Apc Mice Research Articles

IL-33 activates tumor stroma to promote intestinal polyposis

Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

Treg-cell depletion promotes chemokine production and accumulation of CXCR3(+) conventional T cells in intestinal tumors.

Colorectal cancer (CRC) is one of the most prevalent tumor types worldwide and tumor-infiltrating T cells are crucial for anti-tumor immunity. We previously demonstrated that Treg cells from CRC patients inhibit transendothelial migration of conventional T cells. However, it remains unclear if local Treg cells affect lymphocyte migration into colonic tumors. By breeding APC(Min/+) mice with depletion of regulatory T cells mice, expressing the diphtheria toxin receptor under the control of the FoxP3 promoter, we were able to selectively deplete Treg cells in tumor-bearing mice, and investigate the impact of these cells on the infiltration of conventional T cells into intestinal tumors. Short-term Treg-cell depletion led to a substantial increase in the frequencies of T cells in the tumors, attributed by both increased infiltration and proliferation of T cells in the Treg-cell-depleted tumors. We also demonstrate a selective increase of the chemokines CXCL9 and CXCL10 in Treg-cell-depleted tumors, which were accompanied by accumulation of CXCR3(+) T cells, and increased IFN-γ mRNA expression. In conclusion, Treg-cell depletion increases the accumulation of conventional T cells in intestinal tumors, and targeting Treg cells could be a possible anti-tumor immunotherapy, which not only affects T-cell effector functions, but also their recruitment to tumors.

Su2003 Nrip2, a Novel Regulator of the Non-Canonical Wnt Pathway, Promotes the Self-Renewal of Colorectal Cancer Initiating Cells

As normal tissues contain normal tissue stem cells (NSCs), tumors are thought to contain tumor stem cells (TSCs) which perform self-renewal and generate their progenies. TSC can be a therapeutic target; however, cancer therapies targeting TSCs are limited, because most of the TSC markers are shared by NSCs. Therefore, it is important to identify TSC-specific markers. Previously, we reported that doublecortin-like kinase 1 (Dclk1)-positive epithelial cells do not produce any progenies in the normal small intestine but supply progeny tumor cells in the tumors of Apc mice. Selective ablation of Dclk1-positive TSCs collapses the intestinal tumors of Apc mice. Therefore, Dclk1 appears to be a specific marker of TSCs in the mouse intestinal tumors. In the present study, we performed microarray analysis in order to investigate the characteristics of Dclk1-positive TSCs in the tumors of Apc mice. Dclk1-positive tumor cells had higher expression levels of cyclooxygenase 1 (Cox1), leukotriene C4 synthase (Ltc4s), and arachidonate 5-lipoxygenase activating protein (Alox5ap) than Dclk1-negative cells in the tumors of Apc mice, suggesting that Dclk1positive cells in the tumors of Apc mice had higher expression levels of the genes which were related to prostaglandin and leukotriene metabolism. As well, microarray analysis revealed that gustducin alpha and gamma subunits, transient receptor potential cation channel, subfamily M, member 5 (TRPM5), and phospholipase C beta 2 were more highly expressed in Dclk1-positive tumor cells. Thus, Dclk1-positive tumor cells seemed to possess the characteristic of Tuft cells. Furthermore, Dclk1-positive tumor cells had higher expression level of Ras-related C3 botulinum toxin substrate 2 (Rac2) than Dclk1-negative tumor cells in Apc mouse intestine. In order to elucidate the role of Rac2 in Dclk1-positive TSCs, we generated tumor spheroids from Apc mouse tumors, and administrated EHT1864, Rac inhibitor to the spheroids. Following 5-day treatment with EHT1864, the growth of the tumor spheroids was significantly suppressed. Importantly, on day1, EHT1864 treatment significantly reduced the proportion of Dclk1-positive cells in the tumor spheroids. These results suggested that Rac2 played some roles for the maintenance of the intestinal tumor spheroids. In conclusion, this study may provide new insights into the maintenance mechanisms of Dclk1-positive TSC in the intestinal tumors, and give us hints to develop novel therapeutic strategies targeting TSCs against colorectal cancers.

