Inflammation-associated Colon Tumorigenesis Research Articles

Refined fiber inulin promotes inflammation-associated colon tumorigenesis by modulating microbial succinate production.

There is an increased risk of colon cancer associated with inflammatory bowel disease (IBD). Dietary fibers (DFs) naturally present in vegetables and whole grains offer numerous beneficial effects on intestinal health. However, the effects of refined DFs on intestinal health remain unclear. Therefore, we elucidated the impact of the refined DF inulin on colonic inflammation and tumorigenesis. Four-week-old wild-type (WT) mice were fed diets containing insoluble DF cellulose (control) or refined DF inulin for 4 weeks. A subgroup of mice was then switched to drinking water containing dextran sulfate sodium (DSS, 1.4% wt/vol) for colitis induction. In another subgroup of mice, colitis-associated colorectal cancer (CRC) was initiated with three 7-day alternate cycles of DSS following an initial dose of mutagenic substance azoxymethane (AOM; 7.5 mg/kg body weight; i.p.). Post 7 weeks of AOM treatment, mice were euthanized and examined for CRC development. Mice consuming inulin-containing diet exhibited severe colitis upon DSS administration, as evidenced by more body weight loss, rectal bleeding, and increased colonic inflammation than the DSS-treated control group. Correspondingly, histological analysis revealed extensive disruption of colon architecture and massive infiltration of immune cells in the inulin-fed group. We next examined the effect of inulin on CRC development. Surprisingly, significant mortality (~50%) was observed in the inulin-fed but not in the control group during the DSS cycle. Consequently, the remaining inulin-fed mice, which completed the study exhibited extensive colon tumorigenesis. Immunohistochemical characterization showed comparatively high expression of the cell proliferation marker Ki67 and activation of the Wnt signaling in tumor sections obtained from the inulin-fed group. Gut microbiota and metabolite analysis revealed expansion of succinate producers and elevated cecal succinate in inulin-fed mice. Human colorectal carcinoma cells (HCT116) proliferated more rapidly when supplemented with succinate in an inflamed environment, suggesting that elevated luminal succinate may contribute to tumorigenesis. Our study uncovers that supplementation of diet with refined inulin induces abnormal succinate accumulation in the intestinal lumen, which in part contributes to promoting colon inflammation and tumorigenesis.

Cover Image, Volume 61, Issue 10

The cover image is based on the Research Article Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis by Robin Kumar et al., https://doi.org/10.1002/mc.23452.

Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis.

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RBresponsive microbe Bifidobacterium longum), and non-fermented RBwere fed as 10% w/w (diet) to gut microbiota-intactspf or germ-free micegf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed micegf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

Background and AimsMutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.MethodsWe generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis.ResultsVCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.ConclusionsOur results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

The gut microbiome can contribute to colon tumor susceptibility via an effect on CD8+ T cell responses

Abstract There is increasing evidence that colorectal cancer patients have altered gut microbiomes compared to healthy controls, but the mechanisms by which the microbiota contribute to colon carcinogenesis remain to be fully elucidated. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of inflammation-associated colon tumorigenesis, our lab discovered two colonies of specific pathogen free (SPF) C57BL/6J wild type (WT) mice housed in the same mouse room that develop differential tumor burdens. Mice from the “WT1” colony developed five tumors on average while mice from the “WT2” colony developed 15 tumors on average. The increased tumor susceptibility in WT2 mice can be directly attributed to the gut microbiome as germ-free (GF) mice colonized with WT2 bacteria developed more tumors compared to that of GF mice colonized with WT1 bacteria. Additionally, 16S rRNA gene sequencing of fecal bacteria from WT1 and WT2 mice revealed distinct microbiomes with certain bacteria consistently associated with high or low tumor numbers. Furthermore, naïve and acutely-inflamed (day 10 of AOM/DSS) WT2 mice have increased colon lamina propria CD8+ IFNγ+ T cells compared to WT1 mice as measured by flow cytometry. However, in tumor-bearing WT2 mice, there was decreased tumor-infiltrating CD8+ T cells with reduced IFNγ production, possibly due to T cell exhaustion. GF Rag1−/− mice as well as SPF CD8−/− mice inoculated with WT2 gut microbiota developed fewer tumors than SPF WT2 mice, suggesting that the WT2 gut microbiome increases tumor susceptibility, in part, through an effect on CD8+ T cells. Altogether, our data reveal a potential novel role of microbiota in altering colon CD8+ T cell function that ultimately impacts colon cancer risk.

