Plant stanols induce intestinal tumor formation by up-regulating Wnt and EGFR signaling in ApcMin mice

The rate of APC mutations in the intestine increases in middle-age. At the same period of life, plant sterol and stanol enriched functional foods are introduced to diet to lower blood cholesterol. This study examined the effect of plant stanol enriched diet on intestinal adenoma formation in the Apc^Min mouse. Apc^Min mice were fed 0.8% plant stanol diet or control diet for nine weeks. Cholesterol, plant sterols and plant stanols were analyzed from the caecum content and the intestinal mucosa. Levels of β-catenin, cyclin D1, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) were measured from the intestinal mucosa by Western blotting. Gene expression was determined from the intestinal mucosa using Affymetrix and the data were analyzed for enriched categories and pathways. Plant stanols induced adenoma formation in the small intestine, however, the adenoma size was not affected. We saw increased levels of nuclear β-catenin, phosphorylated β-catenin (Ser675 and Ser552), nuclear cyclin D1, total and phosphorylated EGFR and phosphorylated ERK1/2 in the intestinal mucosa after plant stanol feeding. The Affymetrix data demonstrate that several enzymes of cholesterol synthesis pathway were up-regulated, although the cholesterol level in the intestinal mucosa was not altered. We show that plant stanols induce adenoma formation by activating Wnt and EGFR signaling. EGFR signaling seems to have promoted β-catenin phosphorylation and its translocation into the nucleus, where the expression of cyclin D1 was increased. Up-regulated cholesterol synthesis may partly explain the increased EGFR signaling in the plant stanol-fed mice.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

Role of Gadd45a in Wip1-dependent regulation of intestinal tumorigenesis

Conversion of intestinal stem cells into tumor-initiating cells is an early step in Apc^Min-induced polyposis. Wild-type p53-induced phosphatase 1 (Wip1)-dependent activation of a DNA damage response and p53 has a permanent role in suppression of stem cell conversion, and deletion of Wip1 lowers the tumor burden in Apc^Min mice. Here we show that cyclin-dependent kinase inhibitor 2a, checkpoint kinase 2, and growth arrest and DNA damage gene 45a (Gadd45a) exert critical functions in the tumor-resistant phenotype of Wip1-deficient mice. We further identified Gadd45a as a haploinsufficient gene in the regulation of Wip1-dependent tumor resistance in mice. Gadd45a appears to function through its ability to activate the Jnk-dependent signaling pathway that in turn is a necessary mediator of the proapoptotic functions of p53 that respond to activation of the β-catenin signaling pathway. We propose that silencing of Gadd45a is sufficient to override p53 activation in the presence of active β-catenin under conditions of an enhanced DNA damage response.     

Novel roles for MLH3 deficiency and TLE6-like amplification in DNA mismatch repair-deficient gastrointestinal tumorigenesis and progression

DNA mismatch repair suppresses gastrointestinal tumorgenesis. Four mammalian E. coli MutL homologues heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. To understand the mechanistic contributions of MLH3 and PMS2 in gastrointestinal tumor suppression, we generated Mlh3^−/−;Apc^1638N and Mlh3^−/−;Pms2^−/−;Apc^1638N (MPA) mice. Mlh3 nullizygosity significantly increased Apc frameshift mutations and tumor multiplicity. Combined Mlh3;Pms2 nullizygosity further increased Apc base-substitution mutations. The spectrum of MPA tumor mutations was distinct from that observed in Mlh1^−/−;Apc^1638N mice, implicating the first potential role for MLH1/PMS1 in tumor suppression. Because Mlh3;Pms2 deficiency also increased gastrointestinal tumor progression, we used array-CGH to identify a recurrent tumor amplicon. This amplicon contained a previously uncharacterized Transducin enhancer of Split (Tle) family gene, Tle6-like. Expression of Tle6-like, or the similar human TLE6D splice isoform in colon cancer cells increased cell proliferation, colony-formation, cell migration, and xenograft tumorgenicity. Tle6-like;TLE6D directly interact with the gastrointestinal tumor suppressor RUNX3 and antagonize RUNX3 target transactivation. TLE6D is recurrently overexpressed in human colorectal cancers and TLE6D expression correlates with RUNX3 expression. Collectively, these findings provide important insights into the molecular mechanisms of individual MutL homologue tumor suppression and demonstrate an association between TLE mediated antagonism of RUNX3 and accelerated human colorectal cancer progression.     

