Colon Tumor Formation Research Articles

Colonic Injuries Induced by Inhalational Exposure to Particulate-Matter Air Pollution.

Particulate matter (PM) exposure has been associated with intestinal disorders. Therefore, there is an urgent need to understand the precise molecular mechanism involved and explore potential prevention strategies. In this study, inhaled PM is shown to activate inflammatory pathways in murine colon. In a panel study, it is found that ambient PM levels are significantly associated with elevated number of fecal white blood cells in healthy subjects. Acting as a promoter, PM exposure accelerates chemical carcinogenesis‐induced colonic tumor formation in a murine model. Mechanistically, RNA‐seq assays suggest activation of phosphoinositide 3‐kinase (PI3K)/AKT cascades in chronically PM‐exposed human colon mucosal epithelial cells. Ablation of up‐stream driver fibroblast growth factor receptor 4 (FGFR4) effectively inhibits inflammation and neoplasia in PM‐exposed murine colons. Notably, dietary curcumin supplement is shown to protect against PM‐induced colonic injuries in mice. Collectively, these findings identify that PM exposure accelerates colonic tumorigenesis in a PI3K/AKT‐dependent manner and suggests potential nutrient supplement for prevention.

Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic.

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

The role of Parvimonas micra in intestinal tumorigenesis in germ-free and conventional APCmin/+ mice.

531 Background: Our in-house meta-analysis of fecal shotgun metagenomic sequences from colorectal cancer (CRC) and control subjects from four cohorts of various ethnicities identified a higher abundance of Parvimonas micra in CRC. We aimed to investigate the effect of P. micra in colon tumor formation and development. Methods: We collected 309 fecal samples and 259 colon biopsies from patients with CRC, advanced adenoma and healthy subjects. P. micra strain was isolated from the feces of a CRC patient. APC min/+ mice and germ-free (GF) mice were orally gavaged with P. micra, or Esherichia coli. Colon epithelial cell line NCM460 and cancer cell lines HT-29 and Caco-2 were exposed to P. micra or E. coli conditional medium. Results: P. micra was significantly enriched both in the feces (n = 207, p < 0.01) and tissue biopsies (n = 99, p < 0.01) of CRC patients compared with controls (n = 102 for fecal samples, n = 160 for tissues biopsies). APCmin/+ mice gavaged with P. micra exibited significantly higher tumor burden ( p < 0.01) and tumor load ( p < 0.01), compared to mice gavaged with either E. coli or non-bacterial control. Consistently, cell proliferation was significantly higher in the colon tissues of P. micra gavaged GF mice relative to control mice evidenced by increased Ki-67 positive cells ( p < 0.05) and PCNA protein expression ( p < 0.01) at weeks 20 and 32. In line with this, colon cell lines NCM460, HT-29 and Caco-2 exposed to P. micra conditional medium significantly increased proliferation than control group (all p < 0.05). Flow cytometry analyses showed that Th2 and Th17 cells were markedly increased, while Th1 were reduced in the lamina propria of the colon tissues of P. micra gavaged mice compared to control mice (all p < 0.01) . Consistently, P. micra colonization in GF mice was associated with increased expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12 (all p < 0.01). Conclusions: The abundance of P. micra was significantly increased in the feces and tissue biopsies of CRC patients. P. micra promotes intestinal carcinogenesis in APC min/+ mice and increase cell proliferation in GF mice. The tumor promoting effect of P. micra is associated with altered immune responses and enhanced inflammation in the gut.

Zyxin promotes colon cancer tumorigenesis in a mitotic phosphorylation-dependent manner and through CDK8-mediated YAP activation

Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo-YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.

