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Ohashi, Shinya1, 2; Natsuizaka, Mitsuteru1, 2; Nakagawa, Momo1, 2; Kalman, Ross A.1, 2;
Chang, Sanders1, 2; Vega, Maria E.1, 2; Kalabis, Jiri1, 2; Stairs, Douglas B.1, 2; Klein-Szanto,
Andres J.3; Diehl, J. Alan4, 2; Herlyn, Meenhard5; Nakagawa, Hiroshi1, 2
1. GI Division, University of Pennsylvania, Philadelphia, PA, United States.
2. Abramson Cancer Center, Philadelphia, PA, United States.
3. Fox Chase Cancer Center, Philadelphia, PA, United States.
4. Cancer Biology, University of Pennsylvania, Philadelphia, PA, United States.
5. Wistar Institute, Philadelphia, PA, United States.
EGFR and Notch determine esophageal cell fates through an antagonistic
Introduction: Malignant transformation of human esophageal cells by epidermal growth
factor receptor (EGFR) oncogene results in upregulation of Zinc finger E-box binding
proteins ZEB1 and ZEB2 (Cancer Res. 2010;70:4174-84), which are transcription factors
implicated in epithelial-to-mesenchymal transition (EMT) and maintenance of cancer
stem cells. Notch signaling regulates cell fate and differentiation. Notch may act also as
a tumor suppressor in squamous cell carcinomas. We have investigated the interplay
between EGFR and Notch in esophageal carcinogenesis. Methods: EGFR was inhibited
by AG1478, an EGFR-specific tyrosine kinase inhibitor in non-transformed and EGFRtransformed human esophageal cells. Notch was inhibited by a γ-secretase inhibitor
(GSI) or Cre-excisable dominant negative mastermind-like 1 (DNMAML1), a genetic
pan-Notch inhibitor. Functional roles of ZEBs were evaluated by RNA interference
(RNAi). Cells were transduced with ICN1, an active form of NOTCH1 (N1) in a
tetracycline-inducible manner. 8xCSL-luciferase reporter was transfected to assess
Notch activity. Squamous differentiation and cell invasion were assessed in organotypic
3D culture, a form of tissue engineering. Additionally, cell growth was assessed in soft
agar and immunodeficient mice. Gene expression was determined by quantitative RTPCR, Western blotting, immunostaining and flow cyotometry. Results: AG1478
activated N1 in esophageal cells, resulting in activation of CSL-dependent transcription
and squamous differentiation with induction of NOTCH3, HES5 and Involucrin. GSI
prevented AG1478 from activating Notch, implying direct Notch receptor activation.
Interestingly, induction of NOTCH3 and Involucrin by AG1478 was found greater in
EGFR-transformed cells than non-transformed cells. Similar to EGFR overexpression,
DNMAML1 inhibited squamous differentiation and enriched ZEB-upregulated EMT
competent CD44-bright and CD24-dim cells. DNMAML1 augmented cell invasion, colony
formation, tumorigenicity and chemotherapeutic drug resistance. RNAi experiments
implied ZEBs in EMT and drug-sensitivity in Notch inhibited cells. Importantly, Cremediated removal of chromosomally integrated DNMAML1 reactivated Notch and
squamous differentiation. Moreover, Notch activation by ectopically induced ICN1
facilitated squamous differentiation and increased drug sensitivity in esophageal cancer
cells. Conclusions: These data suggest that EGFR contributes to Notch inhibition
during malignant transformation of esophageal cells. Notch inhibition not only limits
squamous differentiation but allows expansion of a unique subset of cells with potential
cancer stem cell characteristics mediated by ZEBs, thus providing novel mechanistic
insights and harboring translational implications for novel therapeutics.