Intestinal Tumorigenesis In Mice Research Articles

Zinc finger protein 280C contributes to colorectal tumorigenesis by maintaining epigenetic repression at H3K27me3-marked loci

Dysregulated epigenetic and transcriptional programming due to abnormalities of transcription factors (TFs) contributes to and sustains the oncogenicity of cancer cells. Here, we unveiled the role of zinc finger protein 280C (ZNF280C), a known DNA damage response protein, as a tumorigenic TF in colorectal cancer (CRC), required for colitis-associated carcinogenesis and Apc deficiency–driven intestinal tumorigenesis in mice. Consistently, ZNF280C silencing in human CRC cells inhibited proliferation, clonogenicity, migration, xenograft growth, and liver metastasis. As a C2H2 (Cys2-His2) zinc finger-containing TF, ZNF280C occupied genomic intervals with both transcriptionally active and repressive states and coincided with CCCTC-binding factor (CTCF) and cohesin binding. Notably, ZNF280C was crucial for the repression program of trimethylation of histone H3 at lysine 27 (H3K27me3)-marked genes and the maintenance of both focal and broad H3K27me3 levels. Mechanistically, ZNF280C counteracted CTCF/cohesin activities and condensed the chromatin environment at the cis elements of certain tumor suppressor genes marked by H3K27me3, at least partially through recruiting the epigenetic repressor structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1). In clinical relevance, ZNF280C was highly expressed in primary CRCs and distant metastases, and a higher ZNF280C level independently predicted worse prognosis of CRC patients. Thus, our study uncovered a contributor with good prognostic value to CRC pathogenesis and also elucidated the essence of DNA-binding TFs in orchestrating the epigenetic programming of gene regulation.

IL22BP Mediates the Antitumor Effects of Lymphotoxin Against Colorectal Tumors in Mice and Humans

Background & AimsUnregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice.MethodsWe obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf–/–, Lta–/–, and Ltb–/– mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times.ResultsLevels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp–/– mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte–derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC.ConclusionsLymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist.

Low and high dose rate heavy ion radiation-induced intestinal and colonic tumorigenesis in APC1638N/+ mice

Ionizing radiation (IR) is a recognized risk factor for colorectal cancer (CRC) and astronauts undertaking long duration space missions are expected to receive IR doses in excess of permissible limits with implications for colorectal carcinogenesis. Exposure to IR in outer space occurs at low doses and dose rates, and energetic heavy ions due to their high linear energy transfer (high-LET) characteristics remain a major concern for CRC risk in astronauts. Previously, we have demonstrated that intestinal tumorigenesis in a mouse model (APC1638N/+) of human colorectal cancer was significantly higher after exposure to high dose rate energetic heavy ions relative to low-LET γ radiation. The purpose of the current study was to compare intestinal tumorigenesis in APC1638N/+ mice after exposure to energetic heavy ions at high (50cGy/min) and relatively low (0.33cGy/min) dose rate. Male and female mice (6–8 weeks old) were exposed to either 10 or 50cGy of 28Si (energy: 300MeV/n; LET: 70keV/μm) or 56Fe (energy: 1000MeV/n; LET: 148keV/μm) ions at NASA Space Radiation Laboratory in Brookhaven National Laboratory. Mice (n=20 mice/group) were euthanized and intestinal and colon tumor frequency and size were counted 150days after radiation exposure. Intestinal tumorigenesis in male mice exposed to 56Fe was similar for high and low dose rate exposures. Although male mice showed a decreasing trend at low dose rate relative to high dose rate exposures, the differences in tumor frequency between the two types of exposures were not statistically significant after 28Si radiation. In female mice, intestinal tumor frequency was similar for both radiation type and dose rates tested. In both male and female mice intestinal tumor size was not different after high and low dose rate radiation exposures. Colon tumor frequency in male and female mice after high and low dose rate energetic heavy ions was also not significantly different. In conclusion, intestinal and colonic tumor frequency and size was similar irrespective of energetic heavy ion radiation dose rate suggesting that carcinogenic potential of energetic heavy ions is independent of dose rate.

