Carmen Z. Michaylira

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.

TLR3-mediated NF-κB signaling in human esophageal epithelial cells

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

Abstract 279: Increased Met receptor activation mediates enhanced cell invasion upon mutant p53 expression in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), a type of squamous cell cancer, 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 mutations and overexpression of oncogenes such as cyclin D1, EGFR, and Met. Using an invasive primary esophageal epithelial cell model system genetically transformed by the overexpression of EGFR and p53 R175H , we find novel evidence of cross-talk between p53 R175H and the Met receptor tyrosine kinase. Increased Met receptor activation is observed upon expression of p53 R175H and is further enhanced upon subsequent overexpression of EGFR. Increased Met activation is observed also in a number of human ESCC cells lines that harbor either p53 genetic mutations or are p53 null. Met phosphorylation can be inhibited using two small molecule compounds (CP31398 and 5-iminodaunorubicin) that have been demonstrated previously to activate wild-type p53 signaling through distinct mechanisms, thereby providing further evidence to a link between these two pathways. Both CP31398 and 5-iminodaunorubicin blocked invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53 R175H ) suggesting that the mechanism of increased invasiveness upon EGFR and p53 R175H expression may be mediated by Met activation. In aggregate, mutant p53 and Met appear to cooperate to foster tumor invasion and this novel linkage has not been reported previously. These results provide further implications to the use of combinatorial therapeutics directed at Met and mutant p53 in ESCC and other squamous cell cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 279.

179 Functional Characterization of Toll-Like Receptor 3 Signaling in Human Esophageal Keratinocytes

cells (EPCs). VEGF and SDF-1 mRNA levels were reduced in KP-1N in the presence of cyclopamine both In Vitro and In Vivo, but the number of circulating EPCs was not affected by Hh inhibition. Host derived Ang-1 and IGF-1 mRNA levels in xenografts were strongly downregulated by cyclopamine administration, which may contribute to the maintenance and maturation of tumor vasculature. In Vitro co-culture experiments demonstrated that KP1N cells induced Ang-1/IGF-1 production in BM-EPCs, and this induction was significantly attenuated either by cyclopamine or neutralizing antibody to Shh. In addition, a matrigel plug assay supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of BM-derived EPCs. This “paracrine” effect of Hh seems to be a late event during pancreatic tumorigenesis, as stromal Patch1 expression was detected within PDAC lesions in Pdx-Cre;Kras;p53lox/+ mice, but not in precursor PanIN lesion in PdxCre;Kras mice. Collectively, Hh derived from cancer cells can have a profound effect on neovascularization through the regulation of EPCs during late stages of pancreatic tumorigenesis, and targeting Hh would be a novel therapeutic approach to inhibit tumor angiogenesis. This work was supported by New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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