Loss of solute carrier family 7 member 2 exacerbates inflammation-associated colon tumorigenesis

Solute carrier family 7 member 2 (SLC7A2, also known as CAT2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in wound repair. We have reported that both SLC7A2 expression and L-Arg availability are decreased in colonic tissues from inflammatory bowel disease patients and that mice lacking Slc7a2 exhibit a more severe disease course when exposed to dextran sulfate sodium (DSS) compared to wild-type (WT) mice. Here, we present evidence that SLC7A2 plays a role in modulating colon tumorigenesis in the azoxymethane(AOM)-DSS model of colitis-associated carcinogenesis (CAC). SLC7A2 was localized predominantly to colonic epithelial cells in WT mice. Utilizing the AOM-DSS model, Slc7a2–/– mice had significantly increased tumor number, burden, and risk of high-grade dysplasia versus WT mice. Tumors from Slc7a2–/– mice exhibited significantly increased levels of the proinflammatory cytokines/chemokines IL-1β, CXCL1, CXCL5, IL-3, CXCL2, CCL3, and CCL4, but decreased levels of IL-4, CXCL9, and CXCL10 compared to tumors from WT mice. This was accompanied by a shift toward pro-tumorigenic M2 macrophage activation in Slc7a2-deficient mice, as marked by increased colonic CD11b+F4/80+ARG1+ cells with no alteration in CD11b+F4/80+NOS2+ cells by flow cytometry and immunofluorescence microscopy. The shift toward M2 macrophage activation was confirmed in bone marrow-derived macrophages from Slc7a2–/– mice. In bone marrow chimeras between Slc7a2–/– and WT mice, the recipient genotype drove the CAC phenotype, suggesting the importance of epithelial SLC7A2 in abrogating neoplastic risk. These data reveal that SLC7A2 has a significant role in the protection from CAC in the setting of chronic colitis, and suggest that the decreased SLC7A2 in inflammatory bowel disease (IBD) may contribute to CAC risk. Strategies to enhance L-Arg availability by supplementing L-Arg and/or increasing L-Arg uptake could represent a therapeutic approach in IBD to reduce the substantial long-term risk of colorectal carcinoma.

Abstract 1265: Protective efficacy of gut microbiome and microbial metabolites after rice bran consumption against colon tumorigenesis

Abstract Colorectal cancer (CRC) is the 2nd leading cause of cancer-related deaths in US. Therefore, practical interventions that reduce incidence of adenomatous polyps and/or prevent their progression to CRC are urgently needed. In this regard, several studies have reported the chemopreventive potential of dietary rice bran [a wasted ‘by-product' of white rice (Oryza sativa) processing] against CRC. Given that high heat can stabilize rice-bran for safe human consumption, our research efforts are evaluating ‘stabilized-rice bran', as a functional food for CRC control and prevention. Rice bran has been previously shown to increase colonization of native gut probiotics and undergo microbiota fermentation. However, little is known about rice bran constituents as prebiotics, microbial substrates that promote healthy gut microbiota, and how these microbes produce novel, bioactive microbial metabolites. In this regard, our studies show that native gut probiotic - fermented rice bran extracts have strong in vitro activity against CRC cells and significantly decrease their colonosphere formation (a stem cell characteristic). Rice bran (fermented and non-fermented) also strongly inhibits TNF alpha-induced NF-kappa B activation and interleukin (IL)- 4-induced expression of CD44, which along with its variant form (CD44 v3-v6) plays an essential role in the adhesion and infiltration of inflammatory cells in CRC. Notably, even the stool extracts from rice bran-fed CRC survivors showed increased inhibitory effects on CRC cell viability compared to the stools collected from the same individuals prior to rice bran consumption. Next, extensive in vivo investigations were performed to determine the relative efficacy of dietary rice bran-modified gut microbiota and microbiota-fermented rice bran on inflammation associated-colon tumorigenesis. First, Bifidobacterium Longum -fermented (fermented with selected rice bran responsive microbe that was induced during rice bran feeding clinical trials) and non-fermented dietary rice bran were fed to conventionally-raised or germ-free mice and evaluated for their comparative efficacy against azoxymethane (AOM)/ dextran sodium sulphate (DSS)-induced CRC. In second set of studies, we ‘humanized' gut of germ-free mice with stool inoculum from rice bran feeding human clinical trials and evaluated their efficacy against AOM/DSS-induced CRC. In conventional mice study, both the rice bran diets showed protective efficacy against CRC. Interestingly, germ-free mice having undergone transfaunation with rice-bran modified stool inoculum showed significantly less cancerous lesions compared to mice humanized with control stool inoculums, indicating that rice bran has the potential to re-structure the ‘high risk' gut microflora of CRC patients towards the colonization of populations with protective effects against CRC growth and progression. Citation Format: Akhilendra K. Maurya, Sushil Kumar, Rama Kant, Dileep Kumar, Bupinder Raina, Anita Yadav, Elizabeth P. Ryan, Komal Raina. Protective efficacy of gut microbiome and microbial metabolites after rice bran consumption against colon tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1265.

