Colitis-associated Colon Cancer Model Research Articles

GPR15 in colon cancer development and anti-tumor immune responses

IntroductionThe chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies.MethodsIn human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry.ResultsOur analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to ‘tumor-free’ surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15+-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden.DiscussionOur findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

Loss of NOD2 in macrophages improves colitis and tumorigenesis in a lysozyme-dependent manner.

Crohn's disease (CD) is a complex and poorly understood myeloid-mediated disorder. Genetic variants with loss of function in the NOD2 gene confer an increased susceptibility to ileal CD. While Nod2 in myeloid cells may confer protection against T-cell mediated ileopathy, it remains unclear whether it may promote resolution of the inflamed colon. In this study, we evaluated the function of Nod2 in myeloid cells in a model of acute colitis and colitis-associated colon cancer (CAC). To ablate Nod2 specifically within the myeloid compartment, we generated LysMCre/+;Nod2fl/fl mice. The role of NOD2 was studied in a setting of Dextran Sodium Sulfate (DSS)-induced colitis and in azoxymethane (AOM)/DSS model. Clinical parameters were quantified by colonoscopy, histological, flow cytometry, and qRT-PCR analysis. Upon DSS colitis model, LysMCre/+;Nod2fl/fl mice lost less weight than control littermates and had less severe damage to the colonic epithelium. In the AOM/DSS model, endoscopic monitoring of tumor progression revealed a lowered number of adenomas within the colon of LysMCre/+;Nod2fl/fl mice, associated with less expression of Tgfb. Mechanistically, lysozyme M was required for the improved disease severity in mice with a defect of NOD2 in myeloid cells. Our results indicate that loss of Nod2 signaling in myeloid cells aids in the tissue repair of the inflamed large intestine through lysozyme secretion by myeloid cells. These results may pave the way to design new therapeutics to limit the inflammatory and tumorigenic functions of NOD2.

Beneficial effects of alpha-1 antitrypsin therapy in a mouse model of colitis-associated colon cancer

BackgroundIt is widely accepted that chronic inflammatory bowel diseases significantly higher a risk for colorectal cancer development. Among different types of treatments for patients with colon cancer, novel protein-based therapeutic strategies are considered.AIMTo explore the effect of human plasma alpha-1 antitrypsin (AAT) protein in the chemically induced mouse model of colorectal cancer.MethodsBALB/c mice with azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colorectal cancer (CAC), we intraperitoneally treated with commercial preparation of human plasma AAT (4 mg per mouse). Effects of this therapy were evaluated histologically, and by immunohistochemical and gene expression assays.ResultsWhen compared with non-treated controls, AOM/DSS mice receiving AAT therapy exhibited significantly longer colons, and less anal bleeding. Concurrently, AAT-treated mice had significantly fewer polyps, and lower numbers of large colon tumors. Immunohistochemical examinations of colon tissues showed significantly lower neutrophil counts, more granzyme B-positive but fewer MMP9 (gelatinase B)-positive cancer cells and lower numbers of apoptotic cells in mice receiving AAT therapy. The expression levels of IL4 were significantly higher while TNFA was slightly reduced in tumor tissues of AOM/DSS mice treated with AAT than in AOM/DSS mice.ConclusionHuman AAT is an acute phase protein with a broad-protease inhibitory and immunomodulatory activities used as a therapeutic for emphysema patients with inherited AAT deficiency. Our results are consistent with previous findings and support an idea that AAT alone and/or in combination with available anti-cancer therapies may represent a new personalized approach for patients with colitis-induced colon cancer.

Biglycan regulated colorectal cancer progress by modulating enteric neuron-derived IL-10 and abundance of Bacteroides thetaiotaomicron

Biglycan (BGN) is a proteoglycan with branch chains and highly expressed in enteric neurons in the tumor tissue of colorectal cancer (CRC), which is negatively associated with survival rates in patients with CRC. However, how the proteoglycan promotes the progress of CRC through interacting with bacteria and regulating the immune response of enteric neurons remains largely unknown. In the present study, we found that biglycan deficiency changed tumor distribution in a colitis-associated colon cancer model. Furthermore, we revealed that BGN deficiency inhibits tumor growth in an allograft tumor model and the migration of cancer cell by upregulating interleukin-10 expression in enteric neurons. Significantly, we demonstrated that biglycan deficiency enriched the abundance of Bacteroides thetaiotaomicron through competing with it for chondroitin sulfate to inhibit CRC progress. Our work provided new insights into the interaction between host proteoglycan and gut microbiota as well as the role of enteric neurons in the tumor microenvironment.

