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Estrogen receptors (ERs) [ERα (Esr1) and ERβ (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERα and ERβ is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERα knockout and ApcMin mouse strains, we demonstrate that ERα deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in ApcMin/+ mice. Within the normal intestinal epithelium of ApcMin/+ mice, ERα deficiency is associated with an accumulation of nuclear β-catenin, an indicator of activation of the Wnt–β-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERα deficiency is associated with activation of Wnt–β-catenin signaling, ERα deficiency in the intestinal epithelium of ApcMin/+ mice also correlated with increased expression of Wnt–β-catenin target genes. Through crosses between an ERβ knockout and ApcMin mouse strains, we observed some evidence that ERβ deficiency is associated with an increased incidence of colon tumors in ApcMin/+ mice. This effect of ERβ deficiency does not involve modulation of Wnt–β-catenin signaling. Our studies suggest that ERα and ERβ signaling modulate colorectal carcinogenesis, and ERα does so, at least in part, by regulating the activity of the Wnt–β-catenin pathway.
Colorectal cancer incidence is 35% lower in women than men (1). Although the basis for this difference is unknown, gonadal steroids are considered a contributing factor. In women, high parity, early age at first pregnancy, oral contraceptive use and estrogen replacement therapy are each associated with decreased colorectal cancer risk (2–4). In the Women's Health Initiative study, hormone replacement therapy conferred a 40% reduction in colorectal cancer risk (5). Altogether, these data suggest that estrogens and/or progestins protect against colorectal cancer.
Consistent with the hypothesis that estrogens protect against colorectal cancer, administration of either a synthetic estrogen or the naturally occurring 17β-estradiol (E2) inhibits carcinogen-induced gastrointestinal tumorigenesis in rats (6,7). Similarly, E2 attenuates intestinal tumorigenesis in ApcMin/+ mice (8,9), in which inheritance of the multiple intestinal neoplasia (Min) mutation in the adenomatous polyposis coli (Apc) gene predisposes carriers to spontaneous intestinal tumorigenesis (10,11). The effects of estrogens are mediated by estrogen receptors (ERs) [ERα (Esr1) and ERβ (Esr2)], which function as ligand-dependent transcription factors. Both receptors are expressed in the intestinal epithelium (12–15), but their function in this tissue is not known. Loss of ERα expression due to promoter hypermethylation occurs in colorectal cancers in both men and women (13), suggesting that ERα functions as a tumor suppressor in the colon. ERα promoter hypermethylation occurs in normal colonic epithelium during aging (13,16) and is postulated to create a microenvironment permissive to carcinogenesis. In contrast, loss of ERβ expression correlates with advanced Dukes stages (17).
Some support for the hypothesis that ERα and ERβ impact intestinal tumorigenesis has been obtained from studies using ApcMin/+ mice. In one study, heterozygosity for an ERα knockout allele (Esr1Pcn1) was associated with increased incidence of colon tumors in ApcMin/+ mice (18). The impact of homozygosity for this ERα knockout allele (ERα−/−) on tumorigenesis in ApcMin/+ mice could not be evaluated because no ERα−/− mice carrying the ApcMin/+ mutation were obtained from the intercross. Although two studies have examined the impact of an ERβ knockout allele (Esr2Pcn1) on tumorigenesis in ApcMin/+ mice, the results of these studies are strikingly different. In one study, heterozygosity and homozygosity for the ERβ knockout allele were associated with increased incidence of colon tumors (18). In the second study, this same ERβ knockout allele had no impact on colon tumorigenesis (19). Rather, these investigators observed that homozygosity for the ERβ knockout was associated with an increase in small intestinal tumor number in ApcMin/+ female but not male mice (19). Altogether, these results do support the idea that disruption of ERα and ERβ signaling promotes intestinal tumorigenesis in ApcMin/+ mice. However, because a crucial genotypic class was absent in the ERα study and the two ERβ studies yielded disparate results, the nature of the impact of ERα and ERβ signaling on intestinal tumorigenesis remains largely unknown.
