Colitis-associated Colon Cancer Model Research Articles

640P - Expression profile of EPHB3 and its prognostic significance in colorectal cancer progression (Running head: Prognostic value of EPHB3 in colorectal cancers)

BackgroundA tumour suppressive role for EPHB receptors in various malignancies has been described, however their expression profile and prognostic significance in human colorectal cancers (CRCs) remain unclear. In this study, we aimed to investigate the expression profile of EPHB3 during CRC progression and determined its prognostic impact in a large cohort of CRC samples. MethodsWe examined the EPHB3 mRNA levels by real time PCR with fresh frozen CRC samples and evaluated protein expression by immunohistochemistry with tissue microarrays containing various benign tumours and 610 CRC samples. AOM-DSS induced colitis model was used to investigate the EPHB3 expression profile during the colitis-associated carcinogenesis. ResultsEPHB3 expression was upregulated in CRCs than in normal colonic mucosa, and showed positive correlations with intestinal stem cell (ISC) markers (EPHB2, OLFM4, LRIG1) and CD44, a candidate cancer stem cell marker. EPHB3 positivity was observed in 24% of 610 CRCs and showed negative correlations with differentiation, lympho-vascular invasions and TNM stages. EPHB3 expression significantly declined during adenoma-carcinoma transition and invasion into deeper layers. In particular, a substantial reduction of EPHB3 was observed in the budding cancer cells at the invasive fronts. In AOM-DSS induced colitis-associated colon cancer model, EPHB3 expression increased along with tumor development. Notably, EPHB3 was positively associated with microsatellite instability (MSI) phenotype but was not associated with CpG island methylator phenotype, KRAS and BRAF mutations. Furthermore, EPHB3 positivity was a prognostic marker for better clinical outcomes in CRC patients. ConclusionsEPHB3 was upregulated in CRCs and showed positive correlations with candidate cancer stem cell marker CD44. EPHB3 expression decreased during adenoma-carcinoma transition and particularly in the budding cancer cells at the invasive fronts. EPHB3 positivity was associated with MSI phenotype and demonstrated to be a good prognostic marker in CRCs. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Helminth-derived molecules inhibit colitis-associated colon cancer development through NF-κB and STAT3 regulation.

Inflammation is currently considered a hallmark of cancer and plays a decisive role in different stages of tumorigenesis, including initiation, promotion, progression, metastasis and resistance to antitumor therapies. Colorectal cancer is a disease widely associated with local chronic inflammation. Additionally, extrinsic factors such as infection may beneficially or detrimentally alter cancer progression. Several reports have noted the ability of various parasitic infections to modulate cancer development, favoring tumor progression in many cases and inhibiting tumorigenesis in others. The aim of our study was to determine the effects of excreted/secreted products of the helminth Taenia crassiceps (TcES) as a treatment in a murine model of colitis-associated colon cancer (CAC). Here, we found that after inducing CAC, treatment with TcES was able to reduce inflammatory cytokines such as IL-1β, TNF-α, IL-33 and IL-17 and significantly attenuate colon tumorigenesis. This effect was associated with the inhibition of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation. Furthermore, we determined that TcES interfered with LPS-induced NF-κB p65 activation in human colonic epithelial cell lines in a Raf-1 proto-oncogene-dependent manner. Moreover, in three-dimensional cultures, TcES promoted reorganization of the actin cytoskeleton, altering cell morphology and forming colonospheres, features associated with a low grade of aggressiveness. Our study demonstrates a remarkable effect of helminth-derived molecules on suppressing ongoing colorectal cancer by downregulating proinflammatory and protumorigenic signaling pathways.

Abstract 352: The effect of anti-TNF-alpha Ab in AOM/DSS-induced colitic cancer mouse model

