Inflammation-associated Colorectal Carcinogenesis Research Articles

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

Dro1/Ccdc80 inactivation promotes AOM/DSS-induced colorectal carcinogenesis and aggravates colitis by DSS in mice.

Colorectal carcinogenesis is a progressive multistep process involving the sequential accumulation of genetic alterations in tumor suppressor genes and oncogenes. Downregulated by oncogenes 1 (Dro1/Ccdc80) has been shown to be a potent tumor suppressor of colorectal carcinogenesis in the genetic ApcMin/+ mouse model. In ApcMin/+ mice, loss of DRO1 strongly increases colonic tumor multiplicity and leads to the regular formation of adenocarcinoma in the colon. To investigate DRO1's role in chemically induced as well as inflammation-associated colorectal carcinogenesis, the effect of Dro1 inactivation was studied in mice subjected to the carcinogen azoxymethane (AOM) and upon combined treatment with AOM and the proinflammatory agent dextran sodium sulfate (DSS), respectively. Loss of DRO1 increases multiplicity of preneoplastic aberrant crypt foci and colonic tumors upon administration of AOM. Combined treatment with AOM and DSS leads to increased colonic tumor number and promotes formation of adenocarcinoma in the colon. Moreover, Dro1 inactivation aggravates histological signs of acute and chronic DSS-induced colitis, strongly enlarges the size of ulcerative lesions in the intestinal lining, and exacerbates clinical signs and morbidity by DSS. Our results demonstrate DRO1 to be a strong tumor suppressor in the chemically induced colon carcinogenic mouse model. Additionally, we demonstrate DRO1 to inhibit colitis-associated colon cancer formation and uncover a novel putative role for DRO1 in inflammatory bowel disease.

Erratum: Tanaka, T. et al. Cimetidine and Clobenpropit Attenuate Inflammation-Associated Colorectal Carcinogenesis in Male ICR Mice. Cancers, 2016, 8, 25.

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Different Susceptibilities between Apoe- and Ldlr-Deficient Mice to Inflammation-Associated Colorectal Carcinogenesis.

Hypercholesterolemia resulting in atherosclerosis is associated with an increased risk of ischemic heart disease and colorectal cancer (CRC). However, the roles of apoliprotein (Apo) E (Apoe) and low-density lipoprotein (Ldl) receptor (Ldlr) in colorectal carcinogenesis have not yet been investigated. In this study, we examined the susceptibility of Apoe-deficient and Ldlr-deficient mice, which are genetic animal models of atherosclerosis to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. In Experiment 1, male Apoe-deficient (n = 20) and wild type (WT) mice (C57BL/6J, n = 21) were treated with a single intraperitoneal (i.p.) injection of AOM (10 mg/kg body weight) and then given 1.5% DSS in drinking water for seven days. They were maintained up to week 20 and sacrificed for the histopathological examination of colorectal tumors. The mRNA expression of cyclooxygenase (Cox)-2, inducible nitric oxide synthase (Nos2), tumor necrosis factor (Tnf)-α interleukin (Il)-1β, and Il-6 was assayed in the colorectal mucosa. In Experiment 2, male Ldlr-deficient (n = 14) and WT mice (C57BL/6J, n = 10) were given a single i.p. injection of AOM (10 mg/kg body weight) and then given 2% DSS in drinking water for seven days. They were sacrificed at week 20 to evaluate their colorectum histopathologically. In Experiment 1, the multiplicity of CRCs was significantly higher in the Apoe-deficient mice (2.75 ± 1.48) than in the WT mice (0.62 ± 0.67). The serum lipoprotein levels in the Apoe-deficient mice were also significantly higher than in the WT mice. In Experiment 2, the incidence (29%) and multiplicity (0.50 ± 0.94) of CRCs in the Ldlr mice were significantly lower than in the WT mice (80% incidence and 3.10 ± 2.38 multiplicity). The mRNA expression of two inducible enzymes and certain pro-inflammatory cytokines in the colorectum of each genotype was greater than in the respective WT mice. The values in the Apoe-deficient mice were much greater than in the Ldlr mice. These findings suggest that Apoe-deficient mice showed increased susceptibility to inflammation-associated colorectal carcinogenesis due to their high reactivity to inflammatory stimuli.