In the present study, we sought to clarify the role of ER signaling in tumorigenesis in the ApcMin/+ mouse by crossing targeted disruptions of ERα and ERβ that are distinct from those used in the previous studies described above with the ApcMin strain. We intercrossed these ERα and ERβ knockout strains with the ApcMin strain and evaluated their impact on intestinal tumorigenesis. The impact of these knockouts on the activity of the Wnt–β-catenin pathway in ApcMin/+ mice was also evaluated. Our data indicate that loss of ERα is associated with increased adenoma multiplicity in ApcMin/+ mice, whereas loss of ERβ increases the incidence of colon tumors in ApcMin/+ mice without significantly altering tumor multiplicity. Loss of ERα but not ERβ is associated with activation of Wnt–β-catenin signaling in the intestinal epithelium of ApcMin/+ mice. These studies suggest that both ERα and ERβ modulate colorectal carcinogenesis, and ERα does so, at least in part, by regulating the Wnt–β-catenin pathway.
The ERα cross and the second ERβ cross were conducted at the University of Nebraska Medical Center. The Institutional Animal Care and Use Committee of the University of Nebraska Medical Center approved all procedures involving live animals in these studies. B6 ApcMin/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The ERα knockout strain (B6.129-Esr1tm1Ksk) (20) was provided by D.Lubahn. The ERβ knockout strain (B6.129-Esr2tm1Ksk) (21) was obtained from Taconic (Hudson, NY). Animals were housed under controlled temperature, humidity and 12 h light–12 h dark lighting conditions in a facility accredited by the American Association for Accreditation of Laboratory Animal Care and operated in accordance with the standards outlined in Guide for the Care and Use of Laboratory Animals (The National Academies Press, 1996). Mice were provided Harlan irradiated rodent diet 7904 (Harlan Teklad, Madison, WI), which contains soy, milk and meat-based protein sources and allowed to feed ad libitum.
B6 females heterozygous for targeted disruption of the ERα gene (ERα+/−) were crossed to B6 ApcMin/+ males. The resulting ERα+/− ApcMin/+ males were crossed to B6 ERα+/− females to produce ERα−/−, ERα+/− and ERα+/+ ApcMin/+ mice. Similar crosses were performed with the ERβ knockout. The ERα and Apc genotypes were determined by polymerase chain reaction (PCR) as described previously (20,22). ERβ genotype was determined by PCR using neo primers (20) and primers that amplify ERβ exon 3 (forward primer 5′-AGAGACCCTGAAGAGGAAGC-3′ and reverse primer 5′-GCTTCTTTTAAAAAAGGCCT-3′) only in the absence of the neo insert.
The first ERβ cross was conducted at the University of Helsinki. The Laboratory Animal Ethics Committee of the Faculty of Agriculture and Forestry, University of Helsinki, approved this study protocol. ApcMin/+ mice on a C57BL/6J (B6) genetic background were obtained from The Jackson Laboratory. Mice from the B6.129-Esr2tm1Ksk strain (21) that was heterozygous for the ERβ knockout (ERβ+/−) mice were serially backcrossed to B6 mice. Mice were genotyped at Apc and ERβ using PCR-based assays as described previously (22,23).
After 10 generations of backcrossing to B6 (N10), ERβ+/− females were intercrossed with ApcMin/+ males to generate ApcMin/+ ERβ+/− mice. Male ApcMin/+ ERβ+/− mice were then bred with female ERβ+/− mice to obtain ApcMin/+ ERβ+/+, ApcMin/+ ERβ+/− and ApcMin/+ ERβ−/− offspring. All mice were fed with Teklad Global 14% Protein Rodent Maintenance Diet 2014 (Harlan Teklad, Horst, The Netherlands), which contains 14% protein, 3.5% oil, 5.5% fiber and no alfalfa or soybean meal. In this diet, the level of natural phytoestrogens is negligible. Animals were housed in plastic cages in a temperature- and humidity-controlled animal facility, with 12 h light–dark cycle. Food and water were provided ad libitum.
