Culture-Independent Microbial Community Analysis Reveals that Inulin in the Diet Primarily Affects Previously Unknown Bacteria in the Mouse Cecum

Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.     

Germline mutations in the 3′ part of APC exon 15 do not result in truncated proteins and are associated with attenuated adenomatous polyposis coli

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer characterized by the development of numerous adenomatous polyps predominantly in the colorectal region. Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for most cases of FAP. Mutations at the 5′ end of APC are known to be associated with a relatively mild form of the disease, called attenuated adenomatous polyposis coli (AAPC). We identified a frameshift mutation in the 3′ part of exon 15, resulting in a stop codon at 1862, in a large Dutch kindred with AAPC. Western blot analysis of lymphoblastoid cell lines derived from affected family members from this kindred, as well as from a previously reported Swiss family carrying a frameshift mutation at codon 1987 and displaying a similar attenuated phenotype, showed only the wild-type APC protein. Our study indicates that chain-terminating mutations located in the 3′ part of APC do not result in detectable truncated polypeptides and we hypothesize that this is likely to be the basis for the observed AAPC phenotype. Received: 18 June 1996 / Revised: 8 July 1996     

More than two decades of Apc modeling in rodents

Mutation of tumor suppressor gene adenomatous polyposis coli (APC) is an initiating step in most colon cancers. This review summarizes Apc models in mice and rats, with particular concentration on those most recently developed, phenotypic variation among different models, and genotype/phenotype correlations.     

Adenomatous polyposis coli-deficient zebrafish are susceptible to digestive tract neoplasia

Truncation of the tumour suppressor adenomatous polyposis coli (APC) constitutively activates the Wnt/beta-catenin signalling pathway. This event constitutes the primary transforming event in sporadic colorectal cancer in humans. Moreover, humans or mice carrying germline truncating mutations in APC develop large numbers of intestinal adenomas. Here, we report that zebrafish that are heterozygous for a truncating APC mutation spontaneously develop intestinal, hepatic and pancreatic neoplasias that are highly proliferative, accumulate beta-catenin and express Wnt target genes. Treatment with the chemical carcinogen 7,12-dimethylbenz[a]anthracene accelerates the induction of these lesions. These observations establish apc-mutant zebrafish as a bona fide model for the study of digestive tract cancer.     

Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development

Mutations and deregulation of adenomatous polyposis coli (APC) and beta-catenin are implicated in specific cancers of the pancreas, but the role of Wnt pathway in normal pancreas development and homeostasis is unknown. This article reports a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two reporter lines to monitor canonical Wnt pathway activity during development and after birth and demonstrate activity in endocrine cells and in the mesenchyme. We have specifically deleted the beta-catenin gene in the epithelium of the pancreas and duodenum by using Pdx1-Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that beta-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, beta-catenin mutant cells have a competitive disadvantage during development that also affects the exocrine compartment. Moreover, the conditional knockout (KO) mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of beta-catenin at the membrane. Mice later recover from pancreatitis and regenerate normal pancreas and duodenal villi from the wild-type (wt) cells that escape beta-catenin deletion.     

Gastro-intestinal tumorigenesis in Smad4 mutant mice

The SMAD4 gene plays a key role in the TGF-beta signaling pathway. We inactivated its mouse homolog Smad4. The homozygous mutants were embryonically lethal, whereas the heterozygotes were viable and fertile. Although young heterozygotes appeared normal, old mice developed gastric and duodenal polyps similar to human juvenile polyps characterized by abundant stroma and eosinophilic infiltrations. These data are consistent with the reports that a subset of human juvenile polyposis kindreds carry germline mutations in the SMAD4 gene. We then introduced the Smad4 mutation into the Apc(Delta716) knockout mice, a model for human familial adenomatous polyposis. Because both Apc and Smad4 are located on mouse chromosome 18, we constructed by meiotic recombination compound heterozygotes carrying both mutations on the same chromosome. In such mice, intestinal polyps developed into more malignant tumors than those in the simple Apc(Delta716) heterozygotes, showing an extensive stromal cell proliferation and strong submucosal invasion. These results indicate that mutations in SMAD4 play a significant role in the malignant progression of colorectal tumors.     

Teaching tumour suppressors new tricks

Examination of the tumour suppressor adenomatous polyposis coli (APC) has shown that it may be multifunctional. Recent work has demonstrated dynamic interactions of APC with the microtubule cytoskeleton, supporting the idea that APC has an important function in cell migration.     