Abstract 1273: Effect of a Western diet on colitis-associated colon tumor formation in BALB/c mice

Abstract Patients with inflammatory bowel disease (IBD) are at increased risk for inflammation associated colorectal cancer (CAC). The incidence of IBD is increasing in the Western industrialized world. Epidemiological studies suggest that a diet, high in fat but low in vitamin D, calcium, fibre and methyl donors is a risk factor for both, IBD and colorectal cancer (CRC). It has been shown previously that a ”New Western Diet”, caused development of sporadic colonic tumours in mice. Our study was performed to evaluate whether a Western Diet (WD) aggravates azoxymethane (AOM) /dextran sodium sulfate salt (DSS)-induced colitis-associated carcinogenesis in mice, and if a switch to the standard purified mouse diet AIN93G after cancer initiation is still able to ameliorate or delay the onset of the disease. Female BALB/c mice received either the WD (0.5 mg/g calcium, 2% fibre, 21 % butter fat, 0.23 μg/g folic acid) or AIN93G standard diet for 5 weeks until AOM was administered at a dose of 12.5 μg/kg intraperitoneally, followed by 3 cycles of DSS (2.5%). In one group the WD was switched to AIN93G diet one day before first administration of DSS. The mice were euthanized 80 days after AOM injection. One part of the colon, small intestine, kidney and liver were frozen in liquid nitrogen, the rest formalin fixed and paraffin embedded for mRNA and immunhistochemical analysis. Feeding constantly the WD shortened the colon (p<0.05) and increased the number (p<0.05) and size (p<0.01) of aberrant colonic crypt foci. The switch to the AIN93G diet ameliorated this effect, leading to reduced colitis and tumor promotion comparable with the AIN93G group. In colon ascendens of the WD group, Ki67 protein levels (p<0.001) and relative mRNA levels of the Wnt target genes c-myc (p<0.05) and axin2 (p<0.001) were reduced while mRNA levels of the vitamin D catabolizing enzyme cyp24a1 (p<0.001) and inos (p<0.05) were increased when compared with the AIN93G group. A healthy diet protects colonic mucosa against AOM/DSS-treatment and is an effective chemopreventive strategy to reduce chronic colonic inflammation and tumorigenesis. This project is funded by the Vienna Science and Technology Fund and the Austrian Research Fund. Citation Format: Charlotte Groeschel, Samawansha Tennakoon, Abhishek Aggarwal, Enikoe Kallay. Effect of a Western diet on colitis-associated colon tumor formation in BALB/c mice [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 1273.

Abstract 2379: PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function

Abstract Department of Genetics & Genome Sciences and Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA. In addition to phosphorylation, tyrosine residues in proteins can have nitration at the 3-carbon position on the phenol ring (Y-NO2). Protein tyrosine nitration occurs under both physiological and pathologic conditions. However, enzymes that add or remove this protein modification remain to be identified. Protein tyrosine phosphatase receptor T (PTPRT) is one of the twelve receptor protein tyrosine phosphatases (RPTPs) that have two catalytic PTP domains in their intracellular parts. While the membrane proximal PTP domains (D1) are protein tyrosine phosphatases, it has been thought that the C-terminal PTP domains (D2) are pseudo-phosphatase that lack enzymatic activity. Here we report that the pseudo-phosphatase domain (D2) of PTPRT is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT normally functions as a tumor suppressor and is frequently mutated in a variety of human cancers including colorectal cancer. Interestingly, a fifth of tumor-derived mutations of PTPRT are located in the D2 pseudo-PTP domain. We demonstrate that some of the tumor-derived mutations located in the pseudo-phosphatase domain impair the denitrase activity. Moreover, PTPRT mutant mice that inactivate the denitrase activity are susceptible to carcinogen-induced colon tumor formation. Our study uncovers a novel enzyme that functions as a tumor suppressor. Citation Format: Yiqing Zhao, Zhenghe Wang. PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function [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 2379.

5-Demethylnobiletin is more effective than nobiletin in preventing AOM/DSS-induced colorectal carcinogenesis in ICR mice

Nobiletin and 5-demethylnobiletin as polymethoxyflavone and hydroxy polymethoxyflavone, especially, have been reported to exhibit various beneficial biological activities for human health. In this study, the effects of NOB and 5-demethylnobiletin (DMNB), on azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colorectal carcinogenesis in Institute for Cancer Research (ICR) mice were compared. We found that NOB and DMNB significantly alleviated the weight loss, colon length and reduced colon tumor formation in AOM/DSS-induced colorectal carcinogenesis mice. At the molecular level, our results from western blot and polymerase chain reaction (PCR) analysis showed that 0.025% of NOB and DMNB presented an anti-inflammation property by reducing cyclooxygenase- 2 (COX-2) and interleukin 6 (IL-6) expression. Taken together, these results demonstrate that DMNB had a better chemo-preventive efficacy than NOB in AOM/DSS-induced colorectal carcinogenesis in ICR mice model.