Abstract Aim: To reveal whether anti TNF-α antibody (Ab) induces colon carcinogenesis or not in colitic cancer models. Methods: The effects of TNF-α and anti TNF-α Ab were analyzed with cell-proliferation, migration, and invasion assays in human colon cancer cell lines, in vitro. Then, the effects of anti TNF-α Ab, infliximab (10 mg/kg, i.p.) were investigated with drug-induced colitis-associated colon cancer model by azoxymethane (AOM: 10 mg/kg, i.p.)/dextran sodium sulfate (DSS: 2.5%, oral) in vivo. Results: 50 ng/ml of TNF-α inhibited cell proliferation, migration, and invasion of HCT8 and COLO205, however, anti TNF-α Ab offset the TNF-α mediated-inhibition in vitro. Infliximab significantly attenuated the development of colon cancers in AOM/DSS treated mice. The microarray analyses revealed that mast cell related genes (mast cell protease1, mast cell protease 2, chymase 1, etc) were down-regulated in cancer tissues of the infliximab-treated AOM/DSS mice. The results of microarray were validated by real-time RT-PCR. Finally, the number of mast cells were also reduced in those mice. Conclusions: Mast cell may have a pivotal role in the development of colitic cancer. Anti TNF-α Ab may prevent colitic cancer in the patients with inflammatory bowel diseases. Citation Format: Shinobu Ohnuma, Wang Dang Yang, Hideyuki Suzuki, Keigo Kanehara, Hideaki Karasawa, Atsushi Kohyama, Kazuhiro Watanabe, Koh Miura, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. The effect of anti-TNF-alpha Ab in AOM/DSS-induced colitic cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 352.

GARP Dampens Cancer Immunity by Sustaining Function and Accumulation of Regulatory T Cells in the Colon.

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFβ. How GARP modulates Treg function in inflammation and cancer remains unclear. Here we demonstrate that loss of GARP in Treg cells leads to spontaneous inflammation with highly activated CD4+ and CD8+ T cells and development of enteritis. Treg cells lacking GARP were unable to suppress pathogenic T-cell responses in multiple models of inflammation, including T-cell transfer colitis. GARP-/- Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the colitis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP-/- Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissue-specific accumulation, highlighting the importance of cell surface TGFβ in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy.Significance: These findings uncover functions of membrane-bound TGFβ and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.

Crocin synergistically enhances the antiproliferative activity of 5-flurouracil through Wnt/PI3K pathway in a mouse model of colitis-associated colorectal cancer.

Colorectal cancer (CRC) is the third most common cause of cancer-related death, and hence there is a need for the identification of novel-agents to improve the efficacy of existing therapies. There is growing evidence for the antitumor activity of crocin, although its activity and molecular mechanisms in CRC remains to be elucidated. Here we explored the therapeutic application of crocin or its combination with 5-flurouracil in a mouse model of colitis-associated colon cancer. The antiproliferative activity of crocin was assessed in two-dimensional and three-dimensional cell-culture models. The migratory behaviors were determined, while the expression levels of several genes were assessed by quantitative reverse transcriptase polymerase chain reaction/Western blot analysis. We examined the anti-inflammatory properties of crocin by pathological evaluation and disease-activity index as well as oxidative or antioxidant markers: malondialdehyde (MDA) and total-thiols (T-SH) levels and superoxide dismutase (SOD) and catalase (CAT) activity. Crocin suppressed cell-growth and the invasive behavior of CRC cells through modulation of the Wnt-pathway and E-cadherin. Moreover, administration of crocin alone, or in combination with 5-FU dramatically reduced the tumor number and tumor size in both distal/mid-colon followed by reduction in disease-activity index. Crocin also suppressed the colonic inflammation induced by dextran-sulfate-sodium and notably recovered the increased levels of MDA, decreased thiol levels and activity of CAT levels. Crocin was able to ameliorate the severe inflammation with mucosal ulcers and high-grade dysplastic crypts as detected by inflammation score, crypt loss, pathological changes and histology scores. We demonstrated an antitumor activity of crocin in CRC and its potential role in improvement of inflammation with mucosal ulcers and high-grade dysplastic crypts, supporting the desireability of further investigations on the therapeutic potential of this approach in CRC.

Walnut phenolic extract inhibits nuclear factor kappaB signaling in intestinal epithelial cells, and ameliorates experimental colitis and colitis-associated colon cancer in mice.

Walnuts (Juglans regia) are known to have anti-cancer and immunomodulatory effects. However, little information is available on the effects of walnut phenolic extract (WPE) on intestinal inflammation and colitis-associated colon cancer. COLO205 cells were pretreated with WPE and then stimulated with tumor necrosis factor (TNF)-α. In the acute colitis model, wild type mice (C57BL/6) were administered 4% dextran sulfate sodium (DSS) for 5 days. In the chronic colitis model, interleukin (IL)-10-/- mice were administered with either the vehicle or WPE (20mg/kg) by oral gavage daily for 2 weeks. In an inflammation-associated tumor model, wild type mice were administered a single intraperitoneal injection of azoxymethane followed by three cycles of 2% DSS for 5 days and 2 weeks of free water consumption. WPE significantly inhibited IL-8 and IL-1α expression in COLO205 cells. WPE attenuated both the TNF-α-induced IκB phosphorylation/degradation and NF-κB DNA binding activity. The administration of oral WPE significantly reduced the severity of colitis in both acute and chronic colitis models, including the IL-10-/- mice. In immunohistochemical staining, WPE attenuated NF-κB signaling in the colons of both colitis models. Finally, WPE also significantly reduced tumor development in a murine model of colitis-associated colon cancer (CAC). WPE ameliorates acute and chronic colitis and CAC in mice, suggesting that WPE may have potentials for the treatment of inflammatory bowel disease.

