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Cancer Research 67, 2366-2372, March 1, 2007. doi: 10.1158/0008-5472.CAN-06-3026
© 2007 American Association for Cancer Research
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Epidemiology and Prevention

Estrogen Receptors {alpha} and ß Are Inhibitory Modifiers of Apc-Dependent Tumorigenesis in the Proximal Colon of Min/+ Mice

Nancy L. Cho1, Sara H. Javid1, Adelaide M. Carothers1, Mark Redston2 and Monica M. Bertagnolli1

Departments of 1 Surgery and 2 Pathology, Brigham and Women's Hospital, Boston, Massachusetts

Requests for reprints: Monica M. Bertagnolli, Department of Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. Phone: 617-732-8991; Fax: 617-582-6177; E-mail: mbertagnolli{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen replacement therapy in postmenopausal women is associated with a reduction in colorectal cancer risk, potentially via interactions between 17ß-estradiol (E2) and the estrogen receptors (ER) {alpha} and ß. To study the role of E2 in intestinal tumor inhibition, we separately crossed C57BL/6J-Min/+ (Min/+) mice with Er{alpha}+/– and Erß+/– 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 Er{alpha}+/–Min/+ mice, suggesting that ER{alpha} 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 E2-treated OvxMin/+ mice (OvxMin/+ +E2), revealed significant differences in crypt architecture, enterocyte proliferation, and goblet cell differentiation relative to Min/+ and Er+/+Apc+/+ (wild-type) controls. The expression of ER{alpha} and ERß was regionally compartmentalized along the colonic crypt axis, suggesting functional antagonism. Our results indicate that ER{alpha} and ERß are inhibitory modifiers of Apc-dependent colon tumorigenesis. As a result, loss of E2 and ER signaling in postmenopausal women may contribute to colorectal cancer development. [Cancer Res 2007;67(5):2366–72]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signaling by 17ß-estradiol (E2) results from the binding of this steroid hormone to two nuclear receptors, estrogen receptor (ER) {alpha} or ß (1). Little is currently known about the role of E2 signaling in the colon, but circumstantial evidence suggests a causal association between the loss of E2 in women after menopause and colorectal cancer (2). Annual age-adjusted colorectal cancer incidence rates in the United States are higher for men than for women (3), and there are gender differences regarding cancer location and type within the colorectum. Women have an excess of right-sided cancers at all ages relative to men (4). Colon cancers in this location more commonly show high levels of methylation at CpG islands (CIMP+), a modification that silences tumor-suppressor genes (5). Methylation-associated silencing is responsible for defects in DNA repair capability due to loss of MLH-1, a condition that accelerates tumor progression. Promoter methylation also disrupts the function of the gene encoding ER{alpha} (5, 6). Finally, data from prospective randomized trials showed that hormone replacement therapy reduced the risk of colorectal cancer in postmenopausal women by 30% to 40% (7). Hormone replacement therapy also conferred protection against the incidence and size of adenomas, the colorectal cancer precursor lesions. Taken together, these data indicate that ER-mediated signaling plays a role in colorectal cancer prevention.

ER{alpha} and ERß affect growth and differentiation by regulating gene transcription. These receptors share conserved DNA and ligand-binding domains, and both activate and repress target gene expression when homodimerized or heterodimerized and bound to the estrogen-response element, their cognate promoter site (1). Estrogen receptors are widely distributed throughout the body, and tissues that simultaneously express both ER{alpha} and ERß exhibit cell type–specific patterns of expression (8). For instance, only ER{alpha} enhances the transactivation of other transcription factors that are associated with cell proliferation, such as activator protein-1 and Sp1 (9). ERß negatively regulates the E2-dependent activities of ER{alpha} that stimulate cell growth. When coexpressed with ER{alpha}, ERß caused a concentration-dependent reduction in ER{alpha}-mediated transcription, suggesting that ERß acts as a dominant regulator of E2 signaling (10, 11). ERß may inhibit colorectal carcinogenesis because its expression suppressed the growth of colon cancer cells and was frequently lost in colon tumors (12, 13).

The C57BL/6-Min/+ mouse carries a germ line mutation in the Apc tumor-suppressor gene and is a model for studying early events in intestinal tumorigenesis and chemoprevention. Min/+ mice develop dozens of adenomas in the small intestine and have an average life span of 16 to 24 weeks. Unlike humans carrying Apc mutations, macroscopic tumors in the colon of Min/+ mice are rare. In previous studies, we showed that E2 suppresses intestinal tumor formation in the Min/+ model. We found that E2 loss produced by ovariectomy of 5-week-old Min/+ mice caused a 77% increase in the small intestinal tumor burden by 3 months of age and that E2 replacement inhibited tumors to the basal number found in age-matched intact Min/+ females (14). We also reported that dietary treatment of ovariectomized Min/+ (OvxMin/+) mice with coumestrol, a phytoestrogen and selective ERß ligand, inhibited tumorigenesis and increased ERß expression in the intestinal mucosa (15).

