The role of NO synthases in arginine-dependent small intestinal and colonic carcinogenesis

Arginine is catabolized by NOS2 and other nitric oxide synthases to form nitric oxide. We evaluated the roles of dietary arginine and Nos2 in Apc-dependent intestinal tumorigenesis in Min mice with and without a functional Nos2 gene. NOS2 protein was expressed only in intestinal tissues of Apc(Min/+) Nos2+/+ mice. NOS3 expression was higher in intestinal tissues of mice lacking Nos2, mainly in the small intestine. When diet was supplemented with arginine (0.2% and 2% in drinking water), lack of Nos2 results in decreased tumorigenesis in both small intestine and colon. In Nos2 knockout mice, supplemental arginine (up to 2%) caused a decrease in small intestinal tumor number and size. The arginine-dependent decrease was associated with an increase in nitrotyrosine formation and apoptosis in the region of intestinal stem cells. Mice expressing Nos2 did not show these changes. These mice did, however, show an arginine-dependent increase in colon tumor number and incidence, while no effect on apoptosis was seen. These changes were associated with increased nitrotyrosine formation in epithelial cells. Mice lacking Nos2 did not show changes in tumorigenesis or nitrotyrosine formation, while demonstrating an arginine-dependent increase in apoptosis. These data suggest that Nos2 and dietary arginine have significant effects on intestinal and colonic tumorigenesis in Min mice. In both tissues, loss of Nos2 is associated with decreased tumorigenesis when mice are supplemented with dietary arginine. In the small intestine, Nos2 prevents the arginine-induced decrease in tumor number and size, which is associated with NOS3 expression and increased apoptosis. In the colon, Nos2 is required for the arginine-induced increase in tumor number and incidence.     

APC-dependent changes in expression of genes influencing polyamine metabolism, and consequences for gastrointestinal carcinogenesis, in the Min mouse.

The colorectal mucosa of pre-symptomatic individuals with familial adenomatous polyposis (FAP) contains elevated levels of the proliferation-associated polyamines. The Min mouse, like humans with FAP, expresses an abnormal genotype for the APC tumor suppressor gene. In order to determine how APC mutation influences intestinal tissue polyamine content, we measured steady-state RNA levels of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, antizyme (AZ), a protein which negatively regulates ODC, and the spermidine/spermine N(1)-acetyltransferase (SSAT), the first enzyme in polyamine catabolism. RNA content was increased 6- to 8-fold in both the small intestine and colon for ODC, decreased significantly in the small intestine but not the colon for AZ and was not statistically different in either intestinal tissue for SSAT in Min mice compared with normal littermates. Consistent with the changes in ODC and AZ gene expression, small intestinal, but not colonic, polyamine content was elevated in Min mice compared with normal littermates. Treatment of Min mice with the specific ODC inhibitor difluoromethylornithine (DFMO) suppressed small intestinal, but not colonic, polyamine content and tumor number. These data indicate that small intestinal tissue polyamine content is elevated in Min mice by a mechanism involving APC-dependent changes in ODC and AZ RNA. Further, ODC enzyme activity, which is influenced by both ODC and AZ RNA levels and inhibited by DFMO, is consequential for small intestinal tumorigenesis in this model. In the FAP population, DFMO may be of value in the chemoprevention of small intestinal adenocarcinoma that remains a risk following colectomy.     

Spermine synthase overexpression in vivo does not increase susceptibility to DMBA/TPA skin carcinogenesis or Min-Apc intestinal tumorigenesis

Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in Apc (Min) (/+) (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N ( 1) -acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss.     

