Number Of Colon Tumors Research Articles

Unlocking the Potential: FKK6 as a Microbial Mimicry-Based Therapy for Chronic Inflammation-Associated Colorectal Cancer in a Murine Model.

Chronic intestinal inflammation significantly contributes to the development of colorectal cancer (CRC) and remains a pertinent clinical challenge, necessitating novel therapeutic approaches. Indole-based microbial metabolite mimics FKK6, which is a ligand and agonist of the pregnane X receptor (PXR), was recently demonstrated to have PXR-dependent anti-inflammatory and protective effects in a mouse model of dextran sodium sulfate (DSS)-induced acute colitis. Here, we examined the therapeutic potential of FKK6 in a mouse model (C57BL/6 FVB humanized PXR mice) of colitis-associated colon cancer (CAC) induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). FKK6 (2 mg/kg) displayed substantial anti-tumor activity, as revealed by reduced size and number of colon tumors, improved colon histopathology, and decreased expression of tumor markers (c-MYC, β-catenin, Ki-67, cyclin D) in the colon. In addition, we carried out the chronic toxicity (30 days) assessment of FKK6 (1 mg/kg and 2 mg/kg) in C57BL/6 mice. Histological examination of tissues, biochemical blood analyses, and immunohistochemical staining for Ki-67 and γ-H2AX showed no difference between FKK6-treated and control mice. Comparative metabolomic analyses in mice exposed for 5 days to DSS and administered with FKK6 (0.4 mg/kg) revealed no significant effects on several classes of metabolites in the mouse fecal metabolome. Ames and micronucleus tests showed no genotoxic and mutagenic potential of FKK6 in vitro . In conclusion, anticancer effects of FKK6 in AOM/DSS-induced CAC, together with FKK6 safety data from in vitro tests and in vivo chronic toxicity study, and comparative metabolomic study, are supportive of the potential therapeutic use of FKK6 in the treatment of CAC.

Banxia Xiexin Decoction inhibits colitis-associated colorectal cancer development by modulating STAT3 signaling and gut microbiota

ObjectiveTo investigate the therapeutic effects of Banxia Xiexin Decoction (BXD), a herbal medicine formula, on inflammation and the imbalance of the gut microbiota in a rat model of colorectal cancer (CRC) induced by azoxymethane (AOM) /dextran sulfate sodium (DSS). MethodsA total of 75 male C57BL/6 mice were randomly divided into five groups: normal control group (NC), model group (MODEL), low-dose BXD treatment group (L-BXD), high-dose BXD treatment (H-BXD) group and MS treatment group (MS). BXD and MS were used in CRC mice at the doses of 3.915 g/kg, 15.66 g/kg, 0.6 g/kg for 3 weeks consecutively.Histopathological changes in the colon were observed using hematoxylin-eosin (HE) staining. The content of inflammatory factors in serum was detected by an enzyme-linked immunosorbent assay (ELISA), and the expression of mRNA and protein of genes related to immunity, apoptosis, inflammation, and inflammatory factors was evaluated. Changes in the intestinal flora of mouse fecal were determined based on high-throughput sequencing of the 16S rRNA microbial gene. ResultsCompared to the model group, the low-dose BXD and high-dose BXD groups decreased the number of colon tumors, reversed weight loss, and shortened colon length of mice. The pathological examination showed that BXD alleviated the malignancy of intestinal tumors. It also suppressed signal transducer and activator of transcription 3 (STAT3), matrix metalloproteinase-9 (MMP-9), and transforming growth factor beta 1 (TGF-β1) expression, while increasing the expression of the tight junction protein ZO-1 in colon tissues. Additionally, the levels of key pathway proteins involved in inflammation (phosphorylated-STAT3, Bcl-2, COX-2) and cell cycle regulatory molecules (c-Myc and PCNA) were reduced. According to 16S rRNA sequence analysis, BXD enhanced the relative abundance of potentially beneficial bacteria, while that of cancer-related bacteria decreased. ConclusionBXD plays a preventive role in developing colorectal cancer; its mechanisms are related to the inhibition of inflammation and tumor proliferation, as well as maintenance of intestinal homeostasis.