Based on these human and animal data, we hypothesized that E2-activated signaling via either ER{alpha} or ERß or both modifies intestinal tumor formation. To test this, we did separate crosses of heterozygous ER{alpha} and ERß mice in a homogeneous genetic background (BL/6) with the Min/+ mouse to produce ER-deficient Min/+ strains. We compared the characteristics of these animals with Er+/+Apc+/+ [wild-type (WT)], Min/+, OvxMin/+, and E2-treated OvxMin/+ (OvxMin/+ +E2) mice. Our results show that E2 and its receptors are inhibitory modifiers that cooperate to regulate colonic tissue homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Er+/+C57BL/6-Min/+, their WT littermates, and OvxMin/+ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Antibodies directed against ß-catenin (clone 14) were from BD Bioscience (San Diego, CA); ERß (PA1-310B) was from Affinity Bioreagents (Golden, CO); ER{alpha} (MC-20) was from Santa Cruz Biotechnology (Santa Cruz, CA); and Ki-67 (clone TEC-3) was from DAKO (Carpinteria, CA). The DNeasy Tissue Kit was from Qiagen (Valencia, CA). The AmpliTaq Gold kit for PCR was from Applied Biosystems (Branchburg, NJ).

ER-deficient Min/+ crosses, tissue collection, and genotyping. C57BL/6 Er{alpha}+/– male and female and Erß–/– male and Erß+/– female mice pairs were generously provided by Dr. Pierre Chambon (Illkrich-Cedex, France) and were bred to produce heterozygous progeny. Both male and female Er{alpha}–/– mice are infertile, and Erß–/– females have significantly impaired fertility (16). A standard maintenance diet was provided ad libitum, and mice were examined daily. Genotyping was done using DNA extracted from tail biopsies of 1- to 2-week-old pups, and new breeding harems of 4- to 5-week-old mice were established to expand the ER-deficient population before initiating separate crosses of Er{alpha}+/– and Erß+/– mice with Min/+ mice. Offspring from these crosses yielded Er{alpha}+/–Min/+ and Erß+/–Min/+ mice, and final breeding harems were separately created to produce Erß+/–Min/+, Erß–/–Min/+, and Er{alpha}+/–Min/+ mice. All mice were sacrificed at ages 3 to 4 months. The entire small intestine and colon of each animal were excised and opened longitudinally. An investigator blinded to the genotype of each animal counted all visible tumors. Primers and PCR conditions for genotyping ER{alpha}- and ERß-deficient Min/+ mice were as detailed (16, 17).

Histology. Serial 4-µm-thick sections of proximal colon (2 cm) were cut perpendicular to the crypt-surface epithelial cuff axis and parallel to the cephalocaudal axis. All work used specimens from three or more different mice of the same treatment group or genotype and included archival blocks of normal proximal colon from Min/+, OvxMin/+, and OvxMin/+ +E2 mice, and pooled small intestinal tumors (n = 3–5) from OvxMin/+ mice. We prepared specimens and did immunohistochemistry by standard procedures.

Statistical analyses. An unpaired t test evaluated tumor multiplicity in both small intestine and colon of ER-deficient mice relative to Min/+ controls. A Fisher's exact test was used to distinguish colon tumor incidence in ER-deficient mice relative to Min/+ controls, and also to compare the incidence of dysplastic aberrant crypt foci in OvxMin/+ and OvxMin/+ +E2 relative to Min/+ controls. For the proliferative index, nuclear Ki-67 staining was recorded as the total number of positive cells in 10 to 15 distinct, well-oriented crypts per strain or treatment (three separate animals of each strain). Proliferative index was expressed as the ratio of positively staining nuclei to the total number of cells present per crypt. An unpaired t test compared results relative to Min/+ controls. A similar approach was used to analyze the number of differentiated goblet cells in specimens.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER-deficient Min/+ mice have increased colon tumors and abnormal mucosal histology. ER-deficient (Erß+/–, Erß–/–, and Er{alpha}+/–) Min/+ mice were generated. None of the viable pups resulting from Er{alpha}+/– x Er{alpha}+/–Min/+ pairings were of Er{alpha}–/–ApcMin/+ genotype. Animals were sacrificed at ages 3 to 4 months, and the entire intestinal tract of each was harvested and examined. At this age, Min/+ mice typically contain 20 to 60 visible tumors in the small intestine, and approximately 1 animal in 20 has a visible tumor in the colon. Tumor numbers in ER-deficient Min/+ mice were not affected by gender (data not shown). The mean number of small intestinal tumors in the ER-deficient Min/+ crosses was similar to Er+/+Min/+ littermate controls (Fig. 1A, top ). However, both ER{alpha} and ERß deficiency resulted in increased tumor formation in the cecum and proximal one third of the colon. Because colon tumors were still relatively few in all animals, this effect was most clearly seen when the number of animals with colonic tumors were compared with those without tumors for each genotype (Fig. 1A, bottom). Colonic adenomas were of both sessile and pedunculated forms. This increase in colon tumors was not previously noted in either ER{alpha}- or ERß-deficient Apc+/+ mice, and E2 loss minimally affected the number of colon tumors in OvxMin/+ mice (16, 18).


