T1177 Tumor Progression, Invasion and Metastasis in a Genetically Defined Model of Esophageal Squamous Carcinogenesis

relocated to the cell membrane in cancer cells. Cathepsin B is recruited from the lysosomes to the membrane during malignant transformation. ZEB1 overexpressing cells showed an increase especially in single cell invasion into the ECM in OTC as well as the spheroids resembling a squamous EMT. Conclusions: In our human cellular model of oral-esophageal carcinogenesis we demonstrated that collagen XVII is differentially expressed during carcinogenesis. Cellular localization of collagen XVII and cathepsin B is defined by the distinct steps of tumor development. ZEB1 overexpression led to an increased invasive potential resembling a squamous EMT phenotype in organotypic culture, which is a prerequisite for a cell to become invasive and metastatic. T1178 Folic Acid Supplementation Alters Expression of Genes Regulating 1-Carbon Metabolism and Inflammation in the Human Rectosigmoid Mucosa Petr Protiva, Joel B. Mason, Zhenhua Liu, Michael E. Hopkins, Celeste G. Nelson, Richard W. Lambrecht, James R. Marshall, Levy Kopelovich, Martin Lipkin, Peter R. Holt BACKGROUND: Folate availability modulates cancer risk. Recent evidence suggests overly abundant folate supplementation in susceptible individuals with normal folate status increases the risk of malignancies, including colorectal cancer (1). The pathways that underlie this effect are unknown but evidence from a colorectal aspirin/folate polyp prevention trial suggests that folate antagonizes the actions of aspirin on circulating inflammation markers (2). AIM: To define molecular pathways altered by supplemental folic acid administration on folate replete in the human colon. METHODS: Following 8 weeks of folate controlled intak folic acid 1 mg/day was administered for 8 weeks under close dietary control to 10 subjects at increased risk of colorectal neoplasia. Primary endpoints studied in the rectosigmoid mucosa were genome wide expression arrays by Illumina RefSeq bead array technology, genomic DNA methylation by LC-mass spectrometry, and p53 gene (exons 5-8) strand breaks by RT-PCR. RESULTS: Serum and rectosigmoid folate levels rose 50-60% (p<0.05) with supplementation. Gene array analysis showed that folic acid supplementation significantly altered expression of several critical enzymes involved in 1-carbon metabolism, including dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS) suggesting that colonic genes were responsive to the intervention. Numerous genes involved in inflammation and immune-related pathways were upregulated including IL6, chemokine 19 and 20 (CCL19, CCL20) and olfactomedin 4 (OLFM4), a gut stem cell marker shown to be over-expressed in inflamed colonic epithelium (3). Key gene changes were confirmed by qRT-PCR. DNA methylation and p53 strand breaks were unchanged in the colon, although a modest decrease in p53 exon 8 breaks occurred in peripheral blood monocyte DNA. CONCLUSIONS: Folic acid supplementation produced substantial increases in systemic and rectosigmoid folate status and altered expression of several genes involved in 1-carbon (‘folate') metabolism. Pronounced upregulation in several inflammation and immune-related pathways represent novel mechanism through which abundant supply of folic acid could enhance colorectal neoplasia risk. References 1. Ebbing M et al. Cancer incidence and mortality after treatment with folic acid… JAMA. 2009 Nov 18;302(19):2119-26. 2. Ho GY et al. Antagonistic Effects of Aspirin and Folic Acid on Inflammation Markers... J Natl Cancer Inst. 2009 Oct 12. [Epub ahead of print] 3. van der Flier LG et al. OLFM4 is a robust marker for stem cells in human intestine…Gastroenterology. 2009 Jul;137(1):15-7.