Abstract 2820: The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation

Abstract Background and Aim: We have reported genetically-engineered mouse models for colonic neoplasia by using our novel CDX2P-Cre mice, in which Cre is restrictedly expressed in colonic epithelial cells. Although colonic tumors are generated in CDX2P-Cre;Apcflox/+ (CPC;Apc) mice with highly penetrant rate, greater number and deeper invasion of colonic tumors were observed in CDX2P-Cre;Apcflox/+;Ptenflox/+ (CPC;Apc+Pten), possibly resulting from haploinsufficiency of Pten gene. We have also confirmed shorter lifespan in CPC;Apc+Pten than that in CPC;Apc (AACR Annual Meeting 2018). Here, we test if these phenotypes come from one copy loss of Pten itself or secondary genetic and/or transcriptomic alterations induced by Pten loss by using whole genome sequence (WGS) and RNA sequence from macroscopically-dissected colonic tumors (bulk RNA-seq). Methods: WGS for five tumors and five livers as counterpart controls in each mouse model was performed by DNBSEQ platforms. Bulk RNA-seq was performed for three tumors in each model. Results: In the analysis using B-allele frequency, copy-neutral LOH occurred in chromosome 18 including Apc loci specifically in colonic neoplasia in both CPC;Apc and CPC;Apc+Pten mice. In contrast, LOH was not observed in chromosome 19 including Pten loci in CPC;Apc+Pten mice. We also confirmed remarkably decreased coverage of reads in reference mapping in Apc locus, and there was a slight decrease of coverage in Pten locus. There were no additional driver gene mutations in CPC;Apc or CPC;Apc+Pten in WGS. All these results suggested just one copy loss of Pten resulting in aggressive tumor phenotype in CPC;Apc+Pten mice. In RNA-seq and subsequent GSEA comparing colonic tumors in CPC;Apc+Pten with those in CPC;Apc revealed upregulation of some signal pathways including mTORC1. Supporting this observation, number of colon tumors, especially adenocarcinomas, were decreased by rapamycin administration to CPC;Apc+Pten. Similarly, rapamycin was more effective in tumorids from CPC;Apc+Pten those from CPC;Apc. Conclusion: WGS reveals one copy loss of Pten locus is the only genetic alteration in CPC;Apc+Pten when compared with CPC;Apc. One copy loss of Pten is sufficient to upregulate mTORC1 pathway in a CPC;Apc+Pten mouse model, resulting in promotion of tumor formation and invasion. Citation Format: Haruki Sada, Hiroaki Niitsu, Hikaru Nakahara, Naoya Sakamoto, Hirotaka Tashiro, Hideki Ohdan, Takao Hinoi. The effect of Pten haploinsufficiency on genomic and transcriptomic profiles in combined genetically-engineered mouse models for colonic neoplasia based on Apc inactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2820.

A4 DNA HYPOMETHYLATION INHIBITS COLITIS-ASSOCIATED CANCER

Abstract Background Colorectal cancer is the second leading cause of cancer death in Canada. A major risk factor for the development of colorectal cancer is chronic inflammation leading to colitis-associated cancer (CAC). We previously described a CAC mouse model in which tumors arise from DCLK1+ cells following loss of the tumor suppressor adenomatous polyposis coli (APC) and induction of colitis. Interestingly, both colitis and CAC are associated with DNA methylation changes that lead to altered gene expression. Moreover, inhibition of DNA methylation has recently been shown to lead to a viral mimicry response. The effects of DNA methylation on colonic tumorigenesis, however, is not known. Aims Thus, we aim to investigate whether inhibition of DNA methylation leads to a viral mimicry response that inhibits colitis-associated tumorigenesis. Methods We first examined the effects of DNA hypomethylation on CAC, by crossing Dclk1-CreERT2/Apcf/f mice to Dnmt1f/f mice which allowed for knock-out of the DNA methyltransferase enzyme DNMT1 in DCLK1+ cells. Dclk1/Apcf/f/Dnmt1f/f and Dclk1/Apcf/f (control group) mice were administered three doses of tamoxifen followed by 2.5% dextran sodium sulfate (DSS) for five days to induce colitis. Fourteen weeks later, we assessed colonic tumor number and size. In a separate cohort of Dclk1/Apcf/f mice treated with DSS, we compared colonic tumor number in mice treated with six doses of 5-AZA-2’-deoxycytidine (5-AZA) versus vehicle. The effects of 5-AZA and DSS on DNA methylation were assessed using the Infinium Mouse Methylation BeadChip Array on colonic epithelial cells isolated from mice treated with vehicle versus 5-AZA in the presence or absence of colitis. RNA expression of viral transposable elements and type I interferon response related genes were measured as a readout of a viral mimicry response. Lastly, we investigated the mechanism by which DNA hypomethylation affects tumorigenesis by crossing Dclk1/Apcf/f/Dnmt1f/f mice to MAVS knockout mice and examining tumor number following tamoxifen and DSS administration as above. Results Deletion of DNMT1 in DCLK1+ cells or treatment with 5-AZA significantly decreased colonic tumor number and size. Treatment with 5-AZA or DNMT1 loss led to DNA hypomethylation and subsequent upregulation of endogenous retroviral expression. Both 5-AZA treatment and DNMT1 knockout in DCLK1+ cells led to increased expression of type I interferon response genes. Knockout of the type I interferon response related gene, MAVS, reversed the anti-tumor effect observed with DNMT1 loss. Conclusions Our findings demonstrate that DNA hypomethylation by 5-AZA or loss of DNMT1 reduces colitis-associated tumorigenesis through activation of a viral mimicry response. Funding Agencies CAG, CIHRCancer Research Society, Canada Foundation for Innovation, Lawson Health Research Institute

Capsaicin and Cold exposure promote EMT-mediated premetastatic niche formation to facilitate colorectal cancer metastasis.

