Adenomatous Polyposis Coli Min Research Articles

Familial adenomatous polyposis and changes in the gut microbiota: New insights into colorectal cancer carcinogenesis.

Patients with familial adenomatous polyposis (FAP), an autosomal dominant hereditary colorectal cancer syndrome, have a lifetime risk of developing cancer of nearly 100%. Recent studies have pointed out that the gut microbiota could play a crucial role in the development of colorectal adenomas and the consequent progression to colorectal cancer. Some gut bacteria, such as Fusobacterium nucleatum, Escherichia coli, Clostridium difficile, Peptostreptococcus, and enterotoxigenic Bacteroides fragilis, could be implicated in colorectal carcinogenesis through different mechanisms, including the maintenance of a chronic inflammatory state, production of bioactive tumorigenic metabolites, and DNA damage. Studies using the adenomatous polyposis coliMin/+ mouse model, which resembles FAP in most respects, have shown that specific changes in the intestinal microbial community could influence a multistep progression, the intestinal “adenoma-carcinoma sequence”, which involves mucosal barrier injury, low-grade inflammation, activation of the Wnt pathway. Therefore, modulation of gut microbiota might represent a novel therapeutic target for patients with FAP. Administration of probiotics, prebiotics, antibiotics, and nonsteroidal anti-inflammatory drugs could potentially prevent the progression of the adenoma-carcinoma sequence in FAP. The aim of this review was to summarize the best available knowledge on the role of gut microbiota in colorectal carcinogenesis in patients with FAP.

Protease‐activated receptor‐1 impedes prostate and intestinal tumor progression in mice

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APCMin/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APCMin/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.

Decreased RXRα is Associated with Increased β-Catenin/TCF4 in (56)Fe-Induced Intestinal Tumors.

Although it is known that accumulation of oncogenic β-catenin is critical for intestinal tumorigenesis, the underlying mechanisms have not yet been fully explored. Post-translational β-catenin level is regulated via the adenomatous polyposis coli (APC)-dependent as well as the APC-independent ubiquitin–proteasome pathway (UPP). Employing an APC-mutant mouse model (APCMin/+) the present study aimed to investigate the status of RXRα, an APC-independent factor involved in targeting β-catenin to UPP for degradation, in tumor-bearing and tumor-free areas of intestine after exposure to energetic 56Fe ions. APCMin/+ mice were exposed to energetic 56Fe ions (4 or 1.6 Gy) and intestinal tumor samples and tumor-free normal intestinal samples were collected 100–110 days after exposure. The status of TCF4, β-catenin, cyclin D1, and RXRα was examined using immunohistochemistry and immunoblots. We observed increased accumulation of the transcription factor TCF4 and its co-activator β-catenin as well as their downstream oncogenic target protein cyclin-D1 in 56Fe ion-induced intestinal tumors. Further, decreased expression of RXRα in tumors as well as in adjacent normal epithelium was indicative of perturbations in β-catenin proteasomal-targeting machinery. This indicates that decreased UPP targeting of β-catenin due to downregulation of RXRα can contribute to further accumulation of β-catenin and to 56Fe-induced tumorigenesis.

Abstract SY43-02: Mismatch repair, gut microbiota, and the etiology of colon cancer

Abstract The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)Msh2-/- and APC(Min/+)Mlh1-/- mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuels hyperproliferation of Msh2-/- and Mlh1-/- colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating β-catenin activity and potentially modulating the differentiation of transit-amplifying cells in the colon. These data show that mismatch repair deficiency sensitizes colon epithelial cells to transformation by gut microbiota, and thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. Citation Format: Alberto Martin. Mismatch repair, gut microbiota, and the etiology of colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY43-02. doi:10.1158/1538-7445.AM2015-SY43-02

Gut Microbial Metabolism Drives Transformation of Msh2-Deficient Colon Epithelial Cells

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating β-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

Inhibition of intestinal adenoma formation in APC(Min/+) mice by Riccardin D, a natural product derived from liverwort plant Dumortiera hirsuta.