Pristimerin, a naturally occurring triterpenoid, attenuates tumorigenesis in experimental colitis-associated colon cancer

BackgroundPristimerin is a quinonemethide triterpenoid with anti-cancer, anti-angiogenic, anti-inflammatory and anti-protozoal activity. However, the therapeutic role of pristimerin in colitis-associated colorectal carcinogenesis is unknown. PurposeWe sought to examine the therapeutic effects of pristimerin on colitis-associated colon cancer induced in mice using azoxymethane (AOM)/dextran sulfate sodium (DSS). The goal was to identify the potential mechanism of action underlying the pharmacological activity of pristimerin. MethodsBALB/c mice were injected with AOM and administered 2% DSS in drinking water. The mice were fed with a diet supplemented with pristimerin (1 to 5 ppm), and colonic tissue was collected at 64 days. The inflammatory status of the colon was assessed by determining the levels of cyclooxygenase-2, inducible nitric oxide synthase and pro-inflammatory cytokines using Western blotting, immunohistochemistry and real-time RT-PCR analyses. Markers of proliferation (proliferating cell nuclear antigen) and apoptosis (TUNEL) were identified in the colon tissues immunohistochemically. The levels of cell cycle-, apoptosis-, and signaling-related proteins were detected by Western blot in colon tissues. ResultsAdministration of pristimerin significantly reduced the formation of colonic tumors. Western blot and immunohistological analyses revealed that dietary pristimerin markedly reduced NF-κB-positive cells and levels of inflammation-related proteins in colon tissue. Pristimerin also reduced cell proliferation, induced apoptosis, and decreased the phosphorylation of AKT and FOXO3a in colon tissue. ConclusionPristimerin administration decreased inflammation and proliferation induced by AOM/DSS in colon tissue. It also induced apoptosis and regulated the AKT/FOXO3a signaling pathway. Overall, this study indicates the potential value of pristimerin in suppressing colon tumorigenesis.

Chemopreventive Effect of Aster glehni on Inflammation-Induced Colorectal Carcinogenesis in Mice.

Although Aster glehni is a common dietary herb that has various bioactivities, including anti-diabetic, anti-adipogenic, and anti-inflammatory effects, A. glehni has not been studied in colon cancer. Therefore, we hypothesized the chemopreventive effects of an ethanol extract of A. glehni (AG) on azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated cancer (CAC) in mice. In this study, we found that treatment with AG significantly attenuated the AOM/DSS-induced enlargement of the spleen and shortening of the colon. In addition, colonic tumor formation, colonic damage, and increased muscle thickness were significantly reduced in AOM/DSS-induced mice fed AG. Treatment with AG also reduced intestinal interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α production and decreased inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expression in mice with AOM/DSS-induced CAC. Furthermore, AG reduced nuclear factor (NF)-κB activation via phosphorylation and degradation of inhibitor of kappa Bα (IκBα), leading to inhibition of NF-κB p65 nuclear translocation. It also downregulated the expression of NF-κB-related proteins, including the B-cell lymphoma 2 (Bcl-2) family and inhibitors of apoptosis proteins (IAPs), in mice with AOM/DSS-induced CAC. Taken together, these findings suggest that the treatment with AG inhibited colitis-associated colon carcinogenesis in mice, and this chemopreventive effect was strongly mediated by suppression of the NF-κB signaling pathway, indicating that AG could be a promising protective agent against CAC.

Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria.

Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of Escherichia coli and Bacteroides fragilis Genes for colibactin (clbB) and Bacteroides fragilis toxin (bft), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with E. coli (expressing colibactin), and enterotoxigenic B. fragilis showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.

PKM2 is not required for colon cancer initiated by APC loss

BackgroundCancer cells express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2). PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in β-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in β-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated.ResultsColon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in pyruvate kinase M1 expression but had no effect on nuclear β-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression.ConclusionsPKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of β-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression.

