Development Of Colitis-associated Cancer Research Articles

Tropisetron suppresses colitis-associated cancer in a mouse model in the remission stage

Patients with inflammatory bowel disease (IBD) have a high risk for development of colitis-associated cancer (CAC). Serotonin is a neurotransmitter produced by enterochromaffin cells of the intestine. Serotonin and its receptors, mainly 5-HT3 receptor, are overexpressed in IBD and promote development of CAC through production of inflammatory cytokines. In the present study, we demonstrated the in vivo activity of tropisetron, a 5-HT3 receptor antagonist, against experimental CAC. CAC was induced by azoxymethane (AOM)/dextran sodium sulfate (DDS) in BALB/c mice. The histopathology of colon tissue was performed. Beta-catenin and Cox-2 expression was evaluated by immunohistochemistry as well as quantitative reverse transcription-PCR (qRT-PCR). Alterations in the expression of 5-HT3 receptor and inflammatory-associated genes such as Il-1β, Tnf-α, Tlr4 and Myd88 were determined by qRT-PCR. Our results showed that tumor development in tropisetron-treated CAC group was significantly lower than the controls. The qRT-PCR analysis demonstrated that the expression of 5-HT3 receptor was significantly increased following CAC induction. In addition, tropisetron reduced expression of β-catenin and Cox-2 in the CAC experimental group. The levels of Il-1β, Tnf-α, Tlr4 and Myd88 were significantly decreased upon tropisetron treatment in the AOM/DSS group. Taken together, our data show that tropisetron inhibits development of CAC probably by attenuation of inflammatory reactions in the colitis.

Changes of macrophages in colitis-associated colonic carcinogenesis

To investigate the changes of quantity and phenotype of macrophages during the progress of colitis-associated carcinogenesis, and to identify the chemokines mediating macrophage recruitment. Colitis-associated cancer was induced by azoxymethane (AOM) combined with dextran sulfate sodium (DSS) in C57BL/6 mice. The three sequential developmental stages of colitis associated cancer in the mice were named AD1, AD2 and AD3, respectively. Colon tissues were collected and digested into single-cell suspension. The percentage and phenotype of macrophages in the colon tissues were determined by fluorescence activated cell sorter (FACS). Protein array and real-time polymerase chain reaction (PCR) were used to predict potential chemotatic factors of macrophages. Colitis-associated cancer was effectively induced in C57BL/6 mice using AOM combined with DSS. The percentage of macrophages was gradually elevated in the AD1, AD2 and AD3 groups [(9.93±1.28)%, (15.42±1.15)%, (21.25±0.62)%], respectively, significantly higher than that of the control group [(2.39±0.54)%, P<0.01]. The macrophages infiltrating the colonic mucosa exhibited mainly a pro-inflammatory phenotype as CD206(-)CD86(+) MHCII(-). The positive rates of CD206 in the AD1, AD2 and AD3 groups were (15.03±1.54)%, (8.11±3.70)%, and (9.06±1.16)%, respectively, significantly lower than that of the control group [(19.43±7.31)%, P<0.01]. The positive rates of CD86 in the AD2 and AD3 groups were (46.73±6.58)% and (76.90±14.32)%, respectively, significantly higher than that of the control group [(19.37±9.69)%, P<0.01)]. The positive rates of MHCⅡ in the AD1, AD2 and AD3 groups were (31.10±2.69)%, (33.93±14.08)%, and (29.93±1.41)%, respectively, significantly lower than that of the control group [(50.30±6.58)%, P<0.01]. Protein array analysis and real-time PCR data revealed that G-CSF was the potential chemokine to recruit macrophages in the AOM-DSS mouse model. Macrophages infiltrate increasingly during the carcinogenesis and development of colitis-associated cancer, which mostly express CD206(-)CD86(+) MHCII(-) and might be potentially recruited by G-CSF.

Macrophage targeting contributes to the inhibitory effects of embelin on colitis-associated cancer.

