Nitroquinoline 1-oxide (4NQO)-induced esophageal squamous cell carcinoma (ESCC) lesions (Supplementary Fig. 1c), suggesting a loss of epithelial characteristics in tumor cells originating from esophageal basal keratinocytes

Summary

Introduction

Notch[1] transactivates Notch[3] to drive terminal differentiation in stratified squamous epithelia. Activated form of Notch[1] promotes xenograft tumor growth when expressed ectopically. We demonstrate that Notch[1] activation and epithelial–mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-β present in the tumor microenvironment. Histopathology of squamous cell carcinomas (SCCs) features squamous-cell differentiation, a process normally regulated via direct transcriptional activation of Notch[3] by ICN1, the activated form of Notch[1], in esophageal epithelia[4]. Epithelial properties are converted to CD44H cells with mesenchymal traits in response to transforming growth factor (TGF)-β30, 31, a potent EMT inducer present in the tumor microenvironment[32]. Notch[1] and Notch[3] cooperate to drive squamous-cell differentiation[4], these Notch paralogs may play opposing roles in EMT and, potentially, regulation of CSC dynamics. The precise molecular mechanisms through which Notch signaling regulates distinct cell fates in a context-dependent manner have yet to fully elucidated

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Conclusion