Mice were euthanized by CO2 asphyxiation. Intestinal tumors were counted and measured as described previously (10,24–26) except that the small intestine was cut into four segments of equal length and the entire length of these segments, along with the colon, was examined. In Omaha crosses, segments of intestinal tissue fixed in 10% neutral buffered formalin were processed, sectioned and stained with hematoxylin and eosin. Stained sections were examined by an observer blinded with respect to the ER genotype. For intestinal tissue used for extraction of RNA or protein, adenomas were excised under the stereomicroscope at ×10 magnification. The remaining normal epithelium was scraped from the submucosal smooth muscle, frozen in liquid nitrogen and stored at −70°C.
Total RNA was prepared using the Absolutely RNA miniprep kit (Stratagene Corporation, La Jolla, CA). Reverse transcription reactions were performed as described previously (27). For Cyclin D1 and c-Myc complementary DNA, PCR was performed using 900 nM each primer and 200 nM probe. The primer and probes sequences were as follows: 5′-TCCGCAAGCATGCACAGA-3′ (Cyclin D1 forward primer); 5′-CTTTGTGGCCCTCTGTGCCACAGA-3′ (Cyclin D1 probe); 5′-GGTGGGTTGGAAATGAACTTCA-3′ (Cyclin D1 reverse primer); 5′-TGAGCCCCTAGTGCTGCAT-3′ (c-Myc forward primer); 5′-AGGAGACACCGCCCACCACCAG-3′ (c-Myc probe) and 5′-CCTCATCTTCTTGCTCTTCTTCAGA-3′ (c-Myc reverse primer). PCR was performed and analyzed using the ABI 7500 Real-Time PCR System and Sequence Detection Software version 1.9 (Applied Biosystems, Foster City, CA) as described previously (27). Transcript abundance was normalized to glyceraldehyde 3′-phosphate dehydrogenase (Gapdh) (27). Neither the ApcMin mutation nor the ERα deficiency impacted Gapdh expression (data not shown).
E2 and progesterone in unextracted sera collected at killing were measured using a coated tube radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA) as described previously (29). Samples were assayed in duplicate. For ERα+/+ Apc+/+ and ERα+/+ ApcMin/+ mice, E2 measurements were obtained during the follicular phase of estrous and progesterone measurements were obtained during the luteal phase. Phase of estrous was determined based on ovarian histology. ERα−/− females do not cycle normally and are anovulatory; E2 measurements were made in these mice without selection for phase of estrous.
Mucosal scrapings were isolated in ice-cold phosphate-buffered saline supplemented with protease and phosphotase inhibitors. Nuclear and cytoplasmic extracts from fresh tissue were prepared using the Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Whole-cell extracts were prepared from frozen tissue and the same kit using manufacturer's instructions. Western blots were performed as described previously (30) except that the Li-Cor Odyssey Imaging system (Li-Cor, Lincoln, NE) was used. The antibodies used were as follows: E-5 β-catenin and MS-3 nucleolin mouse monoclonal antibodies and I-19 actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin mouse monoclonal antibody, clone B-5-1-2 (Sigma Corporation, St. Louis, MO) and secondary antibodies labeled either with Alexa fluor® 680 (Invitrogen Corporation, Carlsbad, CA) or with IRDye800™ (Rockland Immunochemicals, Gilbertsville, PA). Primary and secondary antibodies were used at a dilution of 1:1000 and 1:2000, respectively.
DNA was isolated from normal intestinal epithelium scrapings. The quantitative PCR assay for the Apc locus was performed and analyzed as described previously (31). For this assay, the Apc+ and ApcMin alleles are amplified and digested with HindIII, resulting in a 123 bp product from the Apc+ allele and a 144 bp product from the ApcMin allele. Radiolabeled products were resolved on a 5% denaturing polyacrylamide gel and quantitated using a Molecular Dynamics PhosphorImager and ImageQuaNT 5.0 software (GE Healthcare, Piscataway, NJ).
Average tumor multiplicity, tumor diameter and tumor burden were compared using the Wilcoxon rank sum test. Differences in the distribution of tumors along the intestine were evaluated with the chi-square test. Tumor incidence data were compared using Fisher's exact test. Differences in gene expression were evaluated using a t-test. All statistical analyses were conducted using SPSS software, version 13 (SPSS, Chicago, IL). P ≤ 0.05 was considered to be significant.