Macrophages are a major component of inflammatory and tumor microenvironment. We previously reported that embelin suppresses colitis-associated tumorigenesis. Here, the role of macrophage targeting in the anti-inflammatory and anti-tumor properties of embelin was investigated. By using colitis-associated cancer (CAC) model, we demonstrated that embelin significantly depleted colon macrophages by blocking their recruitment. Moreover, embelin attenuated M2-like polarization of macrophages within the tumor microenvironment and eliminated their tumor-promoting functions during the development of CAC. Embelin potently inhibited NF-κB signaling in macrophages and decreased the production of key pro-inflammatory cytokines and tumorigenic factors involved in CAC, such as TNFα, IL-6 and COX-2. In addition, embelin directly reduced the polarization of M2 macrophages in vitro even in the presence of Th2 cytokines. These results suggested that targeting macrophages is, at least in part, responsible for the anti-tumor activity of embelin in CAC. Our observations strengthen the rationale for future validation of embelin in the prevention and treatment of CAC

Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer.

To date, substantial evidence has shown a significant association between inflammatory bowel diseases (IBD) and development of colitis-associated cancer (CAC). The incidence/prevalence of IBD is higher in western countries including the US, Australia, and the UK. Although CAC development is generally characterized by stepwise accumulation of genetic as well as epigenetic changes, precise mechanisms of how chronic inflammation leads to the development of CAC are largely unknown. Preceding intestinal inflammation is one of the major influential factors for CAC tumorigenesis. Mucosal immune responses including activation of aberrant signaling pathways both in innate and adaptive immune cells play a pivotal role in CAC. Tumor progression and metastasis are shaped by a tightly controlled tumor microenvironment which is orchestrated by several immune cells and stromal cells including macrophages, neutrophils, dendritic cells, myeloid derived suppressor cells, T cells, and myofibroblasts. In this article, we will discuss the contributing factors of epithelial as well as immune cell signaling in initiation of CAC tumorigenesis and mucosal immune regulatory factors in the colonic tumor microenvironment. In depth understanding of these factors is necessary to develop novel anti-inflammatory and anti-cancer therapies for CAC in the near future.

Targeting of MyD88 Homodimerization by Novel Synthetic Inhibitor TJ-M2010-5 in Preventing Colitis-Associated Colorectal Cancer.

The TLR/MyD88 signaling pathway is an important driver of inflammation and cancer and is a possible target for antitumor therapy. We generated a MyD88 inhibitor (TJ-M2010-5), which was designed to bind to the TIR domain of MyD88 to interfere with its homodimerization, and the TLR/MyD88 signal pathway. We utilized a mouse model of azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated cancer (CAC) in combination with TJ-M2010-5 administration to investigate the anti-inflammation-related cancer effect of MyD88 inhibitor in vivo. Data were analyzed with one-way and repeated measures analysis of variance. Differences in survival between groups were compared using the log rank test. All statistical tests were two-sided. TJ-M2010-5 inhibited MyD88 homodimerization in transfected HEK293 cells in a concentration-dependent manner and suppressed MyD88 signaling in LPS-responsive RAW 264.7 cells in vitro. In a 10-week CAC mouse model (n = 30 per group), TJ-M2010-5 treatment statistically significantly reduced AOM/DSS-induced colitis and completely prevented CAC development with less related body mass loss, resulted in 0% mortality of treated mice (compared with 53% mortality of control mice), decreased cell proliferation, and increased apoptosis in colon tissue. TJ-M2010-5 treatment also inhibited production of inflammatory cytokines and chemokines (TNF-α, IL-6,G-CSF, MIP-1β, TGF-β1, IL-11, IL-17A, IL-22 and IL-23) and infiltration of immune cells (macrophages, dendritic cells, neutropihls and CD(+)4 T cells) in colon tissues of mice. Our findings suggest that TLR/MyD88 signaling may be a therapeutic target for CAC intervention and MyD88 inhibitors may be a promising therapeutic modality for treating patients with colitis or CAC.

IMPlicating Mesenchymal Imp1 in Colitis-Associated Cancer.

Chronic inflammation and associated pathways are significant facilitators of many disease states, including malignancies. In the context of cancer, fibroblasts can actively regulate both inflammation and carcinogenesis. In this issue, Hamilton and colleagues describe a fibroblast-specific role of the RNA binding protein Imp1 in suppression of intestinal inflammatory responses and development of colitis-associated cancer.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis-associated cancer by regulating S100A9

Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1 in vivo. Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells.

