Sa1666 DARPP-32 PROMOTES ACTIVATION OF STAT3 THROUGH IGF1R-SRC AXIS IN HUMAN AND MOUSE GASTRIC TUMORIGENESIS Shoumin Zhu, Mohammed Soutto, Zheng Chen, Wael M. El-Rifai Background: We have previously shown that Dopamine and cAMP-regulated phosphopro- tein, Mr 32000 (DARPP-32), is a novel cancer gene overexpressed in 2/3 of gastric cancer patients. We reported that DARPP-32 promotes cancer cell survival, drug resistance, and invasion. Activation of STAT3, signal transducer and activator of transcription 3, is important for tumorigenesis and drug resistance. Among factors that regulate STAT3, SOCS3 acts as a negative regulator of STAT3 to prevent its uncontrolled oncogenic activation. In this study, we aimed to investigate if DARPP-32 regulates STAT3 and determine the underlying molecular mechanisms. Methods: Using human and mouse models, in vitro assays, and 3D organoid gastric gland cultures, we report that DARPP-32 promotes phosphorylation, nuclear localiza- tion, and transcription activation of STAT3. Results: Overexpression of DARPP-32 in AGS gastric cancer cells increased phosphorylation of STAT3 (Y705) and activation of STAT3 luciferase reporter (P<0.01), and decreased SOCS3 protein expression. We provide evidence showing that DARPP-32 promotes activation of IGF1R-SRC axis, critical for phosphorylation and degradation of SOCS3, a negative regulator of STAT3 activity. Proximity ligation and co-immunoprecipitation assays indicated that DARPP-32 and IGF1R co-exist in the same protein complex where DARPP-32 binding to IGF1R promotes its phosphorylation with activation of downstream SRC and STAT3. Using Tff1-knockout (KO) mouse model of gastric cancer, we detected DARPP-32 overexpression and STAT3 phosphorylation in the early stages of gastric tumorigenesis. Although the Darpp32 KO mouse model did not show histological lesions in gastric mucosa, the Darpp32 KO /Tff1 KO gastric mucosa demonstrated lack of phosphorylation and nuclear localization of STAT3 with significant delay in the onset of tumor lesions. Immunohistochemistry analysis confirmed overexpression of DARPP-32 and nuclear localization of STAT3 in human gastric cancers. Conclusion: The in vitro studies indicate that DARPP-32 plays a critical role in activation of STAT3 signaling through regulation of IGF1R and SRC, leading to SOCS3 degradation. In vivo results suggest that lack of DARPP-32 delays the development of tumors in the TFF1 knockout mouse model but is not sufficient alone to abrogate the full carcinogenesis cascade. These novel findings add to the oncogenic functions of DARPP-32 and highlight its potential role in the pro- inflammatory gastric carcinogenesis cascade. Sa1667 PROFILING OF RNA EXPRESSION IN COLITIS ASSOCIATED CANCER (CAC) - A COMPARATIVE STUDY IN IBD AND CAC PATIENTS Maximilian Sehn, Britta Siegmund, Martin E. Kreis, Sefer Elezkurtaj, Michael Hummel, Michael Schumann Introduction Crohn's disease (CD) and ulcerative colitis (UC) are highly prevalent in the western world and are associated with an increased risk for the development of colon carcinoma. Malignant transformation from colitis to colitis-associated carcinoma (CAC) follows pathways different from the adenoma-carcinoma-sequence known from sporadic colon cancer. However, human studies to elucidate CAC-genesis are scarce and most of what is known in CAC-pathogenesis comes from mouse models imitating this complex disease. With our present study we aimed to contribute to the understanding of CAC- development by comparative analysis of gene expression in human samples. Material and Methods: RNA was isolated from microdissections done in surgical colon specimen of 60 patients suffering from either UC-colitis, Crohn's colitis, UC- or CD-related colitis-associated