Cimetidine and Clobenpropit Attenuate Inflammation-Associated Colorectal Carcinogenesis in Male ICR Mice.

Histamine and histamine receptors (Hrhs) have been identified as critical molecules during inflammation and carcinogenesis. This study was conducted to determine the effects of Hrh1-Hrh3 antagonists on inflammation-associated colorectal carcinogenesis. Male ICR mice were treated with azoxymethane (AOM, 10 mg/kg bw, i.p.) and 1.5% dextran sodium sulfate (DSS, drinking water for 7 days) to induce colorectal carcinogenesis. The mice were then fed diets containing test chemical (500 ppm terfenadine, 500 ppm cimetidine or 10 ppm clobenpropit) for 15 weeks. At week 18, feeding with the diets containing cimetidine (Hrh2 antagonist) and clobenpropit (Hrh3 antagonist/inverse agonist) significantly lowered the multiplicity of colonic adenocarcinoma. Terfenadine (Hrh1 antagonist) did not affect AOM-DSS-induced colorectal carcinogenesis. Adenocarcinoma cells immunohistochemically expressed Hrh1, Hrh2, Hrh3 and Hrh4 with varied intensities. Because clobenpropit is also known to be a Hrh4 receptor agonist, Hrh2, Hrh3 and Hrh4 may be involved in inflammation-related colorectal carcinogenesis. Additional data, including the mRNA expression of pro-inflammatory cytokines and inducible inflammatory enzymes in the colonic mucosa, are also presented.

Ghrelin administration suppresses inflammation-associated colorectal carcinogenesis in mice.

Ghrelin is a 28-amino-acid peptide that stimulates the release of pituitary growth hormone. Because of its orexigenic effects, ghrelin is being developed as a therapeutic option for postoperative support and treatment of anorexia-cachexia syndrome of cancer patients. However, ghrelin has a multiplicity of physiological functions, and it also affects cell proliferation. Therefore, the effects of ghrelin administration on carcinogenesis and cancer progression in patients susceptible to cancer should be clarified. In this study, we examined the effects of ghrelin on cancer promotion in vivo using murine intestinal carcinogenesis models. Intestinal tumorigenesis was examined to determine the effects of either exogenous ghrelin administration or ghrelin deficiency following deletion of the Ghrl gene. Two murine intestinal tumorigenesis models were used. The first was the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced inflammation-associated colon carcinogenesis model and the second was the ApcMin/+ genetic cancer susceptibility model. In AOM/DSS-treated mice, administration of ghrelin significantly suppressed tumor formation in the colon. In contrast, ghrelin administration did not affect the number of intestinal tumors formed in ApcMin/+ mice. The absence of endogenous ghrelin did not affect the incidence of intestinal tumors in either AOM/DSS-treated mice or ApcMin/+ mice, though tumor size tended to be larger in Ghrl−/− colons in the AOM/DSS model. No tumor-promoting effect was observed by ghrelin administration in either tumorigenesis model. In summary, this study provides in vivo experimental evidence for the usefulness of ghrelin administration in the chemoprevention of inflammation-associated colorectal carcinogenesis and may suggest its safety in patients under colitis-associated cancer susceptibility conditions.

The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis.

MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh-/- mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh-/- than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh-/- mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh-/- mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh-/- mice provides a good model for MAP.

