Multiple Intestinal Neoplasia Research Articles

Kallikrein-Related Peptidase 6 Contributes to Murine Intestinal Tumorigenesis Driven by a Mutant Adenomatous polyposis coli Gene.

The objective of this study was to assess the role of a secreted serine protease, kallikrein-related peptidase 6 (KLK6), during colorectal tumorigenesis driven by a mutant Adenomatous polyposis coli (APC) tumor suppressor gene. A first analysis of KLK6 expression in the intestinal tract of Apc-mutant multiple intestinal neoplasia (ApcMin/+) mice revealed up to four-fold induction of Klk6 mRNA levels in adenomas relative to its level in the adjacent mucosa. The presence of KLK6 protein in the adenomatous areas was confirmed by immunohistochemistry and optical coherence tomography/laser-induced fluorescence (OCT/LIF) imaging. To assess the contribution of the KLK6 expression on the Apc-mutant intestinal and colon tumorigenesis, we engineered a mouse with floxed alleles of the Klk6 gene (Klk6lox/lox) and crossed it with a mouse expressing the truncated APC protein under control of the intestinal tract-specific human CDX2P9.5-NLS Cre transgene (CPC;Apcfl/fl;Klk6+/+). We found that CPC;Apcfl/fl mice with disrupted Klk6 gene expression (CPC;Apcfl/fl;Klk6fl/fl) had a significantly smaller average size of the small intestinal and colon crypts (p < 0.001 and p = 0.04, respectively) and developed a significantly fewer adenomas (p = 0.01). Moreover, a decrease in high-grade adenomas (p = 0.03) and adenomas with a diameter above 2 mm (p < 0.0001) was noted in CPC;Apcfl/fl;Klk6fl/fl mice. Further molecular analysis showed that Klk6 gene inactivation in the small intestine and colon tissues of CPC;Apcfl/fl;Klk6fl/fl mice resulted in a significant suppression of transforming growth factor β2 (TGF-β2) protein (p ≤ 0.02) and mitogen-activated protein kinase (MAPK) phosphorylation (p ≤ 0.01). These findings demonstrate the oncogenic role of KLK6 in the mutant Apc-mediated intestinal tumorigenesis and suggest the utility of KLK6 for early diagnosis of colorectal tumors.

Single-Cell Profiling Reveals the Impact of Genetic Alterations on the Differentiation of Inflammation-Induced Murine Colon Tumors.

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (MinApcΔ716/+) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAFV600E mutation (BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+, BLM) or knocking out Msh2 (Msh2LoxP/LoxPVil1-creMinApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.

Abstract 4243: Dietary flavonoids prevent colon tumor formation in ApcMin/+ mice in a microbiome- and sex-dependent manner

