Katharine D. Grugan

Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion

Squamous cell cancers comprise the most common type of human epithelial cancers. One subtype, esophageal squamous cell carcinoma (ESCC), is an aggressive cancer with poor prognosis due to late diagnosis and metastasis. Factors derived from the extracellular matrix (ECM) create an environment conducive to tumor growth and invasion. Specialized cancer-associated fibroblasts (CAFs) in the ECM influence tumorigenesis. We have shown previously that the nature and activation state of fibroblasts are critical in modulating the invasive ability of ESCC in an in vivo-like organotypic 3D cell culture, a form of human tissue engineering. Dramatic differences in invasion of transformed esophageal epithelial cells depended on the type of fibroblast in the matrix. We hypothesize that CAFs create an environment primed for growth and invasion through the secretion of factors. We find that fibroblast secretion of hepatocyte growth factor (HGF) fosters the ability of transformed esophageal epithelial cells to invade into the ECM, although other unidentified factors may cooperate with HGF. Genetic modifications of both HGF in fibroblasts and its receptor Met in epithelial cells, along with pharmacologic inhibition of HGF and Met, underscore the importance of this pathway in ESCC invasion and progression. Furthermore, Met activation is increased upon combinatorial overexpression of epidermal growth factor receptor (EGFR) and p53R175H, two common genetic mutations in ESCC. These results highlight the potential benefit of the therapeutic targeting of HGF/Met signaling in ESCC and potentially other squamous cancers where this pathway is deregulated.

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.

A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human cancer with poor prognosis due to late diagnosis and metastasis. Common genomic alterations in ESCC include p53 mutation, p120ctn inactivation, and overexpression of oncogenes such as cyclin D1, EGFR, and c-Met. Using esophageal epithelial cells transformed by the overexpression of EGFR and p53R175H, we find novel evidence of a functional link between p53R175H and the c-Met receptor tyrosine kinase to mediate tumor cell invasion. Increased c-Met receptor activation was observed upon p53R175H expression and enhanced further upon subsequent EGFR overexpression. We inhibited c-Met phosphorylation, resulting in diminished invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53R175H), suggesting that the mechanism of increased invasiveness upon EGFR and p53R175H expression may be the result of increased c-Met activation. These results suggest that the use of therapeutics directed at c-Met in ESCC and other squamous cell cancers.

HER2 amplification in micrometastatic esophageal cancer cells predicts prognosis.

Faten Aberra, Philadelphia, PA Antoni Castells, Catalonia, Spain Naga Chalasani, Indianapolis, IN Raymond T. Chung, Boston, MA Jason Dominitz, Seattle, WA Gary W. Falk, Cleveland, OH James Farrell, Los Angeles, CA Richard J. Farrell, Boston, MA Lauren Gerson, Stanford, CA Michael Goggins, Baltimore, MD John M. Inadomi, San Francisco, CA W. Ray Kim, Rochester, MN Paul J. Limburg, Rochester, MN Uma Mahadevan-Velayos, San Francisco, CA Hiroshi Nakagawa, Philadelphia, PA Oliver G. Opitz, Freiburg, Germany Henry Parkman, Philadelphia, PA Scott Plevy, Chapel Hill, NC Shanthi Sitaraman, Atlanta, GA Christina M. Surawicz, Seattle, WA Jonathan Terdiman, San Francisco, CA George Triadafilopoulos, Stanford, CA Thomas A. Ullman, New York, NY Michael F. Vaezi, Nashville, TN

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.