Abstract 1480: Hot spot 19F MR imaging of inflammation predicts colitis-associated cancer

Abstract Introduction: The causal relationship between inflammation and cancer is well established. In the colon, colitis-associated cancer (CAC) develops through complications of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease. CAC development is directly correlated with the severity and longevity of inflammation in IBD patients. We hypothesized that non-invasive serial MRI of inflammatory sites and measuring their severity may enable earlier diagnosis and prediction of cancer development. Methods: To induce CAC, female A/J mice (n = 5) were injected with azoxymethane (10mg/kg) on day 0 and fed with 2.5% dextran sodium sulfate (DSS) solution for every 3 weeks until week 7. 200 μl of perfluoro-15-crown-5-ether (PFC) emulsion (Celsense Inc., VS-580H), which labels macrophages, was injected through the tail vein 48 hours before the first MRI scan. In vivo MR imaging was performed every 15 days from day 50 to day 110 using a 11.7T MR scanner and a surface coil tunable to 1H and 19F. Conventional 1H (proton) images were acquired using a fast spin echo sequence (FSE) with RARE, TR/TE = 1200/30 ms, matrix size = 256×256, FOV = 3.2×2.0cm, rare factor = 8, and slice thickness = 2mm. “Hot spot” 19F (fluorine) images were acquired using an FSE with RARE, TR/TE = 1000/14ms, matrix size = 64×32, rare factor = 8, and slice thickness = 2mm. Ex vivo images were acquired with a 750 MHz microimaging system and a 25mm volume coil. Fixed colon tissues were paraffin-cut to evaluate tumor development. Multiple longitudinal colon segments were chosen from areas either with or without 19F signal. H&amp;E stained sections from each segment were scored by a blinded pathologist for the severity of inflammation and presence of dysplasia. Results and Discussion: Hot spots of 19F signal were detected in the colon wall after the onset of IBD, with a patchy distribution pattern. Since PFC emulsions specifically label macrophages, these signals represent infiltrated macrophages in the colon wall as a result of inflammation driven by DSS. Tumor outgrowths were observed on MRI at the inflammatory sites starting at day 80. In vivo data were validated by ex vivo imaging of excised colon tissues showing an exact co-localization of tumors with the 19F-positive inflammatory sites. Histologically, tumors were classified as adenomas. CAC develops through the sequence of inflammation-adenoma-carcinoma suggesting that we were able to detect early stages of carcinomatous changes in the inflammatory sites. Histological scores of inflammation and adenoma showed a significant correlation with the intensity of 19F signals. We conclude that hot spot 19F MRI enables early diagnosis of future sites of colon cancer development. Citation Format: Soo-Hyun Shin, Deepak K. Kadayakkara, Jeff W. M Bulte. Hot spot 19F MR imaging of inflammation predicts colitis-associated cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2015-1480