Abstract Background: A diet rich in colorful fruits and vegetables is effective for the prevention of various inflammation-induced cancer types. Mechanistically, this effect has been attributed to the potent antioxidant properties of flavonoid molecules. However, contrary to observations in mice, the success of flavonoids in human dietary cancer prevention trials is often dampened due to large interpersonal variation. Antioxidant activity alone does not explain this variability, suggesting there is an additional important variable that has not been accounted for. Since flavonoids have a poor bioavailability and predominantly passage into the large intestine, we hypothesize that gut microbial metabolism is essential for flavonoid bioactivity in colon cancer prevention. Goals and Objectives: The goal of this study was to evaluate the impact of microbiota composition on the efficacy of dietary flavonoids in preventing intestinal tumorigenesis. Methods: We used the adenomatous polyposis coli - multiple intestinal neoplasia (ApcMin/+) mouse model, which is genetically predisposed to develop adenomas along its gastrointestinal tract. Development of these adenomas and eventual adenocarcinoma formation is further influenced by factors such as inflammation, dietary input and the gut microbiota. Animals were fed a control high fat diet (HFD) or prevention diets consisting of 1% berry extract, 1% purified flavonoid or 1% microbial catabolite, all on the HFD background. The contribution of the microbiota was modelled by including experimental groups that received an antibiotic cocktail in their drinking water throughout the course of the experiment. For these animals, microbiota community composition was determined by 16S rRNA sequencing, plasma was collected for histological profiling and metabolomics analysis and end-point measurements of intestinal tumor numbers and size were performed. Results: Supplementation with berry extract significantly reduced colon tumor numbers and incidence in male, but not in female mice. This effect was negated in mice on antibiotic drinking water, implicating the involvement of the gut microbiota. Community analysis revealed a relative increase in Erysipelotrichaceae, Clostridia and Akkermansia in the berry supplemented versus HFD control mice. In addition, their systemic monocyte counts were reduced. Dietary supplementation with a major gut microbial flavonoid catabolite, did not reduce colon tumor formation, suggesting the functional involvement of different microbial metabolites. Conclusions: Our study demonstrates the involvement of the mouse gut microbiota in dietary prevention of colon tumor formation. These effects impact circulating monocytes and are linked to changes in the plasma metabolome. We are currently elucidating the microbial actors and associated molecular pathways involved. Citation Format: Beckey DeLucia, Jan Claesen. Dietary flavonoids prevent colon tumor formation in ApcMin/+ mice in a microbiome- and sex-dependent manner. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4243.

Dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology and alters metabolism in APCmin mice

Colorectal cancer (CRC) is the second most prevelant malignancy in Europe and diet is an important modifiable risk factor. Processed meat consumption, including meats with preservative salts such as sodium nitrite, have been implicated in CRC pathogenesis. This study investigated how the CRC pathology and metabolic status of adenomatous polyposis coli (APC) multiple intestinal neoplasia (min) mice was perturbed following 8 weeks of pork meat consumption. Dietary inclusions (15%) of either nitrite-free pork, nitrite-free sausage, or nitrite-containing sausage (frankfurter) were compared against a parallel control group (100% chow). Comprehensive studies investigated: gastrointestinal tract histology (tumours), aberrant crypt foci (ACF), mucin deplin foci (MDF), lipid peroxidation (urine and serum), faecal microbiota, and serum metabolomics (599 metabolites). After 8 weeks mice consuming the frankfurter diet had 53% more (P = 0.014) gastrointestinal tumours than control, although ACF and MDF did not differ. Urine and serum lipid peroxidation markers were 59% (P = 0.001) and 108% (P = 0.001) higher, respectively in the frankfurter group. Gut dysbiosis was evident in these mice with comparably fewer Bacteriodes and more Firmicutes. Fasting serum levels of trimethylamine N-oxide (TMAO) and numerous triglycerides were elevated. Various serum phosphotidylcholine species were decreased. These results demonstrate that nitrite-containing sausages may exaccerbate the development of CRC pathology in APCMin mice to a greater extent than nitrite-free sausages, and this is associated with greater lipid peroxidation, wide-ranging metabolic alternation and gut dysbiosis.

Genetic mapping of novel modifiers for ApcMin induced intestinal polyps\u2019 development using the genetic architecture power of the collaborative cross mice

BackgroundFamilial adenomatous polyposis is an inherited genetic disease, characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli (Apc) gene. Mice carrying a nonsense mutation in the Apc gene at R850, which is designated ApcMin/+ (Multiple intestinal neoplasia), develop intestinal adenomas. Several genetic modifier loci of Min (Mom) were previously mapped, but so far, most of the underlying genes have not been identified. To identify novel modifier loci associated with ApcMin/+, we performed quantitative trait loci (QTL) analysis for polyp development using 49 F1 crosses between different Collaborative Cross (CC) lines and C57BL/6 J-ApcMin/+mice. The CC population is a genetic reference panel of recombinant inbred lines, each line independently descended from eight genetically diverse founder strains. C57BL/6 J-ApcMin/+ males were mated with females from 49 CC lines. F1 offspring were terminated at 23 weeks and polyp counts from three sub-regions (SB1–3) of small intestinal and colon were recorded.ResultsThe number of polyps in all these sub-regions and colon varied significantly between the different CC lines. At 95% genome-wide significance, we mapped nine novel QTL for variation in polyp number, with distinct QTL associated with each intestinal sub-region. QTL confidence intervals varied in width between 2.63–17.79 Mb. We extracted all genes in the mapped QTL at 90 and 95% CI levels using the BioInfoMiner online platform to extract, significantly enriched pathways and key linker genes, that act as regulatory and orchestrators of the phenotypic landscape associated with the ApcMin/+ mutation.ConclusionsGenomic structure of the CC lines has allowed us to identify novel modifiers and confirmed some of the previously mapped modifiers. Key genes involved mainly in metabolic and immunological processes were identified. Future steps in this analysis will be to identify regulatory elements – and possible epistatic effects – located in the mapped QTL.