To examine the impact of disrupting ERα in ApcMin/+ mice, we intercrossed the ERα knockout and ApcMin mouse strains. All genotypes of F2 progeny were obtained at the expected frequencies (P > 0.05; data not shown). Mice were killed at 80 days of age. Mean tumor multiplicity in ERα+/+ ApcMin/+ mice was 37.9 (Figure 1A). Mean tumor multiplicity in ERα+/− ApcMin/+ mice was 37.1 and did not differ from that in ERα+/+ ApcMin/+ mice (P > 0.05; Figure 1A). Relative to ERα+/+ ApcMin/+ and ERα+/− ApcMin/+ mice, the mean adenoma multiplicity in ERα−/− ApcMin/+ mice was increased by 50% to 56.4 (P = 0.01; Figure 1A). Gender did not impact tumor multiplicity (Figure 1B; P > 0.05). ERα genotype did not alter the distribution of adenomas along the length of the intestine (Figure 1C; P > 0.05).
To examine the effect of ERα deficiency on tumor size, we focused on small intestinal adenomas because these lesions are flat; therefore, tumor diameter and/or tumor area are used as indicators of tumor size (25). The mean tumor area in ERα+/+ ApcMin/+ mice did not differ significantly from that in ERα+/− ApcMin/+ mice (P > 0.05). In contrast, the average tumor area in ERα−/− ApcMin/+ mice was significantly larger than that in either ERα+/+ or ERα+/− mice (P ≤ 0.001). Combining tumor multiplicity and tumor size data, we calculated tumor burden. The average tumor burden in ERα+/+ ApcMin/+ mice averaged 70 ± 23 mm2. The mean tumor burden in ERα+/− ApcMin/+ mice of 81 ± 14 mm2 did not differ significantly from that in ERα+/+ ApcMin/+ mice (P > 0.05; Figure 1D). In contrast, the mean tumor burden of 143 ± 28 mm2 in ERα−/− ApcMin/+ was significantly greater than that in ERα+/+ and ER+/− mice (P < 0.005; Figure 1D). ERα genotype did not impact adenoma histology in ApcMin/+ mice (Figure 2A and B).
ERα deficiency in female Apc+/+ mice is associated with a 10-fold elevation in serum E2 by 60–120 days of age (32). These observations raised the possibility that ERα deficiency might modulate tumorigenesis via an unknown mechanism driven by high levels of E2. Therefore, we examined serum E2 levels in ERα−/− ApcMin/+ females. We observed that in 80-day-old ERα−/− ApcMin/+ females, the mean serum E2 concentration was 32 ± 7 pg/ml. This value is at the low end of the normal physiological range of E2 in female mice, which varies from 20 to 100 pg/ml depending upon the phase of estrous. Thus, the enhanced intestinal tumorigenesis in female ERα−/− ApcMin/+ mice is not a consequence of high serum E2.
We postulated that ERα−/− ApcMin/+ females might exhibit low levels of serum E2 as a result of deleterious effects of the ApcMin mutation on ovarian function. In the estrus phase, when E2 levels are highest, the mean concentration of serum E2 in 80-day-old ERα+/+ ApcMin/+ mice is <20 pg/ml, the lower limit of detection of the assay used. In contrast with ERα+/+ Apc+/+ females (Figure 2C), ERα+/+ ApcMin/+ females at 80 days of age possess ovaries exhibiting a high degree of follicular degeneration (Figure 2D) and few corpora lutea (Figure 2E; P ≤ 0.05). ERα+/+ ApcMin/+ females also exhibit low-serum progesterone levels (Figure 2F; P ≤ 0.05), indicating reduced ovarian function. Although ovaries in ERα−/− ApcMin/+ mice showed the enlarged, hemorrhagic and cystic follicles (Figure 2G) characteristic of ERα−/− Apc+/+ females [Figure 2H; (32)], these follicles in ERα−/− ApcMin/+ females did not show the hypertrophied theca, indicative of leutinizing hormone stimulation, frequently seen in older ERα−/− Apc+/+ females.