Su2005 DCLK1 Promotes Pancreatic Cancer Cell Clonogenicity and Invasiveness

As normal tissues contain normal tissue stem cells (NSCs), tumors are thought to contain tumor stem cells (TSCs) which perform self-renewal and generate their progenies. TSC can be a therapeutic target; however, cancer therapies targeting TSCs are limited, because most of the TSC markers are shared by NSCs. Therefore, it is important to identify TSC-specific markers. Previously, we reported that doublecortin-like kinase 1 (Dclk1)-positive epithelial cells do not produce any progenies in the normal small intestine but supply progeny tumor cells in the tumors of Apc mice. Selective ablation of Dclk1-positive TSCs collapses the intestinal tumors of Apc mice. Therefore, Dclk1 appears to be a specific marker of TSCs in the mouse intestinal tumors. In the present study, we performed microarray analysis in order to investigate the characteristics of Dclk1-positive TSCs in the tumors of Apc mice. Dclk1-positive tumor cells had higher expression levels of cyclooxygenase 1 (Cox1), leukotriene C4 synthase (Ltc4s), and arachidonate 5-lipoxygenase activating protein (Alox5ap) than Dclk1-negative cells in the tumors of Apc mice, suggesting that Dclk1positive cells in the tumors of Apc mice had higher expression levels of the genes which were related to prostaglandin and leukotriene metabolism. As well, microarray analysis revealed that gustducin alpha and gamma subunits, transient receptor potential cation channel, subfamily M, member 5 (TRPM5), and phospholipase C beta 2 were more highly expressed in Dclk1-positive tumor cells. Thus, Dclk1-positive tumor cells seemed to possess the characteristic of Tuft cells. Furthermore, Dclk1-positive tumor cells had higher expression level of Ras-related C3 botulinum toxin substrate 2 (Rac2) than Dclk1-negative tumor cells in Apc mouse intestine. In order to elucidate the role of Rac2 in Dclk1-positive TSCs, we generated tumor spheroids from Apc mouse tumors, and administrated EHT1864, Rac inhibitor to the spheroids. Following 5-day treatment with EHT1864, the growth of the tumor spheroids was significantly suppressed. Importantly, on day1, EHT1864 treatment significantly reduced the proportion of Dclk1-positive cells in the tumor spheroids. These results suggested that Rac2 played some roles for the maintenance of the intestinal tumor spheroids. In conclusion, this study may provide new insights into the maintenance mechanisms of Dclk1-positive TSC in the intestinal tumors, and give us hints to develop novel therapeutic strategies targeting TSCs against colorectal cancers.

Progression of Cancer Cachexia Alters Muscle Lactate Metabolism In ApcMin/+ Mice

Roughly 100,000 individuals are diagnosed with colorectal cancer a year, 80% of these cases arise from adenomatous polyps. A mutation of the tumor suppressor gene, Adenomatous polyposis coli (Apc) results in multiple intestinal neoplasia (Min), thus the Apc (Min/+) mouse is a common colorectal cancer model. Cancer cells favor a highly glycolytic environment. As such, there is an induction of processes regulating lactate metabolism and lactate shuttling. How colorectal cancer influences skeletal muscle lactate metabolism is uncertain. Purpose: We investigated how muscle lactate metabolism is regulated during the progression of cancer cachexia in Apc (Min/+) mice. Methods: Male Min/+mice (n = 17) were classified by body mass loss: weight stable (WS, 蠄 5%, n = 5), and cachectic (CC, 6‐19%; n = 5). C57BL/6J (B6, n = 6) served as controls. Quadriceps muscle samples were extracted and real‐time PCR was conducted to quantify lactate‐related gene expression. Results: LDH‐A was reduced 47% in the CC mice compared to the WS mice (p < 0.05), while LDH‐B was not changed with cancer cachexia progression (p = 0.09). Monocarboxylate transporter 1 (MCT1) was induced 1.9‐fold in CC mice (p < 0.05) compared to the B6 mice. MCT4 was induced 1.7‐fold and 2.3‐fold in the CC mice compared to B6 and WS mice, respectively. Muscle CD147 was not changed with cancer cachexia progression (p = 0.31).ConclusionThe progression of cachexia in mice with colon cancer alters markers of lactate metabolism that may play a role in muscle wasting associated with cachexia.

Improved innate immune responses by Frondanol A5, a sea cucumber extract, prevent intestinal tumorigenesis.

