Tumor Development In ApcMin Research Articles

Gasdermin D promotes development of intestinal tumors through regulating IL-1β release and gut microbiota composition

The interplay between gut microbiota and host is crucial for maintaining host health. When this balance is broken, various diseases can arise, including colorectal cancer (CRC). However, the mechanism by which gut microbiota and host interactions mediate CRC development remains unclear. Here, we found that Gasdermin D (GSDMD), an inflammasome effector responsible for forming membrane pores to mediate cell pyroptosis, was upregulated in both human and mouse intestinal tumor samples. GSDMD deficiency significantly suppressed intestinal tumor development in Apcmin/+ mice, a spontaneous CRC mouse model. Apcmin/+Gsdmd−/− mice exhibited reduced IL-1β release in the intestine, and the administration of recombinant mouse IL-1β partially restored intestinal tumor development in Apcmin/+Gsdmd−/− mice. Moreover, 16s rRNA sequencing showed a substantial increase in Lactobacillus abundance in the feces of Apcmin/+Gsdmd−/− mice compared to Apcmin/+ mice. Concurrently, Kynurenine (Kyn), a metabolite derived from host tryptophan (Trp) metabolism, was significantly decreased in the feces of Apcmin/+Gsdmd−/− mice, as shown by metabolite analysis. Additionally, Kyn levels were inversely correlated with Lactobacillus abundance. Furthermore, the administration of exogenous Kyn also promoted intestinal tumor development in Apcmin/+Gsdmd−/− mice. Thus, GSDMD promotes spontaneous CRC development through increasing IL-1β release and Kyn production. Our data suggest an association between GSDMD, gut microbiota, the host Trp/Kyn pathway, and CRC development.

LAT1 expression influences Paneth cell number and tumor development in ApcMin/+ mice

BackgroundAmino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood.MethodsTo investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed.ResultsThis study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/β-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production.ConclusionsLAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.

Loss of DRO1/CCDC80 in the tumor microenvironment promotes carcinogenesis.

Tumors are composed of the tumor cells and the surrounding microenvironment. Both are closely interwoven and interact by a complex and multifaceted cross-talk which plays an integral part in tumor initiation, growth, and progression. Dro1/Ccdc80 has been shown to be a potent suppressor of colorectal cancer and ubiquitous inactivation of Dro1/Ccdc80 strongly promoted colorectal carcinogenesis in ApcMin/+ mice and in a chemically-induced colorectal cancer model.The aim of the present study was to investigate whether Dro1/Ccdc80’s tumor suppressive function is tumor-cell-autonomous. Expression of Dro1/Ccdc80 in cancer cells had no effect on both colon tumor development in ApcMin/+ mice and formation of xenograft tumors. In contrast, DRO1/CCDC80 loss in the microenvironment strongly increased tumor growth in xenograft models, inhibited cancer cell apoptosis, and promoted intestinal epithelial cell migration. Moreover, stromal Dro1/Ccdc80 inactivation facilitated formation of intestinal epithelial organoids. Expression analyses showed Dro1/Ccdc80 to be significantly down-regulated in murine gastric cancer associated fibroblasts, in ApcMin/+ colon tumor primary stromal cells and in microdissected stroma from human colorectal cancer compared to normal, non-tumor stroma. Our results demonstrate epithelial derived DRO1/CCDC80 to be dispensable for intestinal tissue homeostasis and identify Dro1/Ccdc80 as tumor suppressor in the tumor microenvironment.

