Abstract 2678: Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma
Authors: Tanaka Koji , Whelan Kelly A. , Rustgi Naryan L. , Chandramouleeswaran Prasanna M. , Naganuma Seiji , Kagawa Shingo , Natsuizaka Mitsuteru , Kita Yoshiaki , Natsugoe Shoji , Jianwen Que , Basu Devraj , Klein-Szanto Andres J. , Bass Adam , Diehl J. Alan , Nakagawa Hiroshi 
Published: July 2016

Abstract
Introduction: Notch signaling may act as a tumor suppressor during the development of squamous cell carcinomas (SCCs); yet, Notch activation promotes tumor growth in a subset of SCC cells. The roles of Notch in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remain elusive.
Methods: Notch1 activation and epithelial-mesenchymal transition (EMT) were determined in an esophageal epithelium-targeted cell-lineage traceable (K5CreERT2-Rosa26tdTomatolsl) mouse model of ESCC induced by 4-nitroquinoline 1-oxide (4NQO), which was coupled with flow cytometry and single cell-derived ESCC organoid formation assays. Tumor-initiating capability was assessed in xenograft transplantation experiments with TE11 human ESCC cells carrying either Crispr-Cas9-deleted Notch1 loci or tetracycline-inducible expression of the activated form of Notch1 (ICN1). Surgically resected primary tumors and adjacent normal mucosa from ESCC patients (n = 152) were analyzed by immunohistochemistry for Notch1 activation and the EMT marker ZEB1.
Results: 4NQO-treated mice developed tdTomato-positive primary and metastatic ESCC tumors with EpCAM-negative ESCC cells displaying traits compatible with EMT. Notch1 activation and ZEB1 expression were co-localized in ESCC cells at the stromal interface, a finding that was further recapitulated in ESCC tumor organoids. Interestingly, Cre-mediated ex vivo Notch1 deletion in a single cell suspension from Notch1loxP/loxP mouse-derived ESCC tumors decreased organoid formation rate. TE11 xenograft tumors appeared to contain a unique ESCC cell fraction containing EpCAM-negative cells, where ICN1 conferred tumorigenicity upon serial transplantation. This population displayed upregulation of Notch1 target genes and the ESCC-lineage survival factor/oncogene SOX2. Moreover, Notch1 deletion in TE11 not only limited tumor formation, but also decreased EMT in culture. A subset of ESCC patients (49/140, 33%) showed ICN1-positive ESCC cells with concurrent ZEB1 expression at the tumor invasive front. The presence of such ESCC cells was associated with poor 5-year survival (P = 0.001), tumor depth (P = 0.01), lymphatic and venous invasion (P = 0.003) and distant metastasis (P = 0.002). Moreover, such ICN1-expressing cells were increased in ESCC patients (7/12, 58%) who received pre-surgical neoadjuvant therapy.
Conclusions: Cell-lineage tracing experiments validate for the first time Notch1 activation and EMT in the 4NQO-induced mouse model of ESCC. Analyses of single cell-derived ESCC tumor organoids, xenograft and primary ESCC tumors reveal that Notch1 activation may be associated with tumor initiating capability, EMT and chemotherapy resistance, implicating Notch1 activation in the pathogenesis of ESCC and potentially other SCCs.
Citation Format: Koji Tanaka, Kelly A. Whelan, Naryan L. Rustgi, Prasanna M. Chandramouleeswaran, Seiji Naganuma, Shingo Kagawa, Mitsuteru Natsuizaka, Yoshiaki Kita, Shoji Natsugoe, Que Jianwen, Devraj Basu, Andres J. Klein-Szanto, Adam Bass, J. Alan Diehl, Hiroshi Nakagawa. Notch1 regulates epithelial-mesenchymal transition and tumor-initiating capability in esophageal squamous-cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2678.