Risk and risk reduction involving arginine intake and meat consumption in colorectal tumorigenesis and survival

Elevated polyamine and nitric oxide levels (both derived from arginine) promote tumorigenesis, whereas non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal cancer (CRC) incidence in experimental and epidemiologic studies. We investigated dietary arginine-induced intestinal tumorigenesis and NSAID-inhibitory effects in Apc(Min/+) mice differentially expressing nitric oxide synthase-2 (Nos2). We also studied effects of estimated arginine exposures through meat consumption on tumor characteristics and survival in human CRC cases. Dietary arginine increased high-grade colon adenoma incidence in Apc(Min/+)Nos2(+/+) mice, but not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N(1)-acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (p = 0.04) and decreased tumor number in the small intestines of Apc(Min/+)Nos2(+/+) mice (p = 0.0003). Five hundred and eleven cases from our NCI-supported CRC gene-environment study were analyzed based on self-reported meat (as a surrogate for arginine) consumption. Familial CRC cases (n = 144) in the highest meat consumption quartile (Q4) had no statistically significant differences in tumor grade compared to cases in Q1-Q3 (p = 0.32); however, they were observed to have decreased overall survival (OS) (10-year OS = 42% vs. 65%; p = 0.017), and increased risk of death in an adjusted analysis (hazards ratio [HR] = 2.24; p = 0.007). No differences in tumor grade, OS or adjusted HR were observed for sporadic CRC cases (n = 367) based on meat consumption. Our results suggest important roles for arginine and meat consumption in CRC pathogenesis, and have implications for CRC prevention.     

Intestinal-specific PPARgamma deficiency enhances tumorigenesis in ApcMin/+ mice

Multiple investigations of the effects of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on colon cancer have produced contradictory results. While some studies demonstrated increased numbers of colonic polyps in Apc(Min/+) mice treated with various thiazolidinedione (TZD) PPARgamma ligands, others reported amelioration of tumor multiplicity and progression in both Apc(Min/+) mice and in mice with chemically-induced colon cancer. Here, we addressed the role of PPARgamma in murine intestinal tumorigenesis using gene knockout methodology. We found that either heterozygous or homozygous intestinal-specific PPARgamma deficiency enhanced the number of Apc(Min/+) tumors in both the small intestine and colon, especially in the colon, where PPARgamma deficiency also modulated tumor incidence. Gender significantly affected tumor multiplicity independent of PPARgamma genotype. Female Apc(Min/+) mice developed more tumors in the small intestine and more tumors overall, whereas male Apc(Min/+) mice developed more tumors in the colon. Nevertheless, intestinal PPARgamma deficiency enhanced tumorigenesis irrespective of gender. Our results suggest that PPARgamma functions as a tumor resistance factor in the mouse intestine and warrant further investigation of the PPARgamma-dependent and independent actions of TZDs in cancer.     

Microadenomatous lesions involving loss of Apc heterozygosity in the colon of adult Apc(Min/+) mice

Mutations in the human adenomatous polyposis coli (APC) gene are causative for familial adenomatous polyposis (FAP), a rare condition in which numerous colonic polyps arise during puberty and, if left untreated, lead to colon cancer. Mouse model for human FAP, Apc(Min/+) mouse, contains a truncating mutation in the Apc gene and spontaneously develops intestinal adenomas. However, the distribution of tumors along the intestine found in Apc(Min/+) mice is different from that in human FAP. A majority of intestinal polyps in the Apc(Min/+) mice is known to be located in the small intestine but rarely detected in the colon. We report here that adult Apc(Min/+) mice develop dozens of microadenomatous lesions in the colon (>20 lesions/colon). Surprisingly, the vast majority of such adenomatous lesions consisting of colonic crypts were <300 microm in their greatest dimension, whereas lesions in the small intestine showed various sizes. The allelic loss analysis revealed that these adenomatous crypts in the colon have already lost the remaining allele of Apc. Such findings suggest that, in contrast to tumorigenesis in the small intestine, loss of heterozygosity of the Apc gene is not sufficient for tumor development in the colon of Apc(Min/+) mice. Our results may give an account for the low incidence of colonic tumors in Apc(Min/+) mice.     