Figure 1
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  		Figure 1. ER-deficient Min/+ mice have significantly increased visible tumors in the proximal colon and abnormal histology in the nontumor mucosa. A, top, comparisons were made between the visible tumor number in Min/+ mice and ER-deficient Min/+ crosses. The number of tumors in each mouse was separately counted in the small intestine and colon. N, number of animals of each genotype examined. An unpaired t test was used to assess results at each location. A, bottom, the number of animals with and without visible colonic tumors was compared for each genotype using a Fisher's exact test. B, representative photomicrographs show H&E staining of proximal colon sections of WT, Min/+, OvxMin/+, OvxMin/+ +E2, Er{alpha}+/–Min/+, and ERß-deficient Min/+ mice. Reduced crypt length is depicted in the proximal colon of OvxMin/+ and Er{alpha}+/–Min/+ mice. Changes in the architecture of the muscularis mucosae are apparent in the colons of OvxMin/+, Er{alpha}+/–Min/+, and Erß–/–Min/+ mice. For all figures, the staining and immunohistochemical procedures were done on all of the specimens at the same time. Original magnification, x200 (Figs. 1–4).

 

Figure 4
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  		Figure 4. Colon adenomas from Er{alpha}+/–Min/+ and Erß–/–Min/+ mice have similar characteristics; however, carcinomas were routinely detected only in the ER{alpha}-deficient crosses. A, representative photomicrographs: H&E staining of large and small bowel sections of carcinomas from different Er{alpha}+/–Min/+ mice. Magnified images, invasive glands. B, representative photomicrographs: immunohistochemical comparisons of similarly sized Er{alpha}+/–Min/+ and Erß–/–Min/+ proximal colon adenomas. Top to bottom, ß-catenin, Ki-67, ER{alpha}, and ERß. Immunohistochemistry for ERß showed cytoplasmic staining of the tumor from Er{alpha}+/–Min/+ and, appropriately, the absence of this staining in the tumor of Erß–/–Min/+ mice.

 
Next, we examined differences in colonic mucosa resulting from either ER deficiency or E2 loss by ovariectomy. First, the histology of proximal colon sections from WT, Min/+, OvxMin/+, OvxMin/+ +E2, Er{alpha}+/–Min/+, Erß+/–Min/+, and Erß–/–Min/+ mice was evaluated by H&E staining (Fig. 1B). Normal crypt morphology was found in the proximal colon of WT, Min/+, and OvxMin/+ +E2, and ERß-deficient Min/+ mice. In the colon of OvxMin/+ mice, however, we observed significant changes in crypt morphology, with reduced crypt length and distortion of the circular shape of surface epithelial cuffs. Architectural changes were also present in the submucosa, including thickening of the muscularis mucosae and enrichment of stromal components, suggesting an increased inflammatory cell infiltrate. Similar morphologic changes were observed in Er{alpha}+/–Min/+ mice, but were less consistent. The presence of mononuclear cell infiltrates was also a common feature in the colons of Erß–/–Min/+ mice.

Compartmentalized E2 signaling by ER{alpha} and ERß modulates enterocyte growth and differentiation within colon crypts. To learn whether changes in the crypt dimensions reflected the relative proliferative index, we did Ki-67 immunohistochemistry using sections from the proximal colons of study animals (Fig. 2A, right ). The number of proliferating cells in the colon of Min/+ mice was similar to that of WT (Fig. 2A, left). Consistent with the observed loss of crypt length, ovariectomy of female Min/+ mice resulted in a 20% reduction in the number of positively stained nuclei relative to colon from intact female and male Min/+ mice. E2 replacement of OvxMin/+ mice reversed this effect, increasing colonocyte proliferation 2-fold. Taken together, these results indicate that signaling via ER{alpha} produces trophic effects on the proximal colonic mucosa. We also examined the effect of ER deficiency on proliferative index. Consistent with the tumor phenotype, ER deficiency was also associated with increased enterocyte proliferation (44–87%) in the colon relative to that of Er+/+Min/+ mice.