We genotyped our B6.129-Esr1tm1Ksk founders using markers distributed across the genome (Table I) to identify residual 129P2 alleles even though this strain had been backcrossed for 12 generations. In these founders, the only 129P2 alleles detected were at three markers linked to ERα on proximal chromosome 10. The 129P2 alleles at these markers were segregating in the F2 intercross mice (Figure 3). Although 70% of ERα−/− ApcMin/+ mice retained the 129 allele at D10Mit152, the marker most closely linked to ERα, linkage disequilibrium dropped off sharply distal of this marker. Genotype at D10Mit51, the next marker distal of D10Mit152, exhibited poor concordance with ERα genotype. Only 10% of ERα−/− mice were homozygous for the 129P2 allele at D10Mit51, indicating that the 129P2 allele at this marker is not in disequilibrium with the 129P2-derived ERα knockout allele. All mice evaluated were homozygous for the B6 allele at markers including and distal to D10Mit87. Thus, <13 Mbp from the 129P2 genome on chromosome 10 is retained in association with the ERα knockout.
The Wnt–β-catenin pathway is constitutively activated in virtually all colon cancers. Although the impact of estrogen on Wnt–β-catenin activity in the intestine is not known, estrogens do influence this pathway in other tissues (33–36). These data, together with our observations that ERα deficiency enhanced intestinal tumorigenesis, led us to postulate that ERα deficiency might activate Wnt–β-catenin signaling in the intestinal epithelium of ApcMin/+ mice. In whole-cell extracts, β-catenin expression was higher in normal intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4A). Nuclear accumulation of β-catenin, a molecular indicator of activation of Wnt–β-catenin signaling, was also evaluated. Quantitative analysis of these western blots indicated that nuclear β-catenin is increased by, on average, 60% in intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4B), consistent with an increased activation of Wnt–β-catenin signaling in the absence of ERα. Expression of Cyclin D1 and c-Myc, two transcriptional targets of the Wnt–β-catenin pathway, was increased on average 2.2-fold and 2.4-fold, respectively, in the intestinal epithelium of ERα−/− ApcMin/+ mice relative to ERα+/+ ApcMin/+ mice (Figure 4C), lending further support to the hypothesis that ERα deficiency is associated with increased Wnt–β-catenin signaling.
To verify that the normal intestinal epithelium specimens used in these analyses did not contain microadenomas, small precursors of adenomas that have lost wild-type Apc allele (Apc+) and thus exhibit increased nuclear β-catenin (37), we used a quantitative assay to determine the relative abundance of the Apc+ and ApcMin alleles in genomic DNA isolated from these specimens. It has been reported that in histologically normal intestinal epithelium of B6 ApcMin/+ mice, the mean Apc+:ApcMin ratio value was 0.83 ± 0.18 (31). Because microadenomas and adenomas in B6 ApcMin/+ mice exhibit loss of the Apc+ allele (31), contamination of normal tissue with these lesions would reduce the Apc+:ApcMin ratio. Here, normal intestinal epithelium samples from ERα+/+ ApcMin/+ mice yielded a mean Apc+:ApcMin ratio value of 0.78 ± 0.24 (Figure 4D). A mean Apc+:ApcMin ratio value of 0.90 ± 0.21 was obtained from normal intestinal epithelium samples from ERα−/− ApcMin/+ mice (Figure 4D). The mean Apc+:ApcMin ratio values from ERα+/+ ApcMin/+ in mice did not differ significantly from the value ERα−/− ApcMin/+ mice (P>0.05), and both means were consistent with the value reported previously (31). This result indicates that the normal epithelium samples used were not contaminated with microadenomas.
