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Dietary Sulforaphane Ameilorates Dextran Sodium Sulfate-Induced Colitis andEnhances Sulindac-Induced Chemoprotection Against Colon Tumor Formationin MiceSohta Inoue, Atsushi Fukumoto, Risa Sumiyama, Akinori Yanaka

Background and Aims: It has been widely accepted that daily intake of NSAIDs affordschemoprevention against colon cancer. However, long term use of NSAIDs frequently causesserious problems, such as GI bleeding and cerebral vascular events. Thus, it is importantto minimize adverse effects of NSAIDs in cancer chemoprevention. Sulforaphane (SFN), asubstance rich in broccoli sprouts, shows anti-inflammatory effects by activating nrf2-medi-ated NF-κB inactivation (Oncogene, 24:4486, 2005). SFN induces apoptosis of cancer cellsIn Vitro (J Biol Chem 280:19911,2005). Thus, daily intake of dietary SFN may be safe anduseful approach to afford long term chemoprotection against inflammation-related coloncancer. Based on these backgrounds, we hypothesized if SFN could strengthen the chemopro-tective effects of NSAIDs, thereby we may be able to minimize the dose and the durationof NSAIDs for chemoprevention. In the present study, we examined if SFN modulatesNSAID-induced chemoprotection against colitis-associated colon tumor in mice. Methods:C57BL/6 female mice were treated twice with 1% azoxymethane (AOM), followed by for 4days treatment with 3% dextran sodium sulfate (DSS), as previously described. These micewere divided into the following 4 groups. Control Group; No further treatment. Sulindac(SLD) Group: treated with SLD alone. SFN Group; treated with SFN alone. SFN+SLD Group:treated with both SFN and SLD. SLD,160 ppm, was orally administrated during the first 8weeks after the DSS treatment. SFN-rich broccoli sprout juice, which contains 2.5 mmmolSFN, was orally administrated as the with ordinary diet throughout the experimental period.Mice were sacrificed at 8 and 16 wks after the DSS treatment. Degree of colitis, size andnumber of colon tumors were quantitatively evaluated. Antioxidant enzyme activity wasevaluated by measuring GST-P1, and HO-1 expression by real time RT-PCR. Apoptosis andcell proliferation were assessed by immunostaining with TUNEL and PCNA, respectively.Mucosal inflammation was assessed by histology and by measuring mucosal levels of TNF-α and IL-1β mRNA. Results: 1. SLD alone did not affect DSS-induced inflammation, butincreased TUNEL-positive cells, decreased PCNA staining, and inhibited tumor formationin colonic mucosa. 2. SFN alone decreased expression of TNF-α and IL-1β, mitigated DSS-induced colitis, and slightly attenuated tumor formation. 3. SFN + SLD attenuated DSS-induced colitis, and markedly suppressed colon tumor formation. Conclusions: Daily intakeof SFN improves DSS-induced colitis and enhances SLD-induced chemoprevention againstcolon tumor in AOM +DSS treated mice.

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MTGR1 Modifies the Tumor Microenvironment in the AOM/DSS ColitisAssociated Carcinoma ModelCaitlyn Whitten, Amanda D. Williams, Mary Kay Washington, Rupesh Chaturvedi, KeithT. Wilson, Scott W. Hiebert, Christopher S. Williams

Background: MTGR1 (Myeloid Translocation Gene, Related-1) is a member of a gene familyoriginally identified as targets of chromosomal translocation in acute myeloid leukemia(AML). Analysis of Mtgr1-/- mice revealed a surprising role for MTGR1 in intestinal differenti-ation, proliferation and wound healing programs. We recently reported that MTGs competewith β-catenin for TCF4 occupancy and repress TCF4 mediated transcriptional programs,potentially contributing to the phenotypes described in the Mtgr1-/- mice. Recently, 6 muta-tions have been identified in MTG family members in association with colorectal carcinoma.Based on these observations we hypothesized that MTGR1may modify inflammatory carcino-genesis(CAC) in the colon.Methods:We selected the azoxymethane(AOM)/dextran sodiumsulfate(DSS) rodent model for our studies as this is a robust model for interrogating geneticmodifiers of CAC. 19 Mtgr1-/- and 22 WT mice were injected with 12 mg/kg AOM followedby four cycles of 3% DSS ad lib. At necropsy, colons were isolated and tumor burden anddistribution scored. IHC for BrdU, β-catenin, COX-2, and immune subsets was performed.Tumor RNA was isolated and transcriptome profile generated and analyzed using the PAN-THER suite. Results: Mtgr1-/- mice had fewer and smaller tumors (1.7 vs 7.5 polyps percolon, p<0.001 and 27 vs 82 mm2, p=0.01). There was no difference in intra-tumoralproliferation, but significantly higher apoptosis in Mtgr1-/- tumors, potentially explainingthe difference in tumor burden (32 vs 13 TUNEL (+) cells/HPF, p<0.001). PANTHER analysisof mRNA expression array data revealed significant upregulation of innate and adaptiveimmune networks in the Mtgr1-/- tumors (p<0.001). In support of this observation, in theMtgr1-/- compared to WT tumors, we found increased T-cell infiltrate (49.4 vs 13.6 CD3+

