The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation

Abstract

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53 R175H, genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.[Keywords: Cell migration and invasion; tumor microenvironment; EGFR; p53; MMP-9; AKT] Supplemental material is available at http://www.genesdev.org. Received February 22, 2007; revised version accepted September 5, 2007. 8 These authors contributed equally to this work. 9Corresponding author. E-MAIL anil2@mail.med.upenn.edu; FAX (215) 573-5412. Article is online at http://www.genesdev.org/cgi

Role of periostin in esophageal, gastric and colon cancer

“…It was previously demonstrated that culturing EPC2-hTERT-EGFR-p53 R175H cells, which overexpress EGFR and contain a p53 missense mutation, in a three-dimensional organotypic culture induced the invasion of these cells into the extracellular matrix (20). Michaylira et al (21) reported that the induction of periostin, which was preferentially expressed in invading cells, was dependent upon EGFR signaling and p53 mutation.…”

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

“…3D cell cultures are well documented to regain intrinsic properties and to better mimic the in vivo situation than cells cultured as monolayers on plastic (Dolznig et al, 2011;Fischbach et al, 2009;Okawa et al, 2007;Pickl and Ries, 2009). Studies have shown that the gene expression profiles (Takagi et al, 2007) as well as the responses to treatment (Desoize and Jardillier, 2000) in the multicellular spheroid 3D models resemble more closely the in vivo situation.…”

The Development and Characterization of an Organotypic Tissue-Engineered Human Esophageal Mucosal Model

“…This approach results in a mature stratified epithelium that is morphologically similar to the native oesophagus and which has been incorporated into a number of experimental procedures [13][14][15][16] . However there is little published characterisation of this model itself and the approach does not include a basement membrane (BM).…”