To evaluate the effect of ERβ deletion on tumor formation in ApcMin/+ mice, we intercrossed ERβ+/− mice with ApcMin/+ mice. In this intercross (Helsinki intercross), tumor multiplicity was evaluated at 90 days of age. ERβ genotype did not have a significant impact on the mean number of adenomas in either the entire intestinal tract (data not shown; P≥0.05) or the small intestine alone (Figure 5A; P≥0.05). Because ERβ is thought to play a role during the later stages of colorectal cancer progression, a second set of intercross mice was produced and evaluated at 140 days of age. This second set of intercross mice (Omaha intercross) was performed at a separate institution. However, both intercrosses involved the same ERβ knockout (Esr2Ksk1) and ApcMin/+ alleles, both of which were extensively backcrossed to the B6 strain. Consistent with our observation in the first set of intercross mice, ERβ deficiency did not impact total tumor multiplicity (data not shown; P≥0.05) or small intestinal tumor multiplicity (Figure 5B; P≥0.05) in the second intercross population.
Analysis of the tumorigenesis in the colon indicated that ERβ genotype had no significant effect on the mean number of adenomas in the colon at 90 days of age (Figure 5C; P=0.06–0.20) or 140 days of age (Figure 5D; P≥0.05). In contrast, the 100% (14/14) incidence of colon tumors in 90-day-old ApcMin/+ ERβ−/− mice was greater than the 57% (8/14) incidence in age-matched ApcMin/+ ERβ+/+ mice (Figure 5E; P<0.01) and the 68% (13/19) incidence in age-matched ApcMin/+ ERβ+/− mice (Figure 5E; P<0.05). ERβ had no impact on colon adenoma incidence in the Omaha intercross mice evaluated at 140 days of age (Figure 5F; P≥0.05).
To assess the potential impact of residual 129P2 alleles present in the ERβ intercross populations, we genotyped the B6.129-Esr2tm1Ksk mice used in each study with markers distributed across the genome (Table I) to identify 129P2-derived regions. In the Helsinki intercross, 129P2 alleles were detected at only two markers—one unlinked to ERβ (D1Mit206) and one linked to ERβ (D12Mit91) (Table I). At these two markers, ApcMin/+ ERβ+/+, ApcMin/+ ERβ+/− and ApcMin/+ ERβ−/− intercross mice were either homozygous for the B6 allele or heterozygous. Genotype at these markers had no significant impact on total adenoma number in the intercross mice (data not shown; P=0.3). Likewise, genotype at these markers did not have a significant impact on colon adenoma number (data not shown; P>0.05).
In the Omaha intercross mice, 129P2 alleles were detected at one marker unlinked to ERβ (D3Mit203) and three markers linked to ERβ (D12Mit60, D12Mit91 and D12Mit118) (Table I). Genotype at D3Mit203 had no impact on tumor multiplicity (P>0.05; data not shown). This analysis is straightforward because D3Mit203 and ERβ are unlinked. In contrast, evaluating the potentially confounding effects of 129P2 alleles at markers linked to the ERβ knockout was complicated by the significant linkage disequilibrium. Genotype at ERβ and the three markers proximal of ERβ on chromosome 12 showed a high degree of concordance as follows: 93% for D12Mit91, 84% for D12Mit60 and 62% for D12Mit182. Thus, as much as 77 Mbp of the 129P2 genome on chromosome 12 is retained in association with the ERβ knockout allele (Table I).
Thus, both the Helsinki and the Omaha intercrosses were performed on a congenic B6 background in which only a single marker unlinked to ERβ was still segregating 129P2 alleles. This uniform genetic background facilitates the interpretation of results because the possible impact of modifier loci unlinked to the ERβ mutation can be discounted. However, there were significant differences between the ERβ knockout mice used in these two crosses with respect to markers linked to ERβ. In the Helsinki cross, the degree of linkage disequilibrium between the ERβ knockout and linked 129P2 alleles is quite small, and the potential for linked modifiers is therefore minimal. In contrast, a larger block of 129P2 alleles that was retained in association with the ERβ knockout in the Omaha cross. Although there is no direct evidence supporting the idea that a locus linked to ERβ modifies tumorigenesis in ApcMin/+ mice, our observations prevent us from discounting this possibility.