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis‐associated colon cancer

Fluorescence tumor imaging using exogenous fluorescent tumor‐targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis‐associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate‐induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31‐immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX‐2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E2 promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast‐enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis‐associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX‐2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.

Prolyl hydroxylase 2 is dispensable for homeostasis of intestinal epithelium in mice

Prolyl hydroxylases (PHD1-3) hydroxylate hypoxia inducible factor α (HIFα), leading to HIFα ubiquitination and degradation. Recent studies indicated that administration of generic inhibitors of PHDs improved mice colitis, suggesting that suppression of PHD activity by these inhibitors may be a potential strategy for the treatment of inflammatory bowel diseases. However, the exact role of each member of PHD family in homeostasis of intestinal epithelium remains elusive. The aim of this work is to study the possible role of PHD2 by using mice with genetic ablation of Phd2 in intestinal epithelial cells (IECs). We found that deletion of PHD2 in IECs did not lead to spontaneous enteritis or colitis in mice. Deletion of PHD2 in IECs did not confer upon mice higher susceptibility to dextran sodium sulfate-induced colitis. Furthermore, in a colitis-associated colon cancer model, the PHD2-conditional knockout mice had similar susceptibility to azoxymethane (AOM)-induced colonic tumorigenesis as control mice did. Our results suggest that PHD2 is dispensable for maintenance of intestinal epithelium homeostasis in mice.

Elevated d-2-hydroxyglutarate during colitis drives progression to colorectal cancer

d-2-hydroxyglutarate (D2HG) is produced in the tricarboxylic acid cycle and is quickly converted to α-ketoglutarate by d-2-hydroxyglutarate dehydrogenase (D2HGDH). In a mouse model of colitis-associated colon cancer (CAC), urine level of D2HG during colitis correlates positively with subsequent polyp counts and severity of dysplasia. The i.p. injection of D2HG results in delayed recovery from colitis and severe tumorigenesis. The colonic expression of D2HGDH is decreased in ulcerative colitis (UC) patients at baseline who progress to cancer. Hypoxia-inducible factor (Hif)-1α is a key regulator of D2HGDH transcription. Our study identifies urine D2HG and tissue D2HGDH expression as biomarkers to identify patients at risk for progressing from colitis to cancer. The D2HG/D2HGDH pathway provides potential therapeutic targets for the treatment of CAC.

P075 Inflammation-induced cancer vs. cancer-induced inflammation is dependent on S1P lyase

The dichotomy of inflammation-induced cancer vs. cancer-induced inflammation is still enigmatic. Since sphingolipids are involved in both tumour cell survival and immune cell differentiation, migration and cytokine pattern we hypothesised their essential role in both mechanism of cancer development. We employed inducible S1P lyase (SGPL1) knockdown bone marrow-chimeric mice in a colitis associated colon cancer model leading to compartment-specific accumulation of sphingolipid levels. We found that, independent of the compartment of SGPL1 deficiency, haematopoietic or extra-haematopoietic, DSS/AOM-treated mice demonstrated tumours with s1pr1, stat3, and IL-6 elevation. However, compartment-specific actions of the SGPL1-S1P axis contributed both morphologically and genetically to a very distinct development of inflammation and carcinogenesis. Haematopoietic SGPL1 knockdown forced severe inflammatory colitis with delayed carcinogenesis characterised by pdcd4-down-regulation. In contrast, extra-haematopoietic SGPL1 knockdown triggered a much more rapid occurrence of epithelial-driven sphingosine kinase 1-positive tumours, exhibiting kras, egfr and IL23-p19 signatures and a weak but distinct Th2 driven immune microenvironment. Moreover, we found that SGPL1 derived from haematopoietic cells is sufficient for S1P-driven lymphocyte sequestration. In this study we demonstrate that compartment-specific sphingolipid modulation clarifies the dichotomy of inflammation-induced cancer vs. cancer-induced inflammation.