Interaction of Muc2 and Apc on Wnt signaling and in intestinal tumorigenesis: potential role of chronic inflammation

Somatic mutations of the adenomatous polyposis coli (APC) gene are initiating events in the majority of sporadic colon cancers. A common characteristic of such tumors is reduction in the number of goblet cells that produce the mucin MUC2, the principal component of intestinal mucus. Consistent with these observations, we showed that Muc2 deficiency results in the spontaneous development of tumors along the entire gastrointestinal tract, independently of deregulated Wnt signaling. To dissect the complex interaction between Muc2 and Apc in intestinal tumorigenesis and to elucidate the mechanisms of tumor formation in Muc2(-/-) mice, we crossed the Muc2(-/-) mouse with two mouse models, Apc(1638N/+) and Apc(Min/+), each of which carries an inactivated Apc allele. The introduction of mutant Muc2 into Apc(1638N/+) and Apc(Min/+) mice greatly increased transformation induced by the Apc mutation and significantly shifted tumor development toward the colon as a function of Muc2 gene dosage. Furthermore, we showed that in compound double mutant mice, deregulation of Wnt signaling was the dominant mechanism of tumor formation. The increased tumor burden in the distal colon of Muc2/Apc double mutant mice was similar to the phenotype observed in Apc(Min/+) mice that are challenged to mount an inflammatory response, and consistent with this, gene expression profiles of epithelial cells from flat mucosa of Muc2-deficient mice suggested that Muc2 deficiency was associated with low levels of subclinical chronic inflammation. We hypothesize that Muc2(-/-) tumors develop through an inflammation-related pathway that is distinct from and can complement mechanisms of tumorigenesis in Apc(+/-) mice.     

Lack of guanylyl cyclase C, the receptor for Escherichia coli heat-stable enterotoxin, results in reduced polyp formation and increased apoptosis in the multiple intestinal neoplasia (Min) mouse model

Guanylyl cyclase C (GC-C), a transmembrane receptor for bacterial heat-stable enterotoxin and the mammalian peptides guanylin and uroguanylin, mediates intestinal ion secretion and affects intestinal cell growth via cyclic GMP signaling. In intestinal tumors, GC-C expression is maintained while guanylin and uroguanylin expression is lost, suggesting a role for GC-C activation in tumor formation or growth. We show by in situ hybridization that GC-C expression is retained in adenomas from multiple intestinal neoplasia (Apc(Min/+)) mice. In order to determine the in vivo role of GC-C in intestinal tumorigenesis, we generated Apc(Min/+) mice homozygous for a targeted deletion of the gene encoding GC-C and hypothesized that these mice would have increased tumor multiplicity and size compared to wild-type Apc(Min/+) mice on the same genetic background. In contrast, the absence of GC-C resulted in a reduction of median polyp number by 55%. There was no change in the median diameter of polyps, suggesting no effect on tumor growth. Somatic loss of the wild-type Apc allele, an initiating event in intestinal tumorigenesis, also occurred in polyps from GC-C-deficient Apc(Min/+) mice. We have found increased levels of apoptosis as well as increased caspase-3 and caspase-7 gene expression in the intestines of GC-C-deficient Apc(Min/+) mice compared with Apc(Min/+) mice. We propose that these alterations are a possible compensatory mechanism by which loss of GC-C signaling also affects tumorigenesis.     

Multistep carcinogenesis of the colon in Apc(Min/+) mouse

Colon cancer arises through different histological stages representing different genetic and epigenetic alterations. The Apc(Min/+) mouse has a point mutation at the Apc gene, and it is considered to be a model for human familial adenomatous polyposis. Our previous studies have revealed the presence of a number of intramucosal microadenomas in the colons of Apc(Min/+) mice, in which only a few macroscopic tumors were recognized. These observations suggest that there are two distinct stages for colon carcinogenesis in Apc(Min/+) mouse, and the Apc(Min/+) mouse is regarded as a good model to study multistage colon carcinogenesis. A number of genes that modify intestinal tumorigenesis have been identified using Apc mutant mice combined with other mutant mice. It has become apparent that epigenetic modification strongly affects intestinal tumorigenesis in Apc(Min/+) mice. We herein describe the different stages of colon tumorigenesis and their modifiers, and discuss the possible application of Apc mutant mice in order to better understand the molecular mechanisms of multistage carcinogenesis in the large bowel of humans.     