cancer or sporadic colon cancer. Inflammation-free colonic mucosa of patients undergoing elective sigmoid resections after diverticulitis served as healthy control tissue. A defined set of 624 immunology barrier and polarity genes was examined using the amplification-free Nanostring nCounter TM technology. Primary data analysis and statistics were performed using the nSolver TM data analysis software. Further analysis included grouping of expression patterns (heatmap), differential expression and pathway analysis. Results: Heatmaps gener- ated from expression data revealed close-to-optimal grouping recapitulating the clinical subgroups. Differential expression comparing Crohn's colitis with Crohn's-associated CAC and UC colitis with UC-associated CAC was adjusted for inflammatory-related expression and identified 203 and 271 differentially expressed genes, respectively. Major upregulated genes (UC-CAC vs. UC and CD-CAC vs. CD) included those forming distinct immune cell populations as osteopontin/SPP1 (8fold and 18fold), the T-cell activating protein Thy-1/ CD90 (2,8 fold only in CD-CAC) and Eotaxin3/CCL26, known to regulate myofibroblasts (2,9 fold and 3,4 fold). Downregulated genes included TACI/TNFRSF13B and BAFF/TNFRSF a member of the TNF-receptor superfamily known to induce NFKB (CAC UC vs UC: -7,3 and CAC CD vs CD -5,4 respectively). Conclusion: We provide a comprehensive quantitative RNA analysis from human colitis associated colon carcinoma tissue. Targets known to influence immunologic as well as oncological processes were for the first time detected in S-347 AGA Abstracts CACs. Their characteristic expression pattern seems to highlight their role in the colitis associated carcinogenesis. Sa1668 EPITHELIAL CELL DERIVED CCL11 PLAYS A KEY ROLE IN COLITIS- ASSOCIATED CARCINOGENESIS Lori A. Coburn, Kshipra Singh, Dina Polosukhina, Daniel Barry, Mohammad Asim, Dana M. Hardbower, Kay Washington, Keith T. Wilson Background: We have shown that CCL11 (eotaxin-1), an eosinophil chemoattractant, is significantly increased in the serum of ulcerative colitis and Crohn's disease patients vs. controls. In addition, CCL11 is increased in dextran sulfate sodium (DSS)-induced murine colitis, and Ccl11 -/- mice are protected in the acute DSS colitis model. We have found that in response to azoxymethane (AOM)-DSS, Ccl11 -/- mice have significantly decreased colonic tumor number and burden compared to wild-type (WT) mice, as well as decreased histologic injury and colonic eosinophil infiltration, and alterations in cytokines/chemokines associated with activated macrophages in the tumor area. Our aim was to assess for baseline alterations in macrophage polarization in Ccl11 -/- mice and to determine if hematopoietic cell- or epithelial cell-derived CCL11 plays a key role in colitis-associated carcinogenesis (CAC). Methods: C57BL/6 WT and Ccl11 -/- mice were exposed to AOM 12.5 mg/kg i.p., followed by 3 cycles of 4% DSS in the drinking water for 5 days followed by 16 days of water, for a total of 77 days. Colonic epithelial cells (CECs) and lamina propria (LP) cells were isolated, and Ccl11 mRNA expression assessed by real-time PCR . Naïve WT and Ccl11 -/- bone marrow- derived macrophages (BMmacs) were exposed to either classic M1 stimuli (LPS and IFN- γ) or M2 stimuli (IL-4 and IL-10) for 24 h, mRNA was isolated and assessed by real-time PCR. Bone marrow transplants were performed where hematopoietic cells from WT or Ccl11 -/- mice were transferred to irradiated WT or Ccl11 -/- mice. After 8 weeks of recovery, mice were exposed to the 77-day AOM-DSS model. Macroscopic tumor number and size were assessed. Results: Exposure to the AOM-DSS model induced a 27-fold increase in Ccl11 mRNA expression in isolated CECs (p<0.001) and a 8-fold increase in LP cells (p<0.05). WT BMmacs exposed to either classical M1 or M2 stimuli led to significant increases in Ccl11 mRNA levels (p<0.05), which were not present in Ccl11 -/- BMmacs. However, WT and Ccl11 -/- BMmacs exhibited similar responses to M1 stimuli or M2 stimuli as assessed by the M1 markers: Nos2 and Tnfa and M2 markers: Arg1 and Relma. In the bone marrow chimeras, Ccl11 -/- recipient mice had significantly reduced tumor number and tumor burden compared to WT recipient mice (p<0.05) ( Table 1). Conclusions: While the decreased tumorigenesis with CCL11 deletion in the AOM-DSS model is associated with decreased histologic injury and decreased macrophage associated-cytokines/chemokines, studies in BMmacs suggest no apparent alteration in M1 or M2 activation patterns. However, studies in bone marrow chimera mice revealed that Ccl11 -/- recipient mice reproduced the pattern of protection with both decreased tumor number and burden. Together these findings indicate that epithelial cell-derived CCL11 is a key mediator of tumorigenesis in CAC. Table 1 *p<0.05 vs. WT to WT AOM-DSS Sa1669 THE SYNTHETIC FLAVAGLINE FL3 PROTECTS AGAINST COLITIS- ASSOCIATED CANCER VIA INHIBITION OF WNT/β-CATENIN SIGNALING Dakota N. Jackson, Jie Han, Hussein Abou-Hamdan, Laurent Desaubry, Xiaofang Huo, Qiuyang Zhang, Rhonda F. Souza, Arianne L. Theiss Introduction: Flavaglines are natural plant products that have anti-inflammatory and anti- cancer effects. In earlier studies, we showed that FL3 (a synthetic flavagline) protected mice from developing colitis induced by dextran sodium sulfate (DSS). Patients with chronic colitis are at risk for developing colitis-associated colorectal cancer (CAC). Hypothesizing that FL3 might protect patients with ulcerative and Crohn's colitis from CAC, we studied the ability of FL3 to prevent CAC development in an azoxymethane (AOM)-DSS mouse model, and we explored the mechanism underlying this CAC-protective effect in vitro . Methods: To induce CAC, 8 week old wild-type C57BL/6 mice were injected with AOM (7.6 mg/kg i.p.) followed by 2 cycles of 3.0% DSS in drinking water for 7 days, followed by 14 or 21 days of recovery. One day before and during DSS, mice were i.p. injected daily with FL3 (0.1 mg/kg) or vehicle. Colons were removed and scored for polyp numbers and size. Colon tumoroids established from AOM-DSS mice were treated with FL3 (10 nM) for 2 hr, followed by RNAseq analysis. After this analysis identified major FL3 effects on Wnt/ β-catenin signaling, we interrogated this pathway using human colon cancer cell lines that have β-catenin mutations (HCT-116), APC mutations affecting β-catenin degradation (SW480), or no such mutations (RKO). Cell lines were treated with FL3 (1 or 10 nM) for 16 hr. We measured β-catenin transcriptional activation by TCF/LEF luciferase reporter; β- catenin, pro-apoptotic Bax, and PCNA protein by Western blot; cell viability by LDH assay; apoptosis by TUNEL staining; and proliferation by MTT assay. Results: FL3-treated AOM- DSS mice developed fewer and smaller polyps than vehicle-treated control mice. Ingenuity Pathway Analysis of RNAseq data from FL3-treated tumoroids identified Wnt/ β-catenin as the canonical pathway most altered by FL3. In RKO (with intact Wnt/ β-catenin signaling), FL3 markedly decreased β-catenin protein expression and its transcriptional activation, inhibited proliferation, and increased apoptosis to the point of complete cell death. In contrast, FL3 had no effect on these readouts in β-catenin-mutated HCT-116 cells. FL3 increased apoptosis and decreased transcriptional β-catenin activation in APC-mutated SW480 cells, but to a lesser extent than in RKO. Conclusions: In a mouse model of CAC, AGA Abstracts