The Impact of Ketogenic Diet on Colorectal Cancer Progression and the Co-evolution of Gut Microbiota: A Research Protocol

Introduction: To date, the ketogenic diet (KD) has been shown to improve certain health conditions such as seizure. These positive effects have been partly mediated through the gut microbiome. However, research on KD’s impact on colorectal cancer (CRC) and the gut microbiome of cancer patients that use KD has been scant. This study aims to investigate the role of KD in the co-evolution of gut microbial composition and CRC progression. We hypothesize that KD alters overall species diversity through either elimination of harmful bacterial genera or perpetuation of beneficial gut microbiota which could ultimately be the mechanism underlying positive CRC outcomes. Methods: In order to test this hypothesis, we propose a murine study using ApcMin/+ (multiple intestinal neoplasia) CRC mouse models in C57BL/6J background. The mice will either be given normal chow (control group) or KD (87% fat) for 8 weeks. Mice will be euthanized at the end of the experiment and analyzed in terms of polyp size and polyp number in the small intestine and colon. Frozen colon tissue will also be used to extract mRNA for quantitative polymerase chain reaction (qPCR) analysis of TH17 cytokine production. Gut microbiome composition will be analyzed by sequencing of 16S rRNA genes. To compare microbiome structure between diet groups, alpha diversity will be used to measure the differences in gut microbial structure in the control and experimental groups. Results: We would expect that mice fed a KD would have altered microbiota diversity, a decreased level of cytokine production, as well as fewer and smaller polyps (as measured in the small intestine and colon). Discussion: Analysis of the gut microbiota post-treatment, in conjunction with assessment of cytokine levels will help to set correlations between microbial gut activity and CRC progression. Conclusion: The results of this experiment could give insight into the impact of KD on development and progression of CRC; which could be used to develop therapeutic or dietary interventions.

Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.

Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in BRAF are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApcΔ716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the BRAF V600E mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized BRAF V600E Lgr5 CreMin (BLM) mice, tumors have similarities to human BRAF V600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with BRAF mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in BRAF V600E Lgr5 CreMin mice, wherein tumors phenocopy aspects of human BRAF-mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.This article is highlighted in the In This Issue feature, p. 1601.

Chemopreventive effect of 4'-hydroxychalcone on intestinal tumorigenesis in ApcMin mice.