Colorectal cancer (CRC) is a common malignant tumor worldwide. Capsaicin and cold exposure were positively correlated with CRC metastasis. However, the mechanisms of action underlying capsaicin and cold exposure in 1,2-dimethylhyrazine (DMH)-induced CRC remain unknown. Multiple assays were utilized in the present study, including methylene blue, hematoxylin eosin (H&E) and immunohistochemistry (IHC) staining, western blotting and Duolink proximity ligation assay (PLA), in order to assess the influence of capsaicin and cold exposure on CRC rat models induced by DMH. The present study reported that capsaicin and cold exposure treatment significantly increased the size and number of colonic tumors, and the CRC metastasis rate in the capsaicin and cold exposure groups was higher than that in DMH model group.Moreover, it was observed that capsaicin and cold exposure increased mRNA and protein expression levels of LAMC2 and integrin-β1 induced by DMH. Duolink PLA results indicated that cold exposure and capsaicin significantly promoted interaction formation between LAMC2 and ITGB1 in CRC rats induced by DMH. Furthermore, western blot and IHC analysis confirmed that cold exposure and capsaicin inhibited DMH-induced decreases in the expression levels of E-cadherin, and increases in the expression levels of p-FAK, Snails, Fibronectin and N-cadherin. In addition, the serum levels of IL-1β and IL-6 in capsaicin and cold exposure group were higher than those of model group. In conclusion, our study suggests that both capsaicin and cold exposure may contribute to EMT-mediated the formation of premetastatic niche, which may lead to CRC metastasis by activating the early interaction between LAMC2 and integrin-β1.

Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1β, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1β mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1β, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1β after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

New gold(III) complexes TGS 121, 404, and 702 show anti-tumor activity in colitis-induced colorectal cancer: an in vitro and in vivo study

BackgroundChronic inflammation in the course of inflammatory bowel disease may result in colon cancer, or colitis-associated colorectal cancer (CACRC). It is well established that CACRC is associated with oxidative stress and secretion of multiple pro-inflammatory cytokines, e.g. tumor necrosis factor-α. Recently, we proved that the administration of gold(III) complexes resulted in the alleviation of acute colitis in mice. The aim of the current study was to assess the antitumor effect of a novel series of gold(III) complexes: TGS 121, 404, 512, 701, 702, and 703.MaterialsAnalyzed gold(III) complexes were screened in the in vitro studies using colorectal cancer and normal colon epithelium cell lines, SW480, HT-29, and CCD 841 CoN, and in vivo, in the CACRC mouse model.ResultsOf all tested complexes, TGS 121, 404, and 702 exhibited the strongest anti-tumor effect in in vitro viability assay of colon cancer cell lines and in in vivo CACRC model, in which these complexes decreased the total number of colonic tumors and macroscopic score. We also evidenced that the mechanism of action was linked to the enzymatic antioxidant system and inflammatory cytokines.ConclusionsTGS 121, 404, and 702 present anti-tumor potential and are an attractive therapeutic option for colorectal cancer.

Altered gut microbiota of obesity subjects promotes colorectal carcinogenesis in mice.

Obesity is a risk factor for colorectal cancer (CRC). The role of gut microbiota in mediating the cancer-promoting effect of obesity is unknown. Azoxymethane (AOM)-treated, ApcMin/+ and germ-free mice were gavaged with feces from obese individuals and control subjects respectively. The colonic tumor load and number were recorded at the endpoint in two carcinogenic models. The gut microbiota composition and colonic transcriptome were assessed by metagenomic sequencing and RNA sequencing, respectively. The anticancer effects of bacteria depleted in fecal samples of obese individuals were validated. Conventional AOM-treated and ApcMin/+ mice receiving feces from obese individuals showed significantly increased colon tumor formation compared with those receiving feces from control subjects. AOM-treated mice receiving feces from obese individuals showed impaired intestinal barrier function and significant upregulation of pro-inflammatory cytokines and activation of oncogenic Wnt signaling pathway. Consistently, transferring feces from obese individuals to germ-free mice led to increased colonic cell proliferation, intestinal barrier function impairment, and induction of oncogenic and proinflammatory gene expression. Moreover, germ-free mice transplanted with feces from obese human donors had increased abundance of potential pathobiont Alistipes finegoldii, and reduced abundance of commensals Bacteroides vulgatus and Akkermansia muciniphila compared with those receiving feces from human donors with normal body mass index (BMI). Validation experiments showed that B.vulgatus and A.muciniphila demonstrated anti-proliferative effects in CRC, while A.finegoldii promoted CRC tumor growth. Our results supported the role of obesity-associated microbiota in colorectal carcinogenesis and identified putative bacterial candidates that may mediate its mechanisms. Microbiota modulation in obese individuals may provide new approaches to prevent or treat obesity-related cancers including CRC. This work was funded by National Key Research and Development Program of China (2020YFA0509200/2020YFA0509203), National Natural Science Foundation of China (81922082), RGC Theme-based Research Scheme Hong Kong (T21-705/20-N), RGC Research Impact Fund Hong Kong (R4632-21F), RGC-CRF Hong Kong (C4039-19GF and C7065-18GF), RGC-GRF Hong Kong (14110819, 14111621), and NTU Start-Up Grant (021337-00001).