AKR1B10 in gastrointestinal diseases

Ulcerative colitis (UC) is characterized by long-standing mucosal inflammation of the large bowel, with increased tendency of developing colorectal cancer (CRC), i.e., colitis-associated cancer (CAC) [1]. The other two predisposing conditions for CRC are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), whose genetic etiology is well defined [1]. The genetic predisposition of UC has not been firmly delineated thus far. The recent work from our laboratory characterized aldo-keto reductase 1B10 (AKR1B10) as a novel risky factor of UC and CAC [2]. Almost all CRCs progress from a dysplastic precursor lesion, but the development of CAC is different in several important aspects from sporadic CRC [3]. Dysplasia in patients with sporadic CRC is usually the adenomatous polyp (adenoma), a discrete neoplastic focus; in contrast, CAC develops from dysplastic lesions that can be polyploid, flat, localized, or multifocal. In addition, the molecular abnormalities of inflamed colonic mucosa in CAC appear present much earlier than any histological evidence (dysplasia or cancer). This raises an important question of how chronic inflammation leads to the neoplastic transformation and CRC pathogenesis. Inflammatory oxidative stress likely plays a causative role. Reactive oxygen species target a wide range of macromolecules, including proteins, DNA and lipids, and induce cellular damage that may be associated with epithelial homeostasis [4]. Under the inflammatory environment, free radicals and other prooxidant molecules generated by neutrophils and macrophages can also inflict lipid peroxidation and biomembrane damage [4]. Lipid peroxides are electrophilic carbonyl compounds and are highly cytotoxic and genotoxic. They may serve as secondary contributors to cellular and DNA damage may target key genes or proteins responsible for dysplasia and subsequent arises of carcinoma [5]. AKR1B10 is primarily expressed in epithelial cells of gastrointestinal tract, and exerts a protective role through eliminating oxidative and carbonyl stresses and promoting epithelial proliferation for damage repair in inflammation. However, AKR1B10 expression is lost or markedly decreased in over 90% UC and CAC [2]. AKR1B8 in the mouse is the ortholog of human AKR1B10. To mimic the phenomenon seen in humans, we disrupted the AKR1B8 locus in mice. Initial findings showed that AKR1B8 deficiency diminished proliferation, migration, and maturation of colonic crypt cells, disrupting the epithelial homeostasis. As a result, the AKR1B8 deficient mice were susceptible to dextran sodium sulfate (DSS)-induced colitis and demonstrated delayed re-epithelialization and epithelial remodeling, leading to more severe inflammatory and neoplastic lesions. In the setting of heightened epithelial corruption, mutagenic assaults and sustained DNA damage caused by oxidative and carbonyl stresses in the AKR1B8 deficient epithelium appear to drive the carcinogenic procession. As such, the process of colitis-neoplasia in the AKR1B8 deficient mice behaves similarly to aging that accumulates DNA damage that fails to repair. Oxidative stress is a leading theory of aging. Progressive oxyradical overloads with aging leading to age-associated physiological function declines [6]. Data from antioxidant studies suggest although the rate of oxidative damage decreases with aging, possibly due to the reducing rate of metabolism, the steady-state levels of oxidative DNA modifications increase due to insufficient repairing [7]. Similarly, oxidative and carbonyl-associated DNA damage/mutations, such as G:C to A:T, are accumulated in AKR1B8 deficient mice. A genome-wide sequencing analysis revealed colitis-associated DNA mutations in up to 28 oncogenes or tumor suppressors uniquely in AKR1B8 deficient colon mucosa. This indicates failure of repairing oxidative damage, such as lipid peroxidation, in AKR1B8 deficient colon, supporting the protective role of AKR1B10 in human colon. It is becoming clear that AKR1B8 deficiency favors tumorigenesis due to increased accumulation of DNA mutations in host cells. At the same time, it is reasonable to speculate that AKR1B8 deficiency may also associate with certain signaling pathways that regulate cell proliferation and survival. Indeed, our work demonstrated that AKR1B8 mediates de novo lipid synthesis [2], which may affect lipid second messenger-mediated cell signaling transducers, such as PI3K/AKT and PKC/ERK. Overall, our studies suggest that AKR1B10 is an important protector in the gastrointestinal epithelium. AKR1B10 deficiency may be a new predisposition of UC and CAC. Human risk of developing gastrointestinal diseases increases with age. It would be interesting to see if AKR1B10 expression in the epithelium declines with aging.

Mo1719 Epithelial NF-κB Activation via TNF Signaling May Be Involved in the Development of Colitis-Associated Carcinogenesis

Background & Aim: Prolonged inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease may promote carcinogenesis in the epithelia. It has been reported that activation of NF-κB pathway in both intestinal epithelial cells and myeloid cells are significant for the development of colitis-associated cancer (CAC). We previously reported that specific up-regulation of the type 2 receptor for tumor necrosis factor (TNFR2) rather than TNFR1 expression was observed in the inflamed colonic epithelia and further in the CAC. It is known that TNFR2 signaling may induce NF-κB activation, but the role of its expression in the setting of CAC has not been elucidated. Here we analyzed TNFR2 signaling in the colonic epithelial cells. Methods & Results: As previously observed in animal models of colitis and CAC, the expression of TNFR2 was up-regulated in an epithelial cell line, MOC1, which was derived from murine colonic ‘non-cancer' tissue, when stimulated with recombinant (r) IFN-γ. MOC1 cells incubated with both rIFN-γ and rTNF showed that NFκB pathway was activated rather than apoptosis pathway. Furthermore, the activated NFκB in MOC1 cells was associated with the expression of myosin light chain kinase (MLCK) as well as disrupted tight junction (TJ) in a rTNF dose-dependent manner. Such MLCK upregulation and TJ disruption in MOC1 cells were abrogated by either anti-TNF mAb (MP6XT22), TNFR2-specific siRNA or even MLCK inhibitor (ML-7). Using an animal model of CAC involving azoxymethane (AOM) and dextran sodium sulfate (DSS), the colonic lamina propria was found to have pro-tumorigenic cytokine production such as IL-1β, IL-6 and MIP-2 in association with epithelial NF-κB activation, TNFR2 and MLCK up-regulations and epithelial TJ disruption. AOM and DSS-administered mice with either MP6-XT22 or ML7 treatment failed to show significant abrogation of colitis severity, however such treatments revealed the restored epithelial TJ and decreased pro-tumorigenic cytokine production in the colonic tissues in association with the reduced CAC development. Conclusions: Our studies showed that epithelial NF-κB activation via TNFR2 signaling in the context of IBD may be involved in the epithelial permeabilization and pro-tumorigenic cytokine production that result in the induction of CAC development.