Chalcones and its derivatives are reported to exhibit anti-cancer effects in several cancer cell lines, including colon cancer cells. However, the in vivo anticancer effects and associated mechanisms of chalcones against intestinal tumorigenesis currently remain unclear. The aim of the present study was to investigate the chemopreventive effect of a chalcone derivative, 4′-hydroxychalcone (4-HC), in a transgenic adenomatous polyposis coli multiple intestinal neoplasia mouse model (ApcMin) of spontaneous intestinal adenomas. ApcMin mice were fed 4-HC (10 mg/kg/day) or the vehicle control by oral gavage starting at 8 weeks of age, and were sacrificed at 20 weeks. The administration of 4-HC significantly decreased the number of colon adenomas by 45% and the size of colon adenomas by 35% compared with the respective controls. Similarly, the number of adenomas in the distal small intestine (DSI) and proximal small intestine also decreased by 35 and 33%, respectively, in 4-HC-treated mice, and adenoma size in the DSI decreased by 39% compared with the respective controls. Treatment with 4-HC strongly decreased proliferation in colon and DSI adenomas, as detected by immunofluorescence staining with the proliferation marker protein Ki-67, and promoted apoptosis in colon adenomas, as detected by TUNEL immunofluorescence staining. In addition, decreased mRNA expression of β-catenin target genes, including c-Myc, Axin2 and CD44, in colon adenomas of 4-HC-treated animals demonstrated the involvement of the Wnt/β-catenin signaling pathway in the initiation and progression of colon neoplasms. Treatment with 4-HC also decreased the protein levels of β-catenin in colon adenomas, as demonstrated by immunofluorescence staining. The results suggested that 4-HC may be a promising candidate for the chemoprevention of intestinal tumorigenesis, and further investigations are required to evaluate its clinical utility.

Intraepithelial Lymphocytes Suppress Intestinal Tumor Growth by Cell-to-Cell Contact via CD103/E-Cadherin Signal.

Background & AimsThe reason why small intestinal cancer is rarer than colorectal cancer is not clear. We hypothesized that intraepithelial lymphocytes (IELs), which are enriched in the small intestine, are the closest immune cells to epithelial cells, exclude tumor cells via cell-to-cell contact.MethodsWe developed DPE-green fluorescent protein (DPE-GFP) × adenomatous polyposis coli; multiple intestinal neoplasia (APCmin ) mice, which is a T-cell–reporter mouse with spontaneous intestinal tumors. We visualized the dynamics of IELs in the intestinal tumor microenvironment and the interaction between IELs and epithelial cells, and the roles of cell-to-cell contact in anti-intestinal tumor immunity using a novel in vivo live-imaging system and a novel in vitro co-culture system.ResultsIn the small intestinal tumor microenvironment, T-cell movement was restricted around blood vessels and the frequency of interaction between IELs and epithelial cells was reduced. Genetic deletion of CD103 decreased the frequency of interaction between IELs and epithelial cells, and increased the number of small intestinal tumors. In the co-culture system, wild-type IELs expanded and infiltrated to intestinal tumor organoids from APCmin mice and reduced the viability of them, which was cell-to-cell contact and CD103 dependent.ConclusionsThe abundance of IELs in the small intestine may contribute to a low number of tumors, although this system may not work in the colon because of the sparseness of IELs. Strategies to increase the number of IELs in the colon or enhance cell-to-cell contact between IELs and epithelial cells may be effective for the prevention of intestinal tumors in patients with a high cancer risk.

Tensin4 (TNS4) is upregulated by Wnt signalling in adenomas in multiple intestinal neoplasia (Min) mice

Summary Apc Min/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. ApcMin/+ mice (n = 11) were sacrificed at approximately 120 days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n = 14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of β‐catenin activity with MSAB or its increase by transfection with a Flag β‐catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT‐qPCR analysis showed approximately 5‐fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, β‐catenin inhibition resulted in reduced TNS4 expression, and conversely, β‐catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the Apc Min/+ mouse represents a good model to study this.

Silymarin, boswellic acid and curcumin enriched dietetic formulation reduces the growth of inherited intestinal polyps in an animal model.