LRRK2 G2019S promotes the development of colon cancer

Abstract LRRK2-G2019S is the most prevalent variant associated with Parkinson’s disease (PD), found in 1–3% of sporadic and 4–8% of familial PD cases. Intriguingly, emerging clinical studies have suggested that LRRK2 G2019S mutation carriers have an overall increased risk of cancer, including colon cancer. However, it remains to be experimentally tested whether and how this mutant directly affects cancer development. Here, by using the AOM/DSS-induced colitis-associated colon cancer model combined with LRRK2 G2019S knock-in (KI) mice, we found that LRRK2 G2019S promoted the pathogenesis of colon cancer, characterized by increased tumor loads and tumor-promoting cytokines compared with those in wild-type controls. Immunoblotting and immunofluorescence indicated that expression levels of Cyclin-D1, Ki-67, β-catenin and p-STAT3 were dramatically increased in the colon of KI mice, and these proteins are well known to promote the pathogenesis of colon cancer. As inflammation is a major factor contributing to the progression of colon cancer, we thus further tested if LRRK2 G2019S promotes colitis in the initiation of the disease. Indeed, we found KI mice were more susceptible to colitis induction, evident by rapid body weight loss and elevated clinical histology score. Mechanistically, LRRK2 G2019S promoted gut inflammasome activation and enhanced the activation of the NF-ƙB and MAPK pathways upon Dectin-1 stimulation. Together, our data for the first time provide direct evidence that gain-of-function of kinase activity in LRRK2 promotes colon cancer development, implicating LRRK2 as a potential target in colon cancer, especially in those LRRK2 G2019S carrier patients and cancer with hyper LRRK2 expression. Startup fund from the Department of pathology at the University of IOWA, NIH R21AG076895, R01 NS104164

Retraction: Id1 Deficiency Protects Against Tumor Formation in ApcMin/+ Mice but not in a Mouse Model of Colitis-associated Colon Cancer.

This article (1) has been retracted at the request of the authors. There was evidence of data falsification or fabrication found in one figure. Suspicious duplications of band images were found in Fig. 3A. A copy of this Retraction Notice was sent to the last known e-mail addresses for the 7 authors. Five authors (Kotha Subbaramaiah, Rhonda K. Yantiss, Xi Kathy Zhou, Robert Benezra and Andrew J. Dannenberg) agreed to the retraction; two authors (Ning Zhang, Yvette Chin) did not respond. The authors apologize to the scientific community and deeply regret any inconveniences or challenges resulting from the publication and subsequent retraction of this article.

Deficiency of Lactoferrin aggravates lipopolysaccharide-induced acute inflammation via recruitment macrophage in mice.

Lactoferrin (Lf), a multiple functional natural immune protein, is widely distributed in mammalian milk and glandular secretions (bile, saliva, tears and nasal mucosal secretions, etc.). In the previous study, we found that Lf plays an anti-inflammatory and anti-tumorigenesis role in AOM/DSS (azoxymethane/dextran sulfate sodium) induced mouse colitis-associated colon cancer model. Although we found that Lf has anti-inflammatory effects in chronic inflammation, its specific role and mechanisms in acute inflammation have not been clarified. Here, we reported that the expression levels of Lf were significantly increased when the organism was infected by Gram-negative bacteria. We then explored the role and potential mechanism of Lf in lipopolysaccharide (LPS)-induced acute inflammation. In the LPS-induced acute abdominal inflammation model, Lf deficiency aggravated inflammatory response and promoted macrophage chemotaxis to the inflammation site. Lf inhibited macrophage chemotaxis by suppressing the expression of macrophage-associated chemokines Ccl2 and Ccl5. Highly activated NF-κB signaling in Lf−/− mice was responsible for the high expression of Ccl2 and Ccl5. Our results suggested that the anti-inflammatory effect of Lf offers a new potential treatment for acute inflammatory diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10534-022-00398-1.

Quxie Capsule Alleviates Colitis-associated Colorectal Cancer Through Modulating the Gut Microbiota and Suppressing A. fumigatus-induced Aerobic Glycolysis.