Maggot extracts chemo-prevent inflammation and tumorigenesis accompanied by changes in the intestinal microbiome and metabolome in AOM/DSS-induced mice.

Inflammatory responses and intestinal microbiome play a crucial role in the progression of colitis-associated carcinoma (CAC). The traditional Chinese medicine maggot has been widely known owing to its clinical application and anti-inflammatory function. In this study, we investigated the preventive effects of maggot extract (ME) by intragastric administration prior to azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced CAC in mice. The results showed that ME had superior advantages in ameliorating disease activity index score and inflammatory phenotype, in comparison with the AOM/DSS group. The number and size of polypoid colonic tumors were decreased after pre-administration of ME. In addition, ME was found to reverse the downregulation of tight junction proteins (zonula occluden-1 and occluding) while suppressing the levels of inflammatory factors (IL-1β and IL-6) in models. Moreover, Toll-like receptor 4 (TLR4) mediated intracellular nuclear factor-κB (NF-κB)-containing signaling cascades, including inducible nitric oxide synthase and cyclooxygenase-2, and exhibited decreasing expression in the mice model after ME pre-administration. 16s rRNA analysis and untargeted-metabolomics profiling of fecal samples inferred that ME revealed ideal prevention of intestinal dysbiosis in CAC mice, accompanied by and correlated with alterations in the composition of metabolites. Overall, ME pre-administration might be a chemo-preventive candidate in the initiation and development of CAC.

Abstract 4243: Dietary flavonoids prevent colon tumor formation in ApcMin/+ mice in a microbiome- and sex-dependent manner

Abstract Background: A diet rich in colorful fruits and vegetables is effective for the prevention of various inflammation-induced cancer types. Mechanistically, this effect has been attributed to the potent antioxidant properties of flavonoid molecules. However, contrary to observations in mice, the success of flavonoids in human dietary cancer prevention trials is often dampened due to large interpersonal variation. Antioxidant activity alone does not explain this variability, suggesting there is an additional important variable that has not been accounted for. Since flavonoids have a poor bioavailability and predominantly passage into the large intestine, we hypothesize that gut microbial metabolism is essential for flavonoid bioactivity in colon cancer prevention. Goals and Objectives: The goal of this study was to evaluate the impact of microbiota composition on the efficacy of dietary flavonoids in preventing intestinal tumorigenesis. Methods: We used the adenomatous polyposis coli - multiple intestinal neoplasia (ApcMin/+) mouse model, which is genetically predisposed to develop adenomas along its gastrointestinal tract. Development of these adenomas and eventual adenocarcinoma formation is further influenced by factors such as inflammation, dietary input and the gut microbiota. Animals were fed a control high fat diet (HFD) or prevention diets consisting of 1% berry extract, 1% purified flavonoid or 1% microbial catabolite, all on the HFD background. The contribution of the microbiota was modelled by including experimental groups that received an antibiotic cocktail in their drinking water throughout the course of the experiment. For these animals, microbiota community composition was determined by 16S rRNA sequencing, plasma was collected for histological profiling and metabolomics analysis and end-point measurements of intestinal tumor numbers and size were performed. Results: Supplementation with berry extract significantly reduced colon tumor numbers and incidence in male, but not in female mice. This effect was negated in mice on antibiotic drinking water, implicating the involvement of the gut microbiota. Community analysis revealed a relative increase in Erysipelotrichaceae, Clostridia and Akkermansia in the berry supplemented versus HFD control mice. In addition, their systemic monocyte counts were reduced. Dietary supplementation with a major gut microbial flavonoid catabolite, did not reduce colon tumor formation, suggesting the functional involvement of different microbial metabolites. Conclusions: Our study demonstrates the involvement of the mouse gut microbiota in dietary prevention of colon tumor formation. These effects impact circulating monocytes and are linked to changes in the plasma metabolome. We are currently elucidating the microbial actors and associated molecular pathways involved. Citation Format: Beckey DeLucia, Jan Claesen. Dietary flavonoids prevent colon tumor formation in ApcMin/+ mice in a microbiome- and sex-dependent manner. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4243.