cells/hpf, p<0.001), M1 and M2 macrophages (29.1 vs 14.1 IL-1β+/F4/80+ cells/hpf, p=0.001, and 15.8 vs 7.2 arginase1+/F4/80+ cells/hpf, p=0.003), B-lymphocytes (15.8 vs 6.0B220+ cells/hpf, p<0.0001), regulatory T cells (51.1 vs 25.0 Foxp3+ cells/hpf, p<0.0001),natural killer cells (51.0 vs 36.0 NKp46+ cells/hpf, p=0.003), andmyeloid-derived suppressorcells (13.3 vs 4.3 Gr1+/CD11b+ cells/hpf, p=0.0097). Interestingly, there were fewer dendriticcells and activated dendritic cells in Mtgr1-/- tumors (14.6 vs 23.7 total CD11c+ cells/hpf,p=0.02 and 7.0 vs 19.0 CD11c+/MHCII+ cells/hpf, p=0.04). Conclusions: These studiesprovide evidence that MTGR1 functions as a tumor modifier in intestinal carcinogenesis.We speculate that these effects may occur via modulation of anti-tumor immunity.

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KrüPpel-Like Factor 5 is a Crucial Mediator of Intestinal Tumorigenesis inMice Harboring Combined APC and KRAS MutationsMandayam O. Nandan, Amr Ghaleb, Beth B. McConnell, Vincent W. Yang

BACKGROUND: Both mutational inactivation of the adenomatous polyposis coli (APC)tumor suppressor gene and activation of the KRAS oncogene are common events in coloncancer. We previously showed that the pro-proliferative transcription factor Krüppel-likefactor 5 (KLF5) is important for mediating transformation upon respective APC inactivationand KRAS activation [Nandan et al. (2008) Gastroenterology 134:120; McConnell et al. (2009)Cancer Res. 69:4125]. AIM: To investigate the In Vivo effect of Klf5 heterozygosity on the

S-32AGA Abstracts

propensity for intestinal tumor formation and target gene expression in mice harboringcombined APC and KRAS mutations. METHODS: ApcMin mice were crossed with Klf5+/- miceto generate double heterozygous ApcMin/Klf5+/- mice. These double heterozygous mice werethen crossed with mice transgenic for an oncogenic KRASV12 under the control of the villinpromoter and the resulting progeny sacrificed at 12 weeks of age. All mice were maintainedon a C57BL/6 background. Intestinal and colonic tissues were collected and prepared forimmunohistochemical analyses for KLF5, β-catenin, cyclin D1, Ki67, pMEK and pERK1/2.RESULTS: At 12 weeks, ApcMin/KRASV12 mice had a 3-fold higher small intestinal adenomaburden than ApcMin mice. The number of tumors per mouse in ApcMin/KRASV12 mice wasreduced on average by 92% when compared to the tri-transgenic ApcMin/KRASV12/Klf5+/-

mice. The tumor number in the tri-transgenic mice was also reduced by 75%when comparedto ApcMin mice. The number of colonic adenomas among the three genotypes was notsignificantly different. The level of Klf5 was elevated in the normal-appearing small intestinalmucosa from both ApcMin and ApcMin/KRASV12 mice compared to wild type mice. Thisincrease was attenuated in the tri-transgenic ApcMin/KRASV12/Klf5+/- mice. There was also adecrease in the levels of β-catenin, cyclin D1 and Ki-67 in the normal-appearing intestinalmucosa of the tri-transgenic mice when compared to the ApcMin/KRASV12 mice. Levels ofpMEK and pERK1/2 were increased in the normal intestinal mucosa of the ApcMin/KRASV12

mice compared to ApcMin and wild type mice. These proteins were modestly reduced in thetri-transgenic mice. Tumor tissues displayed higher levels of Klf5 and β-catenin, irrespectiveof the mouse genotype. CONCLUSIONS: Results of the current study confirm the cumulativeeffect of APC loss and oncogenic KRAS activation on intestinal tumorigenesis. The drasticreduction in tumor burden secondary to Klf5 heterozygosity in ApcMin/KRASV12 mice indicatesa critical function of KLF5 in modulating intestinal tumor formation due to combined APCand KRAS mutations.