Abstract 4507: Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer

Abstract Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor and as an inhibitor of the IGF-1 pathway. Recent data indicate klotho as a tumor suppressor in an array of malignancies, including colorectal cancer (CRC). We aimed to decipher the role of klotho as a tumor suppressor in CRC. Analysis of a public database (Oncomine) indicated reduced expression of klotho in CRC. Two models were employed to study the effect of klotho in vivo. Using the azoxymethane inflammation model, we discovered that klotho inhibited colon polyp formation. Using an orthotopic model consisting of direct injection of MC38 cells to the mice colons, we found that klotho inhibited colonic obstruction and tumor formation. Similarly, klotho inhibited colony formation and proliferation of CRC cell lines. Aberrant activation of the canonical Wnt pathway is implicated in CRC development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. While we did not see an effect of klotho on the IGF-1 pathway, it reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. Yet, the inhibitory effect of klotho on CRC cell colony formation was only partially rescued by a constitutively active β-catenin. These data indicate on additional mechanisms involved in the TS activity of klotho. Thus, we conducted an RNA expression array and observed involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Further studies showed that klotho induced elevation in XBP1 RNA levels, GRP78 protein levels, and eIF2α phosphorylation, all indicative of UPR regulation. Importantly, pharmacologic inhibition of ER stress and UPR overcame the growth suppression effect of klotho in CRC cells. Our data indicate klotho as a potent tumor suppressor in CRC, and suggest its role at very early stages of tumor formation, already at polyp formation. Our data also indicate, for the first time, klotho as a regulator of ER stress and UPR in cancer. These findings may pave the way for the development of novel therapeutic strategies based on enhancement of UPR. Citation Format: Tal Etan, Tammi Rubinstein, Tami Rubinek, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Arbesfeld, Iris Barshack, Ido Wolf. Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2017-4507

Abstract 4804: Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis

Abstract Colorectal cancer (CRC) is the third most common diagnosed cancer and the third leading cause of cancer-related deaths in the United States. It has also been reported that colon cancer incidence and mortality rates are higher in men than woman, but there is yet a determined mechanistic link to show the factors that underlie the sex-specific differences in CRC initiation and progression. Benzo(a)pyrene [B(a)P], a member of the polycyclic aromatic hydrocarbon (PAH) family of compounds is a well-characterized environmental toxicant that has been proven to be a major contributor to the development of sporadic colon cancer. Published studies indicate that Aryl Hydrocarbon Receptor (AhR), a receptor for [B(a)P], bind to estrogen receptor (ER) and negatively affect AhR-target gene transcription. This study aims to elucidate the sex-specific differences in B(a)P-induced colon cancer in adult Polyposis In the Rat Colon (PIRC) model. We hypothesize that sex-specific differences in B(a)P biotransformation modulates the formation of colon tumors in PIRC rats. Groups of female and male PIRC rats (n = 8) received sub-chronic exposure to 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Female and male rats that received no [B(a)P] treatment served as controls. [B(a)P] was shown to have no significant effect on body weight of these rats and female PIRC rats that received 25, 50 and 100 µg B(a)P/kg body wt. showed significant decrease in total polyp count when compared to males with respective treatments. Polyp sizes of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. were increased when compared to males respectively. Histopathological analysis of colon polyps revealed that female animals exhibited low-grade to no dysplasia while high-grade dysplasia was recorded in male animals treated with corresponding doses. Phase 1 enzyme, Cytochrome P450 isoform 1A1 (CYP1A1), and phase 2 enzyme, Sulfotransferase Family 1A Member 1 (SULT1A1), were downregulated in colon tissue of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. when compared to male counterparts. In future studies, by measuring the expression of other phase 1 and phase 2 drug metabolizing enzymes (DME), along with measuring circulating estrogen levels, analyzing [B(a)P] metabolite profile, and probing B(a)P-DNA interactions, we will provide insight into if and how estrogen receptor protects females from developing colon cancer. This research was funded by NIH grants 5RO1CA142845-04, 5R25GM059994-3, and G12MD007586-29. Citation Format: Kenneth J. Harris, Kelly L. Harris, Mary K. Washington, James Amos-Landgraf, Aramandla Ramesh. Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4804. doi:10.1158/1538-7445.AM2017-4804