A27 DNA HYPOMETHYLATION INDUCED BY 5-AZA-CDR OR LOSS OF DNMT1 INHIBITS COLITIS-ASSOCIATED COLORECTAL CANCER

Abstract Background Colorectal cancer is the second leading cause of cancer death in Canada. A major risk factor for the development of colorectal cancer is chronic inflammation leading to colitis-associated cancer (CAC). We previously described a CAC mouse model in which tumors arise from DCLK1+ tuft cells following loss of the tumor suppressor adenomatous polyposis coli (APC) and induction of colitis. Interestingly, both colitis and CAC display epigenetic changes that modulate gene expression. However, the impact of DNA methylation changes on colonic tumorigenesis is not known. Thus, we hypothesize that inhibition of DNA methylation in DCLK1+ tuft cells reduces colonic tumorigenesis. Purpose In this study, we aim to investigate the role of DNA methylation in CAC by inhibiting DNA methylation using genetic and pharmacologic means. Method Using a publicly available dataset (GSE75214) of gene expression data analyzed by microarray from colonic biopsies of patients with ulcerative colitis and Crohn’s Disease with active disease, we examined the expression of DNA methyltransferases (DNMTs). Expression of DNMTs in mice with colitis was additionally examined by RT-qPCR and global DNA methylation levels measured by 5-mC ELISA. In separate experiments, Dclk1-CreERT2/Apcf/f mice were crossed to DNMT1f/f mice to knock-out the DNA methyltransferase DNMT1 in DCLK1+ tuft cells. Dclk1/Apcf/f and Dclk1/Apcf/f/DNMT1f/f mice were then administered three doses of tamoxifen followed by 2.5% dextran sodium sulfate (DSS) for five days to induce colitis. Fourteen weeks later, we assessed colonic tumor number and size. In a separate cohort of Dclk1/Apcf/f mice, we induced colitis and treated the mice with six doses of the DNA de-methylating drug 5-AZA-2’-deoxycytidine (5-AZA) or vehicle, and assessed colonic tumor number. To examine DNA methylation changes, we then treated WT mice with 5-AZA and DSS and isolated intestinal epithelial cells. From the intestinal epithelial cell, we isolated DNA and ran the Infinium MouseMethylation BeadChip Array. Result(s) Patients with IBD were found to have increased expression of DNMT1 compared to healthy controls. Mice treated with DSS similarly had increased DNMT1 expression, as well as, global methylation levels compared to controls. Deletion of DNMT1 in DCLK1+ cells significantly inhibited the number and size of colonic tumors. Treatment of mice with 5-AZA decreased global and gene specific DNA methylation levels, and significantly reduced both the number of mice with tumors, and the average colonic tumor number and size per mouse. Conclusion(s) Our findings demonstrate that colitis in both patients and mice is associated with DNA methylation. Furthermore, DNA hypomethylation by 5-AZA treatment or loss of DNMT1 reduces CAC formation suggesting that altered DNA methylation plays a critical role in colonic tumorigenesis. Disclosure of Interest None Declared

AC1Q3QWB inhibits colorectal cancer progression by modulating the immune response and balancing the structure of the intestinal microbiota

AC1Q3QWB enhances CD8 + T cell response and triggers accumulation of Tregs and B cells. BackgroundColorectal cancer (CRC) is a common malignancy with high mortality and few effective therapeutic measures. Gut microbiota dysbiosis and chronic inflammation might contribute to the development of CRC. The present study aimed to explore the effect of AC1Q3QWB (AQB) on colon carcinogenesis in vivo. MethodsA mouse colon cancer model was constructed by intraperitoneal injection of 10 mg/kg of Azoxymethane (AOM) and 2 % dextran sodium sulfate (DSS) in drinking water. Mice were randomly assigned to four groups: normal control (NC), AOM/DSS (model control, MC), DMSO + AOM/DSS (DMSO), and AQB + AOM/DSS (AQB). Mice in the AQB group were treated with an intraperitoneal injection of AQB (50 mg/kg) after successful modeling. Then, the disease activity index (DAI) of colitis was analyzed. Colon tissues were collected for hematoxylin-eosin, immunohistochemistry, and microscopic and histological evaluation. Stool samples were collected for microbiota analysis by 16S rRNA sequencing. Blood samples were analyzed by flow cytometry to investigate the inflammatory response. ResultsIn AOM/DSS-induced CRC mouse model, AQB treatment dramatically reduced the number and size of colon tumors. AQB treatment enhances CD8++T cell response and triggers the accumulation of CD4++CD25++Foxp3++Regulatory T cells (Tregs) and B cells. AQB regulated the structure and composition of the gut microbiota, which decreased the Firmicutes/Bacteroidetes ratio at the phylum level and increased the abundance of probiotics. ConclusionsAQB has potent antitumor activity against colorectal cancer in vivo by a mechanism that might involve modulation of the immune system and alteration of the intestinal microbiota.