Elevated O-GlcNAcylation promotes colonic inflammation and tumorigenesis by modulating NF-κB signaling.

O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA(+/-) mice have higher susceptibility to DSS-induced colitis than OGA(+/+) mice. OGA(+/-) mice exhibited a higher incidence of colon tumors than OGA(+/+) mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.

Cytokines, IBD, and colitis-associated cancer.

Inflammatory bowel diseases (IBDs) are debilitating conditions that result in intestinal damage due to chronic inflammation. In addition, the perpetual state of inflammation predisposes individuals to the development of colitis-associated cancer. Because of the immense immune cell infiltration into colon, cytokines produced by immune cells are major players in the initiation and progression of IBD and colitis-associated cancer. In this review, we will explore the functions of many key cytokines and their roles in IBD and colitis-associated cancer, as well as their influences on the immune system and stromal cells. Finally, we will briefly discuss current therapies and current clinical trials targeting cytokines in IBD.

MicroRNA targeting for the therapy of colitis-associated colon cancer.

571 Background: Inflammatory Bowel Diseases (IBD) consist of ulcerative colitis (UC) and Crohn’s Disease (CD), which are characterized by activation of inflammatory responses. Patients with longstanding UC are at high risk of developing colorectal cancer. The identification of novel molecular targets with therapeutic potential for UC and UC-related dysplasia are of major importance. Methods: Using a high throughput functional suppressor screen of the human microRNAome, we identified microRNAs involved in the regulation nuclear factor kappa beta (NF-κB). We correlated microRNA expression levels with different clinicopathological parameters in 401 colonic specimens derived from patients with UC, CD, irritable bowel syndrome (IBS), sporadic colon cancer (CRC), colitis-associated cancer (CAC) and control subjects. Bioinformatic and molecular analyses were employed for the study of micoRNA-regulated signaling pathways. A microRNA specific chemical inhibitor was used to treat colonic biopsies ex vivo and murine CAC development in vivo. Results: The microRNA screen identified miR-214 as master regulator of NF-κB. MiR-214 levels are increased in colonic tissues from UC and CAC, but not from CD, IBS and CRC patients and positively correlate with UC disease activity and duration. STAT3 regulates miR-214 expression in colonocytes in vitro and STAT3 and miR-214 levels positively correlate in UC and CAC. MiR-214 regulates the expression of phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2) and both are decreased in colonic tissues of UC and CAC patients. MiR-214 is amplified through a feedback loop circuit and its overexpression increases the tumorigenic and invasive phenotype of colon cancer cells. A chemical miR-214 inhibitor perturbs this circuit in colonic biopsies from UC patients ex vivo while intracolonic delivery suppresses CAC growth in mice. Conclusions: Our findings demonstrate a gene controlling the inflammatory response specifically in UC and CAC. The miR-214 molecular circuit activity correlates with UC disease activity and duration. Activation of this circuit contributes to colitis-associated colon carcinogenesis, and its suppression has therapeutic potential for patients with UC-related dysplasia.

Mechanisms of Immune Signaling in Colitis-Associated Cancer

The inflammatory bowel diseases ulcerative colitis and Crohn’s disease are associated with an increased risk for the development of colorectal cancer. During recent years, several immune signaling pathways have been linked to colitis-associated cancer (CAC), largely owing to the availability of suitable preclinical models. Among these, chronic intestinal inflammation has been shown to support tumor initiation through oxidative stress–induced mutations. A proinflammatory microenvironment that develops, possibly as a result of defective intestinal barrier function and host–microbial interactions, enables tumor promotion. Several molecular pathways such as tumor necrosis factor/nuclear factor-κB or interleukin 6/signal transducer and activator of transcription 3 signaling have been identified as important contributors to CAC development and could be promising therapeutic targets for the prevention and treatment of CAC.