Because ERβ deficiency increased colon tumor incidence, we examined the impact of ERβ deficiency on β-catenin expression and localization, the expression of Wnt–β-catenin target genes including Cyclin D1 and the interaction of β-catenin with the cell adhesion molecule E-cadherin. We used western blotting to analyze the levels of β-catenin, E-cadherin and Cyclin D1 in the nuclear and membrane fraction of the colonic mucosa of ApcMin/+ ERβ+/+ and ApcMin/+ ERβ−/− mice. Additionally, we analyzed the impact of ERβ deficiency on the expression of the ERα. Deletion of ERβ had no effect on the expression of the nuclear β-catenin or Cyclin D1 or membrane β-catenin or E-cadherin (supplementary Figure 1 is available at Carcinogenesis Online). Furthermore, the expression of ERα protein was unaffected (supplementary Figure 1 available at Carcinogenesis Online). Thus, we found no evidence for ERβ-dependent modulation of the Wnt pathway.
In the present study, ERβ deficiency did not increase adenoma multiplicity in ApcMin/+ mice. This lack of effect of ERβ deficiency on total tumor multiplicity or small intestinal tumor multiplicity was confirmed in a second, independent cross involving the same ERβ knockout allele (Esr2Ksk1) and ApcMin/+ mice. This second cross was conducted at a separate institution using distinct husbandry conditions. A similar lack of effect of ERβ deficiency on small intestinal tumorigenesis in ApcMin/+ mice was also reported by Cho et al. (18) in a study using a distinct ERβ knockout allele (Esr2Pcn1) (38). These three observations differ from that of Giroux et al. (19), in which it is reported that deficiency of the Esr2Pcn1 ERβ knockout allele increased tumor multiplicity and size in female but not male ApcMin/+ mice. However, it should be noted that in the study of Giroux et al., female mice had been ovariectomized and implanted with E2 and progesterone pellets that result in continuous serum levels of these hormones in the high physiological range. In the two intercrosses we performed, as well as the study of Cho et al., ovary intact females were used. Consistent with the ovarian defects described previously in ApcMin/+ females (39), we observe that ApcMin/+ females have reduced serum levels of E2 and progesterone. Thus, there are significant differences in hormone levels between mice in these various studies. These differences may contribute to the disparate results observed.
In the present study, we observed that ERβ deficiency was associated with an increased incidence of colon tumors in ApcMin/+ mice. Although our results differ from those described in Giroux et al. (19), in which the Esr2Pcn1 ERβ knockout allele was found to have no impact on colon tumorigenesis in ApcMin/+ mice, our data are consistent with the study of Cho et al. (18), which reported that homozygosity for the Esr2Pcn1 ERβ knockout allele increased colon tumor incidence in ApcMin/+ mice. In our study, we found that the increased incidence in colon tumors in ERβ-deficient ApcMin/+ mice was not associated with enhanced activation of the Wnt–β-catenin pathway. These observations are consistent with a previous report (19) suggesting that ERβ deficiency may promote intestinal tumorigenesis by modulating the transforming growth factor β pathway. Thus, our present studies using ApcMin/+ mice and a distinct ERβ knockout (Esr2Ksk1) help resolve the controversy regarding the impact of ERβ on colon tumorigenesis and provide independent support for the hypothesis that ERβ deficiency may promote tumorigenesis through a Wnt–β-catenin-independent pathway.
We also observed that genetic disruption of ERα in ApcMin/+ mice was associated with an increase in intestinal adenoma multiplicity, size and burden. These data are consistent with the hypothesis that loss of ERα expression contributes to intestinal tumorigenesis. Although the ERα knockout used here is a hypomorph, resulting in a truncated receptor (40) with a deleted amino-terminus and no activation function 1 domain but with some residual ligand binding and/or function, it is clear that disruption of ERα, even if incomplete, is associated with enhanced tumorigenesis ApcMin mice. It is noteworthy that when Cho et al. (18) crossed another ERα knockout (B6.129-Esr1Pcn1), one reported to be a more complete disruption of ERα, with ApcMin/+ mice, no ERα−/− ApcMin/+ mice were recovered. One intriguing interpretation of this result is that there is a synthetic lethal interaction between the ERα allele and the ApcMin mutation. In our study, ERα−/− ApcMin/+ mice were recovered at the expected frequency.