Abstract 1255: PhIP/DSS-induced colon carcinogenesis in CYP1A-humanized mice and its prevention by tocopherols

Abstract In order to establish a more physiologically relevant colorectal cancer model, we recently developed a colon-carcinogenesis model induced by the meat-derived dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and promoted by dextran sodium sulfate (DSS)-induced colitis in the cytochrome P450 1A-humanized (hCYP1A) mice. PhIP/DSS treatments caused rapid destruction of the colonic mucosa with severe inflammation, followed by the presence of reactive changes and low-grade dysplastic lesions, and then manifestation of high-grade dysplastic lesions and finally adenocarcinomas in the middle to distal colon of the hCYP1A mice after 10 weeks. Molecular analysis of the high-grade dysplastic lesions present at early time-points indicates Ctnnb1/β-catenin mutations and β-catenin nuclear accumulation in high-grade dysplastic lesions, but not in low-grade dysplastic lesions or adjacent normal tissues. Using hCYP1A/Lgr5-EGFP mouse, which harbors EGFP-tagged Lgr5 allele, we also investigated the role of Lgr5+ colon stem cells in the PhIP/DSS-induced colon carcinogenesis and found the presence of Lgr5-EGFP-expressing cells amidst ulcerated mucosa, high-grade dysplastic lesions and adenocarcinomas, suggesting a possible role of Lgr5+ stem cells in this colon-carcinogenesis model. In addition, our study demonstrated strong cancer preventive effects of tocopherols (T), the major forms of vitamin E, in PhIP/DSS-induced colon carcinogenesis. Dietary supplementation with δ-T and γ-T significantly reduced colon tumor formation and suppressed the expression of markers of oxidative and nitrosative stress as well as pro-inflammatory mediators in tumors and adjacent tissues. By administering δ-T at different time points, we found that the inhibitory effect of δ-T against colon carcinogenesis was mainly due to protection against early cellular and DNA damages caused by PhIP. α-T was found to be ineffective in inhibiting colon tumorigenesis and less effective in attenuating the molecular changes. Altogether, PhIP/DSS-induced colon carcinogenesis is likely initiated from residual epithelial cells (possibly Lgr5+ colon stem cells) and promoted by colitis, and subsequently developed into high-grade dysplasia and adenocarcinoma. These events are effectively inhibited by δ-T and γ-T, but not α-T. (This work was supported by the US NIH grants RO1 CA133021, RO1 AT007036, and F31 CA168333 as well as shared facilities funded by center grant P30 CA72720 and P30 ES005022). Citation Format: Chung S. Yang, Jayson Chen, Anna B. Liu, Mao-Jung Lee, Hong Wang, Guangxun Li, Lanjing Zhang, Kenneth Reuhl, Nanjoo Suh. PhIP/DSS-induced colon carcinogenesis in CYP1A-humanized mice and its prevention by tocopherols [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2017-1255

Sphingosine kinase 1 expression enhances colon tumor growth

BackgroundAccumulating evidence suggests that sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate pathway plays a pivotal role in colon carcinogenesis.MethodsTo further support the evidence, we investigated the effects of SphK1 using three separate animal models: SphK1 knockout mice, SphK1 overexpressing transgenic mice, and SphK1 overexpression in human colon cancer xenografts. Using azoxymethane (AOM, colon carcinogen), we analyzed colon tumor development in SphK1 KO and SphK1 overexpression in intestinal epithelial cells regulated by a tet-on system. Then, we analyzed subcutaneous tumor growth using xenografts of HT-29 human colon cancer cell. Finally, immunohistochemical analyses for SphK1 and COX-2 were performed on human colon cancer tissue microarray.ResultsSphK1 KO mice, compared to wild-type mice, demonstrated a significant inhibition in colon cancer development induced by AOM (58.6% vs. 96.4%, respectively, P < 0.005). Tumor multiplicity (1.00 vs. 1.64 per colon, respectively, P < 0.05) and tumor volume (14.82 mm3 vs. 29.10 mm3, P < 0.05) were both significantly reduced in SphK1 KO mice compared to wild-type mice. Next, SphK1 overexpression in HT-29 enhanced tumor growth as compared to GFP control in nude mice (229.5 mm3 vs. 90.9 mm3, respectively, P < 0.05). Furthermore, overexpression of SphK1 in intestinal epithelial cells significantly enhances AOM-induced colon tumor formation (P < 0.05). Lastly, SphK1 and COX-2 intensity tended to reduce overall survival of late stage colon cancer patients.ConclusionsSphK1 expression regulates the early stage of colon carcinogenesis and tumor growth, thus inhibition of SphK1 may be an effective strategy for colon cancer chemoprevention.