Figure 2
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  		Figure 2. E2 signaling modulates enterocyte proliferation and differentiation in the proximal colon. A, comparison of Ki-67 proliferative index in E2-replete (Min/+ and OvxMin/+ +E2) mice relative to E2-ablated (OvxMin/+) and ER-deficient Min/+ strains. Nuclear staining by Ki-67 was recorded as the total number of positive cells in 10 to 15 distinct, well-oriented crypts per strain (three separate animals per strain or treatment group). Proliferative index (PI) was expressed as the ratio of positively stained nuclei to the total number of cells present within the crypt. Representative photomicrographs (right) show Ki-67–positive staining in the nuclei of enterocytes in proximal colon sections of WT, Min/+, OvxMin/+, OvxMin/+ +E2, Er{alpha}+/–Min/+, and ERß-deficient Min/+ mice. Immunohistochemistry used the TEC-3 antibody (1:25). B, the colons of ER-deficient Min/+ and OvxMin/+ +E2 exhibit reduced goblet cell maturation. Mature goblet cells were stained by the Alcian blue procedure and scored in well-oriented crypts of proximal colon sections from three different animals per genotype or treatment group. Representative photomicrographs (right) show Alcian blue staining of acidic mucin in goblet cells in colon sections of WT, Min/+, OvxMin/+, OvxMin/+ +E2, Er{alpha}+/–Min/+, and ERß-deficient Min/+ mice. Immature goblet cells found near the base of crypts are small and have a spongy appearance that does not stain strongly. Mature goblet cells found near the top of crypts contain large, spherical vacuoles and stain a deeper blue color.

 
The majority of differentiated cells in the colon are mucin-producing goblet cells, and expression of acidic mucins is detected by Alcian blue staining. We used this technique to assess differentiation in the proximal colons of the study mice and found relatively fewer mature goblet cells in OvxMin/+ +E2, Er{alpha}+/–Min/+, and ERß-deficient Min/+ mice than in the normal colons of WT, Min/+, and OvxMin/+ mice (Fig. 2B, right). Morphologically different, pregoblet cells were especially prevalent in OvxMin/+ +E2 and ERß-deficient Min/+ tissues. The change in the ratio for all ER-deficient Min/+ strains and OvxMin/+ +E2 was significantly reduced (24–42%) relative to Min/+ controls (Fig. 2B, left). In contrast, the goblet cell ratio for OvxMin/+ mice was increased relative to Min/+ mice. Thus, goblet cell maturation was inversely correlated with colonocyte proliferative index.

Because proliferation takes place predominantly in the cells at the base of crypts and mature goblet cells are mostly found near the surface mucosa, we asked whether the expression of ER{alpha} and ERß differed by location within the colonic crypt unit. First, we did immunohistochemistry to examine the cell of origin and location of ER{alpha} in the colons of the study animals (Fig. 3, right ). In all of the specimens, strong nuclear ER{alpha} expression was present in enterocytes extending from the base of crypts to approximately three fourths of their lengths. Confirming our localization of ER{alpha} in intestinal crypts, expression of this receptor was reported in progenitor cells microdissected from the mouse intestine (19). By immunohistochemistry, ER{alpha} expression was not visibly diminished in the colon of Er{alpha}+/–Min/+ mice. Next, we examined the cell of origin and location of ERß in the colons of the study animals (Fig. 3, left). Positive staining for ERß was found in the cytoplasm and nucleus of enterocytes located in the upper regions of colonic crypts and at the surface mucosa. This staining pattern is consistent with the results of others using the same antibody (PA1-310B) on human colon sections (13). ERß staining was scant and cytoplasmic in enterocytes at the base of colonic crypts from Er{alpha}+/–Min/+ mice and absent, as expected, in the Erß–/–Min/+ animals (negative control). The observed difference in receptor location indicates that ER{alpha} and ERß activities are regionally compartmentalized, a result compatible with their functional antagonism in enterocytes undergoing growth arrest. Taken together, these data suggest that the proper balancing of E2-dependent effects on epithelial cell proliferation and differentiation in the colon is achieved by separate and nonredundant activities of ER{alpha} and ERß.