BackgroundMutation of tumor suppressor gene, adenomatous polyposis coli (APC), is the primary molecular event in the development of most intestinal carcinomas. Animal model with APC gene mutation is an effective tool for study of preventive approaches against intestinal carcinomas. We aimed to evaluate the effect of Riccardin D, a macrocyclic bisbibenzyl compound, as a chemopreventive agent against intestinal adenoma formation in APCMin/+ mice.Methods APCMin/+ mice were given Riccardin D by p.o. gavage for 7 weeks. Mice were sacrificed, and the number, size and histopathology of intestinal polyps were examined under a microscope. We performed immunohistochemical staining, western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in intestinal polyps to investigate the mechanism of chemopreventive effect of Riccardin D.ResultsRiccardin D treatment resulted in a significant inhibition of intestinal adenoma formation, showing a reduction of polyp number by 41.7%, 31.1% and 44.4%, respectively, in proximal, middle and distal portions of small intestine. The activity of Riccardin D against polyp formation was more profound in colon, wherein Riccardin D decreased polyp number by 79.3%. Size distribution analysis revealed a significant reduction in large-size polyps (2–3 mm) by 40.0%, 42.5% and 33.3%, respectively, in proximal, middle and distal portions of small intestine, and 77.8% in colon. Histopathological analysis of the intestinal polyps revealed mostly hyperplastic morphology without obvious dysplasia in Riccardin D-treated mice. Molecular analyses of the polyps suggested that the inhibitory effect of Riccardin D on intestinal adenoma formation was associated with its abilities of reduction in cell proliferation, induction of apoptosis, antiangiogenesis, inhibition of the Wnt signaling pathway and suppression of inflammatory mediators in polyps.ConclusionsOur results suggested that Riccardin D exerts its chemopreventive effect against intestinal adenoma formation through multiple mechanisms including anti-proliferative, apoptotic, anti-angiogenic and anti-inflammatory activity.

Effect of Targeting Janus Kinase 3 on the Development of Intestinal Tumors in the Adenomatous Polyposis Colimin Mouse Model of Familial Adenomatous Polyposis

Familial adenomatous polyposis (FAP) is associated with germ-line mutations in the tumor suppressor gene, adenomatous polyposis coli (APC) located on chromosome 5q21. Multiple intestinal neoplasia (Min) in mice resembles FAP in humans, resulting from a single point mutation in the murine homolog of the APC gene. The effects of the rationally-designed Janus kinase 3 (JAK3) inhibitor JANEX-1 (4-(4'-hydroxyphenyl) amino-6,7-dimethoxy-quinazoline, WHI-P131, CAS 202475-60-3) on the development of intestinal tumors in the APC (min/+) mouse model of FAP were examined. The Min mice were fed with rodent chow or chow supplemented with JANEX-1 once a week starting at 1.5 months of age. The cumulative proportions of mice remaining alive at 7 months were 13 +/- 6% for control mice versus 72 +/-12% for JANEX-1 treated mice (P<0.0002). In contrast, Compound DDE24, a synthetically activated genistein, an inhibitor of Epidermal Growth Factor receptor (EGF-R), cellular homologue of oncogene product from Raus Avian sarcoma virus (SRC) and Syk tyrosine kinases, which Thus, selective targeting of JAK3 was highly effective in preventing development of intestinal tumors in Min mice resulting in markedly improved survival outcomes. JAK3 inhibitors may therefore be useful in the prevention of colorectal cancer in individuals with FAP.

Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in the APC(Min/+) mouse using optical colonography.

Mutated adenomatous polyposis coli (APC) genes predispose transformations to neoplasia, progressing to colorectal carcinoma. Early detection facilitates clinical management and therapy. Novel lectin-mediated polymerized targeted liposomes (Rh-I-UEA-1), with polyp specificity and incorporated imaging agents were fabricated to locate and image adenomatous polyps in APC(Min/+) mice. The biomarker α-L-fucose covalently joins the liposomal conjugated lectin Ulexeuropaeus agglutinin (UEA-1), via glycosidic linkage to the polyp mucin layer. Multispectral optical imaging (MSI) corroborated a global perspective of specific binding (rhodamine B 532 nm emission, 590-620 nm excitation) of targeted Rh-I-UEA-1 polymerized liposomes to polyps with 1.4-fold labeling efficiency. High-resolution coregistered optical coherence tomography (OCT) and fluorescence molecular imaging (FMI) reveal the spatial correlation of contrast distribution and tissue morphology. Freshly excised APC(Min) bowels were incubated with targeted liposomes (UEA-1 lectin), control liposomes (no lectin), or iohexol (Omnipaque) and imaged by the three techniques. Computed tomographic quantitative analyses did not confirm that targeted liposomes more strongly bound polyps than nontargeted liposomes or iohexol (Omnipaque) alone. OCT, with anatomic depth capabilities, along with the coregistered FMI, substantiated Rh-I-UEA-1 liposome binding along the mucinous polyp surface. UEA-1 lectin denotes α-l-fucose biomarker carbohydrate expression at the mucin glycoprotein layer; Rh-I-UEA-1 polymerized liposomes target and image adenomatous polyps in APC(Min) mice.