P076 The effect of enalapril on preventing colon cancer in AOM/DSS-induced colitis-associated colon cancer model and xenograft model

P076 The effect of enalapril on preventing colon cancer in AOM/DSS-induced colitis-associated colon cancer model and xenograft model

Early and Partial Reduction in CD4+Foxp3+ Regulatory T Cells during Colitis-Associated Colon Cancer Induces CD4+ and CD8+ T Cell Activation Inhibiting Tumorigenesis

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CRC is associated with inflammatory responses in which intestinal pathology is caused by different cell populations including a T cell dysregulation that concludes in an imbalance between activated T (Tact) and regulatory T (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. A tumor-promoting function for Treg cells has been suggested in CRC, but the kinetics of Treg cells during CRC development are poorly known. Therefore, using a mouse model of colitis-associated colon cancer (CAC) induced by azoxymethane and dextran sodium sulfate, we observed the dynamic and differential kinetics of Treg cells in blood, spleen and mesenteric lymph nodes (MLNs) as CAC progresses, highlighting a significant reduction in Treg cells in blood and spleen during early CAC development, whereas increasing percentages of Treg cells were detected in late stages in MLNs. Interestingly, when Treg cells were decreased, Tact cells were increased and vice versa. Treg cells from late stages of CAC displayed an activated phenotype by expressing PD1, CD127 and Tim-3, suggesting an increased suppressive capacity. Suppression assays showed that T-CD4+ and T-CD8+ cells were suppressed more efficiently by MLN Treg cells from CAC animals. Finally, an antibody-mediated reduction in Treg cells during early CAC development resulted in a better prognostic value, because animals showed a reduction in tumor progression associated with an increased percentage of activated CD4+CD25+Foxp3- and CD8+CD25+ T cells in MLNs, suggesting that Treg cells suppress T cell activation at early steps during CAC development.

Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer

To investigate the contribution of adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we studied the effects of four small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. For each SM, four experimental groups of male mice were used: (i) Control group; (ii) SM group; (iii) DSS group (injected with azoxymethane [AOM] and drank dextran sulfate sodium [DSS]); and (iv) DSS + SM group (treated with AOM, DSS, and the SM). None of the mice in groups i and ii developed tumors, whereas all mice in groups iii and iv developed colon neoplasias. No significant differences were found among mice treated with PAMP modulators (87877 and 106221). Mice that received the AM negative modulator, 16311, had worse colitis symptoms than their control counterparts, whereas mice injected with the AM positive modulator, 145425, had a lower number of tumors than their controls. SM 145425 regulated the expression of proliferation marker Lgr5 and had an impact on microbiota, preventing the DSS-elicited increase of the Bacteroides/Prevotella ratio. These results suggest that treatment with AM or with positive modulator SMs may represent a novel strategy for colon cancer.

Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.

Inflammasomes and Cancer: The Dynamic Role of the Inflammasome in Tumor Development.

Chronic Inflammation in tumor microenvironments is not only associated with various stages of tumor development, but also has significant impacts on tumor immunity and immunotherapy. Inflammasome are an important innate immune pathway critical for the production of active IL-1β and interleukin 18, as well as the induction of pyroptosis. Although extensive studies have demonstrated that inflammasomes play a vital role in infectious and autoimmune diseases, their role in tumor progression remains elusive. Multiple studies using a colitis-associated colon cancer model show that inflammasome components provide protection against the development of colon cancer. However, very recent studies demonstrate that inflammasomes promote tumor progression in skin and breast cancer. These results indicate that inflammasomes can promote and suppress tumor development depending on types of tumors, specific inflammasomes involved, and downstream effector molecules. The complicated role of inflammasomes raises new opportunities and challenges to manipulate inflammasome pathways in the treatment of cancer.