BACKGROUNDSome substances of plant origin have been reported to exert an effect in reducing intestinal neoplasm development, especially in animal models. Adenomatous polyposis coli multiple intestinal neoplasia - ApcMin/+ is the most studied murine model of genetic intestinal carcinogenesis.AIMTo assess whether an enriched nutritional formulation (silymarin, boswellic acid and curcumin) with proven “in vitro” and “in vivo” anti-carcinogenetic properties may prevent inherited intestinal cancer in animal model.METHODSForty adenomatous polyposis coli multiple intestinal neoplasia - ApcMin/+ mice were used for the study of cancer prevention. They were divided into two groups: 20 assumed standard and 20 enriched diet. At the 110th d animals were sacrificed. In each group, four subgroups received intraperitoneal bromodeoxyuridine injection at different times (24, 48, 72 and 96 h before the sacrifice) in order to assess epithelial turnover. Moreover, we evaluated the following parameters: Intestinal polypoid lesion number and size on autoptic tissue, dysplasia and neoplasia areas by histological examination of the whole small intestine, inflammation by histology and cytokine mRNA expression by real-time polymerase chain reaction, bromodeoxyuridine and TUNEL immuno-fluorescence for epithelial turnover and apoptosis, respectively. Additionally, we performed western blotting analysis for the expression of estrogen alpha and beta receptors, cyclin D1 and cleaved caspase 3 in normal and polypoid tissues.RESULTSCompared to standard, enriched diet reduced the total number (203 vs 416) and the mean ± SD/animal (12.6 ± 5.0 vs 26.0 ± 8.8; P < 0.001) of polypoid lesions. In enriched diet group a reduction in polyp size was observed (P < 0.001). Histological inflammation and pro-inflammatory cytokine expression were similar in both groups. Areas of low-grade dysplasia (P < 0.001) and intestinal carcinoma (IC; P < 0.001) were significantly decreased in enriched diet group. IC was observed in 100% in standard and 85% in enriched formulation assuming animals. Enriched diet showed a faster epithelial migration and an increased apoptosis in normal mucosa and low-grade dysplasia areas (P < 0.001). At western blotting, estrogen receptor beta protein was well expressed in normal mucosa of enriched and standard groups, with a more marked trend associated to the first one. Estrogen receptor alpha was similarly expressed in normal and polypoid mucosa of standard and enriched diet group. Cleaved caspase 3 showed in normal mucosa a stronger signal in enriched than in standard diet. Cyclin D1 was more expressed in standard than enriched diet group of both normal and polypoid tissue.CONCLUSIONOur results are suggestive of a chemo-preventive synergic effect of the components (silymarin, boswellic acid and curcumin) of an enriched formulation in inherited IC. This effect may be mediated by the reduction of epithelial proliferation, the increase of apoptosis and the acceleration of villous cell renewal due to dietary formulation intake.

Toll-like receptor 4 regulates spontaneous intestinal tumorigenesis by up-regulating IL-6 and GM-CSF.

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The ApcMin/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, ApcMin/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of ApcMin/+ WT and ApcMin/+ TLR4−/− mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of ApcMin/+ TLR4−/− mice compared with ApcMin/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.

Epigenetic Changes Induced by Bacteroides fragilis Toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) is a Gram-negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of multiple intestinal neoplasia (Apcmin/+ ) mice; tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in colon epithelial cells (CECs) have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in gene expression (transcriptome sequencing [RNA-seq]) and chromatin accessibility (assay for transposase-accessible chromatin using sequencing) induced by treatment of HT29/C1 cells with BFT for 24 and 48 h were examined. Our data show that several genes are differentially expressed after BFT treatment and that these changes relate to the interaction between bacteria and CECs. Further, sites of increased chromatin accessibility are associated with the location of enhancers in CECs and the binding sites of transcription factors in the AP-1/ATF family; they are also enriched for common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation and lay the groundwork for future in vivo studies to explore the impact of BFT on nuclear structure and function.

The application of ApcMin/+ mouse model in colorectal tumor researches.

ApcMin/+ mouse is an excellent animal model bearing multiple intestinal neoplasia, used to simulate human familial adenomatous polyposis and colorectal tumors. The key point of this model is the mutation of Apc gene, which is a significant tumor-suppressor gene in the Wnt signaling pathway. There are also some other possible mechanisms responsible for the development of colorectal tumors in the ApcMin/+ mouse model, such as tumor-associated signaling pathways activation, the changes of tumor-related genes, and the involvement of some related proteins or molecules. The relevant literatures about ApcMin/+ mouse model from PUBMED databases are reviewed in this study. In recent years, increasing studies have focused on the application of ApcMin/+ mouse model in colorectal tumor, trying to find effective therapeutic targets for further use. This article will give a brief review on the related molecular mechanisms of the ApcMin/+ mouse model and its application in colorectal tumor researches.