Abstract LB-4: Ghrelin administration suppresses inflammation-associated colorectal carcinogenesis in mice

Abstract Background and aim: Ghrelin is a 28-amino-acid peptide, capable of stimulating growth hormone release. Because of its orexigenic effect, ghrelin is being developed as a therapeutic option for post-operative support and anorexia-cachexia syndrome of cancer patients. However, ghrelin has a multiplicity of physiological functions in various organs and immune system, and also affects cell proliferation. Therefore, it is important to know the effect of ghrelin administration on carcinogenesis and cancer progression in patients who are susceptible to cancer. This study aimed to test the effect of ghrelin on cancer promotion using murine intestinal carcinogenesis models. Method: Effect of ghrelin knockout or exogenous ghrelin administration (3 nmoles/day, i.p.) on intestinal tumorigenesis was analyzed in two types of murine intestinal tumorigenesis models; ApcMin/+ mice (genetic cancer susceptibility model) and AOM/DSS-treated mice (inflammation-associated colon cancer model). Results: The absence of ghrelin did not affect the number of intestinal tumors formed in ApcMin/+ mice and AOM/DSS-treated mice. Administration of ghrelin did not affect the number of intestinal tumors formed in ApcMin/+ mice, either. However, in AOM/DSS-treated mice, the administration of ghrelin significantly suppressed the formation of colon tumors (p < 0.001). The intestinal mRNA levels of inflammatory cytokines after DSS treatment were also suppressed by the administration of ghrelin. Conclusion: Ghrelin administration suppresses inflammation-associated colorectal carcinogenesis, which is possibly mediated by anti-inflammatory effects of ghrelin. Citation Format: Hiroaki Kataoka, Makiko Kawaguchi, Ai Kanemaru, Tsuyoshi Fukushima, Nobuhiro Matsumoto, Masamitsu Nakazato. Ghrelin administration suppresses inflammation-associated colorectal carcinogenesis in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-4. doi:10.1158/1538-7445.AM2014-LB-4

Apc-Mutant Kyoto Apc Delta (KAD) Rats Are Susceptible to 4-NQO-Induced Tongue Carcinogenesis

Despite widening interest in the possible association between infection/inflammation and cancer development, knowledge of this issue in relation to oral cancer remains inadequate. This study aimed to determine the susceptibility of Apc-mutant Kyoto Apc Delta (KAD) rats, which are vulnerable to developing inflammation-associated colorectal carcinogenesis, to 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in order to clarify the role of inflammation in oral cancer. KAD (20 males and 22 females) and F344/NS1c (22 males and 23 females) rats received drinking water with or without 4-NQO (20 ppm) for eight weeks. Histopathological and immunohistochemical analyses of the tongue were performed at week 20. Additionally, the mRNA expression of inflammatory cytokines in the tongue mucosa was determined at week 8. Tongue squamous cell carcinoma (SCC) developed in the KAD and F344/NS1c rats that received 4-NQO. Regardless of gender, the incidence and multiplicity of tongue SCC were greater in the KAD rats than in the F344/NS1c rats. In addition, the multiplicity of tongue SCC in the female KAD rats was significantly greater than that observed in the male KAD (p < 0.01) and female F344/NS1c rats (p < 0.05). The levels of inflammation and the mRNA expression of inflammatory cytokines in the tongue in the 4-NQO-treated female KAD rats were the highest among the rats given 4-NQO. These results show that KAD rats, particularly females, are susceptible to 4-NQO-induced tongue carcinogenesis, suggesting the utility of models employing KAD rats for investigating the pathobiology of oral (tongue) carcinogenesis associated with inflammation.

NF-κB and Nrf2 signaling pathways contribute to wogonin-mediated inhibition of inflammation-associated colorectal carcinogenesis.