Estradiol Has Differential Effects on Acute Colonic Inflammation in the Presence and Absence of Estrogen Receptor β Expression.

Inflammatory bowel disease (IBD) increases the risk of developing colon cancer. This risk is higher in men compared to women, implicating a role for female hormones in the protection against this disease. Studies from our laboratory demonstrated that estradiol (E2) protects against inflammation-associated colon tumor formation when administered following chemical carcinogen and induction of chronic colitis. This study seeks to better understand the effect of E2 on acute colitis in the presence and absence of estrogen receptor β (ERβ). Inflammation was induced by 2,4,6-trinitrobenzenesulfonic acid in wild-type (WT) and ERβ knockout (ERβKO) mice implanted with a control or E2-containing pellet and killed 5days later. Inflammation and injury were scored by a pathologist. Apoptosis and proliferation were assessed by immunohistochemistry. Cytokines were measured by multiplex analysis. E2 treatment reduced inflammation in the middle colon in WT mice and the distal colon in ERβKO mice compared to control mice. WT mice had reduced IL-6, IL-12, IL-17, GM-CSF, IFN-γ, MCP-1, MIP-1α, and TNF-α, and ERβKO had reduced IL-6 and IFN-γ expression in response to E2. Injury scores were lower in E2-treated ERβKO mice compared to control ERβKO mice. ERβKO mice had increased proliferation in the basal third of crypts in the distal colon and decreased apoptosis in the proximal colon. These data suggest that E2 has differential protective effects against acute colitis in the presence or absence of ERβ and provide insight into how E2 may protect against IBD.

Plant Sterol Esters in Extruded Food Model Inhibits Colon Carcinogenesis by Suppressing Inflammation and Stimulating Apoptosis.

Plant sterols in their free forms are known to inhibit colon cancer. Whether these activities persist when compounds are incorporated into processed food is not reported yet. This study aimed to test the ability of plant sterol esters (PSE) incorporated into a nonpuffed extruded food (NPE) model to inhibit colon carcinogenesis. PSE was added into NPE at four concentrations (0.0%, 0.7%, 1.4%, and 2.1%). PSE-NPE activity was tested in azoxymethane/dextran sodium sulfate-induced Balb/c mice. The groups given PSE-NPE did not show any colon tumor formation. Immunohistochemistry results revealed that the group fed with 1.4% PSE had the lowest histoscore for cyclooxygenase-2 expression and the highest histoscore for cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9expressions. The results of this study indicated that even after incorporation into a food system, which is processed using high pressure and temperature, PSE retained its chemopreventive activity. The proposed mechanisms are by suppressing inflammation and inducing apoptosis.

Interleukin-35 expression is associated with colon cancer progression.

Colon cancer development is closely related to inflammation. Thus, we conducted the present investigation to study the effects of IL-35 (Interleukin 35), a newly identified anti-inflammatory factor, on colon cancer development. We first evaluated the IL-35 expression in 186 pairs of colon cancer samples and paired adjacent normal tissues by qPCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-35 on patient survival rates, colon cancer progression and their sensitivity to chemotherapy drugs were assessed. IL-35 was barely expressed in the colon cancer tissue but highly expressed in the adjacent normal tissue. The down-regulation of IL-35 was significantly associated with the AJCC (American Joint Committee on Cancer) stage, nodal involvement, invasion depth, distant metastasis, differentiation and it was also shown to be an independent prognostic indicator of both disease-free and overall survivals for colon cancer patients. Overexpression of IL-35 in colon cancer cell suppressed cell migration, invasion, proliferation, colony formation and cancer stem cells through suppressing β-catenin. IL-35 inhibited colon tumor formation in the mice model and sensitize the cancer cell to chemotherapy drugs. Our results showed that IL-35 shows an inhibitory effect in colon cancer development and is a novel prognostic indicator. Therefore, IL-35 might be a potential therapeutic target.