Figure 3
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  		Figure 3. ER{alpha} and ERß expression is compartmentalized within the crypt axis of the normal proximal colon. Representative photomicrographs show immunohistochemistry for ER{alpha} and ERß expression in enterocytes of the proximal colon WT, Min/+, OvxMin/+, OvxMin/+ +E2, Er{alpha}+/–Min/+, and ERß-deficient Min/+ mice. Right images, maximal ER{alpha} was located in the nucleus of enterocytes, which are located in the proliferative compartment near the base of crypts. Antibodies for ER{alpha} were used at a 1:1,000 dilution. Left images, maximal ERß expression in Erß+/+ or Erß+/– Min/+ specimens was in the postmitotic compartment near the top of crypts and the surface epithelial cuffs. The appropriate absence of staining of the Erß–/–Min/+ specimen (bottom) indicates that the result seen in the images above was not attributable to an artifact or edge effect. Antibodies for ERß were used at a 1:100 dilution. Bottom, summary of the results, which indicates that ER{alpha} and ERß display decreasing expression gradients with opposite orientations within colon crypts. The relative locations of proliferating and growth-arrested cells in the normal crypt is also noted.

 
Characterization of colon tumors from Er{alpha}+/–Min/+ and Erß–/–Min/+ mice. Invasive tumors are rarely found in Min/+ mice, and when observed are virtually always found in animals older than 3 to 4 months. Microscopic examination of intestinal tumors from Er{alpha}+/–Min/+, Erß+/–Min/+, and Erß–/–Min/+ animals revealed a significant difference in tumor aggressiveness in the former. Er{alpha}+/–Min/+ mice developed from one to three invasive carcinomas each that were located in both the small intestine and colon (Fig. 4A ). Invasive carcinomas were detected in seven of seven (100%) Er{alpha}+/–Min/+ mice, but were not found in any of the other animals examined. To verify the ER{alpha} dependence of this phenotype, we reviewed H&E-stained sections of small intestinal tumors (three to five per mouse) from our archival collection of 19 OvxMin/+ mice (14, 15). No invasive tumors were found in these animals, suggesting that ER{alpha} deficiency accelerated tumor progression independent of E2.

Noninvasive adenomas from Er{alpha}+/–Min/+ and Erß–/–Min/+ mice were histologically similar (Fig. 4B). Typical of Min/+ adenomas, immunostaining showed up-regulated nuclear and cytoplasmic ß-catenin. The number of positively stained nuclei by Ki-67 immunohistochemistry in these tumors was not significantly different (data not shown). Nuclear expression of ER{alpha} was evident in both Er{alpha}+/– and Erß–/– colon tumors. Maintenance of nuclear ER{alpha} expression in Apc-null tumors of the ER-deficient Min/+ mice supports the view that Er{alpha} is a modifier gene and not a tumor suppressor. Finally, immunohistochemistry for ERß in Er{alpha}+/–Min/+ colon tumors revealed low, cytoplasmic expression of this protein. Again, only background staining was apparent in Erß–/– Min/+ tumor cells.

Deficiency of either ER{alpha} or ERß in Min/+ mice stimulates tumor progression without affecting tumor initiation. Colon tumor growth in Min/+ mice requires several genetic and epigenetic events in addition to the loss of Apc function (20). Dysplastic crypt formation is the first step in this process. This lesion occurs spontaneously in the colons of Min/+ mice, arises clonally from a single stem cell that has sustained Apc+ loss of heterozygosity, and each exhibits ß-catenin overexpression (21). Dysplastic crypts are potential but not obligate tumor precursors; in the colon of Min/+ mice, they typically do not progress to become adenomas. We anticipated that dysplastic crypts would be more prevalent in the proximal colon of ER{alpha} and ERß mutant Min/+ strains if these receptors played a role in adenoma initiation. To test this, we stained grossly normal-appearing colon sections using an antibody for ß-catenin to identify microscopic changes associated with adenoma formation. Immunohistochemistry of proximal colon sections from Er{alpha}+/–Min/+ mice revealed dysplastic aberrant crypt foci in the form of single crypts and microadenomas (Fig. 5, top right ). The dysplastic crypt shown on the left is an example of a lesion undergoing crypt fission. The dysplastic microadenoma on the right has already undergone several crypt divisions, and the newly budded crypts remain tethered together beneath the surface mucosa. The incidence and appearance of these lesions were similar in Erß+/– and Erß–/–Min/+ colons (Fig. 5). Next, we used ß-catenin immunohistochemistry to identify dysplastic aberrant crypt foci in our archival proximal colon specimens from 3-month-old female Min/+, OvxMin/+, and OvxMin/+ +E2 mice (Fig. 5, left). In Min/+ colons, 14 dysplastic crypts were found in 10 out of a total of 37 mice. All were single crypts that had not undergone stem cell duplication and further crypt fission. Similar results were obtained using OvxMin/+ +E2 colons in which eight single dysplastic crypts and one microadenoma were found in 5 of 20 mice. Surprisingly, no dysplastic lesions were discovered in any of the 28 OvxMin/+ colon specimens. The incidence of single dysplastic crypts in the ER-deficient Min/+ mice was not significantly different from that of Min/+ mice, but that of microadenomas and visible tumors was significantly greater in the compound mutant animals. As illustrated (Fig. 5, bottom), these results suggest that dysplastic crypts may be products of normal or increased colonocyte proliferation in the Min/+ mouse and that E2 is an important stimulus of colonocyte growth. In addition, our results suggest that ER{alpha} and ERß deficiencies in a heterozygous Apc mutant background promote aberrant crypt fission and microadenoma progression in the proximal colon.