Sea cucumbers are a source of antibacterial, anti-inflammatory, and anticancer compounds. We show that sea cucumber extract Frondanol A5 is capable of enhancing innate immune responses and inhibiting intestinal tumors in APC(Min/+) mice. APC(Min/+) mice were fed semi-purified diets containing 0, 250, or 500 ppm FrondanolA5 for 14 weeks before we assessed intestinal tumor inhibition. Dietary Frondanol A5 suppressed small intestinal polyp sizes and formation up to 30% (P < 0.02) in males and up to 50% (P < 0.01) in females. Importantly, 250 and 500 ppm Frondanol A5 diet suppressed colon tumor multiplicities by 65% (P < 0.007) and 75% (P < 0.0001), compared with untreated male APC(Min/+) mice. In female APC(Min/+) mice, both dose levels of Frondanol A5 suppressed colon tumor multiplicities up to 80% (P < 0.0001). Isolated peritoneal macrophages from treated mice showed increased phagocytosis efficiency (control 24% vs. treated 50%; P < 0.01) and an increase in GILT mRNA expression, indicating increased innate immune responses by these cells in treated animals. Similarly, we observed an increase in GILT expression in treated tumors, compared with untreated tumors. Furthermore, an increase in G-CSF cytokine, a decrease in inflammatory cytokines and marker 5-LOX, its regulator FLAP, proliferation (PCNA), and angiogenesis (VEGF) markers were observed in treatment groups. These data suggest that Frondanol A5 decreased inflammatory angiogenic molecules and increased GILT expression and macrophage phagocytosis. These decreases may have improved the innate immune systems of the treated mice, thus aiding in inhibition of intestinal tumor formation. These results suggest that Frondanol A5 exhibits significant chemopreventive potential against intestinal tumorigenesis.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/β-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of β-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of β-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of β-catenin pathway.

Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis.

Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc(min/+) mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

GLP-1R Agonists Promote Normal and Neoplastic Intestinal Growth through Mechanisms Requiring Fgf7

Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L cells promotes nutrient disposal via the incretin effect. However, the majority of L cells are localized to the distal gut, suggesting additional biological roles for GLP-1. Here, we demonstrate that GLP-1 receptor (GLP-1R) signaling controls mucosal expansion of the small bowel (SB) and colon. These actions did not require the epidermal growth factor (EGF) or intestinal epithelial insulin-like growth factor (IGF1) receptors but were absent in Glp1r(-/-) mice. Polyp number and size were increased in SB of exendin-4-treated Apc(Min/+) mice, whereas polyp number was reduced in SB and colon of Glp1r(-/-):Apc(Min/+) mice. Exendin-4 increased fibroblast growth factor 7 (Fgf7) expression in colonic polyps of Apc(Min/+) mice and failed to increase intestinal growth in mice lacking Fgf7. Exogenous exendin-4 and Fgf7 regulated an overlapping set of genes important for intestinal growth. Thus, gain and loss of GLP-1R signaling regulates gut growth and intestinal tumorigenesis.

Non-cell autonomous effects of targeting inducible PGE2 synthesis during inflammation-associated colon carcinogenesis.

Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc (Δ14/+) in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early cryptal structures.

Treatment with anti-interleukin-6 receptor antibody ameliorates intestinal polyposis in Apc(Min/+) mice under high-fat diet conditions.

The prevalence of colorectal malignancies is increasing in the world. The parallel increase of metabolic syndrome gives a speculation between these two conditions, although the precise mechanism is still unclear. Interleukin-6 (IL-6) is a cytokine known to correlate with obesity and serve as a proinflammatory adipokine. In the present study, we investigated the effect of IL-6 signaling blockade on intestinal polyp formation in obesity using a mouse model of adenomatous polyposis coli (Apc). Male C57BL/6J-Apc(Min/+) mice were fed a high-fat diet from 5 weeks of age, and the overweight mice thus obtained were given a weekly intraperitoneal injection of anti-mouse IL-6 receptor antibody (MR16-1) from 6 to 15 weeks of age, while control mice received IgG or phosphate-buffered saline (PBS). The total number of intestinal polyps was significantly decreased in the MR16-1-injected group (53.1 ± 6.8) relative to the control groups (PBS-injected, 81.3 ± 6.1; rat IgG-injected, 74.7 ± 4.8, p = 0.01), and in particular the number of polyps larger than 2 mm in diameter was markedly decreased. In addition, the mean diameter of polyps in the MR16-1-injected group was significantly smaller than that in the control groups. On the other hand, no significant differences in body weight, epididymal fat pad mass, or the plasma levels of glucose, insulin and triglyceride were observed among the three groups. Thus, treatment with anti-IL-6 receptor antibody suppressed polyp growth in obese Apc(Min/+) mice fed the high-fat diet. We suggest that IL-6 signaling may be responsible for the obesity-associated colorectal tumorigenesis.

Spontaneous Intestinal Tumorigenesis in Apc (/Min+) Mice Requires Altered T Cell Development with IL-17A.