Quxie capsule (QX), a compound of 21 kinds of Traditional Chinese Medicine (TCM) herbs, has been used to treat patients with metastatic colorectal cancer (mCRC) and could suppress the growth of colon cancer. However, the mechanisms of QX inhibiting colorectal cancer remain unclear. In current study, we attempted to determine the anti-colorectal cancer (CRC) effects of QX and the mechanisms of QX in alleviating colorectal cancer. A colitis-associated colon cancer (CAC) model was established by intraperitoneally injecting mice with AOM followed by 3 cycles of 2% DSS in water. During establishment of CAC model, we orally gavaged mice with QX. Hematoxylin and eosin (H&E) and immunohistochemistry were performed to assess lesion of the colonic tumors. The expression of pro-inflammatory cytokines in colonic tumors was measured by qPCR. The proportion of immune cells in colonic tumors was analyzed by flow cytometry. Internal transcribed spacer (ITS) sequencing and 16S rRNA gene sequencing were performed to detect intestinal microbiota. The expression of glycolytic related enzymes, lactate production, and extracellular acidification rate (ECAR) were used to assess the level of aerobic glycolysis. QX markedly inhibited intestinal tumorigenesis by decreasing the expression of pro-inflammatory cytokines and the proportion of myeloid-derived suppressor cells (MDSCs), and increasing the proportion of CD8+ T cells in colon tumors. Fecal microbiota sequencing revealed that QX increased the relative abundances of intestinal symbiotic probiotics, such as, Lactobacillus, Bifidobacterium and Faecalibacterium genera. What's more, opportunistic pathogens, Bacteroides genera and Aspergillus-Aspergillus fumigatus, exhibited remarkably reduced abundances in mice treated with QX compared with untreated CAC mice. Further experiments showed that QX significantly reduced glycolysis of colon tumor and suppressed A. fumigatus-induced glycolytic metabolism of colon cancer cells. QX alleviates the development of CRC at least in part through modulating intestinal microbiota and reducing A. fumigatus-induced aerobic glycolysis of colon cancer cells.

TIPE2 Promotes Tumor Initiation But Inhibits Tumor Progression in Murine Colitis-Associated Colon Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function. The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 -/- and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC. Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 -/- mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells. This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.

Recruitment of M1 Macrophages May Not Be Critical for Protection against Colitis-Associated Tumorigenesis.

A close connection between inflammation and the risk of developing colon cancer has been suggested in the last few years. It has been estimated that patients diagnosed with some types of inflammatory bowel disease, such as ulcerative colitis or Crohn’s disease, have up to a 30% increased risk of developing colon cancer. However, there is also evidence showing that the activation of anti-inflammatory pathways, such as the IL-4 receptor-mediated pathway, may favor the development of colon tumors. Using an experimental model of colitis-associated colon cancer (CAC), we found that the decrease in tumor development in global IL4Rα knockout mice (IL4RαKO) was apparently associated with an inflammatory response mediated by the infiltration of M1 macrophages (F480+TLR2+STAT1+) and iNOS expression in colon tissue. However, when we developed mice with a specific deletion of IL4Rα in macrophages (LysMcreIL4Rα−/lox mice) and subjected them to CAC, it was found that despite presenting a large infiltration of M1 macrophages into the colon, these mice were as susceptible to colon-tumorigenesis as WT mice. These data suggest that in the tumor microenvironment the absence of IL4Rα expression on macrophages, as well as the recruitment of M1 macrophages, may not be directly associated with resistance to developing colon tumors. Therefore, it is possible that IL4Rα expression in other cell types, such as colonic epithelial cells, could have an important role in promoting the development of colitis-associated colon tumorigenesis.

Cardamonin Attenuates Experimental Colitis and Associated Colorectal Cancer.

Cardamonin is a naturally occurring chalcone, majorly from the Zingiberaceae family, which includes a wide range of spices from India. Herein, we investigated the anti-inflammatory property of cardamonin using different in vitro and in vivo systems. In RAW 264.7 cells, treatment with cardamonin showed a reduced nitrous oxide production without affecting the cell viability and decreased the expression of iNOS, TNF-α, and IL-6, and inhibited NF-kB signaling which emphasizes the role of cardamonin as an anti-inflammatory molecule. In a mouse model of dextran sodium sulfate (DSS)-induced colitis, cardamonin treatment protected the mice from colitis. Subsequently, we evaluated the therapeutic potential of this chalcone in a colitis-associated colon cancer model. We performed microRNA profiling in the different groups and observed that cardamonin modulates miRNA expression, thereby inhibiting tumor formation. Together, our findings indicate that cardamonin has the potential to be considered for future therapy against colorectal cancer.