Figure 5
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  		Figure 5. The similar incidence of dysplastic single crypts in the proximal colon of ER-deficient and E2-replete Min/+ mice indicates that ER loss enhances precursor lesion progression to microadenomas and visible tumors. In the table (left top), the incidence of dysplastic single crypts in the proximal colon of the E2-replete (Min/+ and OvxMin/+ +E2) mice was compared with OvxMin/+. Immunohistochemistry (IHC) for ß-catenin was used for aberrant crypt foci detection as described (21). One-centimeter proximal colon specimens (three serial 4-µm sections) from Min/+ mice of the different treatment groups were also used. No adenomas were present in any of the specimens tested. Fisher's exact test was used to compare results from each treatment group. As previously reported (14, 15), ovariectomy of Min/+ mice increased the number of small intestinal tumors but had no effect on the typically low number of colon tumors in these animals; E2 replacement of OvxMin/+ mice reduced small intestinal tumors to the baseline number of Min/+. Examples of dysplastic aberrant crypt foci (ACF) in colons of Er{alpha}+/–Min/+ mice revealed by immunohistochemistry for ß-catenin (right top). Similar lesions were found in ERß-deficient Min/+ (data not shown). A two-step model for dysplastic microadenoma formation in Min/+ mice depicts the proposed role of E2 signaling (bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human colorectal cancer is a heterogeneous disease, and multiple genetic and environmental factors contribute to its development. The Min/+ mouse is a useful colorectal cancer model, as Apc mutation plays an initiating role in the great majority of colorectal cancers. For reasons not understood, intestinal tumors in Min/+ mice occur primarily in the small intestine. The combination of Apc and ER loss, however, produced a phenotype more consistent with that of human colorectal cancer, with development of adenomas and invasive carcinomas in the colon. This condition also occurs in human colorectal cancer, as loss of ER expression is one of the tumor-associated consequences of methylation-associated gene silencing (6). This mouse model, therefore, provides important insights into the role of ER signaling in normal intestinal function and in colorectal carcinogenesis.

Colonic epithelium is a dynamic microenvironment. Progenitor cells in the crypts undergo cell division and give rise to epithelial cells that differentiate into various lineages as they migrate to the crypt lumen. The mucosal architecture is maintained by adhesive and repulsive interactions among enterocytes, signaling to and from stromal elements, and properly timed shedding of senescent or apoptotic cells at the crypt lumen. The studies reported here show that signaling via ER{alpha} and ERß plays important roles in these processes. ERß is thought to functionally antagonize the growth-promoting activities of ER{alpha} (9–11), and our results are consistent with this view. The compartmentalization of ER{alpha} and ERß expression within the crypt axis (Fig. 3) suggests that a negative regulatory function of ERß is directed at ER{alpha} as colonic enterocytes differentiate and undergo growth arrest. The increasing expression of ERß in enterocytes ascending crypts can be expected to cause competition for ligand, as well as coactivators. Also, the repertoire of genes subject to the regulatory control of these receptors may change due to ER{alpha}-ERß heterodimerization, in contrast to the situation in proliferating cells where ER{alpha} abundance may differentially favor its homodimerization. We did not measure serum E2 levels in the animals, as others have shown that serum E2 levels were normal in the Er{alpha}+/–, Erß+/–, and Erß–/– BL/6 mice used in this study (22).

Previously, we reported that E2 loss by ovariectomy increased small intestinal tumor formation in Min/+ mice (14). Here, we show that the effect of ovariectomy on the colon is also significant, with a 20% reduction in proliferative index and architectural changes, suggesting loss of a mucosal trophic factor. Ovariectomy did not increase tumor formation in the colons of Min/+ mice and in fact had the opposite effect, with a reduction in microscopic dysplastic aberrant crypt foci compared with intact or E2-replaced Min/+ mice (Fig. 5). Interestingly, loss of ER{alpha} function in the Er{alpha}+/–Min/+ mouse produced histologic changes similar to that of ovariectomy; however, in this case, the architectural disorder was accompanied by increased proliferative index and loss of goblet cell differentiation. Consistent with these changes, Er{alpha}+/–Min/+ mice developed a spectrum of neoplastic colonic lesions, including dysplastic aberrant crypt foci, visible adenomas, and invasive adenocarcinomas. Taken together, these observations suggest that loss of signaling via ER{alpha} is a major contributor to colorectal cancer development.