Chemopreventive Action by Ethanol-extracted Brazilian Green Propolis on Post-initiation Phase of Inflammation-associated Rat Colon Tumorigenesis.

Propolis has since long been utilized in numerous folk medicines with a variety of medicinal properties. In this study, the effects of ethanol-extracted (EEP) and water-extracted (WEP) Brazilian green propolis on the post-initiation phase of inflammation-associated rat colon tumorigenesis were directly compared. Male F344 rats at 6 weeks of age were subcutaneously injected with 1,2-dimethylhydrazine (DMH) at 40 mg/kg body weight twice during the first week, followed by 1% dextran sodium sulfate (DSS) in drinking water for one week. After a 1-week no-treatment period, animals were administered either basal Oriental MF powdered diet, or 1% EEP or 1% WEP in the basal diet until week 32. Post-initiation treatment with EEP significantly reduced the multiplicity of colorectal carcinomas compared to the control (0.40±0.13/rat vs. 2.29±0.84/rat, respectively, p<0.05), and EEP also reduced the tumor volume. Immunohistochemically, expression of inflammation-associated proteins inducible nitric oxide synthase, tumor necrotic factor alpha, nuclear factor kappa B and glutathione peroxidase-2 were significantly diminished in colorectal tumors from EEP-treated rats. Suppression of inflammation and oxidative stress, which had been triggered by DMH and promoted by DSS, was a primary mechanism by which EEP suppressed carcinogenesis.