YYFZBJS Inhibits Colorectal Tumorigenesis by Remodeling Gut Microbiota and Influence on M2 Macrophage Polarization <i>in vivo</i> and <i>in vitro</i>

Background: The occurrence of CRC is believed to be related to a variety of factors. Accumulating evidence shows that microbiota can influence the outcome of cancer immunotherapy. Our previous studies indicated that the extract of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS), had potent anticancer activities by significantly inhibiting intestinal tumor development in ApcMin/+ mice. However, knowledge regarding the mechanism and effect of YYFZBJS in the prevention of colorectal cancer is limited. Methods: In this study, we investigated the preventive effects of oral administration of YYFZBJS in enterotoxigenic Bacteroides fragilis (ETBF)-colonized mice with azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis. Here, the tumor load, tumor number, histology, and the severity of disease activity index (DAI) scores were reduced as expected. Results: The intragastric administration of YYFZBJS in AOM/DSS model significantly decreased ETBF abundance, immunity and some M2 macrophage markers, including CD206, Arg-1, IL-10, and TGF-β. Additionally, reversing polarized macrophages, which has been modified by YYFZBJS, could suppressed CRC cell proliferation and infiltration, as demonstrated by decreasing some tumor proliferation-related proteins in a dose-dependent manner, including c-Met, cyclinD1 and MMPs. Importantly, ETBF dysbiosis can contribute to the development of colon tumor by stimulating p-STAT3 medicated M2 macrophages polarization to promote chronic inflammation and adenoma malignant transformation, which effectively constrained by YYFZBJS. Conclusion: Altogether, we demonstrate that ETBF dysbiosis may contribute to M2 macrophages-promoted colon carcinogenesis and progression of CRC cells, and indicating that YYFZBJS could be employed as a promising protective agent against ETBF-mediated colorectal cancer. Funding Statement: This research was supported by the National Natural Science Foundation of China (No. 81874399), the Project of Hospital Development Center in Shanghai (No. SHDC2020CR4047). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All animal protocols were approved by the Institutional Animal Use and Care Committee. All experiments and animal care procedures were approved by the Shanghai Medical Experimental Animal Care Commission and were conducted in accordance with the provisions and general recommendations of the Chinese Experimental Animals Administration Legislation. All animals were received humane care according to the National Institutes of Health (USA) guidelines.

Clostridium butyricum, a butyrate-producing probiotic, inhibits intestinal tumor development through modulating Wnt signaling and gut microbiota.

Gut microbiota dysbiosis is closely involved in intestinal carcinogenesis. A marked reduction in butyrate-producing bacteria has been observed in patients with colorectal cancer (CRC); nevertheless, the potential benefit of butyrate-producing bacteria against intestinal tumor development has not been fully investigated. We found that Clostridium butyricum (C. butyricum, one of the commonly used butyrate-producing bacteria in clinical settings) significantly inhibited high-fat diet (HFD)-induced intestinal tumor development in Apcmin/+ mice. Moreover, intestinal tumor cells treated with C. butyricum exhibited decreased proliferation and increased apoptosis. Additionally, C. butyricum suppressed the Wnt/β-catenin signaling pathway and modulated the gut microbiota composition, as demonstrated by decreases in some pathogenic bacteria and bile acid (BA)-biotransforming bacteria and increases in some beneficial bacteria, including short-chain fatty acid (SCFA)-producing bacteria. Accordingly, C. butyricum decreased the fecal secondary BA contents, increased the cecal SCFA quantities, and activated G-protein coupled receptors (GPRs), such as GPR43 and GPR109A. The anti-proliferative effect of C. butyricum was blunted by GPR43 gene silencing using small interfering RNA (siRNA). The analysis of clinical specimens revealed that the expression of GPR43 and GPR109A gradually decreased from human normal colonic tissue to adenoma to carcinoma. Together, our results show that C. butyricum can inhibit intestinal tumor development by modulating Wnt signaling and gut microbiota and thus suggest the potential efficacy of butyrate-producing bacteria against CRC.