The transcriptional factors nuclear factor-κB (NF-κB) and NF-E2-related factor 2 (Nrf2) have been recently reported to have critical roles in protecting various tissues against inflammation and colitis-associated colorectal cancer (aberrant crypt foci). Our previous studies showed that wogonin (5,7-dihydroxy-8-methoxyflavone) possessed anti-neoplastic and anti-inflammatory activities. The present study extended these important earlier findings by exploring the effect of wogonin on the initiation and development of colitis-associated cancer. Wogonin lowered tumor incidence and inhibited the development of colorectal adenomas in azoxymethane- or dextran sulfate sodium-induced mice. We found that wogonin significantly decreased the secretion and expression of IL-6 and IL-1β, reduced cell proliferation and nuclear expression of NF-κB in adenomas and surrounding tissues and promoted Nrf2 nuclear translocation in surrounding tissues, although overexpressed Nrf2 in tumor tissues was independent of wogonin administration. Furthermore, wogonin inhibited the interaction between human monocytic THP-1 cells and human colon cancer HCT116 cells, and significantly downregulated lipopolysaccharide-induced secretion of prototypical pro-inflammatory cytokines IL-6 and IL-1β in THP-1 cells. Further mechanism research revealed that wogonin inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/β, and promoted Nrf2 signaling pathway in HCT116 cells and THP-1 cells. Taken together, the present results indicated that wogonin effectively suppressed inflammation-associated colon carcinogenesis and cancer development, suggesting its potential as a chemopreventive agent against colitis-associated colon cancer.

Abstract 1945: Differential expression of miRNA in inflammation-associated colorectal carcinogenesis assayed by microRNA profiling in Muc2−/− mice.

Abstract Inflammatory bowel disease is an important risk factor for the development of colorectal cancer. How the inflammatory or immunological microenvironment modulates tumor initiation and development is a challenging question. Using an appropriate animal model to accurately reproduce the progression from spontaneous chronic inflammation to tumor formation contributes to better study chronic colitis-associated carcinogenesis and its underlying molecular mechanisms. Previous studies have shown that decreased number of goblet cells and reduced Muc2 expression is commonly observed in ulcerative colitis and colorectal carcinoma. We have demonstrated that targetedly knockout of the Muc2 gene in a mouse causes spontaneous colitis in the early age and carcinogenesis in small intestine, colon and rectum in the late age. Unfortunately, the underlying mechanism is not clear, although it is known that chronic inflammation plays a crucial role in malignancy transformation. Recent studies have revealed that epigenetic changes are involved in colitis-associated cancer formation, among them, microRNA (miRNA) is more important. miRNAs are small molecular noncoding RNAs and are thought to act as post-transcriptional modulators of gene expression by base-pairing with the 3′-untranslated regions of the target mRNAs, functioning in the regulation of development, differentiation, cell proliferation, programmed cell death and fat metabolism. Using the unique Muc2 knockout mouse model of colorectal cancer (Muc2−/−), we conducted miRNA array on intestinal epithelial cells isolated from 3-month old mice (4 Muc2−/− mice and 4 Muc2+/+ mice, respectively). Microarray profiling showed that 29 miRNAs from Muc2−/− mouse intestinal epithelial cells exerted lower expression enrichment and 115 miRNAs displayed higher expression enrichment, in comparison with those in Muc2+/+ mice (fold cutoff ≥2). Interestingly, most of these miRNAs were targeting oncogenes, tumor suppressors, cytokines, autophagy, inflammation, apoptosis, proliferation and differentiation. Notably, approximately 20% of the identified miRNAs showed some degrees of correlation with carcinogenic effects. The inventory of miRNA profiles in the Muc2−/− mice provides an important step toward further elucidation of miRNA function and miRNA-related gene regulatory networks in inflammation-associated cancer formation. (This work is sponsored in part by grant fund 91229115) Citation Format: Yonghua Bao, Zhiguo Chen, Qi Shi, Wenfeng Fang, Xiuli Bi, Huali Dong, Wancai Yang, Wancai Yang. Differential expression of miRNA in inflammation-associated colorectal carcinogenesis assayed by microRNA profiling in Muc2−/− mice. [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 1945. doi:10.1158/1538-7445.AM2013-1945