Based on data from humans implicating ERα as a tumor suppressor in colon, we conclude that ERα deficiency itself is responsible for the enhanced tumorigenesis in ERα−/− ApcMin/+ mice. However, it is impossible to completely exclude the possibility that the enhanced tumorigenesis in ERα−/− ApcMin/+ mice is due to another allele that resides within the 129P2-derived region of <13 Mbp on chromosome 10 that is retained in association with the ERα knockout allele. Less than a dozen genes map to this region, and none of these is known to modulate tumorigenesis in ApcMin/+ mice. The Scc14 locus, which modulates susceptibility to azoxymethane-induced colon cancer in a cross between the BALB/c and the CcS19/Dem strains, is linked to a marker 3.4 Mbp proximal of ERα (41). Nothing is known about the impact of the 129P2 allele of Scc14 on colon cancer. Assuming that ERα and Scc14 represent distinct genes, we cannot exclude the possibility that the 129P2 allele of Scc14 increases tumor multiplicity in our cross. However, our previous studies provide no evidence that 129 substrains carry alleles that enhance tumorigenesis in ApcMin/+ mice. Rather, we have shown that the 129S2/SvPas substrain carries alleles that reduce tumor multiplicity in ApcMin/+ mice (27). Additionally, in Oikarinen et al., in this issue of Carcinogenesis, we report the localization of Mom5, a modifier of tumorigenesis in ApcMin/+ mice; the 129P2 allele of Mom5 is associated with reduced tumor number.
The impact of ERα deficiency was equivalent in ApcMin/+ males and females. These results are consistent with the fact that ERα expression is silenced in colorectal cancers in both men and women (13,42). Although ERα signaling reduces intestinal tumorigenesis in both male and female ApcMin/+ mice, it is unclear whether this effect requires endogenous estrogens or is ligand independent. ApcMin/+ females in our colony exhibit diminished ovarian function and low serum concentrations of E2 and progesterone. Although the physiological basis for the impact of the Min mutation of ovarian function is unknown, these observations, together with our observation that tumorigenesis in ApcMin/+ females is not influenced by ovariectomy (K.A.Gould, unpublished data), suggest that in our colony, ovarian estrogens do not play a role in ERα-dependent processes that attenuate tumorigenesis in ApcMin/+ mice.
We report that ERα deficiency in ApcMin/+ mice is associated with an accumulation of nuclear β-catenin, suggesting that ERα deficiency activates canonical Wnt–β-catenin signaling. The level of β-catenin in normal intestinal epithelium of ERα−/− ApcMin/+ mice is less than that in ApcMin-induced intestinal adenomas (K.A.Gould, unpublished data), suggesting that further activation of Wnt–β-catenin signaling occurs during tumorigenesis, probably as a consequence of loss of Apc+, which occurs at a frequency of 100% in adenomas of B6 ApcMin/+ mice (11,31). Consistent with the hypothesis that ERα deficiency enhances Wnt–β-catenin signaling, the expression of the Wnt–β-catenin target genes Cyclin D1 and c-Myc was increased in the normal intestinal epithelium of ERα−/− ApcMin/+ mice. The expression of Cyclin D1 and c-Myc is regulated by many factors, including estrogens (43,44). Thus, it is possible that the increased expression of Cyclin D1 and c-Myc in ERα−/− ApcMin/+ epithelium is unrelated to Wnt–β-catenin activation. Nonetheless, the upregulation of these genes, which are known to play a role in colorectal cancer, indicates that ERα deficiency in ApcMin/+ mice is associated with increased expression of genes involved in intestinal tumorigenesis.
Although it is known that there is cross talk between the estrogen and the Wnt pathways in estrogen-responsive tissues such as the mammary gland (33), further studies will be required to understand the molecular basis and significance of cross talk between ERα and Wnt signaling in the intestinal epithelium. In contrast to what is observed in the mammary gland, our studies suggest that Wnt and estrogen signaling have opposing effects in intestine, with Wnt signaling promoting tumorigenesis and ERα signaling inhibiting tumorigenesis.
Nebraska Department of Health and Human Services; National Institutes of Health (K01-CA113413 to K.A.G.).
Conflict of Interest Statement: None declared.