Recent advancement in understanding colitis-associated tumorigenesis.

Chronic inflammation predisposes patients with inflammatory bowel disease to the risk of developing colitis-associated cancer (CAC). Growing evidence strongly suggests that CAC development is multifactorial and is attributed to concurrent, dynamic dysregulations in host immunity, enteric microbiota, and epithelial restitution during the course of chronic inflammation. This article discusses the recent advances in understanding the different forms of CAC that may develop in patients with inflammatory bowel disease and animal models, as well as molecular alterations and other processes that orchestrate the development of CAC.

Stress response protein cirp links inflammation and tumorigenesis in colitis-associated cancer.

Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and is reported to be associated with refractory inflammatory bowel disease (IBD). Defective apoptosis of inflammatory cell populations seems to be a relevant pathogenetic mechanism in refractory IBD. We assessed the involvement of stress response protein cold-inducible RNA-binding protein (Cirp) in the development of intestinal inflammation and CAC. In the colonic mucosa of patients with ulcerative colitis, expression of Cirp correlated significantly with the expression of TNFα, IL23/IL17, antiapoptotic proteins Bcl-2 and Bcl-xL, and stem cell markers such as Sox2, Bmi1, and Lgr5. The expression of Cirp and Sox2 was enhanced in the colonic mucosae of refractory ulcerative colitis, suggesting that Cirp expression might be related to increased cancer risk. In human CAC specimens, inflammatory cells expressed Cirp protein. Cirp(-/-) mice given dextran sodium sulfate exhibited decreased susceptibility to colonic inflammation through decreased expression of TNFα, IL23, Bcl-2, and Bcl-xL in colonic lamina propria cells compared with similarly treated wild-type (WT) mice. In the murine CAC model, Cirp deficiency decreased the expression of TNFα, IL23/IL17, Bcl-2, Bcl-xL, and Sox2 and the number of Dclk1(+) cells, leading to attenuated tumorigenic potential. Transplantation of Cirp(-/-) bone marrow into WT mice reduced tumorigenesis, indicating the importance of Cirp in hematopoietic cells. Cirp promotes the development of intestinal inflammation and colorectal tumors through regulating apoptosis and production of TNFα and IL23 in inflammatory cells.

The risk of colorectal cancer in patients with ulcerative colitis.

Ulcerative colitis increases the risk of developing dysplasia and colitis-associated cancer (CAC). The purpose of this study was to determine the risk factors as well as protective measures for disease burden, need for colectomy and the development of CAC in ulcerative colitis (UC) patients. A cohort of n = 434 UC patients was evaluated. Data analysis was performed by univariate and multivariate logistic regression. Odds ratios (OR) and 95 % confidence intervals (CI) were calculated, and significance was assessed by the likelihood ratio test. Mean patient age at UC diagnosis was 45.7 ± 15.1 years which manifested mainly as pancolitis (47 %) or left-sided colitis (45.2 %). CAC was detected in ten patients (2.3 %). UC disease duration was strongly associated with the risk of CAC (P < 0.0014); disease duration between 9 and 15 years: OR of 2.5 (95 % CI 0.2-41.1), more than 15 years: OR of 21.4 (95 % CI 2.6-173.6). The risk of developing dysplasia (low-grade intraepithelial neoplasia, LGIEN and high-grade intraepithelial neoplasia, HGIEN) or the need to undergo colectomy was also significantly related to disease duration (P = 0.006, P = 0.002, respectively). Established anti-inflammatory medication (e.g., 5-ASA, anti-TNF-α) significantly reduced the risk of both dysplasia and CAC (P = 0.02). Despite the use of modern therapies for UC, CAC rates remain high. In our study, risk factors included disease duration while anti-inflammatory therapies reduced the risk. Effective control of the intestinal inflammation also reduced the disease burden as indicated by decreased risk of requiring colectomy, underscoring the need for sufficient surveillance and anti-inflammatory therapies.