Inactivation of the vitamin D receptor in APCmin/+ mice reveals a critical role for the vitamin D receptor in intestinal tumor growth

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/β-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/β-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in β-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and β-catenin signaling pathways in intestinal tumorigenesis.

Rapamycin inhibits oncogenic intestinal ion channels and neoplasia in APCMin/+ mice

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis. Mice with a heterozygous APC(Min) mutation develop multiple intestinal neoplasia (Min) leading to premature death. Early in colorectal carcinogenesis, APC(Min/+) mice show enhanced Akt-mammalian target of rapamycin (mTOR) signaling, which is paralleled by upregulation of oncogenic K(+) channels. In this study, we tested the effect of mTOR inhibition with rapamycin on tumor formation in APC(Min/+) mice and evaluated ion channel regulation. We found that continuous long-term rapamycin treatment of APC(Min/+) mice dramatically inhibits intestinal neoplasia. Moreover, although untreated APC(Min/+) mice lose weight, experience intestinal bleeding and succumb to multiple neoplasia by 22.3+/-1.4 weeks of age, mice treated with rapamycin maintain stable weight and survive long term (39.6+/-3.4 weeks), with more than 30% surviving >1 year. Impressively, abnormalities in colonic electrolyte transport typical for APC(Min/+) mice are abolished, along with the suppression of epithelial Na(+) channel (ENaC) and oncogenic K(+) ion channels BK, Elk1 and Erg1, both functionally and at mRNA levels. These results show that continuous prophylaxis by rapamycin markedly inhibits the development of APC mutation-related polyposis, and suggest a novel contributing mechanism of action through the blockade of intestinal oncogenic ion channels.

Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling

Aberrant Wnt signaling mediated by mutations affecting APC (adenomatous polyposis coli) or beta-catenin initiates the majority of human colorectal cancers (CRC) and drives tumorigenesis through the activation of specific genes such as MYC. We report here a novel association whereby another oncogenic transcription factor, MYB/c-Myb, is necessary for intestinal adenoma development directed by activated Wnt signaling. APC(Min/+) mice in which c-myb is haploinsufficient survive longer than wild-type APC(Min/+) animals due to a delay in adenoma formation. Intestinal adenomas from APC(Min/+) mice were assessed and found to have high levels of c-myc gene expression. We explored the relationship between activated Wnt signaling and MYB in regulating MYC and found activated beta-catenin in combination with MYB induces robust upregulation of MYC promoter activity, as well as endogenous MYC mRNA and protein expression, in human cells. This cooperation occurred through independent binding of MYB and beta-catenin to the MYC promoter. These data highlight a cooperative function for MYB in the context of activated Wnt signaling and provide a molecular basis for the expression of MYC in CRC.

Going with the Wnt? Focus on “Hyperaldosteronism, hypervolemia, and increased blood pressure in mice expressing defective APC”

the wnt signaling system consists of ancient pathways with highly conserved core components tracing back more than 700 million years including insects and nematodes ([20][1]). Wnt signals classically play a crucial role in embryonic development concerning cell fate, motility, and polarity, as well

3,3′‐Diindolylmethane Enhances the Efficacy of Butyrate in Colon Cancer Prevention through Down‐regulation of Survivin

Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively evaluated as a chemoprevention agent for colon cancer. We recently demonstrated that mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC inhibitor‐induced apoptosis in colon cancers (Huang and Guo, Cancer Research, 66(18), 9245‐9251, 2006). Here we show that APC mutation rendered colon cancer cells resistant to butyrate‐induced apoptosis due to the failure of butyrate to down‐regulate survivin in these cells. Another cancer preventive agent, 3,3′‐Diindolylmethane (DIM), was identified to be able to down‐regulate survivin in colon cancers expressing mutant APC. Pre‐treatment with DIM enhanced butyrate‐induced apoptosis. Down‐regulation of survivin by DIM occurred at both transcription and post‐translation level through increased protein degradation. We further demonstrated that DIM was able to down‐regulate survivin and enhance the effects of butyrate in apoptosis induction and prevention of familial adenomatous polyposis in APCmin/+ mice. Thus, the combination of DIM and butyrate is potentially an effective strategy for the prevention of colon cancer.This research was supported by Grants 1R03CA130062, 5R21CA111765 and 2P20RR015566 from the National Institutes of Health (NIH).