We observed stimulation of colonic crypt fission in ER-deficient Min/+ strains, suggesting that ER activities inhibit interactions between stromal and epithelial elements that are necessary for adenoma formation. One possibility is that E2-activated ER{alpha} regulates signaling by bone morphogenetic proteins (BMP2 and BMP4) in the colon. BMP4 is secreted by the stromal mesenchyme and negatively regulates stem cell duplication and intestinal crypt fission (23). Binding of BMP4 to receptors on intestinal stem cells initiates signaling via transforming growth factor ß, leading to phosphorylation of Smad1 and Smad5 and inhibition of Wnt signaling. This mechanism may involve E2 signaling because nuclear interactions between the BMP4 effector, Smad1, and ER{alpha} were inducible by BMP4, and ER{alpha}-dependent induction of BMP4 transcription occurred in response to the selective estrogen response–modifier drug, raloxifene (24, 25). The net effect of these ER{alpha}-mediated activities would be intestinal tumor suppression.

BMP2-dependent mechanisms may also mediate tumor suppression in the colon. This factor enforces the growth arrest of mature enterocytes when secreted by intercrypt fibroblasts or mononuclear leukocytes or by enterocytes via an autocrine route (26, 27). BMP2 expression is strongly dependent on E2-activated ER, and induction of BMP2 transcription was 3-fold greater for ER{alpha} relative to ERß (28). We showed that ER deficiency in Min/+ mice increased proliferation and decreased differentiation (Figs. 2 and 3). Thus, defective growth arrest may have occurred in our compound mutant mice due to reduced BMP2 expression at the surface mucosa. Similarly, BMP6 is expressed in the colon and its gene promoter is E2 dependent (29).

ER deficiency may also promote intestinal tumor formation due to loss of Smad4 activity. Loss of Smad4 in Apc mutant mice is sufficient for carcinoma formation (30). Regulatory cross-talk between ER and Smad4 occurs at the transcriptional level (31), and Smad4 transmits BMP-elicited signals in the nucleus. Finally, E2-activated, ER{alpha}-dependent colorectal cancer inhibition may involve the inhibition of Snail transcription factors. These transcriptional repressors are regulators of the epithelial-mesenchymal transition and are overexpressed in human colorectal cancer (32). Both MTA3 and LIV1 are ER{alpha}-dependent Snail inhibitors and implicated in human cancer (33, 34).

In conclusion, we found that the functions of both ER{alpha} and ERß inhibited Apc-dependent tumor formation in the proximal colon of Min/+ mice. These functions involved coordination of stromal-epithelial interactions to promote maintenance of normal colonic crypt architecture. This work is consistent with results of human clinical trials and cancer epidemiology, indicating that hormone replacement therapy is beneficial for preventing postmenopausal colorectal cancer. In addition, the model presented here provides a framework for further investigating the downstream signaling mechanisms responsible for these antitumor effects.


   Acknowledgments
 
Grant support: Ethicon Society of University Surgeons.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. Pierre Chambon for providing the ER-deficient mice for the breeding.


   Footnotes
 
Note: N.L. Cho and S.H. Javid contributed equally to this work.