Role of p38γ - NFATc4 - IL17A Pathway As a Potential Therapeutic Target in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) is an incurable skin homing T cell malignancy. We have previously reported p38 as therapeutic targets for CTCL.1 However, the mechanism underlying p38 signaling is not completely understood. To further investigate p38 and its downstream signaling components, we examined public database of gene expression and found that p38γ is overexpressed in CTCL as compared to normal T cells. In addition, p38γ has negligible expression in normal lymphoid tissues, with the exception of high level expressed in smooth and cardiac muscle cells. We have demonstrated that p38γ over-expression increases cell proliferation and knockdown of p38γ causes Hut78 cell death. p38γ plays an important role in inflammation-associated tumorigenesis3 and inhibition of its activity has emerged as a strategy to treat a spectrum of cancers.4The transcription factor, NFATc4, downstream of p38γ, is also significantly up-regulated in CTCL cells by microarray analysis, and it is at non-detectable level in normal T cells.1We have demonstrated that shRNA-mediated knockdown of p38γ reduced NFATc4 mRNA levels in Hut78 cells, and that inhibition of NFATc4 by siRNA reduces the proliferation of CTCL cells.We also found that the cytokine IL17A functions downstream of p38γ and NFATc4, as knockdown of either p38γ or NFATc4 significantly reduced IL17A mRNA levels in Hut78 cells. This result suggests that IL17A is a target for transcriptionally activated NFATc4. Previously we have shown that IL17A rescues Hut78 cells from apoptosis induced by combined inhibition of NFAT and NFkB (treated with curcumin and Ly2228820). This implicates IL17A as a key mediator for CTCL survival. Therefore, we propose a novel p38γ - NFATc4 - IL17A signaling pathway in malignant T cells that promotes the survival of CTCL which provides potential therapeutic target against this disease.To further define the role of p38 and identify targets that increase the antitumor efficacy of p38 inhibition, we performed a synthetic lethal RNA interference (RNAi) screen in Hut78 cells treated with 10 µM of the p38 MAPK inhibitor Ly2228820. We transduced control and Ly2228820-treated Hut78 cells with a pooled retroviral RNAi library consisting of 4290 shRNAs that targeted more than 1000 genes involved in human cancers. If a shRNA from the library is not toxic to the control cells, but causes cell death in Ly2228820-treated cells, the gene targeted by this shRNA would be identified by the screen as synthetically lethal to p38 inhibition. Among many hits identified from the screen, we selected UCHL5 for further analysis. UCHL5encodes a deubiquitin enzyme that cleaves K48-linked polyubiquitin chains and plays an important role in the regulation of protein stability. Interestingly, combination of Ly2228820 and b-AP15, a small molecule inhibitor of UCHL5, significantly reduced the protein levels of NFATc4 isoform but not other NFAT isoforms. NFATc4 protein levels are known to be regulated by ubiquitin-proteasome pathway.2 Our finding thus suggests UCHL5 as a potential new regulator that stabilizes NFATc4 protein. Further studies are needed to confirm this prediction. More importantly, combination of Ly2228820 and b-AP15 enhanced apoptosis in CTCL cell lines (HH and Hut78) and primary Sézary cells, but was not toxic in normal PBMC cells.In summary, our findings suggest that the p38γ - NFATc4 - IL17A signaling pathway plays an important role in the survival of CTCL. In addition, improving the efficacy of targeting this pathway via p38 may also benefit from combined inhibition of UCHL5, a potentially important regulator of NFATc4 that needs further characterization.Reference:1 Bliss-Moreau M, Coarfa C, Gunaratne PH, Guitart J, Krett NL, Rosen ST (2015). Identification of p38beta as a therapeutic target for the treatment of Sezary syndrome. The Journal of investigative dermatology135:599-608.2 Fan Y, Xie P, Zhang T, Zhang H, Gu D, She M et al (2008). Regulation of the stability and transcriptional activity of NFATc4 by ubiquitination. FEBS letters582:4008-4014.3 Qi X, Yin N, Ma S, Lepp A, Tang J, Jing W et al (2015). p38gamma MAPK Is a Therapeutic Target for Triple-Negative Breast Cancer by Stimulation of Cancer Stem-Like Cell Expansion. Stem cells33:2738-2747.4 Yin N, Qi X, Tsai S, Lu Y, Basir Z, Oshima K et al (2015). p38gamma MAPK is required for inflammation-associated colon tumorigenesis. Oncogene. DisclosuresQuerfeld:Actelion: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis

Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis. We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1β (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3' untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer+/+ and dicer-/-, and Apobec3+/+ and Apobec3-/- mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy. Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice. We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.

The B-cell tumor promoter Bcl-3 suppresses inflammation-associated colon tumorigenesis in epithelial cells.

Bcl-3 is an atypical member of the IκB family. It associates with p50/NF-κB1 and p52/NF-κB2 homodimers in nuclei where it modulates transcription in a context-dependent manner. A subset of B cell tumors exhibits recurrent translocations of Bcl-3, resulting in overexpression. Elevated expression without translocations is also observed in various B cell lymphomas and even some solid tumors. Here we investigated the role of Bcl-3 in AOM/DSS-induced colon tumors, a mouse model for colitis-associated colorectal cancers in humans. Contrary to expectations, Bcl-3 suppressed colorectal tumor formation: Bcl-3-deficient mice were relatively protected from DSS-induced epithelial damage and developed more polyps after AOM/DSS treatment, though polyp size was unaffected. DSS-challenged mutant mice exhibited increased recruitment of myeloid-derived suppressor cells (MDSCs), consistent with protection of the epithelium. Loss of Bcl-3 in intestinal epithelial cells was sufficient to increase tumorigenesis. The added tumor burden in mutant mice was dependent on TNFα, a tumorigenic, NF-κB-mediated signaling pathway that was dampened by Bcl-3. These findings reveal a tumor-suppressive role for Bcl-3 in this inflammation-associated cancer model. Bcl-3 thus functions as a tumor promoter or suppressor, depending on the cellular and environmental context.