Daily Intake of High-Fat Diet with Lysophosphatidic Acid-Rich Soybean Phospholipids Augments Colon Tumorigenesis in Kyoto Apc Delta Rats.

Oral administration of lysophosphatidic acid (LPA) was shown to attenuate gastric ulceration in rats and mice but aggravate intestinal tumorigenesis in mice. The present study examined whether dietary LPA induces or prevents development of colorectal tumor in rats. Kyoto Apc Delta rats fed high-fat diet with or without an LPA-rich soybean phospholipid mixture (LSP, 0.1 or 1%) were treated with azoxymethane and dextran sodium sulfate to induce colorectal tumorigenesis. Rats were killed 15weeks after azoxymethane treatment, and size, total number, location, and severity of colorectal tumors were assessed. Expression of mRNA of LPA receptors in rat colon tissue was assayed. Rats fed the diet supplemented with 1% LSP had a higher number of tumors 2-4mm long compared than those with or without 0.1% LSP. The mean distance of tumors >4mm long from the anus was significantly higher than those of tumors <2 and 2-4mm long in rats fed 1% LSP-supplemented diet. Supplementation of the diet with 0.1% LSP decreased mRNA expression of LPA5 in colon tumors of rats. Dietary supplementation of LPA-rich phospholipids dose-dependently augmented colorectal tumorigenesis. Decreased expression of LPA5 in colon tumors may be relevant to augmented tumorigenesis.

Abstract 3819: PPAR-delta promotes Wnt/B-catenin-driven colorectal tumorigenesis

Abstract Introduction Aberrant Wnt/B-catenin signaling activation due to mutations in the adenomatous poliposis coli gene, APC, is a critical event in colorectal cancer (CRC). However, to drive CRC tumorigenesis, aberrant Wnt/B-catenin activation requires additional enhancing regulatory mechanisms. The peroxisome proliferator-activated receptor delta gene (PPARD), which is upregulated in CRC, is a downstream target of aberrant Wnt/B-catenin activation in human colon cancer cells. However, PPARD has also been reported to inhibit intestinal tumorigenesis in mice with germline APCmin mutations. Thus, the mechanistic interaction between PPARD and Wnt/β-catenin remains poorly understood. PPARD is a druggable protein for which agonists and antagonists are being developed. Determining PPARD effects on Wnt/B-catenin activation would define the direction of its therapeutic targeting in cancer and other diseases. Methods We developed a novel mouse model that simulates PPARD overexpression in CRC by inducing PPARD overexpression in intestinal epithelial cells via a villin promoter (villin-PPARD mice). We bred villin-PPARD mice with mice that have Apc580 mutation in intestinal epithelial cells induced by CDX2-Cre recombinase expression (Apc580mu mice) to generate Apc580mu-PPARD-Gut mice. Apc580mu-PPARD-Gut mice and Apc580mu control mice were monitored for CRC tumorigenesis. We also assessed the effects of PPARD expression modulation on Wnt/B-catenin activation in human colon cancer cell lines. Results PPARD overexpression in colonic epithelial cells increased CRC tumorigenesis in mice with Apc580 mutation. At 10 weeks of age, 100% of Apc580mu-PPARD-Gut mice but only 60% of Apc580mu mice had tumors of any size, and the mean number of tumors per Apc580mu-PPARD-Gut mouse (1.6 ± 2.5) was significantly higher than that per Apc580mu mouse (0.6 ± 0.25; P = 0.02). Tumors &amp;gt;3.5 mm were present in all Apc580mu-PPARD-Gut mice but only 20% of Apc580mu mice, and the mean number of tumors &amp;gt;3.5 mm per Apc580mu-PPARD-Gut mouse (1.4 ± 2.5) was significantly higher than that per Apc580mu mouse (0.2 ± 0.2; p = 0.005). In mice, colonic epithelial PPARD overexpression increased activated B-catenin protein levels and Wnt/B-catenin target gene mRNA levels. In human colon cancer cells, PPARD increased the levels of activated β-catenin, its nuclear localization and transcriptional activity. Conclusion Our findings indicate that PPARD augments Wnt/B-catenin signaling to promote CRC tumorigenesis and thus support the targeted inhibition of PPARD to suppress CRC tumorigenesis. Citation Format: Xiangsheng Zuo, Rui Tian, Shen Gao, Micheline J. Moussalli, Weidong Chen Chen, Ling Wu, Imad Shureiqi. PPAR-delta promotes Wnt/B-catenin-driven colorectal tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3819.

Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer.

Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity.

A hypermorphic epithelial β-catenin mutation facilitates intestinal tumorigenesis in mice in response to compounding WNT-pathway mutations.

ABSTRACTActivation of the Wnt/β-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant β-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-β-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33ΔN-Bcat mice showed an increase in the constitutive Wnt/β-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33ΔN-Bcat mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7 and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33ΔN-Bcat mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33ΔN-Bcat mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-β-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/β-catenin pathway to facilitate and promote tumorigenesis.

Human cancer xenografts in outbred nude mice can be confounded by polymorphisms in a modifier of tumorigenesis.

Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.

Mismatch Repair Deficient Mice Show Susceptibility to Oxidative Stress-Induced Intestinal Carcinogenesis

We have previously established an experimental system for oxidative DNA damage-induced tumorigenesis in the small intestine of mice. To elucidate the roles of mismatch repair genes in the tumor suppression, we performed oxidative DNA damage-induced tumorigenesis experiments using Msh2-deficient mice. Oral administration of 0.2% Potassium Bromate, KBrO3, effectively induced epithelial tumors in the small intestines of Msh2-deficient mice. We observed a 22.5-fold increase in tumor formation in the small intestines of Msh2-deficient mice compared with the wild type mice. These results indicate that mismatch repair is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. A mutation analysis of the Ctnnb1 gene of the tumors revealed predominant occurrences of G:C to A:T transitions. The TUNEL analysis showed a decreased number of TUNEL-positive cells in the crypts of small intestines from the Msh2-deficient mice compared with the wild type mice after treatment of KBrO3. These results suggest that the mismatch repair system may simultaneously function in both avoiding mutagenesis and inducing cell death to suppress the tumorigenesis induced by oxidative stress in the small intestine of mice.

Abstract 2728: Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice.

Abstract Mutation of the tumor suppressor gene Adenomatous Polyposis Coli (APC) is considered an initiating event in the development of most intestinal tumors. Although much effort has been spent determining functions of the APC protein, to date little is known about the mechanisms that regulate cellular APC levels. Here we report that in cultured cells, induction of a heat-shock response, via heat or compounds such as the HSP90 inhibitor 17-AAG, resulted in increased levels of APC. A novel non-toxic small molecule that we developed, KN1, also induced a heat-shock response and led to increased APC levels in both cultured cells and mice. To investigate the effect of heat-shock response induction and elevation of Apc level in intestinal tumorigenesis, we performed a series of experiments treating mice with either KN1 or 17-AAG. We tested these compounds on two mouse models with different germline Apc mutations, ApcMin/+ and Apc1322T/+. In both cases, a moderate dose of either drug did not change tumor burden, but surprisingly altered the distribution of intestinal polyps. In a third mouse model, colonic tumors were induced via administration of the mutagen azoxymethane (AOM) and colon irritant dextran sodium sulfate (DSS) rather than by germline Apc mutation. In AOM-DSS-treated mice, KN1 reduced tumor incidence, multiplicity, and size. Moreover, KN1-treated mice lost significantly less weight and had smaller spleens than vehicle-treated mice, suggesting KN1 may also suppress colitis. We conclude that induction of the heat-shock response affects intestinal tumorigenesis in germline Apc-mutant and colitis-induced tumor models. Citation Format: Maged Zeineldin, William McGuinness, Brian Blagg, Roger Rajewski, Kristi L. Neufeld. Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2013-2728

Liver X Receptors Inhibit Proliferation of Human Colorectal Cancer Cells and Growth of Intestinal Tumors in Mice