Received 8/15/06. Revised 11/ 7/06. Accepted 12/29/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mansgelsdorf DJ, Thummel C, Beato M, et al. The nuclear receptor superfamily: The second decade. Cell 1995;83:835–9.[CrossRef][Medline]
  2. Butler D, Hassanein K, Dudgeon M, et al. Female gender is a major determinant of changing subsite distribution of colorectal cancer with age. Cancer 1985;56:714–6.[CrossRef][Medline]
  3. Chen MJ, Longnecker MP, Morgenstern H, et al. Recent use of hormone replacement therapy and the prevalence of colorectal adenomas. Cancer Epidemiol Biomarkers Prev 1998;7:227–30.[Abstract]
  4. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–30.[Abstract/Free Full Text]
  5. Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787–93.[CrossRef][Medline]
  6. Issa JP, Ottaviano YL, Celano P, et al. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 1994;7:536–40.[CrossRef][Medline]
  7. Chlebowski RT, Wactawski-Wende J, Ritenbaugh C, et al. Estrogen plus progestin and colorectal cancer in postmenopausal women. N Engl J Med 2004;350:991–1004.[Abstract/Free Full Text]
  8. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001;276:36869–72.[Free Full Text]
  9. Matthews J, Gustafsson J-Å. Estrogen signaling: a subtle balance between ER{alpha} and ERß. Mol Interv 2003;3:181–292.
  10. Liu M-M, Albanese C, Anderson CM, et al. Opposing action of estrogen receptors {alpha} and ß on cyclin D1 gene expression. J Biol Chem 2002;277:24353–60.[Abstract/Free Full Text]
  11. Matthews J, Wihlén B, Tujague M, et al. Estrogen receptor (ER) ß modulates ER{alpha}-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 2006;20:534–43.[Abstract/Free Full Text]
  12. Foley EF, Jazaeri AA, Shupnik MA, et al. Selective loss of estrogen receptor ß in malignant human colon. Cancer Res 2000;60:245–8.[Abstract/Free Full Text]
  13. Campbell-Thompson M, Lynch IJ, Bhardwaj B. Expression of estrogen receptor (ER) subtypes and ERß isoforms in colon cancer. Cancer Res 2001;61:632–40.[Abstract/Free Full Text]
  14. Weyant MJ, Carothers AM, Mahmoud NN, et al. Reciprocal expression of ER{alpha} and ERß is associated with estrogen-mediated modulation of intestinal tumorigenesis. Cancer Res 2001;61:3547–51.
  15. Javid SH, Moran AE, Carothers AM, et al. Modulation of tumor formation and intestinal cell migration by estrogens in the ApcMin/+ mouse model of colorectal cancer. Carcinogenesis 2005;26:587–95.[Abstract/Free Full Text]
  16. Dupont S, Krust A, Gansmuller A, et al. Effect of single and compound knockouts of estrogen receptors {alpha} (ER{alpha}) and ß (ERß) on mouse reproductive phenotypes. Development 2000;127:4277–91.[Abstract]
  17. Su L-K, Kinzler KW, Vogelstein B, et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 1992;256:668–70.[Abstract/Free Full Text]
  18. Wada-Hiraik O, Imamov O, Hiraike H, et al. Role of estrogen receptor ß in colonic epithelium. Proc Natl Acad Sci U S A 2006;103:2959–64.[Abstract/Free Full Text]
  19. Giannakis M, Stappenbeck TS, Mills JC, et al. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches. J Biol Chem 2006;281:11292–300.[Abstract/Free Full Text]
  20. Kim SH, Roth, KA, Moser AR, et al. Transgenic mouse models that explore the multistep hypothesis of intestinal neoplasia. J Cell Biol 1993;123:877–93.[Abstract/Free Full Text]
  21. Yamada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci U S A 2005;102:13580–5.[Abstract/Free Full Text]
  22. Sims NA, Dupont S, Krust A, et al. Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-ß in bone remodeling in females but not in males. Bone 2002;30:18–25.[Medline]
  23. He XC, Zhang J, Tong W-G, et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-ß-catenin signaling. Nat Genet 2004;36:1117–21.[CrossRef][Medline]
  24. Páez-Pereda M, Giacomini D, Refojo D, et al. Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk. Proc Natl Acad Sci U S A 2003;100:1034–9.[Abstract/Free Full Text]
  25. van den Wijngaard A, Muler WR, Dijkema R, et al. Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells. Mol Endocrinol 2000;14:623–33.[Abstract/Free Full Text]
  26. Hardwick JC, van den Brink GR, Bleuming SA, et al. Bone morphogenetic protein 2 is expressed by, and acts upon mature epithelial cells in the colon. Gastroenterology 2004;126:111–21.[CrossRef]
  27. Beck SE, Jung BH, Fiorino A, et al. Bone morphogenetic protein signaling and growth suppression in colon cancer. Am J Physiol Gastrointest Liver Physiol 2006;291:G135–45.[Abstract/Free Full Text]
  28. Zhou S, Turgeman G, Harris SE, et al. Estrogens activate bone morphogenetic protein-2 gene transcription in mouse mesenchymal stem cells. Mol Endocrinol 2003;17:56–66.[Abstract/Free Full Text]
  29. Rickard SJ, Hofbauer LC, Bonde SK, et al. Bone morphogenetic protein-6 in human osteoblastic cell lines: Selective regulation by estrogen. J Clin Invest 1998;101:413–22.[Medline]
  30. Takaku K, Oshima M, Miyoshi H, et al. Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell 1998;92:645–56.[CrossRef][Medline]
  31. Wu L, Wu Y, Gathings B, et al. Smad4 as a transcription corepressor for estrogen receptor {alpha}. J Biol Chem 2003;278:15192–200.[Abstract/Free Full Text]
  32. Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005;132:3151–61.[Abstract/Free Full Text]
  33. Fujita N, Jaye DL, Kajita M, et al. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 2003;113:207–19.[CrossRef][Medline]
  34. Yamahita S, Miyagi C, Fukuda T, et al. Zinc transporter LIV1 controls epithelial-mesenchymal transition in zebrafish gastrula organizer. Nature 2004;429:298–302.[CrossRef][Medline]



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