Abstract 5346: Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil
Authors: Kikuchi Osamu , Ohashi Shinya , Nakai Yukie , Amanuma Yusuke , Yoshioka Masahiro , Miyamoto Shin'ichi , Natsuizaka Mitsuteru , Nakagawa Hiroshi , Chiba Tsutomu , Muto Manabu 
Published: August 2015

Abstract
BACKGROUND: We have shown that Notch3 plays an important role in the regulation of esophageal squamous cell differentiation. However, it remains unclear how Notch3-mediated squamous cell differentiation influences the tumorigenicity of esophageal squamous cell carcinoma (ESCC) or its sensitivity to anti-cancer agents, including 5-Fluorouracil (5-FU).
METHODS: We used 5-FU-resistant human ESCC cells, TE-11R, established by the step-wise continuous exposure of parental TE-11 cells to 5-FU. The TE-11R cells were stably transduced with intracellular domain of Notch3 (ICN3), an active form of Notch3, in a regulatable manner (Tet-On system). The anti-tumor effect of Notch3-overexpression was evaluated by implanting those cells into NOD SCID mice. 5-FU resistance was determined by calculating the 50% inhibitory concentration (IC50) of 5-FU using the WST-1 assay. Gene expression of squamous differentiation markers such as involucrin and cytokeratin 13 (CK13) as well as proliferative activity were determined.
RESULTS: TE-11R cells formed bulky tumors in NOD SCID mice, and the tumor formation rate of TE-11R cells (6/6: 100%) was higher than that of TE-11 cells (1/8: 12.5%). TE-11R-derived tumors showed a less differentiated and more proliferative phenotype represented by fewer ‘keratin pearl’ formations with lower involucrin and Notch3 expressions as well as higher Ki67 expression in comparison with TE-11-derived tumors. Consistent with this, TE-11R cells showed lower gene expressions of Notch3, involucrin, and CK13, and exhibited approximately 1.4-fold higher proliferative activity than parental TE-11 cells in vitro. Notch3 overexpression resulted in the promotion of cell differentiation accompanied by increased expressions of involucrin and CK13, and suppressed cell growth in vitro as well as tumorigenicity in vivo. Finally, it clearly reduced the 5-FU resistance of TE-11R cells in vitro. IC50 values of TE-11R-ICN3 (DOX-) and TE-11R-ICN3 (DOX+) were 96.0 ± 19.7 and 21.6 ± 10.4 μM (P &lt; 0.01), respectively.
CONCLUSION: Our study showed that Notch3-mediated squamous cell differentiation reduced both the tumorigenicity and 5-FU resistance of ESCC cells. We suggest that a strategy to promote squamous cell differentiation may ameliorate the outcome associated with 5-FU-resistant ESCC.
Citation Format: Osamu Kikuchi, Shinya Ohashi, Yukie Nakai, Yusuke Amanuma, Masahiro Yoshioka, Shin'ichi Miyamoto, Mitsuteru Natsuizaka, Hiroshi Nakagawa, Tsutomu Chiba, Manabu Muto. Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2015-5346