Liver X receptors (LXRs) are transcriptional regulators of cholesterol metabolism, controlling cholesterol flow into cells, catabolism, and efflux. Cholesterol controls cell proliferation; disruptions in cholesterol metabolism have been associated with the development of colon cancer. We investigated whether expression of activated LXR protects against intestinal tumorigenesis in mice. We analyzed the development of colon cancer in mice that express a constitutive active form of LXRα only in the intestinal epithelium, under the control of villin promoter (iVP16LXRα). These mice were crossed with adenomatous polyposis coli (Apc)(min/+) mice, or given azoxymethane followed by dextran sodium sulfate, to assess intestinal tumor formation. We also assessed proliferation and apoptosis of a human colorectal cancer cell line (HT29) transfected with an adenoviral vector that expressed Ad VP16hLXRα, compared with cells expressing AdVP16 (control), and their ability to form xenograft tumors in mice. HT29 cells also were incubated with the LXR ligand GW3965. In human colorectal cancer cells, ligand-induced activation of LXR or transfection with Ad VP16hLXRα blocked the G1 phase, increased caspase-dependent apoptosis, and slowed growth of xenograft tumors in mice. iVP16LXRα mice formed fewer, smaller tumors than VP16 (control) mice after administration of azoxymethane and dextran sodium sulfate. APC(min/+)/iVP16LXRα mice also developed fewer, smaller intestinal tumors than APC(min/+)/iVP16 mice. Gene expression analysis indicated that activation of LXRα affected lipid metabolic networks and increased cholesterol efflux in the intestine. Expression of activated LXRα blocks proliferation of human colorectal cancer cells and slows the growth of xenograft tumors in mice. It also reduces intestinal tumor formation after administration of chemical carcinogens, and in Apc(min/+) mice. LXR agonists therefore might be developed as therapeutic treatments for colorectal cancer.

Krüppel-Like Factor 10 Expression as a Prognostic Indicator for Pancreatic Adenocarcinoma

Deregulation of transforming growth factor (TGF)-β function is a common feature of pancreatic cancer, rendering these cancers unresponsive to TGF-β-stimulated growth inhibition. Recent findings have supported a primary role for Krüppel-like factor 10 (KLF10) as an important transcription factor involved in mediating TGF-β1 signaling. The aim of this study was to evaluate the correlation between KLF10 expression and the clinical and pathologic features of pancreatic cancer. Tissue specimens from patients with pancreatic adenocarcinoma were retrospectively collected for immunohistochemical analysis. To demonstrate that Klf10 expression was primarily regulated by methylation status, the Klf10 promoter was examined by methylation-specific PCR using a pancreatic cancer cell line (Panc-1). DNA methyltransferase (DNMT) inhibitor and small-interfering RNA depletion of DNMT genes were used to reverse KLF10 expression in the Panc-1 cells. In parallel, DNMT1 expression was evaluated in the pancreatic cancer tissue specimens. In 95 pancreatic cancer tissue specimens, KLF10 expression was inversely correlated with pancreatic cancer stage (P = 0.01). Multivariable analysis revealed that, in addition to the presence of distant metastasis at diagnosis (P = 0.001 and 0.001, respectively), KLF10 was another independent prognostic factor related to progression-free and overall survival (P = 0.018 and 0.037, respectively). The loss of KLF10 expression in advanced pancreatic cancer is correlated with altered methylation status, which seems to be regulated by DNMT1. Our results suggest that KLF10 is a potential clinical predictor for progression of pancreatic cancer.