Abstract 3220: Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon cancer

Abstract Adrenomedullin (AM) expression is elevated in colon cancer patients and a correlation between higher AM levels and lower disease-free survival has been described in Japan. Nevertheless, the influence of AM in colon cancer is somewhat controversial: on the one hand, antibodies against either the peptide or the receptor reduce the growth of xenografted tumors, whereas application of the peptide clearly reduces inflammation and clinical severity in human and mouse models of colitis, which is an important risk factor for colon cancer. To investigate the contribution of AM and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), to the progression and potential treatment of colon cancer we have studied the effects of several small molecules (SM) related to AM and PAMP on a mouse model of colon cancer. Colon tumors were induced by intraperitoneal injection of a carcinogen: 10 mg/Kg azoxymethane, followed by 3 cycles of a colitis-inducing chemical: 2.5% dextran sulfate sodium (DSS) in the drinking water for a week followed by 2 weeks of regular water. Four SM were tested: 16311 (a negative modulator of AM), 145425 (a positive modulator of AM), 87877 (a negative modulator of PAMP), and 106221 (a positive modulator of PAMP). For each SM, 4 experimental groups of male C57BL6 mice were used: i) Control group (injected with vehicle and drank regular water); ii) Control+SM (vehicle, regular water, and injected with the SM 3 times a week at a concentration of 20 nm/Kg); iii) Cancer group (injected with AOM and drank DSS); and iv) Cancer+SM (treated with AOM, DSS, and the SM). At the end of the experimental procedures, animals were sacrificed and the colon was extracted and analyzed. None of the mice belonging to groups i and ii developed any tumor or had any pathological finding, indicating that the SMs do not present overt toxicity. All mice in groups iii and iv developed colon neoplasias ranging from carcinomas in situ to adenocarcinomas. No significant differences were found among mice treated with PAMP modulators indicating that PAMP may not play a major role in colon cancer or that the PAMP-related SMs were not very effective. On the other hand, mice that received SM 16311 had a higher severity index (p<0.001) and weight loss (p<0.05) than their control counterparts, whereas mice injected with SM 145425 had a lower number of tumors than their controls (p<0.001). These results suggest that AM may have a protective role during the progression phase of colon cancer, and that treatment with AM or with positive modulator SMs may represent a novel treatment for colon cancer. This study was funded by Instituto de Salud Carlos III (PI13/02166), FEDER, and Junta Provincial de La Rioja de la Asociación Española Contra el Cáncer (AECC). Citation Format: Laura Ochoa-Callejero, Josune García-Sanmartín, Sonia Martínez-Herrero, Susana Rubio-Mediavilla, Alfredo Martínez. Small molecules related to adrenomedullin reduce tumor burden in a mouse model of colitis-associated colon 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 3220. doi:10.1158/1538-7445.AM2017-3220

Cysteinyl leukotriene receptor 1 facilitates tumorigenesis in a mouse model of colitis-associated colon cancer.

Cysteinyl leukotriene receptor 1 (CysLT1R) has been shown to be up-regulated in the adenocarcinomas of colorectal cancer patients, which is associated with a poor prognosis. In a spontaneous model of colon cancer, CysLT1R disruption was associated with a reduced tumor burden in double-mutant female mice (ApcMin/+/Cysltr1−/−) compared to ApcMin/+ littermates. In the current study, we utilized a genetic approach to investigate the effect of CysLT1R in the induced azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis-associated colon cancer. We found that AOM/DSS female mice with a global disruption of the Cysltr1 gene (Cysltr1−/−) had a higher relative body weight, a more normal weight/length colon ratio and smaller-sized colonic polyps compared to AOM/DSS wild-type counterparts. The Cysltr1−/− colonic polyps exhibited low-grade dysplasia, while wild-type polyps had an adenoma-like phenotype. The Cysltr1−/− colonic polyps exhibited significant decreases in nuclear β-catenin and COX-2 protein expression, while the normal crypts surrounding the polyps exhibited increased Mucin 2 expression. Furthermore, Cysltr1−/− mice exhibited an overall reduction in inflammation, with a significant decrease in proinflammatory cytokines, polyp 5-LOX expression and infiltration of CD45 leukocytes and F4/80 macrophages. In conclusion, the present genetic approach in an AOM/DSS model further supports an important role for CysLT1R in colon tumorigenesis.

