AGA Abstracts following tamoxifen induction. Once again, lineage tracing of all the epithelial cells within marked glands was observed up to 52 weeks following tamoxifen induction, suggesting that K19 likely marks a common progenitor or stem cell in the antrum, intestine and colon. Interestingly, in the small intestine and antrum K19 positive cells do not overlap with Lgr5- GFP+ cells yet clearly demonstrate stem cell properties. Conclusions. Using K19CreERT2/ Rosa26r mice, we show that K19 marks pit progenitors in the gastric corpus, as well as stem cells within the antrum, intestine and colon. These findings identify a novel population of stem cells for the lineage tracing of gastric pit cells following gastric injury or cancer, and for tracing epithelial cells in the development of intestinal or colonic cancer. Tu1769 Villin-Marked Gastric Epithelial Progenitor Cells Do Not Give Rise to Tumors or Metaplastic Cells in a Variety of Mouse Models Xiaotan T. Qiao, Linda C. Samuelson, Andrzej Dlugosz, Juanita L. Merchant, Deborah L. Gumucio The gastric epithelium constantly self-renews, but until recently, the cells that drive gastric gland homeostasis and regeneration were unknown. Two markers of gastric stem cells have now been identified (Lgr5 and the villin promoter). The villin promoter is active in single cells in a subpopulation of antral glands. These villin promoter-marked cells are normally quiescent, but after stimulation with interferon gamma, they divide first symmetrically and later asymmetrically to replenish entire gastric glands. Thus, villin-marked cells are gastric progenitor cells (V-GPC) that appear to respond to stress conditions, but are likely not the major stem cells responsible for homeostatic maintenance of the antral epithelium. Lgr5- positive cells also repopulate entire glands In Vivo and isolated Lgr5-positive cells ex vivo grow into organoid units that resemble antral glands. Thus, at least some Lgr5 positive cells are also gastric progenitors (L-GPC); their higher proliferative activity and greater number indicates that these cells likely maintain antral glands under normal physiologic conditions. Interestingly, both L-GPC and V-GPC are enriched at the squamous-glandular border, a finding that may be relevant to the tendency of this region to develop metaplasia and adenocarcinoma. In the antrum, single V-GPC reside primarily at the isthmus and migrate to the gland base after division, while L-GPC always appear at the gland base, an environment characterized by high Wnt signaling. Indeed, increased Wnt signaling stimulates V-GPC to divide and APC loss in L-GPCs results in pyloric adenomas. We have now tested the ability of V-GPC to give rise to tumors or metaplasias in a variety of settings (H. pylori infection, gastrin null mice, Cdx2 overexpressing mice, H/K-Atpase-Ctox7 mice, MNU treated mice). We find no evidence that V-GPC can contribute to metaplasia or tumors in these settings. Thus, V-GPC proliferate in response to different stresses, but do not appear to represent cancer stem cells. Tu1770 Celecoxib Inhibits CD133-Positive Cell Migration via Reduction of CCR2 in Helicobacter pylori-Infected Mongolian Gerbils Seiji Futagami, Hiroyuki Nagoya, Mayumi Shimpuku, Tetsuro Kawagoe, Tomotaka Shindo, Masafumi Kusunoki, Katya Gudis, Kazumasa Miyake, Choitsu Sakamoto Background/Aims: To see whether celecoxib prevents gastric cancer occurrence by disrupting the progression of chronic gastritis into gastric carcinoma through its inhibition of the migration of CD133-positive cells, one of the surface markers of bone marrow derived cells, in H. pylori-infected gerbils. Methods: 70 gerbils were divided into six groups. Group 1 gerbils served as control (n=6). 10 gerbils were given N-Methyl-N-nitrosourea (MNU), 30ppm (Group 2). 6 short-term H. pylori-infected gerbils (Group 3) were sacrificed after 8 weeks of H. pylori infection and 6 long-term H. pylori-infected gerbils were sacrificed after 42 weeks of H. pylori infection (Group 4). 