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A Novel Hybrid In Vitro - Orthotopic Transplant Model for Assessment ofExperimental Colon Cancer TherapeuticsLydia Lee, Larissa Georgeon Richard, Michael P. Richardson, Wei Y. Chen, Jatin Roper,Eric S. Martin, Kenneth E. Hung

Careful assessment of experimental therapeutics is crucial prior to clinical trials. Whereasmany discovery programs rely on In Vitro screening of human colon cancer cell lines, thesemay not ultimately translate to robust efficacy in patients. This is not surprising consideringthat many validation studies are performed in xenograft models that transplant high passagecell lines into immunodeficient mice at ectopic sites different from the endogenous colonicmicroenvironment. To address this, we have developed a novel discovery program comprisedof In Vitro screening of low passage mouse colon cancer cell lines followed by In Vivovalidation by orthotopic transplant in the distal colonic mucosa of immunocompetent mice.To generate tumors that are robust surrogates for human sporadic colon cancer, we usedgenetically engineered mouse models similar to those that we have previously described inwhich floxed Apc mice are surgically modified by adenovirus expressing cre recombinase,resulting in discrete tumors restricted to the distal colon. To dissect the efficacy of experi-mental therapeutics in the context of Kras mutant and wild-type tumors, we infected micebearing floxed Apc and p53 alleles (A/P) and an additional latent mutant Kras allele (A/K/P). 13/15 (87%) of the resulting tumors were identified as adenocarcinomas. PCR genotypingconfirmed the inactivation of the Apc and p53 genes in primary A/P colonic tumors andthe additional activation of the latent Kras allele in A/K/P. Western blot analysis demonstratedincreased p-ERK in A/K/P tumors. To generate cell lines, tumors were individually harvestedand enzymatically dissociated. Single cell suspensions and cellular aggregates of isolatedcolonic epithelial cells were plated on collagen-coated dishes; passaged for several weeks toexpand the epithelial tumor cell population under defined media and culture conditions;and assessed for growth, viability, and tumorigenicity. Biochemical and genetic characteriza-tion was performed as with the respective primary tumors. To create an In Vivo orthotopictransplant model, respective tumor cell lines were harvested in log phase growth andtransplanted to the distal colon employing similar protocols as with the adenoviral infection.Colonic tumors were visualized by murine colonoscopy within three weeks. The findingsof combination drug therapy using this composite platform will be presented. In summary,we have developed a hybrid screening program composed of high throughput In Vitroscreening and In Vivo orthotopic transplant in the native colonic environment. These toolsprovide an ideal drug discovery platform for future colon cancer therapeutics.

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Changing Patterns of Clarithromycin and Metronidazole Resistance AmongstHelicobacter pylori Strains in a Reference CentreAnthony O'Connor, Ikue Taneike, Asghar Qasim, Humphrey J. O'Connor, Barbara M.Ryan, Niall Breslin, Colm A. O'Morain

Introduction: Helicobacter pylori causes much pathology including peptic ulcers and gastriccancer. Eradication rates have fallen over recent years. Antibiotic resistance is thought tobe rising and understanding this phenomenon is important in efforts to eradicate H. pylori.Aims: Antibiotic treatment should be tailored to resistance levels. Our study examined levelsof resistance to metronidazole and clarithromycin in H. pylori isolates in a reference centrein Ireland from 2007 and 2008 and compared them to a similar cohort from a study in 1997.Method: Antimicrobial susceptibilities were tested by E-test. Frequencies of spontaneousmetronidazole and clarithromycin resistance were measured on agar plate containing theantibiotics at concentrations of 2x and 4x MIC value. Clinical data was obtained from charts,laboratory and endoscopy reports. Results: 222 patients were analysed, 98 were females.37 had prior attempts at eradication therapy (all with amoxicillin-clarithromycin-PPI) .31.5% had strains resistant to Metronidazole. 13.2% were noted to have strains resistant toclarithromycin. 8.6% had strains resistant to both agents. Clarithromycin resistance is 9.3%in those who have not had prior eradication therapy compared to 32.4% of those who had.Of those who previously had unsuccessful treatment, 67.6% were sensitive to clarithromycinIn Vitro, suggesting poor compliance is a major factor in failures. Clarithromycin resistanceincreased from 3.9% amongst treatment naive subjects in 1997 to 9.3% in our study,correlating with a 26% increase in the rate of macrolide prescribing for respiratory tract