Expression of the novel adipokine C1qTNF-related protein 4 (CTRP4) suppresses colitis and colitis-associated colorectal cancer in mice.

Inflammatory bowel disease (IBD) is an important factor in the induction of colon cancer, but its mechanism is unclear. Colitis and colitis-associated colorectal cancer (CAC) models induced using both dextran sulfate sodium (DSS) and the azoxymethane/DSS protocol were established in wild-type (WT) and CTRP4 transgenic (CTRP4-tg) C57BL6/J mice. Body weight, stool consistency and the presence of blood in the stool were analyzed; tumor quantity, size and histological characteristics were analyzed during the development of CAC. The CTRP4-tg mice exhibited significantly reduced colitis and developed far fewer macroscopic tumors; these tumors were smaller in size, and a majority of the colon tumors in these mice were restricted to the superficial mucosa. Tumors of lower grades were observed in the CTRP4-tg mice. Interleukin-6 was markedly downregulated in the CTRP4-tg mice during CAC tumorigenesis. The phosphorylation of ERK, signal transducer and activator of transcription 3 and Akt in the colon and the proliferation of intestinal epithelial cells were decreased in the CTRP4-tg mice. The injection of recombinant CTRP4 protein significantly reduced the colitis symptoms of the WT mice. CTRP4 plays an important role in inflammation and inflammation-associated colon tumorigenesis, and our research may provide a novel method for the treatment of IBD and CAC.

P38γ MAPK is required for inflammation-associated colon tumorigenesis.

Chronic inflammation has long been considered to causatively link to colon cancer development. However, signal transduction pathways involved remain largely unidentified. Here, we report that p38γ mitogen-activated protein kinase mediates inflammatory signaling to promote colon tumorigenesis. Inflammation activates p38γ in mouse colon tissues and intestinal epithelial cell-specific p38γ knockout (KO) attenuates colitis and inhibits pro-inflammatory cytokine expression. Significantly, p38γ KO inhibits tumorigenesis in a colitis-associated mouse model. The specific p38γ pharmacological inhibitor pirfenidone also suppresses pro-inflammatory cytokine expression and colon tumorigenesis. The tumor-promoting activity of epithelial p38γ was further demonstrated by xenograft studies. In addition, p38γ is required for β-catenin/Wnt activities and p38γ stimulates Wnt transcription by phosphorylating β-catenin at Ser605. These results show that p38γ activation links inflammation and colon tumorigenesis. Targeting p38γ may be a novel strategy for colon cancer prevention and treatment.

Promotion of inflammation‐associated colon tumorigenesis by the total Western diet in the APC min/+ mouse

Our research team developed a new total Western diet (TWD) for rodents that mimics typical human nutrition patterns with respect to macronutrient composition (amounts and types of sugar, fat, protein) and micronutrient content (vitamins and minerals) on a nutrient density basis. Previously, we showed that consumption of the TWD markedly enhanced development of colon tumors in mice initiated with azoxymethane and provided 1% DSS via drinking water to promote colitis. The objective of the present study was to determine if the TWD affected tumor development using the APCmin/+ mouse model of sporadic intestinal tumorigenesis. Mice were fed the TWD, an obesogenic diet containing 45% fat (DIO) or a diet optimized for rodent health (AIN93G) for 12 weeks; half of each group was also given 1% DSS for 1 week to induce inflammation in the colon. Remarkably, consumption of the TWD significantly increased colon tumor multiplicity and volume by nearly 6-fold compared to mice fed AIN93G or DIO, but only in mice that experienced colitis. Compared to mice fed AIN93G, tumorigenesis in the small intestine (SI) was not markedly affected by the TWD, whereas mice fed DIO had significantly more SI tumors. These results extend prior observations by our group showing that the TWD promotes inflammation-associated colon tumorigenesis. Support was provided by the Utah Agricultural Experiment Station.