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1^−/− compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1^−/− compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.Key words: cell cycle, D-type cyclins, skin, carcinogenesis, epidermis     

Identification of Mom12 and Mom13, two novel modifier loci of ApcMin-mediated intestinal tumorigenesis

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc^Min/+ mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc^Min/+ mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc^Min model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc^Min-mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc^Min/+ mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc^Min/+ controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: (1) Mom12, a dominant modifier linked to the congenic region on chromosome 6 and (2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.Key words: adenomatous polyposis coli, modifier of min, congenic mice, caveolin-1, cancer susceptibility     

Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling.KEY WORDS: APC, Wnt, mTOR, mTORC1, Zebrafish, Colon cancer, Polyposis, GSK-3     

The Somatic Mutation Hit on Top of Genetic APC mutations Cause Skin Tumor

Inactivation of the adenomatous polyposis coli (APC) gene is the initiating event in familial adenomatous polyposis (FAP) patients. Up to 90% of FAP patients show intestinal tumors and other extracolonic malignancies including hepatoblastomas, desmoid tumors, and brain cancer. APC mutation mice (Apc^Min/+ mice) develop benign polyps in the intestinal tract. It has been reported that small numbers of Apc^Min/+ mice develop breast carcinomas. Here, we found that approximately 1.6% of Apc^Min/+ mice suffered skin neoplasm. The results demonstrated that these skin tumors are not derived from intestinal adenomas. Sequencing of skin tumors of Apc^Min/+ mice and Apc^Min/+ mice skin. The data showed that somatic mutations and gene expression levels changed greatly in skin tumors compared to control. Similarly, APC mutation accounts for 27% in the patients of nonmelanoma skin carcinomas in cancer database, and two above genes mutation coexist was observed in all patients. Furthermore, using gene mutation reagent (DMBA)–treated Apc^Min/+ mice skin, the skin epithelium and glandular begin hyperplasia in Apc^Min/+ mice. These findings revealed that the somatic mutation hit on the germline mutation increase the tumor incidence, suggesting that the somatic mutation should be avoided if the germline mutation exists in one body.     

Systemic TM4SF5 overexpression in ApcMin/+ mice promotes hepatic portal hypertension associated with fibrosis

Mutation of the gene for adenomatous polyposis coli (APC), as seen in Apc^Min/+ mice, leads to intestinal adenomas and carcinomas via stabilization of β-catenin. Transmembrane 4 L six family member 5 (TM4SF5) is involved in the development of non-alcoholic fatty liver disease, fibrosis, and cancer. However, the functional linkage between TM4SF5 and APC or β-catenin has not been investigated for pathological outcomes. After interbreeding Apc^Min/+ with TM4SF5-overexpressing transgenic (Tg^TM4SF5) mice, we explored pathological outcomes in the intestines and livers of the offspring. The intestines of 26-week-old dual-transgenic mice (Apc^Min/+:Tg^TM4SF5) had intramucosal adenocarcinomas beyond the single-crypt adenomas in Apc^Min/+ mice. Additional TM4SF5 overexpression increased the stabilization of β-catenin via reduced glycogen synthase kinase 3β (GSK3β) phosphorylation on Ser9. Additionally, the livers of the dual-transgenic mice showed distinct sinusoidal dilatation and features of hepatic portal hypertension associated with fibrosis, more than did the relatively normal livers in Apc^Min/+ mice. Interestingly, TM4SF5 overexpression in the liver was positively linked to increased GSK3β phosphorylation (opposite to that seen in the colon), β-catenin level, and extracellular matrix (ECM) protein expression, indicating fibrotic phenotypes. Consistent with these results, 78-week-old Tg^TM4SF5 mice similarly had sinusoidal dilatation, immune cell infiltration, and fibrosis. Altogether, systemic overexpression of TM4SF5 aggravates pathological abnormalities in both the colon and the liver.     

Acarbose improved survival for Apc+/Min mice

Acarbose blocks the digestion of complex carbohydrates, and the NIA Intervention Testing Program (ITP) found that it improved survival when fed to mice. Yet, we do not know if lifespan extension was caused by its effect on metabolism with regard to the soma or cancer suppression. Cancer caused death for ~80% of ITP mice. The ITP found rapamycin, an inhibitor to the pro‐growth mTORC1 (mechanistic target of rapamycin complex 1) pathway, improved survival and it suppressed tumors in Apc^+/Min mice providing a plausible rationale to ask if acarbose had a similar effect. Apc^+/Min is a mouse model prone to intestinal polyposis and a mimic of familial adenomatous polyposis in people. Polyp‐associated anemia contributed to their death. To address this knowledge gap, we fed two doses of acarbose to Apc^+/Min mice. Acarbose improved median survival at both doses. A cross‐sectional analysis was performed next. At both doses, ACA fed mice exhibited reduced intestinal crypt depth, weight loss despite increased food consumption and reduced postprandial blood glucose and plasma insulin, indicative of improved insulin sensitivity. Dose‐independent and dose‐dependent compensatory liver responses were observed for AMPK and mTORC1 activities, respectively. Only mice fed the high dose diet exhibited reductions in tumor number with higher hematocrits. Because low‐dose acarbose improved lifespan but failed to reduced tumors, its effects seem to be independent of cancer. These data implicate the importance of improved carbohydrate metabolism on survival.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc^+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc^+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.     

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/β-Catenin Pathway In Vivo

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apc^min mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc^min mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions.     