T1176 Role of Recombinase (RAD51) in Ongoing Genomic Instability and Proliferation in Barrett's Adenocarcinoma Cells Jagannath Pal, Robert C. Bertheau, Leutz Buon, Aamer Qazi, Ramesh B. Batchu, Rouba Ali-Fehmi, Saem Lee, David G. Beer, Donald W. Weaver, Nikhil C. Munshi, Raj K. Goyal, Masood A. Shammas

Introduction: A prominent feature of most cancers including Barrett's adenocarcinoma (BEAC) is the genetic instability. Genetic instability is characterized by ongoing mutational and karyotypic changes in the genome and is associated not only with pregression of Barrett's esophagus to adenocarcinoma, but also in the progression and drug resistance in already developed adenocarcinoma. The purpose of this study was to investigate the role of recombinase (RAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and continued proliferation of Barrett's adenocarcinoma cells. Methods: Studies were performed on normal cell strains (diploid fibroblasts, normal primary esophageal epithelial cells) and BEAC cell lines (FLO-1 and OE33). Cell viability was confirmed by MTT assays. Expression of RAD51 was monitored by western blot analysis. RAD51 was suppressed by lentiviruses producing RAD51-specific shRNAs. Genomic instability was monitored by evaluating changes in copy number and heterozygosity throughout genome, using single nucleotide polymorphism (SNP) arrays (Affymetrix) and dChip software. Results: Recombinase protein was elevated 3.5 - 6-fold in BEAC cell lines relative to corresponding normal cells. HR activity, as assessed by a plasmid based assay, was also elevated in BEAC cells. We also demonstrated that BEAC cells in culture, continue to acquire genomic rearrangements over time. To evaluate the impact of elevated recombinase on evolving genomic rearrangements, BEAC cells were transduced with lentiviruses producing control or recombinase-specific shRNAs and cultured for various durations. In three independent experiments, the transduction with recombinase shRNAs and subsequent decrease in recombinase expression and HR activity, led to an average of 67% reduction in the appearance of new loss of heterozygosity loci and 70% reduction in the acquisition of copy number changes, throughout genome (P < 0.006). In all three experiments, the proliferation rate of cells transduced with recombinase shRNA was consistently reduced by 20-30% relative to control cells, and in one instance the cells could not continue to grow beyond 60 days and had apoptotic cell death. Conclusions: These studies show that recombinase, RAD51 (a key component of homologous recombination), is constitutively elevated in BEAC cells. Suppression of RAD51 leads to suppression of ongoing genomic rearrangements and reduced rate of BEAC cell proliferation. RAD51 may be a promising target for prevention of disease progression and treatment of BEAC.

T1179 Estrogens Promote Both Tumor Initiation and Progression in a Mouse Model of Colitis Associated Cancer Jarom Heijmans, Rutger J. Jacobs, Willemijn A. van Dop, Johanna van der Zon, Daniel W. Hommes, James C. Hardwick, Izak Biemond, Twan A.G. Edevireen, Patrick G. Groothuis, Vanesa Muncan, Gijs R. van den Brink Introduction: Female hormones have differential effects on multiple forms of cancer. The women's health initiative studies have shown that a combination of estrogen and a progestin protect against the incidence of colorectal cancer in postmenopausal women. However the mechanism of this protective effect and individual role of the two hormones has yet to be elucidated. Here we set out to examine the effect of estrogen in a mouse model of colitis and a model of inflammation associated colorectal cancer. Methods: We treated ovariectomized (OVX) mice with either placebo of 17β-estradiol (E2, 12 ug/kg/day), using sham operated mice as controls and exposed them to 2.5% DSS in drinking water for 7 days. Mice were analyzed for standard clinical parameters and cytokine production. In a similar setup mice were exposed to a single i.p. injection of 10 mg/kg azoxymethane (AOM) followed by three 5 day cycles of 1.5% DSS and sacrificed at 11 weeks after the injection of AOM. Mice were analyzed for tumor size and number. Results: In the DSS alone experiment E2 significantly aggravated the severity of colitis. Production of IL-6 but not IFNγ or TNFα was markedly increased by E2. In the AOM-DSS model E2 increased both tumor number and size. Tumor number was 8 fold increased in estrogen treated OVX animals compared to OVX controls (16 vs. 2 tumors respectively, P < 0.001.). Tumor size was also significantly increased in E2 versus placebo treated OVX mice (2.7 mm vs. 1 mm respectively, P < 0.005). Conclusion: E2 aggravates the severity of disease in the DSS model of colitis. In the AOM-DSS model of colitis associated cancer E2 drives both tumor initiation and progression. The selective effect on IL-6 production suggests that E2 modulates specific inflammatory pathways rather than acting as a general modulator of the inflammatory response.