20 gerbils were given MNU pretreatment and long-term H. pylori infection (Group 5). In addition, after H. pylori inoculation, 22 gerbils also received a celecoxib in their diet (Group 6). CD133 and CCR2 expression in gastric tissues was evaluated by western blot analysis and immunostaining. Results: CD133-positive cells were mainly localized in the bottom of the gastric epithelial cells. CD133-positive cells also migrated into gastric cancer tissues in this model. CD133-positive cells in MNU- pretreated H. pylori-infected gerbils were significantly increased than those in H. pylori short-term infected gerbils. Celecoxib treatment significantly reduced CD133-positive cell migration and CCR2 expression levels. CD133- and CCR2-positive cells were co-localized in H. pylori-infected gastritis and gastric cancer tissues. Celecoxib treatment significantly reduced the number of CD133 and CCR2 positive cells. Conclusions: Celecoxib inhibits CD133-positive cell migration via the reduction of CCR2 in this model. Further studies are needed to clarify the precise mechanisms driving H. pylori infection-induced CD133-positive cell migration and its link to the progression of chronic gastritis into gastric cancer. Tu1771 The Discovery of CD44R as a Marker for Gastric Cancer Stem Cells Eileen Teng, Wen Min Lau, Hui Shan Chong, Kirsten A. Lopez, Amy Y. Tay, Manuel Salto-Tellez, Ting Ting Wang, Bok Yan J. So, Shing Leng Chan The cancer stem cell hypothesis proposed that in many cancers, cells in the tumors are hierarchically organized and sustained by cancer stem cells (CSCs) that are implicated in chemoresistance, radioresistance and tumors relapse. Gastric CSCs are yet to be identified and characterized from clinical samples. In this project a candidate approach was taken to isolate putative gastric cancer stem cells from gastric cancer specimens. Methods: To expand the tumor material, we generated xenograft tumors directly from fresh tumor pieces. Histolog- ical analyses verified that the xenograft tumors recapitulate those in the patient samples. Cells dissociated from the xenograft tumor were then subjected to CSC identification assays. Results: Three lines of xenograft tumors were generated from tumor tissues of well, moderately and poorly differentiated histological subtype of gastric cancers. Analysis of cells isolated from the various xenografts reveals different frequencies of CD44-expressing cells that are characteristic of each xenograft line. We showed that gastric CSCs are robustly enriched by S-830 AGA Abstracts a combination of two surface markers: EpCAM and a spliced variant of CD44, CD44R1. In GC16 xenograft line where the frequency of CD44 expressing cells is 2%, sorting the cells using CD44 will enrich the cancer initiating cells by more than 100 fold. Whereas in GC 21 and GC38 where frequency of CD44 cells is 35% and 26% respectively, CD44 marker only enriches the cancer initiating cells by 2 and 3.5 fold. The frequency of EpCAM+CD44R1+ cells appears to be highly variable between different gastric tumors and it predicts the tumor initiation potency of unsorted cells isolated from these tumors. In contrast to total CD44, CD44 standard and CD44v6, CD44R1 is uniquely up-regulated in cells isolated from tumor sites of gastric cancer samples. Conclusion: CD44 expression predicts poor prognosis for gastric cancer patients, but CD44 antibodies cross react with many other cell types such as leukocytes and myofibroblasts. The presence of CD44R1 may present a window in the targeting of gastric cancer stem cells. Tu1772 Cancer Stem Cells: Implications for Age-Related Rise in Colorectal Carcinogenesis Jyoti Nautiyal, Shailender S. Kanwar, Bonita J. Leavell, Jianhua Du, Bhaumik B. Patel, Edi Levi, Adhip P. Majumdar One of the most consistent pathological conditions in the gastrointestinal (GI) tract with advancing age is malignancy, particularly GI cancers, the incidence of which increases sharply during aging. The regulatory mechanisms for this phenomenon are poorly understood. However, recent evidence supports the contention that epithelial cancers including colorectal cancer are diseases driven by self-renewing cancer stem cells (CSCs). Recently, we reported the presence of colon CSCs in macroscopically normal mucosa in patients with adenomatous polyps which increase with aging (BBRC-2009-378:344), suggesting a role for CSCs in the development of colorectal neoplasia. In support of this, we have observed that mucosal cells isolated from macroscopically normal colonic mucosa of patients with adenomatous polyps form spheres In Vitro in a defined stem cell media (one of the properties of CSCs) and that the number of spheres formed is higher in patients with ≥ 3 polyps than those with < 3 polyps. Although the reasons for the age-related rise in