The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression. Here we address the inhibitory role of Tregs on intestinal tumorigenesis in the Apc /Min+ mouse model that resembles human familial adenomatous polyposis (FAP). Apc /Min+ mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria. To address the role of heterozygous Apc gene mutation in Tregs, we generated Foxp3-Cre, Apc flox/+ mice. Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transfer into Apc /Min+ mice, demonstrating that the heterozygous Apc gene mutation in Tregs does not induce the loss of control over tumor microenvironment. Adoptive transfer of in vitro generated Apc /Min+ iTregs (inducible Tregs) failed to inhibit intestinal tumorigenesis, suggesting that naïve CD4 T cells generated from Apc /Min+ mice thymus were impaired. We also showed that adoptively transferred IL-17A-deficient Apc /Min+ Tregs inhibited tumor growth, suggesting that IL-17A was critical to impair the tumor regression function of Apc /Min+ Tregs. Taken together, our results suggest that both T cell development in a functional thymus and IL-17A control the ability of Treg to inhibit intestinal tumorigenesis in Apc /Min+ mice.

Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.

The Enhancer of Zeste Homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and ApcMin/+ mice were infected with CR (108cfu); BLT1−/−ApcMin/+ mice, AOM/DSS-treated mice and de-identified human adenocarcinoma samples were models of colon cancer. Following infection with wild type but not mutant CR, elevated EZH2 levels in the crypt at days-6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3’s occupancy on WIF1 (Wnt Inhibitory Factor-1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via siRNA or EZH2-inhibitor DZNep either alone or in combination with HDAC inhibitor SAHA, WIF1 promoter activity increased significantly while overexpression of EZH2 attenuated WIF1-reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels while H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, while β-catenin levels were lower in EZH2-knocked-down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days-6 and 12 post-infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected ApcMin/+ mice paralleled changes recorded in BLT1−/−ApcMin/+, AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.

Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles

Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not β-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

Loss of Abhd5 Promotes Colorectal Tumor Development and Progression by Inducing Aerobic Glycolysis and Epithelial-Mesenchymal Transition

How cancer cells shift metabolism to aerobic glycolysis is largely unknown. Here, we show that deficiency of α/β-hydrolase domain-containing 5 (Abhd5), an intracellular lipolytic activator that is also known as comparative gene identification 58 (CGI-58), promotes this metabolic shift and enhances malignancies of colorectal carcinomas (CRCs). Silencing of Abhd5 in normal fibroblasts induces malignant transformation. Intestine-specific knockout of Abhd5 in Apc(Min/+) mice robustly increases tumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells, Abhd5 deficiency induces epithelial-mesenchymal transition by suppressing the AMPKα-p53 pathway, which is attributable to increased aerobic glycolysis. In human CRCs, Abhd5 expression falls substantially and correlates negatively with malignant features. Our findings link Abhd5 to CRC pathogenesis and suggest that cancer cells develop aerobic glycolysis by suppressing Abhd5-mediated intracellular lipolysis.

Deficiency of interferon-gamma or its receptor promotes colorectal cancer development.

Genetic variations in interferon-gamma (IFN-γ) and its receptor (IFNγR) subunits are closely associated with the risk of colorectal cancer (CRC) and survival after diagnosis. However, the role of loss of IFN-γ or IFNγR function in the pathogenesis of CRC remains unclear. Here, we investigated the role of endogenous IFN-γ deficiency in adenomatous polyposis coli (Apc)-mediated intestinal tumor by developing a variant of Apc(Min/+) mice. The Apc(Min/+)IFN-γ(+/-) mice presented with increased number and size of adenomas, and 41.7% of these mice developed adenocarcinoma. Molecular analyses of the adenomas suggested that heterozygous deletion of IFN-γ promoted EGFR/Erk1/2 and Wnt/β-catenin signaling. In vitro, IFN-γ administration inhibited Apc-mutated HT-29 colon cancer cell proliferation and had no effect on the proliferation of HCT-116 colon cancer cells that express wild-type Apc. Besides, we challenged HT-29 cells with small interfering RNA targeting one of its receptor subunits IFNγR1. We found that knockdown of IFNγR1 in HT-29 cells stimulated cell proliferation and colony formation, which was also related to the regulation of EGFR/Erk1/2 and Wnt/β-catenin signaling. Thus, our results strongly support the notion that IFN-γ and IFNγR1 act as a rate-limiting factor in the development of CRC, uncovering a novel role for them in cancer biology.

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc(min/+) mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc(min/+) mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.