<i>Clostridium butyricum</i>, a Butyrate-Producing Probiotic, Inhibits Intestinal Tumor Development Through Modulating Wnt Signaling and Gut Microbiota

Background: Gut microbiota dysbiosis is closely involved in intestinal carcinogenesis. A marked reduction of butyrate-producing bacteria has been reported in patients with colorectal cancer (CRC), nevertheless, the potential benefit of supplement of butyrate-producing bacteria against intestinal tumor development has not been fully investigated. Methods: The effects of Clostridium butyricum (C. butyricum, one of the clinically commonly used butyrate-producing bacteria) on CRC cells were examined. Apcmin/+ mice were fed a basal diet (BD) or high-fat diet (HFD) with or without C. butyricum (2×109 CFU 3-times a week) by gavage for 12 weeks. Human colonic tissues were collected to evaluate butyric acid recognition receptors, namely G-protein coupled receptors (GPRs) in relation to CRC. Findings: C. butyricum significantly inhibited HFD-induced intestinal tumor development in Apcmin/+ mice. Moreover, decreased proliferation and increased apoptosis were found in intestinal tumor cells treated with C. butyricum. Additionally, C. butyricum suppressed Wnt/β-catenin signaling pathway. C. butyricum modulated gut microbiota composition, which decreased some pathogenic bacteria and bile acids (BAs)-biotransforming bacteria, and increased some beneficial bacteria including short-chain fatty acids (SCFAs)-producing bacteria as shown by Illumina 16S rRNA sequencing. Accordingly, C. butyricum decreased fecal secondary BAs contents, increased caecal SCFAs quantities, and activated butyric acid recognition receptors, GPR43 and GPR109A. Clinically, we also observed that the expression of GPR43 and GPR109A was gradually down-regulated from human normal colonic tissue, adenoma, to carcinoma. Interpretation: Our results showed that C. butyricum could inhibit intestinal tumor development via modulating Wnt signaling and gut microbiota, suggesting the potential efficacy of butyrate-producing bacteria against CRC. Funding Statement: This study is supported by the grants (81570478, 81741075, and 81700456) from the National Natural Science Foundation of China, the grant (17JCYBJC24900) from Natural Science Foundation of Tianjin, the grant (2019M651049) from Postdoctoral Science Foundation of China, and the grant (2018242755) from Medical and Health Science and Technology Project of Zhejiang. Declaration of Interests: The authors declare no conflicts interest. Ethics Approval Statement: The written informed consents were obtained from all individuals prior to participation, and the study was approved by the Ethics Committee of General Hospital, Tianjin Medical University, China.

Extrinsic intestinal denervation modulates tumor development in the small intestine of Apc(Min/+) mice.

BackgroundInnervation interacts with enteric immune responses. Chronic intestinal inflammation is associated with increased risk of colorectal cancer. We aimed to study potential extrinsic neuronal modulation of intestinal tumor development in a mouse model.MethodsExperiments were performed with male ApcMin/+ or wild type mice (4 weeks old, body weight approximately 20 g). Subgroups with subdiaphragmatic vagotomy (apcV/wtV), sympathetic denervation of the small intestine (apcS/wtS) or sham operated controls (apcC/wtC) were investigated (n = 6-14 per group). Three months after surgical manipulation, 10 cm of terminal ileum were excised, fixed for 48 h in 4% paraformaldehyde and all tumors were counted and their area determined in mm2 (mean ± standard error of the mean (SEM)). Whole mounts of the muscularis of terminal ileum and duodenum (internal positive control) were also stained for tyrosine hydroxylase to confirm successful sympathetic denervation.ResultsTumor count in ApcMin/+ mice was 62 ± 8 (apcC), 46 ± 11 (apcV) and 54 ± 8 (apcS) which was increased compared to wildtype controls with 4 ± 0.5 (wtC), 5 ± 0.5 (wtV) and 5 ± 0.6 (wtS; all p < 0.05). For ApcMin/+ groups, vagotomized animals showed a trend towards decreased tumor counts compared to sham operated ApcMin/+ controls while sympathetic denervation was similar to sham ApcMin/+. Area covered by tumors in ApcMin/+ mice was 55 ± 10 (apcC), 31 ± 8 (apcV) and 42 ± 8 (apcS) mm2, which was generally increased compared to wildtype controls with 7 ± 0.6 (wtC), 7 ± 0.4 (wtV) and 7 ± 0.6 (wtS) mm2 (all p < 0.05). In ApcMin/+ groups, tumor area was decreased in vagotomized animals compared to sham operated controls (p < 0.05) while sympathetically denervated mice showed a minor trend to decreased tumor area compared to controls.ConclusionsExtrinsic innervation of the small bowel is likely to modulate tumor development in ApcMin/+ mice. Interrupted vagal innervation, but not sympathetic denervation, seems to inhibit tumor growth.