Prediction and Testing of Biological Networks Underlying Intestinal Cancer

Colorectal cancer progresses through an accumulation of somatic mutations, some of which reside in so-called “driver” genes that provide a growth advantage to the tumor. To identify points of intersection between driver gene pathways, we implemented a network analysis framework using protein interactions to predict likely connections – both precedented and novel – between key driver genes in cancer. We applied the framework to find significant connections between two genes, Apc and Cdkn1a (p21), known to be synergistic in tumorigenesis in mouse models. We then assessed the functional coherence of the resulting Apc-Cdkn1a network by engineering in vivo single node perturbations of the network: mouse models mutated individually at Apc (Apc^1638N+/−) or Cdkn1a (Cdkn1a^−/−), followed by measurements of protein and gene expression changes in intestinal epithelial tissue. We hypothesized that if the predicted network is biologically coherent (functional), then the predicted nodes should associate more specifically with dysregulated genes and proteins than stochastically selected genes and proteins. The predicted Apc-Cdkn1a network was significantly perturbed at the mRNA-level by both single gene knockouts, and the predictions were also strongly supported based on physical proximity and mRNA coexpression of proteomic targets. These results support the functional coherence of the proposed Apc-Cdkn1a network and also demonstrate how network-based predictions can be statistically tested using high-throughput biological data.     

Rapamycin Inhibition of Polyposis and Progression to Dysplasia in a Mouse Model

Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/− 2 vs 56.5+/− 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.     

Large intestine intraepithelial lymphocytes from Apc+/+ and Apc+/Min mice and their modulation by indigestible carbohydrates: the IL-15/IL-15Rα complex and CD4+CD25+ T cells are the main targets

We have shown recently that some indigestible carbohydrate (short-chain fructo-oligosaccharides [sc-FOS]) reduced colon tumor incidence in Apc+/Min mice, and that this effect depended on a functional local immune system. In addition, IL-15 mRNA was concomitantly modulated in the mucosa. Since intraepithelial lymphocytes (IELs) are in close contact with intestinal epithelial cells, these cells are the candidates most likely to be involved in early cancer immunosurveillance. The present study documents the effects of sc-FOS on large intestine IELs (LI-IELs) from Apc+/+ or Apc+/Min mice by analyzing markers related to their phenotype, their activation status, and the cell surface IL-15/IL-15R. In the colons of Apc+/Min mice, fewer LI-IELs expressed surface IL-15/IL-15R. In addition, a lower number of CD4⁺ LI-IELs expressed CD25, although more LI-IELs expressed CD69, as compared to normal mice. The sc-FOS–enriched diet caused a decrease in the proportion of CD25⁺ LI-IELs and an increase in the percentage of LI-IELs bearing surface IL-15/IL-15R, independently of the Apc gene status. The IL-15/IL-15R increase was, however, higher in Min mice, and returned to a level very similar to that of Apc+/+ mice when the latter mice were fed a low-fiber diet. The sc-FOS-enriched diet specifically induced an increase in CD69⁺ cells in Apc+/+ mice, and a decrease in the proportion of CD4⁺CD25⁺ LI-IELs in Apc+/Min mice. Some of these modulations could contribute to the development of a better immune anticancer response in the early steps of cancer development.     

Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

Increased angiogenesis in Cdk4R24C/R24C:Apc+/Min intestinal tumors

Cyclin dependent kinase 4 (Cdk4) is a cell cycle regulator involved in early G1 cell cycle progression and has been indirectly implicated in angiogenesis in the Min mouse system, a mouse that harbors a mutation in the Apc gene. Apc^+/Min mice when crossed with Ink4a/arf-/- mice, exhibited increased angiogenesis of colorectal tumors suggesting that dysregulation of Cdk4 (due to loss of Ink4a-mediated suppression) may contribute to enhanced angiogenesis. To demonstrate a direct role for Cdk4 in angiogenesis, we crossed mice that have an activated Cdk4, Cdk4^R24C/R24C mice, with Apc^+/Min mice and examined levels of angiogenesis in intestinal tumors formed. Our results show an increase in the percentage of highly vascularized tumors in Cdk4^R24C/R24C:Apc^Min/+ and Cdk4^+/R24C:Apc^Min/+ mice compared to Cdk4^+/+:Apc^Min/+ mice. In addition immunohistochemical analysis showed an increase in CD-31 staining localized to endothelial cells of Cdk4^R24C/R24C:Apc^Min/+ mouse tumors, supporting the hypothesis of increased vasculature in these tumors. Further analysis showed an increase in the expression of the E2F1 target proteins Vegf-b and Cyclin A in Cdk4^R24C/R24C:Apc^+/Min intestinal tumors. Together these data suggest that the dysregulated Cdk4 gene plays an important role in angiogenesis during intestinal tumor formation and may in part act via increasing E2F1 target proteins. This is the first report to show that Cdk4 has a direct role in angiogenesis in vivo and may be an important drug target to reduce or prevent angiogenesis during intestinal tumor formation.