A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase.

Although valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene - either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model's small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors - unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.

Aldose Reductase Inhibitor, Fidarestat Prevents High-fat Diet-induced Intestinal Polyps in ApcMin/+ Mice

Recent epidemiological and experimental studies have shown that obesity is a major risk factor for Colorectal Cancer (CRC). Regular intake of high fat-containing diet can promote obesity and metabolic syndrome by increasing the insulin resistance and inflammatory response which contribute to carcinogenesis. Previously, we have shown that inhibition of polyol pathway enzyme aldose reductase (AR) prevents carcinogens- and inflammatory growth factorsinduced CRC. However, the effect of AR inhibition on a high-fat diet (HFD)-induced formation of intestinal polyps in Apc-deficient Min (multiple intestinal neoplasia; ApcMin/+) mice is not known. We examined the effect of AR inhibitor, fidarestat on the HFD-induced formation of preneoplastic intestinal polyps in ApcMin/+ mice which is an excellent model of colon cancer. APCMin/+ mice fed for 12 weeks of HFD caused a significant increase in the formation of polyps in the small and large intestines and fidarestat given along with the HFD prevented the number of intestinal polyps. Fidarestat also decreased the size of the polyps in the intestines of HFDtreated APC Min mice. Further, the expression levels of beta-catenin, PCNA, PKC-β2, P-AKT, Pp65, COX-2, and iNOS in the small and large intestines of HFD-treated mice significantly increased, and AR inhibitor prevented it. Our results thus suggest that fidarestat could be used as a potential chemopreventive drug for intestinal cancers due to APC gene mutations.

Non-toxigenic Bacteroides fragilis (NTBF) administration reduces bacteria-driven chronic colitis and tumor development independent of polysaccharide A.

Polysaccharide A (PSA), an immunogenic capsular component of non-toxigenic Bacteroides fragilis (NTBF) strain NCTC 9343, is reported to promote mucosal immune development and suppress colitis. Contrastingly, enterotoxigenic Bacteroides fragilis (ETBF) is highly associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), rapidly inducing IL-17-dependent murine colitis and tumorigenesis. In specific-pathogen-free (SPF) C57BL/6 wild type (WT) and multiple intestinal neoplasia (MinApc716+/−) mice, we show that sequential treatment of the NTBF strain, 9343, followed by the ETBF strain, 86–5443-2–2 (86), diminished colitis and tumorigenesis. Mice treated simultaneously with 9343 and 86 exhibited both severe colitis and tumorigenesis. Abrogated disease severity in sequentially treated mice was attributed to 9343 strain dominance and decreased IL-17A, but 86 colonization prior to or simultaneous with 9343 mitigated the anti-inflammatory effect of 9343. Remarkably, 9343-mediated protection was independent of PSA, as sequentially treated mice receiving ΔPSA 9343 exhibited similar protection. Further, SPF WT and Min mice colonized with PSA-competent or PSA-deficient 9343 exhibited similar IL-10, IL-17, and IFN-γ responses. Treatment of 86-colonized mice with 9343 failed to disrupt 86 pathogenesis. Our findings demonstrate 9343 colonization, independent of PSA, offers prophylaxis against colitis-inducing 86 but may not be a valid therapy once colitis is established.