Immune Chaperone gp96 Drives the Contributions of Macrophages to Inflammatory Colon Tumorigenesis

Macrophages are important drivers in the development of inflammation-associated colon cancers, but the mechanistic underpinnings for their contributions are not fully understood. Furthermore, Toll-like receptors have been implicated in colon cancer, but their relevant cellular sites of action are obscure. In this study, we show that the endoplasmic reticulum chaperone gp96 is essential in tumor-associated macrophages (TAM) to license their contributions to inflammatory colon tumorigenesis. Mice where gp96 was genetically deleted in a macrophage-specific manner exhibited reduced colitis and inflammation-associated colon tumorigenesis. Attenuation of colon cancer in these mice correlated strikingly with reduced mutation rates of β-catenin, increased efficiency of the DNA repair machinery, and reduced expression of proinflammatory cytokines, including interleukin (IL)-17 and IL-23 in the tumor microenvironment. The genotoxic nature of TAM-associated inflammation was evident by increased expression of genes in the DNA repair pathway. Our work deepens understanding of how TAM promote oncogenesis by altering the molecular oncogenic program within epithelial cells, and it identifies gp96 as a lynchpin chaperone needed in TAM to license their function and impact on expression of critical inflammatory cytokines in colon tumorigenesis.

Abstract 2866: Functional impairment of microRNAs induced by chronic inflammation is the cause of inflammation-associated colon tumorigenesis.

Abstract Background: A widespread downregulation of microRNAs is commonly observed in human cancers and is involved in cellular transformation and tumorigenesis. Because we previously reported that microRNA functions are inhibited under various kinds of cellular stresses including inflammatory cytokines in vitro, we hypothesized that chronic inflammation in vivo may also cause chronic functional impairment of microRNAs, which mimics a global reduction of microRNA abundance, resulting in a causal role in inflammation-associated tumorigenesis. Methods &amp; Results: 1) To examine this hypothesis, we first confirmed the functional impairment of microRNAs under inflammatory stimuli in vitro using artificial reporter constructs. The derepression of microRNA-targeting reporter activities by inflammatory stimuli could also be observed using reporter constructs containing natural 3’UTR sequences of c-myc and Lin28B genes, which are targeted by endogenous let-7. 2) Next, we constructed reporter transgenic mice bearing CMV-promoter driven GFP construct carrying target sequences of microRNAs in its 3’UTR, and monitored the changes of microRNA functions in vivo during the course of a Dextran Sulfate Sodium (DSS) induced colitis-associated colon tumor model. The expression levels of GFP were significantly increased during colitis, suggesting that microRNA functions were impaired during the chronic inflammation in vivo. The expression levels of microRNA-target oncogenes such as c-myc and Lin28B in the colonic mucosa were indeed upregulated. 3) Lastly, to determine the consequences of the global functional impairment of microRNAs during the colitis, we examined Dicer-deficient mice, which have globally low levels of microRNA expression and may mimic the functional impairment of global microRNAs induced by chronic inflammation described above. As expected, Dicer deficient mice were more liable to have the colitis-associated tumors. This indicates that the global decrease of microRNAs leads to oncogenesis, suggesting that the functional impairment of global miRNAs during chronic inflammation can also be a cause of the inflammation-associated carcinogenesis. Conclusion: In conclusion, we propose a novel concept that the attenuation of global microRNA functions by chronic inflammation is the cause of inflammation-associated tumorigenesis. Citation Format: Takeshi Yoshikawa. Functional impairment of microRNAs induced by chronic inflammation is the cause of inflammation-associated colon tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2866. doi:10.1158/1538-7445.AM2013-2866

Effective suppression of azoxymethane‐induced aberrant crypt foci formation in mice with citrus peel flavonoids

Citrus peel or its extract has been reported to exhibit a broad spectrum of biological activity. Herein, we report the first investigation of inhibitory effects of a formulated product from citrus peel extract, gold lotion (GL), on azoxymethane-induced colonic tumorigenesis. We have demonstrated that oral feeding of GL decreased the number of aberrant crypt foci (ACF), particularly large size of ACF in colonic tissues of mice. Both gene and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were suppressed by GL treatment. The in vivo data have revealed for the first time that the citrus peel extract-GL-is an effective antitumor agent mechanistically downregulating the protein levels of iNOS, COX-2, ornithine decarboxylase, vascular endothelial growth factor, and matrix metallopeptidase 9 in colonic tissues of mice, suggesting that GL is a novel functional natural product capable of preventing inflammation-associated colon tumorigenesis.