Spindle misorientation in tumors from APCmin/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the majority of colon cancers, and its mutation may initiate cancer by multiple mechanisms. Recently, abnormal mitotic spindle orientation was shown in normal-appearing tissues from mice heterozygous for APC mutation. To determine the effect of APC mutation on spindle orientation in tumors, and to better understand its mechanism, we measured mitotic spindle orientation in intestinal tumors from APC mutant mice, with three-dimensionally reconstructed confocal stacks of microtubule immunofluorescence images. We found spindle angles were increased in crypts heterozygous for the APC(min) mutation, and further increased in tumors. Astral microtubules of these spindles were clearly evident, suggesting astral microtubule loss is not a major mechanism of spindle misorientation in intestinal cells lacking wild-type APC. beta-Catenin staining was markedly abnormal in crypts and tumors from the mutant mice, suggesting a possible role in spindle orientation. Spindle angles in colon tumors with wild-type APC were equivalent to those in wild-type mice, showing that spindle misorientation is specific to APC mutation and not a general feature of tumors. We suggest spindle misorientation may contribute to the loss of normal tissue organization during tumor formation and could offer new insights into early carcinogenic events.

Src transformation of colonic epithelial cells: Enhanced anchorage‐independent growth in an Apc+/min background

Colon cancer arises through a multistep process involving inactivation of tumor suppressor proteins and activation of oncogene-encoded proteins. Development of colon cancer frequently involves mutation of the adenomatous polyposis coli (APC) tumor suppressor. The activity of the proto-oncogene-encoded Src tyrosine kinase is commonly elevated in colon cancer, with higher activity observed as tumors progress and metastasize. Both APC and Src are multifunctional proteins that have been implicated in the control of cell proliferation, but also as regulators of cytoskeletal changes associated with cell motility and invasion. To investigate the potential for biological cooperativity between APC partial loss-of-function and Src gain-of-function, oncogenic Src was stably expressed in mouse colon epithelial cell lines IMCE (APC(+/min)) and YAMC (APC(+/+)). Under permissive growth conditions, these lines are conditionally immortalized through inactivation of p53. Irrespective of the APC genotype or p53 status, oncogenic Src expression led to morphologic transformation associated with loss of cell-cell junctions, cytoskeletal disorganization, and acquisition of invasive properties. However IMCE cells that carry one copy of the mutant APC(min) allele exhibited increased capacity for Src-mediated anchorage-independent proliferation as compared to the YAMC cells, and this property was enhanced under permissive growth conditions. beta-catenin levels and transcriptional activity were also elevated in the Src-transformed IMCE cells. The selective Src inhibitor, AZD0530, was found to be effective in blocking both cell invasion and anchorage-independent proliferation. These findings suggest that the combined effects of elevated Src activity and APC partial loss-of-function may contribute to the growth of colon tumors.

Targeted Suppression of β-Catenin Blocks Intestinal Adenoma Formation in APC Min Mice

IntroductionMutations involving the adenomatous polyposis coli (APC) tumor suppressor gene leading to activation of β-catenin have been identified in the majority of sporadic colonic adenocarcinomas and in essentially all colonic tumors from patients with Familial Adenomatous Polyposis. The C57BL/6J-APCmin (Min) mouse, which carries a germ line mutation in the murine homolog of the APC gene is a useful model for intestinal adenoma formation linked to loss of APC activity. One of the critical downstream molecules regulated by APC is β-catenin; molecular targeting of β-catenin is, thus, an attractive chemopreventative strategy in colon cancer. Antisense oligodeoxynucleotides (AODNs) capable of downregulating murine β-catenin have been identified. Analysis of β-catenin Protein Expression in Liver Tissue and Intestinal AdenomasAdenomas harvested from mice treated for 7 days with β-catenin AODNs demonstrated clear downregulation of β-catenin expression, which was accompanied by a significant reduction in proliferation. There was no effect on proliferation in normal intestinal epithelium. Min mice treated systemically with β-catenin AODNs over a 6-week period had a statistically significant reduction in the number of intestinal adenomas. These studies provide direct evidence that targeted suppression of β-catenin inhibits the formation of intestinal adenomas in APC-mutant mice. Furthermore, these studies suggest that molecular targeting of β-catenin holds significant promise as a chemopreventative strategy in colon cancer.

APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC +/− embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC −/− embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC +/− zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC Min/+ mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.