Abstract 5055: Expression of Tn and SialylTn tumor antigens in intestinal epithelia in APC-mutant mice promotes colorectal carcinogenesis and progression

Abstract Colorectal cancer (CRC) is a leading cause of cancer deaths in the Western world, and several different animal models for CRC have been established to understand its initiation, progression, and metastasis. Mice with mutations in APC (APCm) is widely accepted in the field because APC is the predominant mutated gene in human CRC. Mice carrying APCm only develop multiple intestinal neoplasia, but not carcinoma. Importantly, emerging evidence shows that tumor associated carbohydrate antigens, Tn and sialylTn (STn) appear at an early stage of colon carcinogenesis, and are associated with progression and metastasis. However, the molecular mechanisms underlying the role of Tn/STn antigens in CRC tumorigenesis and progression are unknown. We identified a novel X-linked gene Cosmc (C1GalT1C1) which encodes a molecular chaperone Cosmc regulating O-glycosylation through assisting correct folding of core 1 β3-galactosyltransferase (T-synthase, C1GalT1). T-synthase is the key enzyme that converts Tn antigen to normal core 1 O-glycans on glycoproteins in all animal cells. Cosmc mutations lead to loss of T-synthase activity and resultant expression of Tn/STn antigens in human carcinoma cells and in other diseases. Moreover, in our study, intestinal epithelial cell (IEC)-specific Cosmc-knockout (IEC-Cosmc-KO) mice spontaneously developed invasive tumors mainly in the rectum from 3-12 months of age through complex pathways. To further investigate the role of Tn/STn antigens in CRC formation, progression and metastasis in a context of APC mutations, we generated IEC-Cosmcnull:APC-/+ (double-knockout, DKO) by gene targeting. Totally, 22 DKO mice (13 male and 9 female) and similar numbers of control mouse groups (APC-KO, Cosmc-KO and WT) were generated. Among them, the DKO male and female mice grew slower companied with stress signs and some mortalities around 3-4 months of age. Importantly, both APC-KO and DKO mice had multiple gross tumors in the small intestine, and all DKO mice had more and larger gross tumors in colorectum while only 15% of APC-KO mice developed tumors with smaller sizes in colon. Consistent with our findings, thickened rectum was observed in Cosmc-KO mice. Furthermore, the enlarged inguinal lymph nodes were also found in the DKO mice. Histology confirmed the Tn/STn expression, as well as adenomas and adenocarcinomas in the DKOs, and adenomas in APC-KOs and Cosmc-KO male mice. The distant metastasis of the CRC was not yet observed in these young DKO-mice, since it may require a longer time to develop. These results clearly indicate that the Cosmc deletion resulted in Tn and STn expression, promotes the colorectal carcinogenesis in APC-mutant mice. We are generating more elder DKO and control mice to continue investigating the role of Tn/STn in CRC progression and metastasis by performing detailed molecular analyses. Citation Format: Yuliang Jiang, Su-Ryun Kim, Shweta Kotian, Sean R. Stowell, Guangyu An, Tongzhong Ju. Expression of Tn and SialylTn tumor antigens in intestinal epithelia in APC-mutant mice promotes colorectal carcinogenesis and progression [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 5055.