colon carcinogenesis are not fully understood, we hypothesize that aging increases susceptibility of the colon to carcinogen(s)/ toxicant(s) leading to increased CSCs in the colonic mucosa. We have observed that mucosal cells isolated from the colon of aging Fischer-344 rats show higher propensity to grow in stem cell media and proliferate to form bigger clusters/spheres than those derived from young rats. This phenotypic change, representative of CSC, becomes more pronounced in aged animals in response to the colonic carcinogen dimethylhydrazine (DMH). Furthermore, the proportion of CSCs in colonic crypts increases significantly with aging as evidenced by increased expression of CD44 and CD166 in the colonic mucosa and that administration of DMH causes further increase in these parameters. Although CD44 and CD166 are putative markers of CSCs, their co-expression is considered to be more tumorigenic. We have observed that DMH treatment leads to increased co-localization of CD44 and CD166 in the colonic crypts of aged rats. Moreover, DMH treatment leads to a significantly greater proportion of colonic mucosal cells that exhibit dual expression of CD44 and CD166 in aged than in young animals, compared to their corresponding controls. In conclusion, our data suggest that aging leads to increase in CSCs that may play a pivotal role in the age-related rise in colorectal cancer. Tu1773 Non-Canonical Bone Morphogenetic Protein Signaling Induces Epithelial to Mesenchymal Transition in SMAD4 Deficient Colorectal Cancers Philip W. Voorneveld, Liudmila L. Kodach, Rutger J. Jacobs, Daniel W. Hommes, Gijs R. van den Brink, Maikel P. Peppelenbosch, James C. Hardwick INTRODUCTION/OBJECTIVES: Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is induced during carcinoma progression allowing cancer cells to invade and metastasize. In patients with colorectal cancer (CRC) negative expression of SMAD4, the main component of canonical SMAD-dependent Transforming Growth Factor (TGF)-β and Bone Morphogenetic Protein (BMP) signaling, has been correlated with poor survival. It is believed that SMAD4-negative cancers metastasize due to loss of function of the canonical tumour-supressor arm of the BMP and TGF-β pathways, but the role of non-canonical pathways in this process is poorly understood and underscored. AIMS & METHODS: In this study we have for the first time extensively characterized non-canonical BMP signaling and investigated its role in EMT induction in the context of SMAD4 deficiency. HCT116 SMAD4-/- and SW480 (mutant for SMAD4) cells were transfected with a plasmid encoding BMPR2 to overactivate non-canonical BMP signaling. Kinome arrays were performed to generate a comprehensive description of non-canonical BMP signaling employing a peptide array containing 1176 kinase consensus substrates and these results were validated with immunoblotting and luciferase assays. The induction of EMT was analysed by phase contrast microscopy, immunofluorescence, qRT-PCR and immunoblotting. Migration and invasion was evaluated by wound healing and invasion through matrigel. RESULTS: Induction of non-canonical BMP signaling results in the activation of well known cell migration pathways Rac-MKK-p38MAPK, Rac-MKK-JNK, RHO-Rock and RAS-B-RAF-ERK. Moreover, AKT and WNT pathways, responsible for survival and proliferation of cancer cells, are significantly upregulated. Activation of non-canonical BMP signaling induces changes in cell morphology and gene expression consistent with EMT. SW480 and HCT116 SMAD4-deficient cells but not parental HCT116 SMAD4-proficient cells respond with inhibition of the E-cadherin expression, upregulation of the Vimentin expression and activation of transcription factors responsible for EMT (ZEB1, SNAIL1, SLUG and SIP1), and increased migration and invasion in functional assays on BMPR2 transfection. CONCLUSION: Non-canonical BMP pathway activation in SMAD4-negative cancer cells results in an activation of a signaling network potentiating migration, increasing cancer cell survival and facilitating invasion and metastasis. Our data suggest that the intact non-canonical BMP signaling that remains after loss of canonical BMP signaling due to loss of SMAD4 is responsible for the poor survival of patients with SMAD4-negative CRC. Inhibitors of non-canonical BMP signaling could be a new promising therapeutic option for patients with SMAD4-negative CRC.