Mechanisms of electroacupuncture at "Zusanli"(ST36) in delaying colon "inflammation-cancer transformation"

To investigate the mechanism of electroacupuncture (EA) "Zusanli" (ST36) in delaying colon "inflammation-cancer transformation" in mice by anti-inflammatory. C57BL/6J mice were randomly divided into normal, model and EA groups, with 12 mice in each group. The mouse model of colorectal cancer (CRC) was established by intrape-ritoneal injection of azomethane (AOM) and feeding dextran sodium sulfate (DSS). At the beginning of the 2nd cycle, EA was applied to bilateral ST36 for 30 min once every other day for 12 times. The number of colon tumors in each group was observed, and the weight and length of colon were recorded. The contents of interleukin (IL)-1β, IL-6, IL-17A, IL-23, tumor necrosis factor (TNF)-α and CXC chemokine ligand 1 (CXCL1) of serum and colon tissue were detected by MSD multifactorial assay.The apoptosis of local cells in colon tumor was observed by TUNEL staining. Cell proliferation in colon tumor was observed by immunohistochemistry. Compared with the normal group, the colon length was significantly shortened (P<0.05) and the colon mass was significantly increased (P<0.001) in the model group, the contents of IL-1β, IL-6, TNF-α, IL-17A and CXCL1 of serum and colon tissue were significantly increased (P<0.05, P<0.01, P<0.001), and the content of IL-23 was increased in colon tissue (P<0.05) in the model group. Compared with the model group, the colon mass was decreased (P<0.05) and the contents of IL-1β, IL-6, TNF-α and IL-17A in serum were decreased (P<0.05), while the contents of IL-17A, CXCL1 and IL-23 in colon tissue were decreased (P<0.05) in the EA group, the percentage of local apoptotic cells in the EA group was increased (P<0.001), the percentage of PCNA positive cells was decreased (P<0.001), the number of tumors and the tumor volume were significantly decreased (P<0.01, P<0.05). The contents of IL-6, TNF-α, IL-17A and IL-23 in serum of CRC mice were positively correlated with tumor burden (P<0.05).The contents of IL-1β, TNF-α, CXCL1 and IL-23 in colon tissue of CRC mice were positively correlated with tumor burden (P<0.05). Electroacupuncture at ST36 can inhibit the inflammatory response of AOM/DSS inflammatory associated CRC mice and delay the "inflammation-cancer transformation" of colon.

Sleeve Gastrectomy Increases Colitis-Associated Colon Tumor Growth Independent of Diet

INTRODUCTION: We have previously found that sleeve gastrectomy (SG) causes increased numbers of tumors in a diet-induced obesity mouse model of colitis-associated cancer (CAC). Here we examine the effects of SG on CAC in lean mice fed a regular diet. METHODS: Twelve-week-old lean C57BL/6J mice were maintained on a normal chow diet and randomized to SG or sham operation. Five weeks postoperatively, CAC was established via one-time injection of azoxymethane (AOM) followed by 2-week-long periods of dextran sulfate sodium (DSS) administration. Colitis severity was scored from 0 to 12 by measuring weight loss, stool consistency, and hematochezia. The number of colonic tumors and total tumor surface area were measured at sacrifice 46 days after DSS and compared using Welch’s t-test. RESULTS: CAC tumors were significantly larger in lean SG mice compared with sham (0.67 cm2 vs 0.29 cm2; p = 0.046; Figs. 1A, 1B). However, lean SG mice developed similar levels of colitis as sham (maximum colitis severity 4.7 vs 4.1; data not shown) and similar numbers of tumors (4.4 vs 3.6; p = 0.61; Fig. 1C). In contrast, in obese mice, SG leads to increased tumor numbers (5.5 vs 1.4; p = 0.0003; Fig. 1D) as well as increased colitis severity and larger tumors (data not shown).Figure 1CONCLUSION: SG increases tumor size in a mouse CAC model independent of diet and obesity. SG-induced increases in tumor number and colitis severity are present in diet-induced obese but not lean mice. These data suggest that surgery, diet, and obesity have distinct roles in colon cancer initiation and growth.

Potential Chemoprevention of Paeoniflorin in Colitis-Associated Colorectal Cancer by Network Pharmacology, Molecular Docking, and In Vivo Experiment.

Chronic inflammation plays a positive role in the development and progression of colitis-associated colorectal cancer (CAC). Medicinal plants and their extracts with anti-inflammatory and immunoregulatory properties may be an effective treatment and prevention strategy for CAC. This research aimed to explore the potential chemoprevention of paeoniflorin (PF) for CAC by network pharmacology, molecular docking technology, and in vivo experiments. The results showed that interleukin-6 (IL-6) is a key target of PF against CAC. In the CAC mouse model, PF increased the survival rate of mice and decreased the number and size of colon tumors. Moreover, reduced histological score of colitis and expression of Ki-67 and PCNA were observed in PF-treated mice. In addition, the chemoprevention mechanisms of PF in CAC may be associated with suppression of the IL-6/STAT3 signaling pathway and the IL-17 level. This research provides experimental evidence of potential chemoprevention strategies for CAC treatment.