CXCR4 inhibitor, MSX‐122 suppresses AOM‐induced colon cancer in Apc+/Min mouse

IntroductionIn prior studies, we showed that EGFR is required for tumor promotion by Western diet in the azoxymethane (AOM) model of sporadic colon cancer and the AOM/DSS model of inflammation‐associated colon cancer. We also demonstrated that ADAM17, the metalloprotease responsible for shedding membrane‐bound EGFR ligands, controls tumor development in both the AOM and the Apc+/mutant multiple intestinal neoplasia (Min) mouse model. Furthermore, we established that Sdf1a‐CXCR4 signals activated EGFR by an ADAM17‐dependent mechanism in colon cancer cells; and that Western diet up‐regulated Sdf1a‐CXCR4 and ADAM17 in colonic mucosa. We hypothesized that CXCR4 blockade might inhibit colon cancer development. The aim of this study was to test the ability of a novel CXCR4 antagonist, MSX‐122 (substituted benzylamine) to inhibit AOM‐promoted colon cancer in Apc+/Min mice. Min mice without AOM treatment mostly develop small intestinal tumors, whereas AOM‐treated Apc+/Min mice develop colon cancers (Suzui et al 2002).MethodsIn studies in cell culture, we examined the ability of MSX‐122 to block Sdf1a‐induced EGFR transactivation. In vivo, Apc+/Min mice were given AOM (10 mg i.p./kg body weight) and two weeks later mice were started on a Western diet (20% fat). Mice received MSX‐122 i.p. (2.5 mg/kg body weight, n=12) or vehicle (DMSO, n=13) three times weekly and were killed after 10 wks.ResultsMSX‐122 blocked EGFR transactivation by Sdf1a in HT29 colon cancer cells as assessed by reductions in pErbB2, pAKT and pERK. In vivo, MSX‐122 was well tolerated with mice showing comparable weight gain to vehicle‐treated mice. As expected, AOM induced colon tumors in Apc+/Min mice. MSX‐122 significantly reduced colon tumor incidence (92.3% vs. 50.0%, p=0.025) and cancer incidence (69.2% vs. 16.7%, p=0.015). MSX‐122 also reduced tumor burden as assessed by total tumor multiplicity (1.8 vs. 1.0 tumor per TBM; p=0.02), tumor volume (169.5 mm3 vs. 61.2 mm3, p&lt;0.023) and tumor proliferation as assessed by Ki67 staining (37.2±3.6% vs. 25.3±5.8%, p&lt;0.005). Cancer multiplicity also appeared to be decreased (1.6 vs. 1.0 tumor/TBM, p=0.17).ConclusionsMSX‐122 blocked EGFR transactivation by CXCR4 agonist Sdf1a in colon cancer cells. MSX‐122 also inhibited colon tumor development and progression in the AOM‐promoted Apc+/Min mouse model. We speculate that MSX‐122, which was well tolerated in a phase 1b clinical trial (Painter, W. 2015), could serve as a chemopreventive agent in individuals at increased risk for colon cancer.Support or Funding InformationNIH R01CA164124This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modified Shenlingbaizhu decoction reduces intestinal adenoma formation in adenomatous polyposis coli multiple intestinal neoplasia mice by suppression of hypoxia-inducible factor 1α-induced CD4 + CD25+ forkhead box P3 regulatory T cells

OBJECTIVETo test the hypothesis that modified Shenlingbaizhu decoction (MSD) attenuates the formation of intestinal adenomas by regulating activation of CD4+CD25+ forkhead box P3 (FoxP3) regulatory T cells (Tregs) by downregulation of hypoxia-inducible factor 1α (HIF-1α). METHODSChemical fingerprints of ginsenoside Rb1, ginsenoside Rc, paeoniflorin, and dioscin in standard extractions were used as material bases of MSD. Adenomatous polyposis coli multiple intestinal neoplasia (ApcMin/+) mice, which harbor a mutation in adenomatous polyposis coli, were used to host intestinal adenomas. Peripheral blood and spleen Tregs were analyzed by flow cytometry. Protein expression was analyzed by immunohistochemistry and Western blotting. RESULTSThe number and size of intestinal adenomas were significantly reduced by MSD treatment. Mucosal thickening and the spleen size were also substantially decreased by MSD. The carcinogenesis process in ApcMin/+ mice resembled that of human colorectal cancer. Molecular markers of neoplasms, such as β-catenin, cyclooxygenase-2, proliferating cell nuclear antigen, and p53, were substantially ameliorated by MSD treatment. Moreover, MSD downregulated peripheral and spleen CD4 + CD25+FoxP3+Tregs and reduced in situ expression of CD4, CD25, and FoxP3 in intestinal adenomas. MSD also suppressed HIF-1α expression in the intestinal adenomas, and HIF-1α inhibition decreased expression of FoxP3 in Jurkat T cells under hypoxic conditions. CONCLUSIONMSD is a valid prescription to control the formation of intestinal adenomas in ApcMin/+ mice. It exerts anti-cancer effects partially through suppression of HIF-1α that induced activation of CD4+CD25+FoxP3+ Tregs in vivo and in vitro.