Effects of moxibustion on the P2X7R/STAT3/VEGF pathway in rats with colitis-associated colon cancer

To observe the effects of herb-partitioned moxibustion and ginger-partitioned moxibustion on the growth of colon tumors in rats with colitis-associated colon cancer (CACC), and explore the mechanism of moxibustion intervening CACC through the purinergic receptor P2X ligand-gated ion channel 7 (P2X7R)/signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) pathway. A total of 26 male Sprague-Dawley rats were selected. According to the random number table method, 6 rats were selected as the normal group. The remaining 20 rats were injected intraperitoneally with azoxymethane (AOM) combined with oral dextran sodium sulfate (DSS) to prepare the CACC model. After the model was successfully established, 2 rats were randomly selected for model identification. The remaining 18 rats which were successfully modeled were randomly divided into a model group, a herb-partitioned moxibustion group and a ginger-partitioned moxibustion group, with 6 rats in each group. Moxibustion intervention was performed in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group at Qihai (CV 6) and bilateral Tianshu (ST 25). Moxibustion was performed twice at each point each time, once a day, at a 1-day interval after 6 consecutive interventions, for a total of 30 interventions. After intervention, the colon tumor load, pathological change and histopathological score were observed. Immunohistochemistry was used to detect the expressions of VEGF, P2X7R, phospho-STAT3 (p-STAT3), and nuclear factor-kappa B p65 (NF-κB p65) proteins in rat colon tissue. Western blot was used to detect the levels of p-STAT3 and NF-κB p65 proteins in rat colon tissue. Compared with the normal group, the colon tumor load and histopathological score in the model group were significantly increased (both P<0.001), and different grades of dysplasia were observed in colon tissue from the model group, reaching the degree of adenocarcinoma; the expression level of P2X7R protein in colon tissue was significantly decreased (P<0.001), and the expression levels of p-STAT3, NF-κB p65 and VEGF proteins were significantly increased (all P<0.001) in the model group. Compared with the model group, the colon tumor load, colon histopathological score and the levels of p-STAT3, NF-κB p65 and VEGF proteins in colon tissue were significantly decreased (all P<0.05) in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group while the expression levels of P2X7R protein in colon tissue were significantly increased (both P<0.05). Both herb-partitioned moxibustion and ginger-partitioned moxibustion can reduce the colon tumor load in CACC rats and delay the progression of colon adenomas. The mechanism may be mediated by the P2X7R/STAT3 pathway to inhibit STAT3 phosphorylation, thereby reducing VEGF protein expression.

Eleutherine palmifolia (L.) Merr. Extract Increases The Crypts and Caspase-3 Expression in Colitis-Associated Colon Cancer Model

It is known that Eleutherine palmifolia (L.) Merr contains flavonoid and polyphenol as bioactive compounds that have the ability as chemopreventive agents. This study aims to examine the effect of Eleutherine palmifolia (L.) Merr extract (EPE) on colon histopathology and the enhancement of caspase-3 expression in BALB/c mice of colitis-associated colon cancer (CAC) model. Thirty Balb/c female mice were used in this study and were divided into six groups. Five mice in one group were normal or negative control (given phosphate-buffered saline), and twenty-five mice were induced intraperitoneally with 10 mg/kg BW AOM (Azoxymethane), and it was followed by the administration of 5% DSS (Dextran Sodium Sulfate) every two weeks for 20 weeks. At the sixth week, one mice in each group was sacrificed for the Fecal Occult Bold Test (FOBT) and serum amyloid α (SAA) test to ensure that CAC was indeed formed. The administration of EPE with varying doses was started from the eighth week and was continued until the 21st week. The length of the colonic crypt was measured through histology appearance using Hematoxylin-eosin (HE) staining while the immunohistochemical method was used to observe apoptotic activity through the enhancement of caspase-3 expression. The results showed that the increase in EPE dosage also increased the crypt colon length compared to the positive control group. The administration of 1.00 mg/20gBW EPE significantly increased cell apoptosis which can be observed through caspase-3 expression compared to the positive control group (p&lt;0.05). Based on these data, the extract of EPE can be developed as phytotherapy for chemopreventive.&#x0D; &#x0D;

Activity and Toxicity of Eleutherine palmifolia (L.) Merr. Extract on BALB/c Mice Colitis-Associated Colon Cancer Model.