Abstract 819: ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors

Abstract ERBB4 is commonly over-expressed in human CRC, however the utility of ERBB4 as a target for colorectal cancer (CRC) therapeutics is largely unexplored. Our group recently identified a molecular subtype of CRC that arises independent of EGFR and displays a more aggressive growth phenotype than those dependent on EGFR. The robust growth phenotype of EGFR-independent tumors can be in part attributed to upregulation of IL10 signaling. In addition, EGFR-independent tumors display a significant upregulation of Erbb2 and Erbb4 transcripts in both spontaneous and sporadic CRC mouse models, perhaps compensating for the loss of EGFR. To investigate the importance of other ERBB receptors in EGFR-independent CRC tumor progression, we ablated Erbb4 to interrogate its influence on tumor development in vivo using a conditional allele of Erbb4tm1Fej (Erbb4f). ERBB4-deficient ApcMin/+ mice (ApcMin/+, ErbB4f/f, Tg(Vil1-Cre)) were established and used to show that ERBB4 ablation in the intestinal epithelia results in a significant decrease in the number of intestinal and colon tumors. Polyps lacking ERBB4 were also significantly reduced size, contrary to what is observed with loss of EGFR. We also recently developed ERBB2-deficient ApcMin/+ mice, and preliminary results suggest that Erbb2 ablation also results in a decrease in intestinal and colon tumor number and size. Consistent with data from EGFR-independent tumors, transcriptomic analysis of ERBB4-deficient intestinal tumors predicted down-regulation of IL10 signaling, which was validated through Il10 and Socs3 qPCR. To complement our findings, the observed down-regulation of Il10 and Socs3 at the transcript level corresponded with a significant decrease in IL10 levels in the serum of mice harboring ERBB4 deficient tumors when compared to mice without tumors. Furthermore, we found that transcript levels of Erbb4 significantly decreased after anti-IL10 treatment of EGFR-independent tumors in vivo. This illustrates a possible negative feedback loop whereby neutralizing IL10 also leads to a reduction of Erbb4 expression, further reducing IL10 signaling. Taken together, the data suggest that the absence of EGFR triggers Erbb4 upregulation, implicating an important role for ERBB4 in EGFR-independent colon tumor growth. These results suggest that therapeutic targeting of ERBB4 may lead to a reduction in IL10 signaling and reduced intestinal polyp multiplicity and size in EGFR-independent tumors and inform the development of novel approaches to treat CRC that exhibit aberrant expression of EGFR and ERBB4. Citation Format: Michael P. McGill, Carolina Mantilla Rojas, David W. Threadgill. ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 819.

Abstract 2539: Macrophage derived WNTs regulate immune checkpoint expression and inflammation induced colon cancer

Abstract Colorectal cancer is the third most common cancer in the world. Patients with inflammatory bowel disease are at an elevated risk for developing colon cancer. Macrophages are important members of tumor microenvironment (TME) and promote inhibition of anti-tumor immunity by activation of immune checkpoints and other neo-antigens expressions. However, paracrine signals regulating these checkpoint expressions in macrophages need to be determined. Macrophage-derived WNT ligands play important role in tissue homeostasis in multiple organs. In the present study we have demonstrated that inhibition of macrophage-derived WNT promotes inflammation-induced colon cancer. In both the mice model and human in vitro model we have shown that the absence of macrophage-derived WNT promotes immunosuppressive microenvironment with upregulation of immune checkpoint expressions in tumor-associated macrophages and T cells. Release of WNT ligands expressed in macrophages was inhibited by using Csf1r.iCre; Porcn fl/fl mice where gene expressing PORCUPINE involved in post translational modification and release of WNT ligands was deleted in macrophages using Cre recombinase expressed under Csf1r promoter. In these mice and wild type littermates, Inflammation-induced colon cancer was developed using AOM-DSS treatment. Mice were treated with a single dose of 16mg/kg of body weight Azoxymethane (AOM) (i.p.) and subsequent three cycles of 3.5% of Dextran sodium sulfate (DSS) (supplementation in drinking water). Development of colon cancer was monitored using small animal colonoscopy. After 3 cycles of DSS Csf1r.iCre; Porcn fl/fl mice demonstrated a significantly higher number of colon tumors compared to wild type littermate (Macro size: p&amp;lt;0.0005, Micro size: p&amp;lt;0.013). Moreover, flow cytometric analysis of tumor tissue demonstrated that in Csf1r.iCre; Porcn fl/fl mice both PD1 and PDL1 expression was high in tumor-associated macrophages compared to wild type mice. PDL1 expression was also high in colon tumor tissue, myeloid-derived suppressor cells (MDSCs) and helper T cells in Csf1r.iCre; Porcn fl/fl mice. Similar observation was made in ex-vivo co-culture model using human pan T cells, HTB-38 human colon cancer cells and human peripheral blood mononuclear cell (PBMC) derived macrophages pre-treated with PORCUPINE inhibitor C59. Therefore, our present study demonstrated for the first time that macrophage-derived WNT is required to modulate the immunosuppressive role of macrophages in inflammation-induced colon cancer. Inhibition of WNT release specific from macrophages promotes PDL1 expression in colon cancer cells and other cell types in TME including MDSCs, and T helper cells. Further studies are ongoing to determine the mechanism of autocrine action of WNT ligands in tumor-associated macrophages to maintain anti-tumor immunity. Citation Format: Subhrajit Saha, Pooja Gupta, Rishi Man Chugh, Ximena Diaz Olea, Tanu Arora, Payel Bhanja. Macrophage derived WNTs regulate immune checkpoint expression and inflammation induced colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2539.