Mast cells and inflammation-associated colorectal carcinogenesis

Close association between chronic inflammation and cancer has been recently highlighted. Indeed, inflammatory bowel disease (IBD) has been strongly linked with an increased risk of development of colorectal cancer (CRC). Inflammatory cell-produced inflammatory mediators, such as proinflammatory cytokines and inducible enzymes, contribute to this association. In an inflammatory microenvironment, infiltrating macrophages and mast cells mediate production of these inflammatory mediators to promote growth of tumors in target tissues. In contrast to macrophages, contribution of mast cells to CRC development in inflamed colon is not well understood. This study aimed to determine the role of mast cells in inflammation-associated colorectal carcinogenesis. CRC was induced by administration of the colonic carcinogen, azoxymethane (AOM), and the tumor promoter dextran sodium sulfate (DSS) in male mast cell-deficient WBBF(1)-kit (W/W-v) (W/W(v)) and mast cell-normal WBB6F(1)-+/+(WT) mice. At week 12, the W/W(v) mice had markedly lower inflammation scores in the colon when compared with WT mice. The mRNA levels of colonic proinflammatory cytokines and inducible enzymes were also decreased in W/W(V) mice at weeks 12 and 20, when compared with WT counterparts. Colorectal tumors, including CRC, were identified by histopathological analysis performed 20weeks thereafter. Importantly, there were less neoplastic and preneoplastic colonic lesions in the W/W(v) mice compared with the WT mice. Thus, for the first time, our study shows that mice lacking mast cells are less susceptible to inflammation-associated colorectal carcinogenesis. Our findings also suggest that mast cells and their selected cytokines could play an important role in inflammation-mediated tumorigenesis through regulation of proinflammatory cytokines and inducible inflammatory enzymes.

Abstract 957: Dietary flavonols protect against colorectal carcinogenesis in an AOM-DSS mouse model for colorectal cancer

Abstract Background: Epidemiological studies suggest that a flavonol-rich diet inhibits colorectal carcinogenesis. Flavonols are a subgroup of polyphenols, which are found in fruits and vegetables usually glycosylated and are in particularly high concentrations in dry beans, onions, apples, and tea. The most consumed individual flavonols are in this order quercetin and one of its glycosylated forms rutin, kaempferol, myricetin, and isorhamnetin. Aim: To determine which individual flavonol is most protective against colorectal carcinogenesis in an inflammation-associated mouse colorectal carcinogenesis model (azoxymethane[AOM]/dextran sodium sulfate[DSS]-induced FVB mice). Methods: At 6 weeks of age, mice were injected intraperitoneally with AOM (10 mg/kg body weight). Mice then received for 1 week 2% DSS in the drinking water (1 or 1 and 4 weeks after AOM injection). Three days after the last DSS cycle, mice either remained on the AIN-93G diet or were switched to one enriched with 500 mg of kaempferol or equimolar concentrations of isorhamnetin, myricetin, quercetin, or rutin/kg of diet. Mice (n=32/group) remained for 85 days on the diets unless they had to be sacrificed earlier because of morbidity. A subset of mice was imaged using magnetic resonance imaging (MRI) at various times throughout the study. Results: Among the diets, mice on the control diet had the highest morbidity (50%) and mice on the isorhamnetin diet had the lowest morbidity (22%). Similarly, lesion number and size were lowest in mice on the isorhamnetin (16/mouse and 8.8 mm3/lesion) and the kaempferol (15/mouse and 7.4 mm3/lesion) diet compared to the control diet (21/mouse and 11.6 mm3/lesion). Image parameters detected different stages of colorectal carcinogenesis that can be modified by diet. Conclusion: All individual flavonols showed some protection against colorectal carcinogenesis with isorhamnetin and kaempferol being most efficacious. Studies are underway to identify serum, tissue, and imaging biomarkers of efficacy at earlier stages of colorectal carcinogenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 957.

Increased Susceptibility of Nrf2 Knockout Mice to Colitis-Associated Colorectal Cancer

The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)-induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/DSS-induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS-treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E(2) and leukotriene B(4) levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.