Am J Cancer Res 2012;2(4):2012;2(4):459-475
Original Article
Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling
in invasive esophageal squamous cell carcinoma
Seiji Naganuma, Kelly A Whelan, Mitsuteru Natsuizaka, Shingo Kagawa, Hideaki Kinugasa, Sanders Chang, Harry
Subramanian, Ben Rhoades, Shinya Ohashi, Hiroshi Itoh, Meenhard Herlyn, J Alan Diehl, Phyllis A Gimotty, Andres J
Klein-Szanto, Hiroshi Nakagawa
Gastroenterology Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Abramson Cancer
Center, University of Pennsylvania, Philadelphia, Pennsylvania; Division of Tumor Pathology, Department of Pathological
Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui, Japan; Division of Gastroenterology
and Hepatology, Digestive Disease Center, The Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan;
Wistar Institute, Philadelphia, Pennsylvania; Department of Cancer Biology, University of Pennsylvania, Philadelphia,
Pennsylvania; Division of Biostatistics, Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania;
Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA, USA
Received May 25, 2012; accepted June 17, 2012; Epub June 28, 2012; Published July 15, 2012
Abstract: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas.
Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in
negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch
and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we
tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of
human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1
(DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal
carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice.
In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in
transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those
committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent
activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated
squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the
development of a tumor-promoting inflammatory microenvironment. (AJCR0000125).
Keywords: Esophageal squamous cell carcinoma, organotypic 3D culture, EGFR, P53, cyclin D1, Wnt, notch, squamous-cell
differentiation, invasion, 4-Nitroquinoline 1-oxide
Address all correspondence to:
Dr. Hiroshi Nakagawa
Division of Gastroenterology
University of Pennsylvania
638B Clinical Research Building
415 Curie Boulevard
Philadelphia, PA 19104, USA.
Phone: 215-573-1867, Fax: 215-573-2024
E-mail: nakagawh@mail.med.upenn.edu
Original Article
Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling
in invasive esophageal squamous cell carcinoma
Seiji Naganuma, Kelly A Whelan, Mitsuteru Natsuizaka, Shingo Kagawa, Hideaki Kinugasa, Sanders Chang, Harry
Subramanian, Ben Rhoades, Shinya Ohashi, Hiroshi Itoh, Meenhard Herlyn, J Alan Diehl, Phyllis A Gimotty, Andres J
Klein-Szanto, Hiroshi Nakagawa
Gastroenterology Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Abramson Cancer
Center, University of Pennsylvania, Philadelphia, Pennsylvania; Division of Tumor Pathology, Department of Pathological
Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui, Japan; Division of Gastroenterology
and Hepatology, Digestive Disease Center, The Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan;
Wistar Institute, Philadelphia, Pennsylvania; Department of Cancer Biology, University of Pennsylvania, Philadelphia,
Pennsylvania; Division of Biostatistics, Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania;
Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA, USA
Received May 25, 2012; accepted June 17, 2012; Epub June 28, 2012; Published July 15, 2012
Abstract: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas.
Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in
negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch
and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we
tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of
human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1
(DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal
carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice.
In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in
transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those
committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent
activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated
squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the
development of a tumor-promoting inflammatory microenvironment. (AJCR0000125).
Keywords: Esophageal squamous cell carcinoma, organotypic 3D culture, EGFR, P53, cyclin D1, Wnt, notch, squamous-cell
differentiation, invasion, 4-Nitroquinoline 1-oxide
Address all correspondence to:
Dr. Hiroshi Nakagawa
Division of Gastroenterology
University of Pennsylvania
638B Clinical Research Building
415 Curie Boulevard
Philadelphia, PA 19104, USA.
Phone: 215-573-1867, Fax: 215-573-2024
E-mail: nakagawh@mail.med.upenn.edu
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