This Month in Gastroenterology

This Month in Gastroenterology

Genetic Evidence That Intestinal Notch Functions Vary Regionally and Operate through a Common Mechanism of Math1 Repression

Notch signaling is active in many sites, and its diverse activities must require tissue-specific intermediaries, which are largely unknown. In the intestinal epithelium, Notch promotes crypt cell proliferation and inhibits goblet cell differentiation. Pharmacologic studies suggest that the latter effect occurs through the transcription factor Math1/Atoh1, which specifies all intestinal secretory cells. We tested this hypothesis using mouse mutants. Genetic loss of the Notch effector RBP-Jκ alone increases all intestinal secretory lineages, with variation between proximal and distal gut segments. This secretory cell excess observed with RBP-Jκ loss was blocked in the absence of Math1 in RBP-Jκ(Fl/Fl);Math1(Fl/Fl);Villin-Cre((ER-T2)) mice. Loss of both factors also restored progenitor replication, proving that Math1 is epistatic to Notch signaling in both secretory cell differentiation and crypt cell proliferation. Investigating mechanisms downstream of Math1, we found that expression of the known Notch effector protein Hes1 was predictably lost in RBP-Jκ(-/-) mice but surprisingly recovered in RBP-Jκ;Math1 compound conditional mutants. Furthermore, the cell cycle inhibitors p27(Kip1) and p57(Kip2) were selectively overexpressed in duodenal and ileal crypts, respectively, in RBP-Jκ-deficient mice. Regional activation of these products was completely abrogated in the absence of Math1. Thus, all intestinal Notch effects channel through the tissue-restricted factor Math1, which promotes secretory differentiation and cell cycle exit by regionally distinct mechanisms. Our data further suggest that, besides transmitting Notch signals, the transcription factor Hes1 acts downstream of Math1 to regulate expression of cell cycle inhibitors and intestinal crypt cell replication.

176 KrüPpel-Like Factor 5 is a Crucial Mediator of Intestinal Tumorigenesis in Mice Harboring Combined APC and KRAS Mutations

176 KrüPpel-Like Factor 5 is a Crucial Mediator of Intestinal Tumorigenesis in Mice Harboring Combined APC and KRAS Mutations

Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined Apc Min and KRASV 12mutations

BackgroundBoth mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene and activation of the KRAS oncogene are implicated in the pathogenesis of colorectal cancer. Mice harboring a germline ApcMin mutation or intestine-specific expression of the KRASV12 gene have been developed. Both mouse strains develop spontaneous intestinal tumors, including adenoma and carcinoma, though at a different age. The zinc finger transcription factor Krüppel-like factor 5 (KLF5) has previously been shown to promote proliferation of intestinal epithelial cells and modulate intestinal tumorigenesis. Here we investigated the in vivo effect of Klf5 heterozygosity on the propensity of ApcMin/KRASV12 double transgenic mice to develop intestinal tumors.ResultsAt 12 weeks of age, ApcMin/KRASV12 mice had three times as many intestinal tumors as ApcMin mice. This increase in tumor number was reduced by 92% in triple transgenic ApcMin/KRASV12/Klf5+/- mice. The reduction in tumor number in ApcMin/KRASV12/Klf5+/- mice was also statistically significant compared to ApcMin mice alone, with a 75% decrease. Compared with ApcMin/KRASV12, tumors from both ApcMin/KRASV12/Klf5+/- and ApcMin mice were smaller. In addition, tumors from ApcMin mice were more distally distributed in the intestine as contrasted by the more proximal distribution in ApcMin/KRASV12 and ApcMin/KRASV12/Klf5+/- mice. Klf5 levels in the normal-appearing intestinal mucosa were higher in both ApcMin and ApcMin/KRASV12 mice but were attenuated in ApcMin/KRASV12/Klf5+/- mice. The levels of β-catenin, cyclin D1 and Ki-67 were also reduced in the normal-appearing intestinal mucosa of ApcMin/KRASV12/Klf5+/- mice when compared to ApcMin/KRASV12 mice. Levels of pMek and pErk1/2 were elevated in the normal-appearing mucosa of ApcMin/KRASV12 mice and modestly reduced in ApcMin/KRASV12/Klf5+/- mice. Tumor tissues displayed higher levels of both Klf5 and β-catenin, irrespective of the mouse genotype from which tumors were derived.ConclusionsResults of the current study confirm the cumulative effect of Apc loss and oncogenic KRAS activation on intestinal tumorigenesis. The drastic reduction in tumor number and size due to Klf5 heterozygosity in ApcMin/KRASV12 mice indicate a critical function of KLF5 in modulating intestinal tumor initiation and progression.

