- Email: [email protected]
Models of Esophageal Carcinogenesis
Models of Esophageal Carcinogenesis Anil K. Rustgi Attempting to formulate a model for the development of esophageal cancer, we have undertaken three interrelated projects: (1) identification and characterization of stem cells as a basis to understand lineage specification in the normal esophageal squamous epithelium and possibly transdifferentiation into an intestinal metaplastic phenotype that defines Barrett’s esophagus; (2) elucidation of the role of epidermal growth factor receptor and signaling mechanisms in proliferation and transformation in squamous cell carcinoma; and (3) modeling transformation in organotypic culture, a 3-dimensional culture system, as a platform for investigating epithelial-stromal interactions. It is hoped that development of these models for carcinogenesis will permit formulation of effective chemoprevention, diagnostic, and therapeutic strategies. Semin Oncol 33(suppl 11):S57-S58 © 2006 Elsevier Inc. All rights reserved.
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sophageal squamous cell cancer is common worldwide and among the 10 most frequent cancers in the United States. Esophageal adenocarcinoma is also among the top 10 cancers in the United States.1 The etiology of squamous cell carcinoma includes environmental factors (eg, cigarettes, alcohol) and genetic alterations resulting in, for example, epidermal growth factor receptor (EGFR) and cyclin D1 overexpression and p16 and p53 inactivation. The etiology of esophageal adenocarcinoma includes environmental factors (eg, acid reflux) and Barrett’s esophagus, an intestinal metaplasia of the normal squamous epithelium. Many patients present with advanced stages of esophageal cancer and have a poor prognosis. There is a need for improved diagnostic markers and therapeutic approaches to disease. The esophageal mucosa is a stratified squamous epithelium that is shared across many sites, such as the skin, oral cavity, pharynx, larynx, and anogenital tract. The epithelium sits atop a submucosa (mesenchymal compartment) consisting of stromal cells that are mostly fibroblasts. The epithelium consists of a proliferating basal cell layer, differentiating suprabasal cell layer, and terminally differentiated superficial squamous cell layer (Fig 1). Cells migrate in an outward direction from the basal layer to the superficial squamous layer, and eventually undergo desquamation caused by senescence and apoptosis, with this process being continuously renewed. The proliferative compartment is characterized by
University of Pennsylvania, Philadelphia, PA. Address reprint requests to Anil K. Rustgi, MD, Department of Gastroenterology, University of Pennsylvania, 600 CRB, 415 Curie Blvd, Philadelphia, PA 19104. E-mail: [email protected]
0093-7754/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2006.10.013
activity of the transcription factors KLF4 and KLF5, EGFR, and cytokeratins; as the cells migrate and differentiate, other keratins, cytoskeletal components, and transcription factors are expressed.
Molecular Regulation of ProliferationDifferentiation Gradient EGFR is a central component in cell proliferation, differentiation, and survival. To determine whether overexpression of EGFR is critical for squamous cell carcinogenesis, we have performed studies in which human immortalized esophagus cells are grown in an organotypic culture system, a 3-dimensional cell culture system with a matrix mimicking the submucosa.2 When differentiation is induced, reconstitution of the normal epithelium is observed in this 3-dimensional system. When EGFR is overexpressed in this system, cells that overexpress EGFR exhibit basal cell hyperplasia and increased proliferation, with attenuation of the phenotype resulting from treatment with an EGFR inhibitor. Overall, these studies have indicated that EGFR activation induces migration of cells from the basal cell compartment to the suprabasal cell compartment through induction of matrix metalloproteinases; through activation of E-cadherin, the cells are signaled to stop migrating and to communicate with and adhere to neighboring cells. These findings indicate that EGFR overexpression is likely a necessary condition but not always a sufficient condition, for carcinogenesis. S57
A.K. Rustgi
S58
p53-deficient status, with these findings suggesting that cyclin D1 overexpression and p53 inactivation are sufficient to induce cancer. Lymph-node metastases have been found in approximately one third of mice tested, and only cells derived from those tumors with cyclin D1 overexpression and p53 inactivation resulted in tumors when introduced into immunodeficient mice. This model may also be useful for development of chemoprevention strategies. For example, we found that treatment with sulindac reduces dysplasia in the cyclin D1 overexpressing/p53-deficient mice.3
Conclusion Figure 1 Stratified squamous epithelium of the esophagus.
Modeling Carcinogenesis: Interplay of Oncogenes and Tumor Suppressor Genes In the in vivo setting, we used the Epstein-Barr virus ED-L2 promoter to target genes specifically to the oral-esophageal epithelium in mice. When the cyclin D gene is targeted for upregulation, squamous dysplasia results. When the L2cyclin D1 mice are bred into a p53-deficient background, there is acceleration in dysplasia over 12 months in the oral cavity and upper and lower esophagus. Emergence of squamous cancer is observed at 6 to 12 months in mice with
A model of esophageal squamous carcinogenesis emerging from our studies is that EGFR overexpression drives cells into a preneoplastic state, with such events as overexpression of cyclin D1, p53 inactivation, and telomerase activation being necessary to produce cancer. It is hoped that this model will permit development of effective chemopreventive, diagnostic, and therapeutic strategies.