Expression of tyrosine and threonine protein kinase in carcinogenic process of colorectal cancer and its relationship with prognosis

Objective: To investigate the expression of TTK (tyrosine and threonine protein kinase) in the process of colorectal cancer (CRC) development and its relationship to prognosis in CRC patients. Methods: Colitis-associated colon cancer model was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Mice at four different stages of colon cancer development were obtained, named AD1 (inflammation of the recovery), AD2 (mild dysplasia), AD3 (adenoma) and AD4 (adenocarcinoma), as well as negative control (no treatment). The expression of TTK was measured by real time fluorescent quantitative PCR (qPCR) and immunohistochemical staining in mouse colon tissues and 24 pairs of CRC specimens. The relationship between TTK and prognosis was analyzed in a set of CRC genome-wide gene expression microarray data that was obtained from Gene Expression Omnibus (GEO) of National Center for Biotechnology Information (NCBI). Results: The genome-wide microarray data from mouse AOM-DSS model indicated that the expression of TTK mRNA was gradually elevated during the development of colon cancer. The subsequent qPCR results showed that TTK mRNA levels in negative control, AD1, AD2, AD3 and AD4 groups were 1.05±0.42, 1.10±0.03, 1.38±0.15, 1.33±0.17 and 2.12±0.22, respectively. And TTK expression in AD2, AD3 and AD4 groups were significantly higher than that in negative control (P<0.05). The protein expression of TTK showed by immunohistochemical staining had similar tendency as the results of TTK mRNA. Besides that, the TTK mRNA levels in tumor tissues (0.71±0.10) from 24 CRC patients were significantly higher than those in paired adjacent normal tissues (0.18±0.04; P<0.001). The positive expression rate of TTK protein in 5 pairs of CRC clinical samples was 80.0%, and it was significantly higher than that in adjacent normal tissues (30.8%, P=0.014). Furthermore, according to a public transcriptomic data (GSE17536), the high levels of TTK were associated with poor prognosis in CRC patients. Conclusions: Elevated expression of TTK is related to colonic carcinogenesis in both of mouse model and human CRC samples. TTK is a poor prognostic factor in CRC.

Abstract P3-06-07: Combination of doxorubicin with S1P signaling modulator FTY720 significantly suppressed obesity-associated breast cancer

Abstract Background: Obesity is one of the biggest health issues in the US. It has been shown that obesity-associated breast cancer is more aggressive with poor prognosis, which is partly explained by the low-grade inflammation caused by obesity. Recently we have published that sphingosine-1-phosphate (S1P), a signaling lipid mediator, link inflammation and cancer in colitis-associated colon cancer model. We hypothesized that addition of S1P modulator that block S1P signaling thus suppress the effect of obesity-mediated inflammation should enhance anti-cancer effect of doxorubicin, which is a typical anti-cancer drug for breast cancer used as a standard of care. Methods: Female B6.cg-Lepob (OB/OB) mice fed with high fat diet for 2 weeks prior to implantation of cancer cells were used as an obesity model, and litter mate control mice fed with normal diet were used as a control. 1 x 106 murine mammary adenocarcinoma E0771 cells were inoculated into #2 rt. fat pads as previously described (Katsuta et al JSR 2016). 9 days after inoculation, both OB/OB and control mice were randomized into 4 groups in each group; vehicle, Doxorubicin, FTY720 and Combination of Doxorubicin and FTY720. Doxorubicin was administrated by i.p. injection at a dose of 5 mg/kg on Day 0 and 3. FTY720 was administered everyday by gavage at a dose of 1 mg/kg during the entire course. Tumor growths were measured daily by caliper measurements. Tumor weights were measured on 21 days after cell inoculation. Results: The body weight of obesity model was significantly heavier than control mice at the time of cancer cell inoculation (44.1 g vs 19.4 g; p &amp;lt; 0.001). In non-treatment group, tumor weight in obesity group was significant heavier than control mice[KT1] (1232 mg vs 966 mg; p = 0.049), which is consistent with the dogma that obesity worsen cancer progression. As expected, tumor weight in non-treatment group is heavier than any treatment group, and that in combination treatment of doxorubicin and FTY720 is lightest in both of obesity group and control group. Interestingly, tumor reduction rate in obesity group compared with non-treatment group is significant greater than control group (Doxorubicin: 83% vs 19%, p = 0.001; FTY720: 80% vs 46%, p = 0.027, Doxorubicin + FTY720: 93% vs 64%, p = 0.011). Over 15 % weight loss were seen in obesity doxorubicin group and obesity combination treatment group. Conclusion: Modification of S1P signaling by FTY720 was shown to enhance the effect of doxorubicin particularly in obese mice, which implicate a novel approach to treat obesity-associated breast cancer. Citation Format: Katsuta E, Takabe K. Combination of doxorubicin with S1P signaling modulator FTY720 significantly suppressed obesity-associated breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-07.

Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer.

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12−/− mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox−/− mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox−/− mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox−/− mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19−/− bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19−/− mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.