Impact of Dnmt1 deficiency,with and without low folate diets,on tumor numbers and DNA methylation in Min mice

Although a number of studies have suggested that diets with low intake of folate, an important methyl donor, are associated with increased risks of colon cancer and its precursor the adenomatous polyp, the underlying mechanisms are poorly understood. Dysregulation and instability of DNA methylation and alterations in the levels of the predominant DNA methylating enzyme, DNA (cytosine-5)-methyltransferase 1 (Dnmt1), have also been linked to tumorigenesis. We have used a combination of genetic and dietary manipulation to assess the effects of reduced Dnmt1 expression with and without folate deficiency on tumor induction in the Apc(Min) mouse. Apc(Min) mice with a reduction in Dnmt1 expression (Apc(Min/+)/Dnmt1(C/+)) had significantly lower tumor numbers than Apc(Min) mice with normal Dnmt1 (Apc(Min/+)/Dnmt1(+/+)). Dietary folate deficiency from weaning to 13 weeks of age did not affect tumor number or size in Apc(Min/+)/Dnmt(+/+) mice. However, in Apc(Min/+)/Dnmt1(C/+) mice with high baseline tumor numbers (41 +/- 4), folate deficiency was associated with a decreased absolute number of tumors (27 +/- 3), but a higher proportion of larger tumors as compared with mice on the control diet. In the repeat experiment, Apc(Min/+)/Dnmt1(C/+) mice had low baseline tumor numbers (20 +/- 2) and folate deficiency did not affect tumor number (23 +/- 4) or size as compared with the same mice on the control diet. These results suggest that, in the presence of Dnmt1 deficiency, the effects of folate deficiency on tumor number and size may depend on the stage of adenoma development when folate deficiency is initiated. We also show that folate deficiency with or without reductions in Dnmt1 did not affect overall genomic DNA methylation or the methylation levels of two candidate genes, E-cadherin or p53, in normal or neoplastic intestinal tissue. In conclusion, genetic deficiency in Dnmt1 with or without folate deficiency decreases tumor number in the Apc(Min) mouse model, but this effect may not be mediated by changes in SAM or SAH levels, nor by alterations in global methylation in the pre-neoplastic intestinal tissue.     

Peroxisome proliferator-activated receptor gamma agonist troglitazone induces colon tumors in normal C57BL/6J mice and enhances colonic carcinogenesis in Apc1638 N/+ Mlh1+/- double mutant mice

The role of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in colon tumorigenesis remains controversial. Notwithstanding evidence that PPAR-gamma ligands impede murine colorectal carcinogenesis, PPAR-gamma agonists have been shown to enhance in vivo tumor formation in mouse models of human colon cancer. Our study was designed to determine whether troglitazone (TGZ) induces colonic tumor formation in normal C57BL/6J mice and enhances colorectal carcinogenesis in double mutant Apc1638N/+ Mlh1+/- mice fed a standard AIN-76A diet. We report herein that not only does TGZ enhance carcinogenesis in the large intestine of mutant mice predisposed to intestinal carcinogenesis but TGZ also induces colonic tumors in normal mice without gene targeting or carcinogen administration. This observation indicates that preexisting mutational events are not necessary for induction of colonic tumors by activated PPAR-gamma in vivo.     