PUMA Suppresses Intestinal Tumorigenesis in Mice

Defective apoptosis contributes to tumorigenesis, although the critical molecular targets remain to be fully characterized. PUMA, a BH3-only protein essential for p53-dependent apoptosis, has been shown to suppress lymphomagenesis. In this study, we investigated the role of PUMA in intestinal tumorigenesis using two animal models. In the azoxymethane (AOM)/dextran sulfate sodium salt model, PUMA deficiency increased the multiplicity and size of colon tumors but reduced the frequency of beta-catenin hotspot mutations. The absence of PUMA led to a significantly elevated incidence of precursor lesions induced by AOM. AOM was found to induce p53-dependent PUMA expression and PUMA-dependent apoptosis in the colonic crypts and stem cell compartment. Furthermore, PUMA deficiency significantly enhanced the formation of spontaneous macroadenomas and microadenomas in the distal small intestine and colon of APC(Min/+) mice. These results show an essential role of PUMA-mediated apoptosis in suppressing intestinal tumorigenesis in mice.

The role of the transcription factor AP-1 in colitis-associated and β-catenin-dependent intestinal tumorigenesis in mice

Chronic inflammation is an important cancer risk factor but the molecular pathways linking inflammation and cancer are incompletely understood. The transcription factor c-Jun/AP-1 (activator protein 1) is involved in inflammatory responses and tumorigenesis and has been proposed as an essential mediator of oncogenic beta-catenin signaling in the intestine. Here, we examined the functions of c-Jun in two distinct mouse models of conditional and intestine-specific activation of beta-catenin. c-Jun is strongly expressed in the small intestine of mutant mice. However, beta-catenin-dependent cell proliferation is surprisingly not affected in mice lacking c-jun in intestinal epithelium, suggesting that c-Jun is not an essential immediate target of beta-catenin signaling in the small intestine. To examine the functions of Jun and Fos proteins during inflammation and cancer in the colon, colitis-associated tumors were induced chemically in the respective knockout mice. Tumors were characterized by activated beta-catenin and strongly expressed c-Jun and JunB. However, tumorigenesis was not affected by inactivation of c-Jun in either intestinal epithelium or myeloid cells. Moreover, tumorigenesis was not altered in mice lacking junB, junD, c-fos, fra-1 or fra-2, suggesting that inhibition of c-Jun or other single AP-1 proteins is not a determining factor in colitis-associated cancer in mice.

Inhibition of Intestinal Polyposis with Reduced Angiogenesis in ApcMin/+ Mice Due to Decreases in c-Myc Expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of beta-catenin and increases in beta-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc(Min/+) mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc(+/-) mice to Apc(Min/+) to generate Apc(Min/+) c-myc(+/-) animals. The compound Apc(Min/+) c-myc(+/-) mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc(Min/+) phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc(Min/+) c-myc(+/-) mice compared with control Apc(Min/+) c-myc(+/+) mice. In addition, we observed one to three polyps per colon in Apc(Min/+) c-myc(+/+) mice, whereas only two lesions were observed in the colons of Apc(Min/+) mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc(Min/+) c-myc(+/-) mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer.

Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.

Ectopic expression of certain Wnt genes in mouse mammary tissue is tumorigenic, and mutations that stabilize beta-catenin are found in various human cancers including colorectal cancer. To determine the role of stabilized beta-catenin in intestinal tumorigenesis in mice, we constructed by embryonic stem (ES) cell-mediated homologous recombination, a mutant beta-catenin allele whose exon 3 was sandwiched by loxP sequences. When the germline heterozygotes were crossed with mice expressing Cre recombinase in the intestines, the serines and threonine encoded by exon 3 and to be phosphorylated by glycogen synthase kinase 3beta (GSK3beta) were deleted in the offspring intestines, which caused adenomatous intestinal polyps resembling those in Apc(Delta716) knockout mice. Some nascent microadenomas were also found in the colon. These results present experimental genetic evidence that activation of the Wnt signaling pathway can cause intestinal and colonic tumors.