Interleukin-37 mediates the antitumor activity in colon cancer through β-catenin suppression.

The occurrence and development of colon cancer is closely related to inflammation. Thus, we conducted the present retrospective study to investigate the effects of IL-37 (Interleukin 37), a newly identified anti-inflammatory factor, on colon cancer development. We first evaluated the IL-37 expression in 186 pairs of colon cancer samples and their adjacent normal mucosa by real-time PCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-37 on patient survival rates, colon cancer progression and their sensitivity to chemotherapy drugs were assessed. IL-37 was barely expressed in the colon cancer tissue but highly expressed in the adjacent normal tissue. The down-regulation of IL-37 was significantly correlated with the results of American Joint Committee on Cancer stage, nodal involvement, invasion depth, distant metastasis, differentiation and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with colon cancer. Overexpression of IL-37 in colon cancer cell suppressed cell migration, invasion, proliferation, colony formation and cancer stem cells through suppressing β-catenin. IL-37 inhibited colon tumor formation in the mice model and sensitize the cancer cell to chemotherapy drugs. Our results showed that IL-37 plays an inhibitory role in colon cancer development and function as a novel prognostic indicator and a potential therapeutic target.

SPDEF Induces Quiescence of Colorectal Cancer Cells by Changing the Transcriptional Targets of β-catenin

The canonical Wnt signaling pathway activates the transcriptional activity of β-catenin. This pathway is often activated in colorectal cancer cells, but strategies to block it in tumors have not been effective. The SAM pointed domain containing ETS transcription factor (SPDEF) suppresses formation of colon tumors by unclear mechanisms. We investigated these mechanisms and the effects of SPDEF on β-catenin activity in mouse models of colorectal cancer (CRC), CRC celllines, and mouse and human normal and cancer colonoids. We performed studies of Lgr5CreERT2; β-cateninexon3; Rosa26LSL-rtta-ires-EGFP; TRE-Spdef mice, which express an oncogenic form of β-catenin in Lgr5-positive ISCs upon administration of tamoxifen and SPDEF upon administration of tetracycline. CRC lines (HCT116 and SW480) were engineered to express inducible tagged SPDEF or vector (control) and subcutaneously injected into immunodeficient NSG mice. We generated SPDEF-inducible human colonoids, including a line derived from normal rectal mucosa (control) and an adenocarcinoma line derived from a patient with germline MUTYH mutation. Full-length and truncated forms of SPDEF were expressed in CRC cells; cells were assayed for β-catenin activity and studied in immunoprecipitation and chromatin immunoprecipitation assays. Expression of SPDEF was sufficient to inhibit intestinal tumorigenesis by activated β-catenin, block tumor cell proliferation, and restrict growth of established tumors. In tumor cells with activated β -catenin, expression of SPDEF induced a quiescent state, which was reversed when SPDEF expression was stopped. In mouse and human normal and tumor-derived enteroids/colonoids, those that expressed SPDEF for 3 days were significantly smaller. SPDEF inhibited the transcriptional activity of β-catenin via a protein-protein interaction, independent of SPDEF DNA binding capacity. SPDEF disrupted β-catenin binding to TCF1 and TCF3, displacing β-catenin from enhancer regions of genes that regulate the cell cycle but not genes that regulate stem cell activities. In studies of mice and human CRC, we found that SPDEF induces a quiescent state in CRC cells by disrupting binding of β-catenin to TCF1 and TCF3 and regulation of genes that control the cell cycle. In this model, β-catenin activity determines the proliferation or quiescence of CRC cells based on the absence or presence of SPDEF.