beta-Catenin-accumulated crypts in the colonic mucosa of juvenile ApcMin/+ mice

Although Apc(Min/+) mice are widely used for an animal model of human familial adenomatous polyposis (FAP), a majority of intestinal polyps locate in the small intestine. We recently reported that numerous beta-catenin-accumulated crypts (BCAC), which are reliable precursor lesions for colonic adenocarcinoma, develop in the large bowel of aged Apc(Min/+) mice. In this study, we determined the presence and location of BCAC in the large intestine of juvenile Apc(Min/+) mice (3 and 5 weeks of age). Surprisingly, BCAC were noted in the colon of even Apc(Min/+) mice of both ages, and mainly located in the distal and middle segments of the colon. Also, a few microadenomas were detected in Apc(Min/+) mice of 5-week old. Our results may indicate need of further investigation of the colorectal mucosa of Apc(Min/+) mice for examining colorectal carcinogenesis using Apc(Min/+) mice.     

Chemopreventive efficacy of combined piroxicam and difluoromethylornithine treatment of Apc mutant Min mouse adenomas, and selective toxicity against Apc mutant embryos

Genetic knockout or pharmacological inhibition of cyclooxygenase-2 decreases the number and size of adenomas in mouse models of familial adenomatous polyposis. Epidemiological and clinical studies in humans indicate that the entire class of nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both COX-1 and COX-2 enzymes are promising colon cancer chemopreventive agents. We used the Apc mutant Min mouse model to test combinations of agents that might maximize preventive benefit with minimal toxicity because they act via different mechanisms. Min mice (n = 144) were exposed to low doses of the nonselective COX inhibitor piroxicam and the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO), beginning at the time they were weaned and continuing throughout the duration of the experiment. Piroxicam at 12, 25, and 50 ppm in the diet caused dose-dependent decreases in the number of tumors in the middle and distal portions of the small intestine. This decrease in tumor multiplicity was associated with a striking decrease in the size of those tumors that did grow out. In contrast, none of the doses of piroxicam alone decreased tumor multiplicity in the proximal portion of the intestine (duodenum). Exposure to DFMO (0.5 or 1.0% in water) caused a dose-dependent decrease in tumor multiplicity in the middle and distal portions of the small intestine. However, this decreased multiplicity was not associated with a striking decrease in the size of the tumors. Combined treatment of mice with piroxicam plus DFMO was much more effective than either agent alone and resulted in a significant number of mice totally free of any intestinal adenomas (P < 0.001), in contrast to the 100% incidence and high multiplicity in control Min mice. In addition to this profound effectiveness in reducing tumor number, the few residual tumors in mice treated with the combined drugs were markedly smaller in size than tumors that arose from control Min mice. These experiments suggest that selective COX-2 inhibition combined with ODC inhibition is a very promising approach for colon cancer prevention. These COX-2 and ODC inhibitor drugs were not overtly toxic at the doses used when administered to mice after weaning. However, when treatment was begun in utero, the Mendelian expected progeny ratio of 1:1 that we routinely obtained in untreated control litters was no longer observed. Apc(min)/+ progeny of pregnant dams treated with piroxicam and/or DFMO were reduced in number and their ratio to Apc+/+ progeny was decreased to approximately 0.28:1. Thus, these agents are effective against adenomas that have homozygous mutation of the APC gene and also select against fetuses bearing a heterozygous mutation in the APC gene.     

Increased survival of L1210 leukemic mice by prevention of the utilization of extracellular polyamines. Studies using a polyamine-uptake mutant, antibiotics and a polyamine-deficient diet.