Abstract SY40-01: Tumor elicited inflammation and malignant progression in colorectal cancer

Abstract Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the US and other Western countries. Whereas 2% of CRC arises in the context of pre-existing inflammatory bowel disease (IBD), especially ulcerative colitis (UC), and is known as colitis associated cancer (CAC), most CRC, including familial and sporadic cases, is found in individuals that do not show any signs of IBD. Yet, expression profiling has revealed the same inflammatory gene signature, which depends on activation of NF-κB and STAT3, in both CAC and CRC, findings that generate a question regarding the origin of the CRC-elicited inflammatory response. In early experiments we have shown that activation of NF-κB, which leads to production of the pro-inflammatory cytokine IL-6, a potent activator of STAT3, plays a critical role in the development of CAC. More recently we investigated the origin and role of inflammation in the development and progression of sporadic CRC, most of which is driven by loss of the tumor suppressor APC and activation of the β catenin signaling pathway. We focused our studies on IL-23, a heterodimeric cytokine, composed of a unique p19 subunit and a p40 subunit which it shares with IL-12. As observed by others, we also found that IL-23 expression is strongly elevated in CRC relative to adjacent non-tumor tissue and have extended these findings to the CPC-APC mouse model of colorectal tumorigenesis. In CPC-APC mice, the major source of IL-23 expression in colorectal adenomas are tumor associated macrophages (TAM). Importantly, ablation of IL-23 p19 either in all cells or only in bone marrow (BM)-derived cells attenuates the development and slows down the progression of CRC in CPC-APC mice and similar results were observed upon ablation of IL-23 receptor (IL-23R). As IL-23R is not expressed on adenoma epithelial cells, IL-23 must exert its pro-tumorigenic effect via an indirect mechanism. Indeed, IL-23 signaling promotes the polarization of IL-17 producing T cells (Th17) and the production of IL-6, both of which contribute to the development and progression of sporadic colorectal tumors in mice. Importantly, molecular epidemiological studies carried out by Galon, Fridman and their co-workers have revealed that an “IL-23-Th17” signature that is upregulated in about 10% of human CRC patients, and that the presence of this signature in stage I/II CRC is associated with very poor prognosis and a marked decrease in disease free survival. We investigated the mechanism responsible for the specific induction of IL-23 in TAM and found it to depend on Toll like receptor (TLR) - MyD88 signaling, which appears to be activated in response to components of the colonic microflora that permeate the adenomas. We also detected eubacterial 16S RNA in both mouse and human colonic adenomas, as well as increased penetrance of bacterial endotoxin (LPS) into adenomas relative to surrounding non-cancerous tissue. The development of colorectal adenomas in both mice and men is associated with loss of protective mucins and junctional adhesion proteins and this is likely to be the primary mechanism that accounts for the selective entry of microbial products into the tumor and induction of the tumor promoting “IL-23-Th17” signature. Future studies should focus on the therapeutic value of anti-IL-23 or anti-IL-17 interventions and the genetic or environmental causes of the large variation in the magnitude of IL-17 production amongst human CRC patients.

Abstract 2472: Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4

Abstract NDST4 is one member of N-deacetylase/N-sulfotransferase (heparan glucosaminyl) (NDST) family, which are responsible for heparan sulfate (HS) biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs). The HS chains of HSPGs bind to and regulate their functions of various growth factors, cytokines and chemokines. HSPGs ubiquitously reside on cell surface, inside the cell, and in the extracellular matrix. Importantly, the content and distribution of HSPGs are altered during tumorigenesis. In the study, 30 (57.7%) of 52 primary colorectal carcinomas showed a dramatic reduction in NDST4 RNA transcripts, and genetic loss of NDST4 was significantly associated with poor survival of patients with colorectal cancer. We further generated an Ndst4-knockout mouse strain, which could develop and reproduce normally. Notably, using the dextran sodium sulfate mouse model of acute colitis, Ndst4 deficiency resulted in a significantly higher disease activity index and obviously hyperemic appearance in the cecum. In the development of colitis-associated cancer, the size of colonic tumors induced by azoxymethane/dextran sodium sulfate was significantly increased in Ndst4-deficient mice compared with wild-type mice. Taken together, loss-of-function of NDST4 might impair the modification of HS chains of specific HSPGs leading to tumor-promoting inflammation in the colon. The full spectrum of signaling pathways contributed by NDST4 to colitis and tumor progression remains to be elucidated. Citation Format: Tzu-Ming Jao, Ming-Hong Tsai, Chi-Yen Huang, Sheng-Tai Tzeng, Jing-Xing Lee, Wei-Chen Lo, Che-Wei Chang, Ya-Chien Yang. Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4. [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 2472. doi:10.1158/1538-7445.AM2014-2472