Mo1593 Functional Impairment of MicroRNAs Induced by Chronic Inflammation is the Cause of Inflammation-Associated Colon Tumorigenesis

Mo1593 Functional Impairment of MicroRNAs Induced by Chronic Inflammation is the Cause of Inflammation-Associated Colon Tumorigenesis

Abstract LB-462: Chemopreventive effects of freeze-dried strawberries in inflammation-associated mouse colon tumorigenesis

Abstract Colorectal cancer (CRC) is one of the most common malignant neoplasms in the United States. The risk for CRC is associated with extent and duration of inflammatory bowel disease (IBD). Patients with IBD are at increased risk of developing CRC when compared to the general population. Therefore, primary cancer chemoprevention is an important strategy to reduce the risk of CRC in IBD patients and thus, this approach has recently received more attention. Our laboratory has conducted extensive research on cancer chemoprevention in GI tract. In our previous study, we found that dietary freeze-dried black raspberries significantly inhibited adenoma and adenocarcinoma formation and decreased the level of oxidative stress in an azoxymethane (AOM)-induced rat CRC model. In a carcinogen-induced rat esophageal cancer model, we found that dietary freeze-dried strawberries significantly inhibited tumor development. However, the effects of strawberries in inflammation-associated CRC have not been investigated as yet. The principal objective of this study is to investigate the inhibitory effects of dietary freeze-dried strawberries in AOM/Dextran Sodium Sulfate (DSS)-induced mouse colon carcinogenesis. At 4–5 weeks of age, male Crj: CD-1 mice were given a single intraperitoneal injection of AOM (10mg/kg body weight), followed by 2% DSS in their drinking water for seven days, starting one week after the AOM injection. After 3 weeks, animals were fed AIN-76A diet or AIN-76A diet containing 10% freeze-dried strawberries. At week 20, mice were sacrificed for histopathological examination. We found that strawberries significantly decreased tumor incidence from 100% in mice treated with AOM/DSS only to 44.4% in mice treated with AOM/DSS plus strawberries (55.6% reduction, P = 0.02). We also found that the average animal body weight was significantly higher (P = 0.036) in animals fed strawberries (41.9 ± 5.3 g) when compared to animals fed control diet (38.7 ± 3.7 g). A large-scale investigation of the inhibitory effects of strawberries on biomarkers associated with colon carcinogenesis is under way. (Supported by California Strawberry Commissions) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-462. doi:10.1158/1538-7445.AM2011-LB-462

Pterostilbene Inhibits Colorectal Aberrant Crypt Foci (ACF) and Colon Carcinogenesis via Suppression of Multiple Signal Transduction Pathways in Azoxymethane-Treated Mice

Pterostilbene (PS), a natural dimethylated analogue of resveratrol, is known to have diverse pharmacologic activities including anticancer, anti-inflammation, antioxidant, apoptosis, antiproliferation, and analgesic potential. This paper reports the inhibitory effect of dietary administration of pterostilbene against the formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) preneoplastic lesions and adenomas in male ICR mice and delineates its possible molecular mechanisms. ICR mice were given two AOM injections intraperitoneal and continuously fed a 50 or 250 ppm pterostilbene diet for 6 or 23 weeks. It was found that the dietary administration of pterostilbene effectively reduced AOM-induced formation of ACF and adenomas and inhibited the transcriptional activation of iNOS and COX-2 mRNA and proteins in mouse colon stimulated by AOM. Treatment with pterostilbene resulted in the induction of apoptosis in mouse colon. Moreover, administration of pterostilbene for 23 weeks significantly suppressed AOM-induced GSK3beta phosphorylation and Wnt/beta-catenin signaling. It was also found that pterostilbene significantly inhibited AOM-induced expression of VEGF, cyclin D1, and MMPs in mouse colon. Furthermore, pterostilbene markedly inhibited AOM-induced activation of Ras, phosphatidylinositol 3 kinase/Akt, and EGFR signaling pathways. All of these results revealed that pterostilbene is an effective antitumor agent as well as its inhibitory effect through the down-regulation of inflammatory iNOS and COX-2 gene expression and up-regulation of apoptosis in mouse colon, suggesting that pterostilbene is a novel functional agent capable of preventing inflammation-associated colon tumorigenesis.