Download Abstract - University of Pennsylvania

Mitsuteru Natsuizaka, Shinya Ohashi, Seiji Naganuma, Ross A. Kalman, Asami
Ohyama, Ben Rhoades, Maria E. Vega, Douglas B. Stairs, Andres J. Klein-Szanto, J.
Alan Diehl, Devraj Basu, Meenhard Herlyn, Warren S. Pear, and Hiroshi Nakagawa
Gastroenterology Division, Department of Medicine
Abramson Cancer Center, University of Pennsylvania
Notch regulates squamous differentiation, cell plasticity and tumor heterogeneity
in esophageal carcinoma
Short Title: Notch regulates tumor heterogeneity
Introduction: The Notch receptor family regulates cell fates and may act as a tumor
suppressor in the squamous epithelia. While squamous differentiation is maintained in
esophageal squamous cell carcinoma (ESCC) and its precursor lesions, the roles of
Notch in esophageal tumor biology remain elusive.
Methods: Transformed human esophageal cells expressing EGFR, p53R175H and
cyclin D1 (EPC2-T) and ESCC cell lines were stably transduced with ICN1, an active
form of NOTCH1 (N1) in a regulatable manner (Tet-On system). Notch was inhibited by
dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or
gammasecretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected
to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to
knockdown ZEB1 and ZEB2. Squamous epithelium was reconstituted in organotypic
(3D) culture, a form of tissue engineering. In addition, cell growth was assessed in soft
agar and immunodeficient mice. Quantitative RT-PCR, Western blotting,
immunohistochemistry (IHC) and flow cytometry were done to determine gene
expression. Primary ESCC tissues (n=20) were analyzed by IHC.
Results: In primary ESCC, N1 was localized to well-differentiated tumor cell nests
expressing involucrin (IVL), a squamous differentiation marker, while downregulated in
poorly-differentiated cells lacking IVL within a single tumor tissue, implying N1 in tumor
heterogeneity. In cell lines, ICN1 activated Notch-dependent transcription and
squamous differentiation in the absence of DNMAML1. In 3D culture, DNMAML1 and
GSI not only blocked squamous differentiation but induced massive invasion and
epithelial-to-mesenchymal transition (EMT) with Wnt activation as suggested by loss of
E-cadherin and nuclear localization of β-catenin. In agreement, DNMAML1 upregulated
LEF1, Zinc finger E-box binding proteins ZEB1 and ZEB2, transcription factors all
essential in EMT and maintenance of cancer stem cells, facilitating transforming growth
factor (TGF)-β1-induced EMT in EPC2-T cells. Moreover, DNMAML1 enriched a unique
subset of CD44-bright and CD24-dim cells and augmented colony formation,
tumorigenicity as well as chemotherapeutic drug resistance against Cisplatin.
Importantly, Cre-mediated removal of chromosomally integrated floxed DNMAML1
reactivated Notch to allow terminal differentiation with IVL induction. Moreover, ZEB
knockdown greatly impaired TGF-β-mediated EMT while restoring chemotherapeutic
drug sensitivity.
Conclusions: These data indicate that Notch activation promotes squamous
differentiation whereas Notch inhibition enriches poorly differentiated tumor cells with
cancer stem cell potential with drug resistance, which involves ZEB and Wnt activation,
thus providing a novel mechanistic insight into how Notch signaling may contribute to
tumor heterogeneity in ESCC.