Effect of genistein on the bioavailability and intestinal cancer chemopreventive activity of (-)-epigallocatechin-3-gallate

The green tea (Camellia sinensis) catechin, (-)-epigallocatechin-3-gallate (EGCG), has shown cancer-preventive activity in animal models. Previously, we have reported the bioavailability of EGCG in rats and mice. Here, we report that cotreatment of HT-29 human colon cancer cells with genistein (from soy) increased cytosolic EGCG by 2- to 5-fold compared with treatment with EGCG only. Inclusion of genistein, at non-cytotoxic concentrations, increased the growth inhibitory effects of EGCG against HT-29 cells (up to 2-fold at 20 microM genistein). Intragastric coadministration of EGCG (75 mg/kg) and genistein (200 mg/kg) to CF-1 mice resulted in an increase in plasma half-life (t(1/2) 148.7 +/- 16.4 versus 111.5 +/- 23.4 min) and exposure (AUC(0-->infinity) 183.9 +/- 20.2 versus 125.8 +/- 26.4 microg/ml x min) of EGCG. Cotreatment with genistein also increased the maximal concentration (C(max)), 6 h exposure (AUC(0-->360 min)), and t(1/2) of EGCG in the small intestine by 2.0-, 4.7- and 1.4-fold, respectively, compared with mice treated with EGCG only. Contrary to our expectations, the combination of 0.01% EGCG in the drinking fluid and 0.2% genistein in the diet enhanced intestinal tumorigenesis in male adenomatous polyposis coli (APC)(min/+) mice. This combination increased the multiplicity of tumors in the medial and distal small intestine: the largest increase was in tumors >2 mm in diameter (4.3-fold compared with controls). This study demonstrates that although genistein can enhance EGCG bioavailability and in vitro growth inhibitory activity, this combination enhances tumorigenesis in the APC(min/+) mouse. These results further show the need for careful cancer prevention studies in animal models and for caution when interpreting data from in vitro studies.

Upregulation of colonic ion channels in APC Min/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in almost all human colonic cancers. Disturbances in Na(+) absorption have been observed in colonic cancer, and ion channels such as ether a go-go (Eag) or Ca(2+)-sensitive BK channels have been recognized for their oncogenic potential. APC ( Min/+ ) mice have reduced APC expression and develop multiple intestinal neoplasias (Min). Ion channels in the colonic epithelium were examined using electrophysiology and molecular techniques. APC ( Min/+ ) mice developed intestinal neoplasia and experienced a significant weight loss. Due to intestinal bleedings, the hematocrit was largely reduced and plasma aldosterone levels were enhanced. Rectal potential measurements in vivo indicated an increase in amiloride-sensitive Na(+) absorption in APC ( Min/+ ) mice. Quantitative Ussing chamber studies demonstrated enhanced Na(+) absorption via epithelial Na(+) channels (ENaC) and suggested enhanced activity of oncogenic BK and Eag-1 channels. Patch clamp and fluorescence measurements on isolated crypts suggested enhanced K(+) channel activity in the surface epithelium. ENaC-mRNA and membrane protein expression was enhanced in colonic surface epithelial cells. The data suggest that reduced expression of the APC gene with upregulation of the downstream proteins Akt and mTOR and subsequent hyperaldosteronism is paralleled by upregulation of oncogenic potassium channels and enhanced colonic Na(+) absorption.

Genetic Enhancement of the Lis1+/– Phenotype by a Heterozygous Mutation in the Adenomatous Polyposis Coli Gene

Hemizygous Lis1 mutations cause type 1 lissencephaly, a neuronal migration disorder in humans. The Lis1+/– mouse is a model for lissencephaly; mice exhibit neuronal migration defects but are viable and fertile. On an inbred genetic background, 20% of Lis1+/– mice develop hydrocephalus and die prematurely. Lis1 functions with the microtubule motor cytoplasmic dynein. Because dynactin, a dynein regulator, interacts with end-binding protein 1 (EB1) and β-catenin, two known binding partners of the adenomatous polyposis coli (APC) protein, we looked for a genetic interaction between Lis1 and APC. Mice with a heterozygous truncating mutation in APC (Min mutation) do not exhibit neuronal migration defects or develop hydrocephalus. However, the presence of the APC mutation increases the migration deficit and the incidence of hydrocephalus in Lis1+/– animals. Lis1 and dynein distribution is altered in cells derived from Min mice, and both Lis1 and dynein interact with the C terminus of APC in vitro. Together, our findings point to a previously unknown interaction between APC and Lis1 during mammalian brain development.