p120-Catenin Down-Regulation and Epidermal Growth Factor Receptor Overexpression Results in a Transformed Epithelium That Mimics Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis due to its highly invasive and metastatic potential. The molecular pathogenesis underlying the invasive mechanism of ESCC is not well known because of the lack of existing models to study this disease. p120-Catenin (p120ctn) and the epidermal growth factor receptor (EGFR) have each been implicated in several cancers, including ESCC. p120ctn is down-regulated in 60% of ESCC tumors, whereas EGFR is the most commonly overexpressed oncogene in ESCC. For these reasons, we investigated the cooperation between p120ctn and EGFR and its effect on ESCC invasion. We show that p120ctn down-regulation is commonly associated with EGFR overexpression. By using a three-dimensional culture system, we demonstrate that the inverse relationship between p120ctn and EGFR has biological implications. Specifically, p120ctn down-regulation coupled with EGFR overexpression in human esophageal keratinocytes (EPC1-PE) was required to promote invasion. Morphological comparison of EPC1-PE cells grown in three-dimensional culture and human ESCC revealed identical features, including significantly increased cellularity, nuclear grade, and proliferation. Molecular characteristics were measured by keratin expression patterns, which were nearly identical between EPC1-PE cells in three-dimensional culture and ESCC samples. Altogether, our analyses have demonstrated that p120ctn down-regulation and EGFR overexpression are able to mimic human ESCC in a relevant three-dimensional culture model.Esophageal cancer is the eighth most common cancer type¹ in the United States, ranking as the seventh leading cause of cancer-related mortality in the United States² and fifth worldwide.³ Esophageal cancers are classified into two distinct histological subtypes with unique clinical behaviors: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC is a highly aggressive malignancy that is typically diagnosed at an advanced tumor stage.^3–5 The disease develops as the result of a multistep process, arising as squamous dysplasia in the stratified squamous epithelium of the esophageal mucosa and subsequently invading through the submucosa and muscle layers of the esophagus.⁶ ESCC is aggressive, with a high rate of direct local invasion to adjacent organs, such as the aorta, respiratory tract, and lungs.⁷ The underlying molecular pathogenesis and biological features of this invasive mechanism in ESCC are not known and are understudied because of the lack of existing models available.p120-Catenin (p120ctn; alias CTNND1) is a tumor suppressor gene whose down-regulation is correlated with poorly differentiated tumors and a metastatic phenotype in several cancers, including prostate, lung, and adenocarcinoma of the gastroesophageal junction.^8–10 It has been shown previously that p120ctn is either down-regulated or lost in 35% to 60% of ESCC tumors.^11,12 Epidermal growth factor receptor (EGFR) is the most commonly overexpressed oncogene in many cancer types, including ESCC,¹³ with the ability to modulate signal transduction pathways involved in tumor cell migration, proliferation, angiogenesis, and inhibition of apoptosis.^14,15 EGFR is overexpressed in 43% to 97% of ESCC patients,^15–18 and its overexpression is significantly correlated with the depth of tumor invasion.¹⁵Because p120ctn is so often down-regulated or lost and EGFR is so often overexpressed in ESCC, we focused on investigating the cooperation between these pathways in ESCC. In addition, we used a novel and relevant three-dimensional (3D) tissue culture model using immortalized human esophageal keratinocytes (EPC1-hTERT) grown to form an epithelium on an extracellular matrix embedded with human esophageal fibroblasts¹⁹ with which to study the disease.This model constitutes a complete stratified squamous epithelium, histologically resembles in vivo esophageal epithelium, and recreates the normal differentiation program of the esophagus.^19–21 We assessed the intersection of two genetic events (namely, down-regulation of the tumor suppressor p120ctn and overexpression of the oncogene EGFR), and its ability to lead to an invasive ESCC phenotype. Our data suggest that p120ctn down-regulation and EGFR overexpression in our 3D model are able to mimic human ESCC.