The cell adhesion molecule E-cadherin maintains epithelial homeostasis and is frequently downregulated during development and tumorigenesis to facilitate epithelial cell invasion. Being a central node in epithelial biology, we investigatedthe role of E-cadherin in esophageal cell invasion. We could show that E-cadherin interacts with TGF2 receptor II and that coordinated loss of both molecules is a frequent event in esophageal cancer. Our recent work demonstrates that the crosstalk of the stromal compartment with esophageal keratinocytes expressing dominant-negative mutants of E-cadherin and TGF2 receptor I is necesary to induce epithelial cell invasion in a three-dimensional organotypic reconstruct model. Furthermore, in this study we identified molecules that are overexpressed in response to E-cadherin and TGF2 receptor I loss (e.g. cathepsin B and CD44) which, in part, mediate the observed cell invasion. An important finding of the studies carried out under the K01 is that increased secretion of TGF2 by the epithelial cels leads to fibroblast activation as a prerequisite to epithelial cell invasion. We are now elucidating the cell signaling pathways and mechanisms regulating epithelial cell invasion in response to E-cadherin and TGF2 receptor II loss. This R03 application builds on the data collected during the K01 entitled "E-cadherin regulates TGF2 receptor II biology and function" and addresses the role of Activin A in esophageal cell invasion and tumorigenesis. We observe high levels of pSmad2 in the absence of functional E-cadherin and T2RII in our model as well as in tumor tissues leading us to hypothesize that Activin A signaling is activated. Our preliminary data show increased secretion of Activin A in invasive organotypic cultures and enhanced cell invasion in Boyden chamber assays after Activin A stimulation. We will utilize three-dimensional organotypic cultures as wel as novel tisue recombination models (renal capsule xenograft) to clarify the function of Activin A in epithelial-mesenchymal transition and tumorigenesis. In addition, we will determine if Activin A alone can induce tumorigenesis or if this event is fibroblast-dependent.
The Specific Aim addressed here will be the basis for a R01 proposal as a continuation of the K01, and will include the analysis of the receptor signaling complex in the absence of functional TGF2 receptor II (original K01 Subaim), the analysis of the effects of Activin A on fibroblasts and the generation of a novel mouse model to analyze the role of Activin A in esophageal keratinocyte biology. !

Public Health Relevance

We established a three-dimensional organotypic culture model using esophageal keratinocytes expressing dominant-negative mutant E-cadherin and TGF2 receptor and demonstrated fibroblast-dependent esophageal cell invasion mediated by cathepsin B and the CD44-MMP9 axis. We observe high levels of pSmad2 in the absence of functional E-cadherin and T2RII in our model as well as in tumor tissues leading us to hypothesize that Activin A signaling, which intersects with TGF2 signaling at the level of Smad2 and Smad3, is activated. The overaching hypothesis of this proposal is that invading keratinocytes undergo epithelial- mesenchymal transition in response to autocrine Activin A signaling resulting in the observed cell invasion and tumorigenesis which we will address in vitro and in vivo using state-of-the-art xenograft models. !

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Small Research Grants (R03)
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Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
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Podskalny, Judith M,
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Le Bras, Grégoire F; Taylor, Chase; Koumangoye, Rainelli B et al. (2015) TGF? loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion. Exp Cell Res 330:29-42
Le Bras, Grégoire F; Loomans, Holli A; Taylor, Chase J et al. (2014) Activin A balance regulates epithelial invasiveness and tumorigenesis. Lab Invest 94:1134-46