Invariant natural killer T (iNKT) lymphocytes promote the development of intestinal tumors (TUM2P.914)

Abstract CD1d-restricted NKT cells are known as potent regulatory cells of immune responses. Invariant NKT cells, which are activated by the artificial lipid ligand alpha-galactosylceramide, have anti-tumor functions in several murine experimental models, and iNKT cells can naturally protect against spontaneous tumors, as recently described in two different murine models. APCmin/+ mice develop intestinal tumors due to a mutation in the adenomatous polyposis coli gene, mutated in human colorectal cancer. To investigate whether iNKT cells influence tumor formation in APCmin/+ mice, this strain was crossed with Jalpha281-/- mice or CD1d-/- mice that lack iNKT cells or all NKT cells, respectively. We show that absence of iNKT cells decreased the number of tumors/polyps more than 50%, compared with the iNKT cell sufficient APCmin/+ control litter mate mice. This indicates that iNKT cells naturally promote tumor development in APCmin/+ mice. Moreover, we investigated the modulation of tumor development by iNKT cell directed therapy in preclinical studies. This study was performed by systemically activating iNKT cells with aGalCer, its TH2 skewing analogue C20:2 or the TH1 skewing analogue C-glycoside. The results revealed a decreased number of tumors after treatment with aGalCer. In contrast, in particular the TH2 skewing ligand C20:2 promoted tumor development, which suggests that iNKT cells may promote tumors through TH2 cytokines in this model.

Altered CD4+ T cell subset balance in lymphoid organs of mouse model of colorectal cancer (397.2)

The adenomatous polyposis coli (APC) gene is a known tumor suppressor gene, and mutations resulting in APC inactivation leads to the formation of multiple intestinal neoplasms (Min) within mutant mice (APCMin/+). Thus, the APCMin/+ mouse is used as a model of human colorectal cancer (CRC). The intestinal compartment is especially susceptible to tumor formation due the high turnover rate of epithelial cells. Also, the presence of the intestinal microbiota makes the intestines a very immunologically active site. Reports show that tumor associated CD4+ T cell derived IL‐17 promotes intestinal tumor development in APCMin/+ mice. To further investigate the role of the APCMin/+ mutation on CD4+ T cell function, we performed flow cytometric, histochemical, and immunofluorescent studies on both wild type (APC+/+) and APCMin/+ mice. We identified increased levels of IFN‐+ CD4+ and IL‐17A+ CD4+ cells, and decreased levels of IFN‐+IL‐17+ double positive CD4+ cells in the mesenteric lymph nodes and Peyer’s patches of APCMin/+mice. Culture of naïve CD4+ T cells from the spleens of APCMin/+mice lead to increased regulatory T cell development and lower IL‐17A expression. These results suggest an altered T cell balance in APCMin/+ mice. This intrinsic imbalance in CD4+ T cell subsets may disrupt the intestinal immune environment, inhibiting intestinal tumor destruction in the APCMin/+mouse.Grant Funding Source: UAB Comprehensive Cancer Center

Chemopreventive effects of berberine on intestinal tumor development in Apc min/+mice