References 1. Sun W, Rustgi AK: Esophageal neoplasms, in: Yamada T, Alpers DH, Kaplowitz N, et al (eds): Textbook of Gastroenterology. Ed 4. Philadelphia, PA, Lippincott, Williams & Wilkins, 2001, pp 1238-1261 2. Andl CD, Mizushima T, Nakagawa H, et al: Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J Biol Chem 278: 1824-1830, 2003 3. Opitz OG, Harada H, Suliman Y, et al: A mouse genetic model of human oral-esophageal cancer. J Clin Invest 110:761-769, 2002
E
sophageal squamous cell cancer is common worldwide and among the 10 most frequent cancers in the United States. Esophageal adenocarcinoma is also among the top 10 cancers in the United States.1 The etiology of squamous cell carcinoma includes environmental factors (eg, cigarettes, alcohol) and genetic alterations resulting in, for example, epidermal growth factor receptor (EGFR) and cyclin D1 overexpression and p16 and p53 inactivation. The etiology of esophageal adenocarcinoma includes environmental factors (eg, acid reflux) and Barrett’s esophagus, an intestinal metaplasia of the normal squamous epithelium. Many patients present with advanced stages of esophageal cancer and have a poor prognosis. There is a need for improved diagnostic markers and therapeutic approaches to disease. The esophageal mucosa is a stratified squamous epithelium that is shared across many sites, such as the skin, oral cavity, pharynx, larynx, and anogenital tract. The epithelium sits atop a submucosa (mesenchymal compartment) consisting of stromal cells that are mostly fibroblasts. The epithelium consists of a proliferating basal cell layer, differentiating suprabasal cell layer, and terminally differentiated superficial squamous cell layer (Fig 1). Cells migrate in an outward direction from the basal layer to the superficial squamous layer, and eventually undergo desquamation caused by senescence and apoptosis, with this process being continuously renewed. The proliferative compartment is characterized by
University of Pennsylvania, Philadelphia, PA. Address reprint requests to Anil K. Rustgi, MD, Department of Gastroenterology, University of Pennsylvania, 600 CRB, 415 Curie Blvd, Philadelphia, PA 19104. E-mail: [email protected]
0093-7754/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2006.10.013
activity of the transcription factors KLF4 and KLF5, EGFR, and cytokeratins; as the cells migrate and differentiate, other keratins, cytoskeletal components, and transcription factors are expressed.
Molecular Regulation of ProliferationDifferentiation Gradient EGFR is a central component in cell proliferation, differentiation, and survival. To determine whether overexpression of EGFR is critical for squamous cell carcinogenesis, we have performed studies in which human immortalized esophagus cells are grown in an organotypic culture system, a 3-dimensional cell culture system with a matrix mimicking the submucosa.2 When differentiation is induced, reconstitution of the normal epithelium is observed in this 3-dimensional system. When EGFR is overexpressed in this system, cells that overexpress EGFR exhibit basal cell hyperplasia and increased proliferation, with attenuation of the phenotype resulting from treatment with an EGFR inhibitor. Overall, these studies have indicated that EGFR activation induces migration of cells from the basal cell compartment to the suprabasal cell compartment through induction of matrix metalloproteinases; through activation of E-cadherin, the cells are signaled to stop migrating and to communicate with and adhere to neighboring cells. These findings indicate that EGFR overexpression is likely a necessary condition but not always a sufficient condition, for carcinogenesis. S57
A.K. Rustgi
S58
p53-deficient status, with these findings suggesting that cyclin D1 overexpression and p53 inactivation are sufficient to induce cancer. Lymph-node metastases have been found in approximately one third of mice tested, and only cells derived from those tumors with cyclin D1 overexpression and p53 inactivation resulted in tumors when introduced into immunodeficient mice. This model may also be useful for development of chemoprevention strategies. For example, we found that treatment with sulindac reduces dysplasia in the cyclin D1 overexpressing/p53-deficient mice.3
Conclusion Figure 1 Stratified squamous epithelium of the esophagus.
Modeling Carcinogenesis: Interplay of Oncogenes and Tumor Suppressor Genes In the in vivo setting, we used the Epstein-Barr virus ED-L2 promoter to target genes specifically to the oral-esophageal epithelium in mice. When the cyclin D gene is targeted for upregulation, squamous dysplasia results. When the L2cyclin D1 mice are bred into a p53-deficient background, there is acceleration in dysplasia over 12 months in the oral cavity and upper and lower esophagus. Emergence of squamous cancer is observed at 6 to 12 months in mice with
A model of esophageal squamous carcinogenesis emerging from our studies is that EGFR overexpression drives cells into a preneoplastic state, with such events as overexpression of cyclin D1, p53 inactivation, and telomerase activation being necessary to produce cancer. It is hoped that this model will permit development of effective chemopreventive, diagnostic, and therapeutic strategies.
References 1. Sun W, Rustgi AK: Esophageal neoplasms, in: Yamada T, Alpers DH, Kaplowitz N, et al (eds): Textbook of Gastroenterology. Ed 4. Philadelphia, PA, Lippincott, Williams & Wilkins, 2001, pp 1238-1261 2. Andl CD, Mizushima T, Nakagawa H, et al: Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J Biol Chem 278: 1824-1830, 2003 3. Opitz OG, Harada H, Suliman Y, et al: A mouse genetic model of human oral-esophageal cancer. J Clin Invest 110:761-769, 2002