Supplementation with High or Low Iron Reduces Colitis Severity in an AOM/DSS Mouse Model.

The relationship between colitis-associated colorectal cancer (CAC) and the dysregulation of iron metabolism has been implicated. However, studies on the influence of dietary iron deficiency on the incidence of CAC are limited. This study investigated the effects of dietary iron deficiency and dietary non-heme iron on CAC development in an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. The four-week-old mice were divided into the following groups: iron control (IC; 35 ppm iron/kg) + normal (NOR), IC + AOM/DSS, iron deficient (ID; <5 ppm iron/kg diet) + AOM/DSS, and iron overload (IOL; approximately 2000 ppm iron/kg) + AOM/DSS. The mice were fed the respective diets for 13 weeks, and the AOM/DSS model was established at week five. FTH1 expression increased in the mice’s colons in the IC + AOM/DSS group compared with that observed in the ID and IOL + AOM/DSS groups. The reduced number of colonic tumors in the ID + AOM/DSS and IOL + AOM/DSS groups was accompanied by the downregulated expression of cell proliferation regulators (PCNA, cyclin D1, and c-Myc). Iron overload inhibited the increase in the expression of NF-κB and its downstream inflammatory cytokines (IL-6, TNFα, iNOS, COX2, and IL-1β), likely due to the elevated expression of antioxidant genes (SOD1, TXN, GPX1, GPX4, CAT, HMOX1, and NQO1). ID + AOM/DSS may hinder tumor development in the AOM/DSS model by inhibiting the PI3K/AKT pathway by increasing the expression of Ndrg1. Our study suggests that ID and IOL diets suppress AOM/DSS-induced tumors and that long-term iron deficiency or overload may negate CAC progression.

P‐cadherin regulates intestinal epithelial repair, but is dispensable for colitis associated colon cancer development

Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases (IBD), triggers significant changes in the intestinal epithelial homeostasis. These changes include leakiness of the gut barrier, formation of mucosal wounds and, in most severe cases, oncogenic transformation of colonic epithelium resulting in colitis‐associated colon cancer (CAC). Altered structure and dynamics of epithelial junctions is a hallmark of intestinal inflammation, mediating epithelial barrier injury and repair. P‐cadherin (gene name: CDH3) is an important component of adherens junctions (AJ), which is poorly expressed in normal intestinal epithelium, but could be induced in inflamed and injured mucosa. The goal of this his study was to investigate the roles of P‐cadherin in regulating intestinal inflammation, mucosal repair and CAC.P‐cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. Roles of P‐cadherin in intestinal inflammation and tumorigenesis in vivo were investigated using a mouse strain with total P‐cadherin knockout. Dextran sulfate sodium (DSS)‐induced colitis was utilized to study mucosal inflammation, whereas CAC was established using a classical azoxymetane (AOM)/DSS model. Severity of acute DSS colitis was not affected by P‐cadherin knockout, however, P‐cadherin null mice exhibited faster recovery after DSS withdrawal indicating accelerated repair of injured mucosa. No significant differences in the number and size of colonic tumors was observed in P‐cadherin null and control mice after AOM/DSS induced CAC. Consistently, CRISPR/Cas9‐mediated knockout of P‐cadherin in SK‐CO15 and HCA7 human colonic epithelial cell lines accelerated epithelial wound healing without affecting cell proliferation. The faster migration of P‐cadherin depleted cells was associated with diminished cell‐matrix adhesions and increased cell spreading. Loss of P‐cadherin resulted in activation of Src kinase and the pro‐migratory phenotype of P‐cadherin depleted cell was reversed by pharmacological inhibition of Src. Furthermore, accelerated epithelial wound healing caused by loss of P‐cadherin was prevented by Rac1 inhibition. Our findings highlight P‐cadherin as a negative regulator of intestinal epithelial would heling in vitro and mucosal repair in vivo. By contrast, this AJ protein appears to be dispensable for intestinal epithelial cell proliferation and CAC development.

P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer.

Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development.