Stromal Indian Hedgehog Signaling Is Required for Intestinal Adenoma Formation in Mice

Indian hedgehog (IHH) is an epithelial-derived signal in the intestinal stroma, inducing factors that restrict epithelial proliferation and suppress activation of the immune system. In addition to these rapid effects of IHH signaling, IHH is required to maintain a stromal phenotype in which myofibroblasts and smooth muscle cells predominate. We investigated the role of IHH signaling during development of intestinal neoplasia in mice. Glioma-associated oncogene (Gli1)-CreERT2 and Patched (Ptch)-lacZ reporter mice were crossed with Apc(Min) mice to generate Gli1CreERT2-Rosa26-ZSGreen-Apc(Min) and Ptch-lacZ-Apc(Min) mice, which were used to identify hedgehog-responsive cells. Cyp1a1Cre-Apc (Apc(HET)) mice, which develop adenomas after administration of β-naphthoflavone, were crossed with mice with conditional disruption of Ihh in the small intestine epithelium. Apc(Min) mice were crossed with mice in which sonic hedgehog (SHH) was overexpressed specifically in the intestinal epithelium. Intestinal tissues were collected and analyzed histologically and by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. We also analyzed levels of IHH messenger RNA and expression of IHH gene targets in intestinal tissues from patients with familial adenomatous polyposis (n = 18) or sessile serrated adenomas (n = 15) and normal colonic tissue from control patients (n = 12). Expression of IHH messenger RNA and its targets were increased in intestinal adenomas from patients and mice compared with control colon tissues. In mice, IHH signaling was exclusively paracrine, from the epithelium to the stroma. Loss of IHH from Apc(HET) mice almost completely blocked adenoma development, and overexpression of SHH increased the number and size of adenomas that developed. Loss of IHH from Apc(HET) mice changed the composition of the adenoma stroma; cells that expressed α-smooth muscle actin or desmin were lost, along with expression of cyclooxygenase-2, and the number of vimentin-positive cells increased. Apc mutant epithelial cells secrete IHH to maintain an intestinal stromal phenotype that is required for adenoma development in mice.

Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Abstract 250: Sulindac delayed and suppressed the tumor progression, was not effective on inhibition of tumor initiation in a human colorectal cancer mouse model (CPC;Apc mouse)

Abstract Purpose Non-steroidal anti-inflammatory drugs (NSAIDs) have shown potential as chemopreventive agents against cancer formation, especially colorectal cancers. However, the mechanisms by which these drugs act are not fully understood. In this study, CPC;Apc mice, a human colorectal cancer mouse model treated with sulindac, were evaluated according to tumor initiation and prognosis and gene expression profile of the tumors were analyzed. Methods CPC;Apc mice, originating from ApcFlox/wt mice harbor a Cdx2-Cre transgene in which colorectal tumorigenesis was driven by Apc allelic loss and represent a genetic model of human colorectal cancer (CRC) were treated with control (n=20) or sulindac (160ppm, n=22) for 12 weeks and their colorectal tumors were evaluated. Gene expression analysis was performed to identify genes differentially expressed in tumor cells from sulindac-treated mice. The gene expression was validated through quantitative real-time polymerase chain reaction in vivo. Results Body weight of mice with control or sulindac was no significantly difference ( P = 0.801). However, we observed that sulindac significantly suppressed the tumor progression relative to untreated controls (the colon tumors size per mouse: median of 4.2mm, range 2.3-5.9 versus 5.0mm, range 3.1-8.7, respectively ; P = 0.007 ), but not the initiation (the colon tumor numbers per mouse: median of 1.5, range 0-5 versus 1.0 tumors, range 0-8 respectively ; P = 0.979) in CPC;Apc mice. We identified some upregulated (n = 6, &amp;gt;1.4 fold ) and downregulated (n = 5, &amp;gt;1.4 fold) target genes by microarray assay. We validated expression of these candidate genes in human colorectal cancer. Conclusion We have identified for the first time that sulindac affected tumor progression by delayed and suppressing it, but had no effect on the initiation of tumor in CRC mice model. Citation Format: Tomohiro Adachi, Takao Hinoi, Yuu Sasaki, Manabu Shimomura, Yasufumi Saito, Masashi Miguchi, Hiroaki Niitsu, Yuusuke Sotomaru, Naohide Oue, Wataru Yasui, Hideki Ohdan. Sulindac delayed and suppressed the tumor progression, was not effective on inhibition of tumor initiation in a human colorectal cancer mouse model (CPC;Apc mouse). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 250. doi:10.1158/1538-7445.AM2014-250