T1177 Tumor Progression, Invasion and Metastasis in a Genetically Defined Model of Esophageal Squamous Carcinogenesis Heike Kunert, Sarah Hauss, Angela Queisser, Thomas Reinheckel, Leena BrucknerTudermann, Christoph Peters, Thomas Brabletz, Oliver G. Opitz Introduction: Invasion and metastasis are the key characteristics of tumor progression. Epithelial-mesenchymal-transition (EMT) is a developmental program that is often activated during cancer invasion and metastasis, especially at the invasive front. To investigate the role of a master regulator of EMT and metastasis we overexpressed the transcriptional repressor ZEB1 in our cellular model of human oral-esophageal carcinogenesis. In this stepwise model cyclin D1 overexpression and p53 inactivation (OKF6D1/d.n.p53) led to immortalization, additional EGFR overexpression (OKF6D1/d.n.p53/EGFR) induced an In Vitro transformed phenotype, whereas additional c-myc overexpression (OKF6D1/d.n.p53/ EGFR/c-myc) resulted in invasive cancer cells. Methods: The expression of collagen XVII, cathepsin B and E-cadherin as a ZEB1 target genes was assayed in the distinct cell types by RT-PCR and western blot analysis. The cellular distribution was studied by immunofluorescence (IF). Functional relevance of ZEB1 overexpression was analyzed in an organotypic 3D culture system (OTC) of the distinct cell types representing the different steps of squamous carcinogenesis. To further study invasiveness spheroid cultures using the hanging-drop method were established. Results: RT-PCR revealed a downregulation of collagen XVII expression in In Vitro transformed cells followed by an upregulation in cancer cells. IF demonstrated that the cellular localization of collagen XVII and cathepsin B is influenced by malignant transformation, i.e. collagen XVII is first translocated to the cytoplasm and

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AGA Abstracts

AGA Abstracts

and phosphorylates FAK, facilitating FAK activation, while FAK in turn potentiates Akt activity. Exposure to 15 mmHg increased extracellular pressure stimulated serine phosphorylation of FAK (p-FAK) in Caco-2 and primary human colon cancer cells isolated from surgical specimens. This pressure-induced increase in serine p-FAK was blocked by Akt inhibitor and by siRNA silencing of Akt1 but not by silencing Akt2. Co-precipitation demonstrated that Akt associates directly with FAK. Akt-FAK association was increased by pressure and this increased association was blocked by inhibiting FAK or silencing Akt1 but not Akt2. Scanning the FAK sequence with Scansite software revealed three serine-containing consensus sequences for AKT phosphorylation in the FAK sequence. We therefore constructed a FAK non-phosphorylatable mutant with point mutations (S®A) at these three putative serine phosphorylation sites (S517/601/695) of FAK by Akt to investigate their relevance to pressurestimulated cell adhesion and tyrosine p-FAK. Indeed, overexpression of the triple mutant of FAK (S517/601/695®A) in Caco-2 cells, in contrast to wild type FAK, prevented the increase in p-FAK at Y397 and cancer cell adhesion induced by extracellular pressure. These results suggest that Akt regulates pressure-induced cancer cell adhesion by binding directly to and phosphorylating FAK at S509/601/695. This serine phosphorylation, in turn, permits the pressure-dependent tyrosine autophosphorylation of p-FAK at Y397, the conventional initiator of FAK activation. Although Akt is therefore required for FAK activation in response to pressure, further studies demonstrated that FAK also potentiates Akt activation. Blocking or silencing FAK by three different FAK-specific siRNA sequences prevented the increases in serine p-Akt (S473) and tyrosine p-FAK(Y397) induced by increased pressure. Thus, FAK and Akt bind directly and potentiate each other's activation. This novel mechanism of FAKAkt interaction suggests that FAK and Akt1 may be important dual therapeutic targets for preventing cancer cell adhesion, and eventually cancer metastasis.