Objective: Eleutherine palmifolia (L.) Merr. extract (EPE) containing isoliquiritigenin and oxyresveratrol is believed to be an anticancer agent. This study evaluates colon histopathology, TNF-α, TGF-β, and hepatotoxicity on BALB/c mice colitis-associated colon cancer (CAC) model treated with EPE. Methods:In vivo study was performed on BALB/c mice CAC model induced by 10 mg/kgBW AOM on the first day followed by administration that each cycle consisted of 5% DSS in water for seven days and regular water for seven days. The indicators of the formation of CAC were observed by a fecal occult blood test (FOBT) and serum amyloid α (SAA) test. The treatment was conducted once a week started from the seventh week up to the twentieth week with six treatment groups: I was administrated by regular water only (negative control), II was administrated by AOM and DSS only (positive control), III was administrated by doxorubicin, IV-VI were treated by EPE (0.25 mg/kg BW, 0.50 mg/kg BW, and 1.00 mg/kg BW) respectively. The colon and liver’s histopathology was observed using hematoxylin-eosin (HE) staining, TNF-α with immunohistochemistry (IHC), and level measurement of TGF-β colon with ELISA reader. The data were used one-way ANOVA followed by post hoc as statistical analysis. Results:The administration of EPE increased the expression of TNF-α, the total of goblet cells of the colon, and decreased the level of TGF-β. Administration of EPE 0.50 mg/20g BW decreased a liver histopathological score but induced a histopathological alteration of the liver at a dose of 1.00 mg/20g BW. Conclusion:This study indicate that EPE could be recommended as a colon anticancer through increase the goblet cells, induce apoptosis through increase TNF-α, and decrease TGF-β.

Stroma-derived ANGPTL2 establishes an anti-tumor microenvironment during intestinal tumorigenesis.

Previous studies show that tumor cell-derived angiopoietin-like protein 2 (ANGPTL2) functions as a tumor promoter in some cancer contexts. However, we recently reported that host ANGPTL2 also shows tumor suppressive activity by enhancing dendritic cell-mediated CD8+ T cell anti-tumor immune responses in mouse kidney cancer and murine syngeneic models. However, mechanisms underlying ANGPTL2-mediated tumor suppression are complex and not well known. Here, we investigated ANGPTL2 tumor suppressive function in chemically-induced intestinal tumorigenesis. ANGPTL2 deficiency enhanced intestinal tumor growth in an experimental mouse colitis-associated colon cancer (CAC) model. Angptl2-deficient mice also showed a decrease not only in CD8+ T cell responses but in CD4+ T cell responses during intestinal tumorigenesis. Furthermore, we show that stroma-derived ANGPTL2 can activate the myeloid immune response. Notably, ANGPTL2 drove generation of immunostimulatory macrophages via the NF-κB pathway, accelerating CD4+ T helper 1 (Th1) cell activation. These findings overall provide novel insight into the complex mechanisms underlying ANGPTL2 anti-tumor function in cancer pathology.

Inhibition of Aurora-A recruited CD8+ T cells infiltration via mediating IL-10 production of cancer cells

Abstract It has been shown that approximately 10% of infiltrated CD8+ T cells could be the tumor-specific intratumoral CD8+ T cells in colorectal cancer. While, inhibition of Aurora-A, a member of serine/threonine Aurora kinase family, by alisertib promoted the percentage of infiltrated intratumoral CD8+ T cells. We therefore assumed that targeting Aurora-A could be a therapeutic strategy for recruitment of intratumoral CD8+ T cells. In CT-26 tumor model, blockade of Aurora-A with alisertib slowed the tumor growth in association with an increased infiltration, activation and expansion of intratumoral CD8+ T cells. We further analyzed the role of Aurora-A in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model, and found that AURKA deficiency significantly decreased tumor formation, in parallel with an increase in infiltration of intratumoral CD8+ T cells. Gene expression profiling indicated that Il10ra expression was obviously increased in intratumoral CD8+ T cells sorted from alisertib-treated tumors. Antibody-mediated blockade of IL-10Ra dramatically attenuated the anti-tumor ability of alisertib in association with reducing the percentage of intratumoral CD8+ T cells. IL-10Ra was the key mediator of IL-10. We herein examined the levels of IL-10 and found that the levels of IL-10 in the serum of AURKA deficiency mice treated with AOM/DSS were elevated. Additionally, IL-10 obviously promoted migration of CD8+ T cells in vitro. Blockade of Aurora-A increased the transcriptional levels of IL-10. In conclusion, inhibition of Aurora-A could be useful for recruiting infiltration of intratumoral CD8+ T cell by mediating IL-10 production.

