Jiri Kalabis

Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene.

p120-catenin (p120ctn) interacts with E-cadherin, but to our knowledge, no formal proof that p120ctn functions as a bona fide tumor suppressor gene has emerged to date. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop preneoplastic and neoplastic lesions in the oral cavity, esophagus, and squamous forestomach. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.

A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification

The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label-retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.

Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

Reciprocal regulation of MelCAM and AKT in human melanoma

Alteration in the expression of invasion/metastasis-related melanoma cell adhesion molecule (MelCAM) is strongly associated with the acquisition of malignancy by human melanoma. However, little is known about the molecular and biochemical mechanisms that regulate the expression and function of MelCAM, or its downstream signaling transduction. In this study, we show that there is a reciprocal regulation loop between AKT and MelCAM. Pharmacological inhibition of AKT in human melanoma cell lines substantially reduced the expression of MelCAM. Overexpression of constitutively active AKT upregulated the levels of MelCAM in melanoma cell lines, whereas expression of a dominant-negative PI-3 kinase downregulated MelCAM. On the other hand, overexpression of MelCAM activated endogenous AKT and inhibited proapoptotic protein BAD in melanoma cells, leading to increased survival under stress conditions. Constitutive activation of AKT was observed in most melanoma cell lines and tumor samples of different progression stages. These data link AKT activation with MelCAM expression, and implicate that intervention of MelCAM-AKT signaling axis in melanoma is a potential therapeutical approach.

Stimulation of human colonic epithelial cells by leukemia inhibitory factor is dependent on collagen-embedded fibroblasts in organotypic culture

The colonic epithelium undergoes a continuous cycle of proliferation, differentiation, and apoptosis. To characterize factors important for colonic homeostasis and its dysregulation, human fetal colonic epithelial cells were isolated and seeded on a collagen type I matrix with embedded colonic fibroblasts. The epithelial cells rapidly spread from clusters and proliferated, and within 3 days, a columnar layer of polarized epithelium surrounded the surface of the constricted collagen matrix. The polarized enterocytes developed brush borders, tight junctions and desmosomes, and goblet and enteroendocrine cells were present. A balance of growth and differentiation was maintained for several weeks in the presence of collagen‐embedded fibroblasts and a complex mixture of growth factors. Leukemia inhibitory factor (LIF) was critical for proliferation of enterocytes and inhibited expression of the differentiation marker carbonic anhydrase II. In the presence of LIF, the relative number of goblet cells remained stable, whereas enteroendocrine relative cell number declined. LIF‐stimulated epithelial cells remained dependent on the presence of fibroblasts in the matrix. In combination with stem cell factor and endothelin 3, LIF induced formation of disorganized structures of stratified and semi‐stratified cells, suggesting that the homeostatic balance in the normal human colon requires cooperation with differentiation‐inducing factors.

Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.

Abstract CN04-03: Esophageal cancer and the tumor microenvironment

Squamous cell cancers are the most common type of epithelially derived human cancers and arise from a number of diverse sites. They share a number of common genetic alterations and environmental exposures. Employing oral and esophageal cancers as prototypes, we have developed three‐dimensional (3D) or organoptypic models that represent a form of human tissue engineering (Genes and Development 2007; Journal of Clinical Investigation 2008). These approaches underscore the combinatorial roles of EGFR signaling and p53 mutation in fostering tumor cell migration and invasion in the microenvironment. Microarray analyses reveal specific effectors in the adhesion family of genes that might be exploited as biomarkers for detection and targets for new therapeutics. We have also generated genetically engineered mouse models that reveal the effects of cyclin D1 overexpression in concert with p53 loss, as well as the role of p120‐catenin loss in the initiation and progression of cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN04-03.

838 Synergism Between EGFR and Mutant p53 Facilitates Epithelial-Mesenchymal Transition By Negating Oncogene-Activated Cellular Senescence in Transformed Human Esophageal Cells

Preoperative Biliary Drainage Versus Direct Operation for Pancreatic Tumors Causing Obstructive Jaundice Niels Anthony Van Der Gaag, Erik Rauws, Casper H. van Eijck, Marco Bruno, Erwin van der Harst, Josephus J. Gerritsen, J. W. M. Greve, Michael F. Gerhards, Ignace H. de Hingh, Jean H. Klinkenbijl, Chung Y. Nio, Steve M. de Castro, Olivier R. Busch, Thomas M. Van Gulik, Patrick M. Bossuyt, Dirk J. Gouma

Abstract A3: Facilitating epithelial to mesenchymal transition through inhibition of senescence checkpoint functions

Transforming growth factor (TGF)‐β is a potent inducer of epithelial to mesenchymal transition (EMT). However, EMT is not a sole consequence of TGF‐β mediated stimulation. It remains unclear as to what determines the underlying cellular capacity to undergo EMT in response to TGF‐β. Overexpression of epidermal growth factor receptor (EGFR) and mutations in tumor suppressor p53 are common in the early stage of esophageal carcinogenesis and required for transformation and invasive growth of human esophageal epithelial cells. EGFR overexpression and mutant p53 appear to be key determinants in facilitating TGF‐β-induced EMT. EGFR tyrosine kinase activity was dispensable for EMT. However, EGFR overexpression fated the esophageal cells to EMT through induction of ZEB1 and ZEB2, zinc finger E‐box binding proteins which evade EGFR‐mediated oncogenic as well as the TGF‐β-activated senescence responses by inhibiting p15 INK4b and p16 INK4a . Mutant p53 limits TGF‐β-induced senescence by suppressing the p53‐dependent senescence checkpoint function. When senescence was negated, TGF‐β prompted EMT through induction of ZEB1 and ZEB2 along with concomitant downregulation of the microRNA‐200 family. Thus, genetic lesions may involve not only suppression of senescence checkpoint functions but also reactivation of the cellular EMT program in the early stages of carcinogenesis. Citation Information: Cancer Res 2009;69(23 Suppl):A3.