BackgroundBerberine, an isoquinoline alkaloid, has shown inhibitory effects on growth of several tumor cell lines in vitro. The aim of this study was to investigate chemopreventive effects of berberine on intestinal tumor development in Apcmin/+ mice.MethodsFour-week old Apcmin/+ mice were treated with 0.05% or 0.1% berberine in drinking water for twelve weeks. The number and the size of tumors were measured to evaluate intestinal tumor development. Tissue sections were prepared for PCNA and Ki-67 immunostaining to detect cell proliferation, and TUNEL assay and cleaved caspase-3 immunostaining for apoptosis. Western blot analysis and immunostaining were performed to detect the activation of Wnt and epidermal growth factor receptor (EGFR) signaling pathways and COX-2 expression in the intestinal tumor cells. The prostaglandin E2 level in the small intestine was detected using ELISA.ResultsCompared with untreated Apcmin/+ mice, the total numbers of tumors in the small intestine and the colon were reduced by 39.6% and 62.5% in 0.05% and 0.1% berberine-treated mice, respectively. The numbers of tumors in proximal, middle, and distal segments of the small intestine in 0.1% berberine-treated mice were significantly reduced by 53.7%, 55.3%, and 76.5% respectively. Berberine treatment also decreased the numbers of all sizes of tumors (>2 mm, 1–2 mm, and <1 mm) in the small intestine. Berberine suppressed tumor cell proliferation and increased apoptosis. Furthermore, berberine decreased the activation levels of Wnt and EGFR signaling pathways, and down-regulated COX-2 expression in intestinal tumor cells and prostaglandin E2 production in the small intestine.ConclusionsBerberine inhibits intestinal tumor development, which is correlated with its activity to suppress tumor cell proliferation and increase apoptosis in Apcmin/+ mice. Down-regulation of Wnt and EGFR signaling pathways and COX-2 expression by berberine may be involved in its anti-tumorigenic effects.

Targeted Cox2 gene deletion in intestinal epithelial cells decreases tumorigenesis in female, but not male, ApcMin/+ mice

Mice heterozygous for mutations in the adenomatous polyposis coli gene (Apc+/− mice) develop intestinal neoplasia. Apc+/− tumor formation is thought to be dependent on cyclooxygenase 2 (COX2) expression; both pharmacologic COX2 inhibition and global Cox2 gene deletion reduce the number of intestinal tumors in Apc+/− mice. COX2 expression is reported in epithelial cells, fibroblasts, macrophages and endothelial cells of Apc+/− mouse polyps. However, the cell type(s) in which COX2 expression is required for Apc+/− tumor induction is not known. To address this question, we developed ApcMin/+ mice in which the Cox2 gene is specifically deleted either in intestinal epithelial cells or in myeloid cells. There is no significant difference in intestinal polyp number between ApcMin/+ mice with a targeted Cox2 gene deletion in myeloid cells and their control littermate ApcMin/+ mice. In contrast, ApcMin/+ mice with a targeted Cox2 deletion in intestinal epithelial cells have reduced intestinal tumorigenesis when compared to their littermate control ApcMin/+ mice. However, two gender-specific effects are notable. First, female ApcMin/+ mice developed more intestinal tumors than male ApcMin/+ mice. Second, targeted intestinal epithelial cell Cox2 deletion decreased tumorigenesis in female, but not in male, ApcMin/+ mice. Considered in the light of pharmacologic studies and studies with global Cox2 gene knockout mice, our data suggest that (i) intrinsic COX2 expression in intestinal epithelial cells plays a gender-specific role in tumor development in ApcMin/+ mice, and (ii) COX2 expression in cell type(s) other than intestinal epithelial cells also modulates intestinal tumorigenesis in ApcMin/+ mice, by a paracrine process.

Influence of apple pomace and its fractions on intestinal tumor development in APC Min/+ mice

Consumption of apples has been demonstrated to lower the risk of chronic diseases due to the presence of bioactive components such as fiber and polyphenols. The aims of this study were to determine the effects of apple pomace, a major waste product of apple juice processing, and its fractions on tumor development in APCMin/+ mice. Mice were assigned to different treatment groups consisting of powdered AIN‐93G diet supplemented with apple pomace, apple juice, apple ethanol extract or apple residue. Mice consuming the apple juice‐containing diet had the fewest adenomas (P&lt;0.05) in the small intestine and mice consuming apple residue the greatest number. No statistically significant differences in adenoma numbers were observed in the colon. However, male mice had significantly higher incidence of colonic adenomas (79.7%) than female mice (59.3%). In cecum, mice consuming diets containing apple juice had the smallest average adenoma size and mice consuming apple residue the greatest average adenoma size. These results demonstrate that fractions derived from apple pomace differentially influence intestinal adenoma development in APCMin/+ mice, but further research is required to determine the mechanisms for the observed effects. This research was supported in part by the Michigan Apple Committee.