Abstract B93: Investigating the role of the hematopoietic-specific DNMT3A mutations in the aggressive phenotype of colon cancer

Abstract Clonal hematopoiesis (CH) is a nonmalignant condition characterized by clonal expansion of one or few hematopoietic stem cells. CH is associated with advanced age and presence of presumed leukemic driver mutations, such as in the DNMT3A gene. Clinical studies show that presence of CH mutations in the bone marrow in patients with solid tumors is associated with unfavorable prognosis and shorter survival. Moreover, CH mutations are enriched in tumor-infiltrating leukocytes compared to peripheral blood. While these observations have profound clinical implications, the understanding of the relationship between mutations in the blood system and aggressive phenotype of solid tumors is still lacking. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A contribute to tumor-promoting microenvironment through immune imbalance. To this end, we established a mouse model of CH driven by Dnmt3a alterations and are investigating its effect on a well-established colitis-associated colon cancer model (CAC) as a prototypical solid tumor. Wild-type recipient mice were transplanted with bone marrow from animals with: a) heterozygous loss of Dnmt3a (Dnmt3a+/−); b) Dnmt3a R878H point-mutation (Dnmt3aRH); c) wild-type controls (Dnmt3a+/+). Since inflammation is known to promote tumorigenesis, we are testing the sensitivity of engrafted mice to experimental colitis induced by 2.5% dextran sulfate sodium salt (DSS) in drinking water. Animals of all 3 experimental groups will be co-housed to control for cage-to-cage variability in microbiota. We will use total body weight loss, histopathology of harvested colons (swiss roll method)/colitis scores, colon size, and appearance of intestinal contents as a readout for colitis severity. Next, we will investigate AOM/DSS-induced tumorigenesis in CH model, including tumor burden and extent of inflammation between experimental groups. Finally, we will characterize tumor-infiltrating leukocytes that contribute to aggressive CAC phenotype to identify immune populations that engender this tumor-modulating function, including epigenetic and gene expression profiles. Our goal is to understand the relationship between mutations in the blood system and aggressive phenotype of nonhematologic cancers and to nominate potential targets for therapeutic intervention. Citation Format: Yang Feng, Daniil E. Shabashvili, Luisa M. Posada, Kathryn I. Krajcik, Chamara Gunaratne, Olga A. Guryanova. Investigating the role of the hematopoietic-specific DNMT3A mutations in the aggressive phenotype of colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B93.

Use of STAT6 Phosphorylation Inhibitor and Trimethylglycine as New Adjuvant Therapies for 5-Fluorouracil in Colitis-Associated Tumorigenesis.

Colorectal cancer (CRC) is one of the most widespread and deadly types of neoplasia around the world, where the inflammatory microenvironment has critical importance in the process of tumor growth, metastasis, and drug resistance. Despite its limited effectiveness, 5-fluorouracil (5-FU) is the main drug utilized for CRC treatment. The combination of 5-FU with other agents modestly increases its effectiveness in patients. Here, we evaluated the anti-inflammatory Trimethylglycine and the Signal transducer and activator of transcription (STAT6) inhibitor AS1517499, as possible adjuvants to 5-FU in already established cancers, using a model of colitis-associated colon cancer (CAC). We found that these adjuvant therapies induced a remarkable reduction of tumor growth when administrated together with 5-FU, correlating with a reduction in STAT6-phosphorylation. This reduction upgraded the effect of 5-FU by increasing both levels of apoptosis and markers of cell adhesion such as E-cadherin, whereas decreased epithelial–mesenchymal transition markers were associated with aggressive phenotypes and drug resistance, such as β-catenin nuclear translocation and Zinc finger protein SNAI1 (SNAI1). Additionally, Il-10, Tgf-β, and Il-17a, critical pro-tumorigenic cytokines, were downmodulated in the colon by these adjuvant therapies. In vitro assays on human colon cancer cells showed that Trimethylglycine also reduced STAT6-phosphorylation. Our study is relatively unique in focusing on the effects of the combined administration of AS1517499 and Trimethylglycine together with 5-FU on already established CAC which synergizes to markedly reduce the colon tumor load. Together, these data point to STAT6 as a valuable target for adjuvant therapy in colon cancer.

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.