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sdiffered between Responders and Non-Responders at the P<0.05 level. OTUs and metaboliteswhich were found to share significant correlations (q < 0.10) are highlighted in the Figure.Conclusions: Fecal metabolite composition differed between Responders and Non-Respond-ers to a low fermentable substrate dietary intervention and are associated with differencesin microbiome composition in children with IBS. Microbial factors such as gut microbiomecomposition and fecal metabolites may relate to dietary intervention efficacy. Further studiesare needed to investigate the potential relationship between fecal metabolites and symptomgeneration/amelioration.

Metabolite and Microbial OTU Correlations seen in Responders vs. Non-Responders to aLow Fermentable Substrate Diet

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Primocolonizing Escherichia coli Impact Colonic Epithelial Homeostasis inGnotobiotic RatsJulie Tomas, Julie Reygner, Camille Mayeur, Robert Ducroc, Chantal Bridonneau, Jean-Baptiste Cavin Cavin, Muriel Thomas, Philippe Langella, Claire Cherbuy

At every birth associations with the gut microbiota are challenged as the newborn passesfrom a protected site to a densely colonized environment. The first cells that to be confrontedby this first colonization are intestinal epithelial cells, mainly in the colon where the bacterialdensity reaches highest values. Our aim was to investigate the effects of primocolonizingbacteria on the colonic epithelium. We isolated five dominant bacteria from the intestinalcontents of conventional suckling rats under selective media, Escherichia coli, Enterococcusfaecalis, Lactobacillus intestinalis, Clostridium innocuum and Fusobacterium varium, andtransferred them in different combinations into germfree (GF) adult rats. Colonic epithelialcell responses were monitored at various time points up to 21 days. Proliferative cell markers(Ki67, PCNA, PH3, cyclin A) were higher in rats monocolonized with E. coli than in GFat all time points. In parallel, the MUC2 content in goblet cells rapidly declined andmucus layer doubled suggesting the release of MUC2 cellular contents shortly after E. coliadministration. Seven days post-inoculation, the epithelial MUC2 content returned to base-line, following an increase in KLF4 and in the cell cycle arrest-related proteins p21CIP1and p27KIP1. Markers of colonic differentiated cells involved in electrolyte (CAII andslc26A3) and water (aqp3) transport, and secretory responses to carbachol were also modu-lated after E. coli inoculation suggesting that ion transport dynamics was also affected. E.coli had a larger effect than the other isolated bacteria on colonic proliferation. Indeed theproliferative effect was observed when E. coli was associated with the four other isolatedstrains whereas there was no such effect in the absence of E. coli. We report the prominentrole of E. coli relative to other primocolonizing bacteria we have isolated and gain newinsights on the involvement of the epithelial responses in the dynamic of homeostatic host-microbe interactions. We show that administration of E. coli elicits sequential remodelingof the colonic epithelium affecting the dynamics of proliferation, of mucus productionand of ionic movements without altering colonic epithelial barrier but resulting in a newhomeostatic state.

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Investigating the Mechanisms That Drive Invasion in p120-Catenin KnockoutEsophageal KeratinocytesJulie Masse, Patricia A. Welsh, Heather L. Lehman, Douglas B. Stairs

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis due to its detection ata late stage combined with the highly locally invasive and metastatic nature of the disease.Thus, it is crucial to understand the mechanisms involved in the very invasive nature ofESCC. To that end, we have focused our research on a cell adhesion protein, p120-catenin(p120ctn). p120ctn is a tumor suppressor that is frequently down-regulated in many tumors,suggesting a central role for this protein in the development of cancer, including ESCC. Inthe conditional p120ctn knockout mouse, loss of p120ctn in the esophagus results in thedevelopment of invasive squamous cell carcinoma. Given this observation and that 60% ofhuman ESCC has decreased or lost p120ctn expression, we are interested in exploring thepathways that lead from p120ctn depletion to invasion. In order to mechanistically studythis, we generated cell lines from esophagi of p120ctnloxP/loxP mice and deleted p120ctnthrough expression of Cre recombinase in these cells (p120ctnKO cells). These p120ctnnull cells have increased migration and invasion capabilities compared with control cells,supporting a role for p120ctn loss in tumor progression. To explore gene expression changesthat occur after p120ctn loss, we performed a gene expression microarray and identifiedcandidate genes that regulate cellular invasion. Our analysis identified increases in ITGA2and MMP11, genes known to be involved in invasion in other cancer types. Based on thesegenes, we created a novel, putative signaling pathway that links p120ctn loss to invasion