ASC-associated inflammation promotes cecal tumorigenesis in aryl hydrocarbon receptor-deficient mice

The aryl hydrocarbon receptor (AhR) plays a suppressive role in cecal carcinogenesis by CUL4B/AhR-mediated ubiquitylation and degradation of β-catenin, which is activated by xenobiotics and natural ligands. AhR-deficient (AhR(-)(/-)) mice develop cecal tumors with severe inflammation. To elucidate whether the tumors develop autonomously in AhR(-/-) mice due to impaired β-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR(-/-) mice and compound mutant mice lacking genes for AhR and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which plays an essential role in caspase-1 activation in inflammasomes. Both GF AhR(-/-) and AhR(-/-)•ASC(-/-) mice showed considerably reduced tumor development compared with that in AhR(-/-) mice albeit in a 'cancer-prone' state with aberrant β-catenin accumulation. Blocking of the interleukin (IL)-1β signaling pathway by treatment with a caspase-1 inhibitor, YVAD, reduced cecal tumorigenesis in AhR(-/-) mice. Signal transducers and activators of transcription 3 (STAT3) activation was detected in the cecal epithelium of the AhR(-/-) mice due to enhanced IL-6 production. An inhibitor of the STAT3 signaling pathway, AG490 suppressed the tumor formation. ASC-mediated inflammation was also found to play a critical role in tumor development in Apc(Min/+) mice, a mouse model of familial adenomatous polyposis. Collectively, these results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in the intestinal tumorigenesis of mice and suggest a possible chemical therapeutic intervention, including AhR ligands and inhibitors of the inflammation pathway.

Targeting Tumor-Expressing CCR6 Inhibits Colorectal Cancer Progression in Mice (127.44)

Abstract Background: CCR6/CCL20 expressed by colorectal cancer (CRC) cells. Although their interactions may play an essential role in proliferation and migration of CRC, the in vivo evidence is lacking. Methods: Expression of CCR6/CCL20 was evaluated in 130 primary tumor specimens from CRC patients. Mouse models was established by the injection of CMT93 transfected with luciferase in CCR6-/- mice, or by crossing CCR6-/- mice with APCmin/+ mice. Intratumor injection of anti-CCR6 was performed in CCR6-/- mice grafted luciferase-labeled CMT93. Luciferase intensity of CMT93 was monitored using the IVIS Imaging System. Intestinal tumor development in APCmin/+ mice was studied with a dissecting microscope. Migration of CMT-93 treated with anti-CCR6 was investigated by the Real-Time Cell Analyzer. Results: In contrast to paratumor tissues, CCR6/CCL20 predominantly expressed by tumor cells in CRC patients of all tumor stages. CCR6/CCL20 was consistently identified in CMT-93. Injection of anti-CCR6, significantly inhibited the growth of CRC in CCR6-/- mice. Knock-out of CCR6 resulted in a decrease in numbers and size of intestinal adenomas in APCmin/+ mice. Anti-CCR6 treatment markedly inhibited the migration of CMT-93 in vitro. Conclusion: The CCR6/CCL20 interaction plays an essential role in proliferation and migration of colorectal cancer cells via the autocrine mechanism in mice, and selectively targeting tumor-expressing CCR6 may harbor a novel therapeutic strategy for human CRC.