When L1210 leukemia cells are inhibited in their polyamine synthesis by treatment with alpha-difluoromethylornithine (DFMO), their growth in culture is strongly suppressed. In striking contrast, the survival of L1210 leukemic mice is only marginally prolonged by DFMO treatment. This inconsistency is due to the fact, that in the mouse the tumor cells can utilize extracellular polyamines to compensate for the decrease in putrescine and spermidine synthesis caused by DFMO treatment. In the present study, we demonstrate that a reduction in the transport of polyamines into the tumor cells is a more effective means of increasing the therapeutic effect of DFMO than is a reduction in the supply of extracellular polyamines. DFMO treatment cured 30-75% of leukemic mice bearing mutant L1210-MGBGr cells deficient in polyamine uptake, but only slightly increased the survival time of leukemic mice bearing the parental L1210 cells despite the fact that the supply of extracellular polyamines was reduced (by feeding the mice a polyamine-deficient diet containing antibiotics). The effectiveness by which DFMO cured leukemic mice bearing L1210-MGBGr cells appeared to be sex dependent. Thus, 58% of the female mice, as compared to 30% of the male mice, were cured by DFMO treatment.     

Potent modulation of intestinal tumorigenesis in Apcmin/+ mice by the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.     

Polyethylene glycol inhibits intestinal neoplasia and induces epithelial apoptosis in Apc(min) mice

Efficacy of a safe and clinically utilized polyethylene glycol formulation (PEG-3350) to suppress intestinal tumors was investigated in the Apc(min) mouse-model of experimental carcinogenesis. Furthermore, based on our previous finding on the induction of apoptosis in HT-29 cells by PEG, we evaluated its ability to stimulate epithelial cell apoptosis in both Apc(min) mouse as well as AOM-treated rat as a potential molecular mechanism of chemoprevention. Twenty-two Apc(min) mice were randomized equally to PEG or vehicle (control) supplementation. Tumors were scored and uninvolved intestinal mucosal apoptosis was assayed using a modified terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay and by immunohistochemical detection of cleaved caspase-3. Supplementation of Apc(min) mice with 10% PEG 3350 (in drinking water) resulted in a 48% (P<0.05) reduction in intestinal tumor burden and induced 2-3 fold increase in mucosal apoptosis. Dietary supplementation of polyethylene glycol (5%) also stimulated colonic mucosal apoptosis 4-5 fold in AOM-treated rats, the regimen that we previously reported to reduce tumor burden by 76% (P<0.05). In summary, we demonstrate, for the first time, that PEG does protect against Apc(min) mouse tumorigenesis. The correlation between pro-apoptotic actions and chemopreventive efficacy of PEG in these models strongly implicates induction of apoptosis as one of the impending mechanisms of chemoprevention.     

Estrogen receptors alpha and beta are inhibitory modifiers of Apc-dependent tumorigenesis in the proximal colon of Min/+ mice

Estrogen replacement therapy in postmenopausal women is associated with a reduction in colorectal cancer risk, potentially via interactions between 17beta-estradiol (E(2)) and the estrogen receptors (ER) alpha and beta. To study the role of E(2) in intestinal tumor inhibition, we separately crossed C57BL/6J-Min/+ (Min/+) mice with Eralpha(+/-) and Erbeta(+/-) mice to generate ER-deficient Min/+ progeny. We found an increased incidence of visible colon tumors and dysplastic microadenomas in ER-deficient Min/+ relative to Er(+/+)Min/+ controls. Small intestinal tumor numbers were unaffected. Invasive carcinomas were found only in Eralpha(+/-)Min/+ mice, suggesting that ERalpha plays additional non-cell autonomous roles that limit tumor progression. Histologic analyses of ER-deficient Min/+ colons, as well as colons from ovariectomized Min/+ mice (OvxMin/+) and E(2)-treated OvxMin/+ mice (OvxMin/+ +E(2)), revealed significant differences in crypt architecture, enterocyte proliferation, and goblet cell differentiation relative to Min/+ and Er(+/+)Apc(+/+) (wild-type) controls. The expression of ERalpha and ERbeta was regionally compartmentalized along the colonic crypt axis, suggesting functional antagonism. Our results indicate that ERalpha and ERbeta are inhibitory modifiers of Apc-dependent colon tumorigenesis. As a result, loss of E(2) and ER signaling in postmenopausal women may contribute to colorectal cancer development.