Down-regulation of beta-catenin by the colorectal tumor suppressor APC requires association with Axin and beta-catenin

The tumor suppressor adenomatous polyposis coli (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. APC forms a complex with beta-catenin, Axin, and glycogen synthase kinase-3beta and induces the degradation of beta-catenin. In the present study, we examined whether APC association with Axin is required for degradation of beta-catenin. We found that a fragment of APC that induces beta-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Also, overexpression of an Axin fragment spanning the regulator of the G-protein signaling domain inhibited APC-mediated beta-catenin degradation. An APC fragment with mutated beta-catenin-binding sites but intact Axin-binding sites also failed to induce degradation of beta-catenin. These results suggest that APC requires interaction with Axin and beta-catenin to down-regulate beta-catenin.     

Gastrointestinal tumorigenesis in Smad4 (Dpc4) mutant mice

The SMAD4 (Dpc4) gene plays a key role in the TGF-beta signaling pathway. We recently inactivated the mouse homolog Smad4. The homozygous mutants were embryonic lethals, whereas the heterozygotes were viable and fertile. Although young heterozygotes were normal, old mice developed gastric and duodenal polyps similar to those found in human juvenile polyps characterized by abundant stroma and eosinophilic infiltrations. These data are consistent with the reports that a subset of human juvenile polyposis kindreds carry germline mutations in the SMAD4 gene. We then introduced the Smad4 mutation into the Apc delta 716 knockout mice, a model for human familial adenomatous polyposis. Because both Apc and Smad4 are located on mouse chromosome 18, we constructed by meiotic recombination, compound heterozygotes carrying both mutations on the same chromosome. In such mice, intestinal polyps developed into more malignant tumors than those in the simple Apc delta 716 heterozygotes, showing an extensive stromal cell proliferation and strong submucosal invasion. These results indicate that mutations in SMAD4 play a significant role in the malignant progression of colorectal tumors.     

Examination of actin and microtubule dependent APC localisations in living mammalian cells

Background  

The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented.     

Colon polyps: a damaged developmental system and a precursor to cancer.

The observation that the attenuated form of adenomatous polyposis coli (AAPC) has a different mutational spectrum in both the adenomatous polyp and the carcinoma than the classical form of APC has led to the hypothesis that AAPC alleles retain some residual APC activity associated with a greatly reduced number of polyps. It is suggested that further progression to carcinoma may be dependent upon either loss of the AAPC allele (loss of heterozygosity), followed by an APC mutation in the wild-type allele, or the occurrence of APC mutations within both the AAPC and wild-type alleles.     

End-binding protein 1 (EB1) up-regulation is an early event in colorectal carcinogenesis

End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa (“field effect”) of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis.     

Frequent polymorphism in exon 15 of the adenomatous polyposis coli gene

In the course of a study of tumor suppressor gene mutation in hepatoblastoma, a frequent neutral polymorphism was identified at codon 1493 in exon 15 of the gene causing adenomatous polyposis coli (APC). As the polymorphism introduces a new BsaJ1 site, DNA amplified by the polymerase chain reaction (PCR) can be rapidly screened for this polymorphism. This polymorphism can be used in cosegregation studies for presymptomatic diagnosis of APC and family studies.     

The oncogenic activation of beta-catenin

The activation of beta-catenin to an oncogenic state can result from the inactivation of the tumor suppressor adenomatous polyposis coli (APC), by direct mutation in the beta-catenin gene, or by the activation of wnt receptors. Once activated, beta-catenin most likely promotes tumor progression through its persistent interaction with one or more of its numerous downstream targets.     

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of β-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef^−/− mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.     

Role of Smad4 (DPC4) inactivation in human cancer

The tumor suppressor gene Smad4 (DPC4) at chromosome 18q21.1 belongs to the Smad family, which mediates the TGFbeta signaling pathway suppressing epithelial cell growth. This review summarizes the mutational events of the Smad4 gene in human cancer. The Smad4 gene is genetically responsible for familial juvenile polyposis, an autosomal dominant disease characterized by predisposition to gastrointestinal polyps and cancer. In this syndrome, polyps are formed by inactivation of the Smad4 gene through germline mutation and loss of the unaffected wild-type allele. In pancreatic and colorectal cancer, inactivation of the Smad4 gene through homozygous deletion or intragenic mutation occurs frequently in association with malignant progression. However, mutation of this gene is seen only occasionally in the rest of human cancers. The majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2), which interfere with the homo-oligomer formation of Smad4 protein and the hetero-oligomer formation between Smad4 and Smad2 proteins, resulting in disruption of TGFbeta signaling. Supporting evidence for the above observation was provided by genetically manipulated mice carrying either a heterozygote of the Smad4 gene or a compound heterozygote of the Smad4 and APC genes, which develop either gastrointestinal polyps/cancer mimicking familial juvenile polyposis or progressed colorectal cancer, respectively.     

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.