Abstract 5223: The role of bone-marrow derived cells in the intestinal tumor microenvironment of apcmin/+ mouse on tumor response to inhibitors of thymidylate synthase

Abstract The vast majority of chemotherapeutic agents have been developed to specifically target actively dividing neoplastic cells. However, tumors are not exclusively composed of a heterogeneous population of neoplastic cells. They also include a heterogeneous collection of host derived infiltrating non-neoplastic cells that together with the extracellular matrix and secreted molecules compose the tumor stroma or microenvironment. Multiple interactions between stromal and tumor cells play a critical role in tumor growth and progression. Therefore, tumor response to chemotherapy and recurrence post-therapy must also be influenced by these interactions. For many decades, inhibitors of the enzyme thymidylate synthase (TS), such as fluoropyrimidines, have been used in the clinical management of various cancers; however, their clinical efficacy continues to be limited by their toxicity to normal cells and acquired resistance by tumor cells. Therapeutic efficacy is often measured by its impact on tumor burden, however, the impact of TS inhibitors on tumor stromal cells and their role on tumor response to therapy is largely unknown. The goal of this project is to determine the role of bone marrow derived tumor stromal cells (BMDCs) in mediating tumor response to systemic therapy with TS inhibitors. We hypothesize that, in addition to targeting tumor cells, TS inhibitors also directly target stromal cells and that the sensitivity of stromal cells to the therapy determines tumor response. Using the ApcMin/+ mouse as a model of intestinal tumorigenesis, we identified and characterized BMDCs in the intestinal tumor stroma. These included Macrophages, Fibroblasts, Pan T cells, Helper T cells, Leukocytes, Mast cells, T-regulatory cells (T-reg) and Myeloid derived suppressor cells (MDSCs), among others. We generated chimeric mice wherein the chemosensitivity of intestinal tumor cells to TS inhibitors was distinct from that of the BMDCs by transplanting eGFP positive bone marrow cells that were genetically modified to express a drug resistant variant of TS. We examined the effect of 5-FU on MDSCs, mast cells, and T-regs, that have previously been shown to promote intestinal tumor development in ApcMin/+ mice. The results showed that MDSCs were specifically sensitive to 5-FU due to its apoptotic effect on these cells. Current studies are aimed at identifying the combination of stromal cells that mediate tumor response to TS inhibitors in order to identify cellular targets for combined therapy that would enhance reduction of tumor burden or delay tumor recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5223. doi:1538-7445.AM2012-5223

Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice

BackgroundThe adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E2 (PGE2) - PI-3 kinase pathways. Recent reports show that PGE2-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE2 on β-catenin homeostasis.FindingsTreatment of ApcMin/+ mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE2-induced β-catenin phosphorylation and c-Myc upregulation.ConclusionBased on our findings we suggest that PGE2 act through PKA to promote β-catenin nuclear translocation and tumor development in ApcMin/+ mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in Apc Min /+ mice with natural ligands

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

Identification of Novel Modifier Loci of ApcMin Affecting Mammary Tumor Development

Genetic background affects the susceptibility to mammary tumor development in Apc(Min/+) mice. Here we report the identification of four novel modifier loci that influence different aspects of mammary tumor development in Apc(Min/+) mice. Analysis of tumor development in a backcross of (FVBB6 Apc(Min/+)) x B6 Apc(Min/+) mice has identified a modifier on chromosome 9 that significantly affects tumor multiplicity, and a modifier on chromosome 4 that significantly affects tumor latency and affects tumor number with suggestive significance. This modifier was also identified in a backcross involving 129X1/SvJ and B6 Apc(Min/+) mice. A modifier on chromosome 18 specifically affects tumor latency but not tumor number. Kaplan-Meier analysis suggests there is at least an additive interaction affecting tumor latency between the loci on chromosomes 4 and 18. We also identified a modifier locus on chromosome 6 that interacts with the loci on chromosome 4 and chromosome 9 to affect tumor number. These results suggest that multiple genetic loci control different aspects of mammary tumor development. None of these modifiers is associated with intestinal tumor susceptibility, which indicates that these modifiers act on tumor development in a tissue-specific manner.