S-14AGA Abstracts

(Figure 1). Loss of p120ctn was previously known to activate the transcription factor AP-1 through phosphorylation of c-jun and Western blot analysis of p120ctnKO cells revealsan increase in phospho-c-jun levels compared with control cells. ChIP experiments demon-strate that phospho-c-jun binding on the ITGA2 promoter is increased in p120ctnKO cells.ITGA2 expression is subsequently increased in p120ctn null cells as observed by Westernblot. Likewise, MMP11 protein expression is increased in p120ctnKO cells. ShRNA-mediatedknockdown of either ITGA2 or MMP11 results in decreased invasion of p120ctnKO cells.Conversely, overexpression of ITGA2 in normal esophageal keratinocytes results in increasedinvasion. To extend these in vitro experiments, we analyzed ITGA2, MMP11 and phospho-c-jun protein expression by immunohistochemistry (IHC) in our p120ctn knockout mice.Increased expression of all three proteins is detected in tumors arising in our mice. IHC onhuman ESCC and normal esophagi demonstrate an enriched expression of ITGA2 andMMP11 when p120ctn is decreased or lost. In summary, we have identified a novel, keypathway involving the loss of p120ctn that may mediate cancer cell invasion in human ESCC.

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PTK6 Antagonizes the EMT in Colon Cancer Cell LinesPriya S. Mathur, Rosa M Xicola, Xavier Llor, Angela L. Tyner

Background & Aims: Protein Tyrosine Kinase 6 (PTK6, also referred to as BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinaltract, and prostate. Dedifferentiation, loss of adhesive constraints, and enhanced motilityare hallmarks of metastatic cells, driven by the epithelial-mesenchymal transition (EMT).The human colon adenocarcinoma cell lines SW480 and SW620 represent an epithelial-like primary colon tumor and a mesenchymal-like metastasis from the same patient, respec-tively. PTK6 is highly expressed in epithelial-like SW480 cells and significantly reduced inmesenchymal-like SW620 cells. Stable PTK6 knockdown in SW480 cells results in decreasedE-cadherin expression and increased ZEB-1 and Vimentin, similar to SW620 cells. Wehypothesize that PTK6 promotes the epithelial phenotype and its loss leads to the EMT andmetastasis in colorectal cancer. We will investigate the role of PTK6 in EMT through (1)manipulating its expression in cell lines, (2) injecting PTK6-knockdown and overexpressingcells into immune-compromised mice for in vivo metastasis studies, and (3) analyzing patientcolon tissue and tumor samples for correlation of PTK6 expression with tumor invasiveness.Results: Knockdown of PTK6 in SW480 cells drives a mesenchymal and metastatic phenotypecharacterized by increased proliferation, migration, and invasion; as well as resistance toanoikis and robust anchorage-independent growth. Ptk6 knockout in immortalized youngadult mouse colon (YAMC) epithelial cells also yields an EMT phenotype. Reintroductionof PTK6 in metastatic SW620 cells shows a potential reversal of the EMT phenotype, restoringexpression of E-Cadherin and retarding cell proliferation. Analysis of tissue samples fromcolon cancer patients finds decreased PTK6 expression in high-grade tumors. Xenograftstudies with engineered cell lines are ongoing. Conclusions: In colon epithelial and cancercells, PTK6 is required for the maintenance of the epithelial phenotype, which is characterizedby markers of differentiation, contact-inhibited growth, a lack of migratory and invasivebehavior, and sensitivity to anoikis. Loss of PTK6 results in EMT characterized by a loss ofdifferentiation and increased invasiveness. The EMT provides a mechanism for tumor metas-tasis. This study may provide insight into the mechanisms that control colon cancer metastasisand could lead to the development of novel therapeutic approaches.