Cancer Research Landon Prizes for Basic and Translational Cancer Research AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weyant, M. J.
Right arrow Articles by Bertagnolli, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weyant, M. J.
Right arrow Articles by Bertagnolli, M. M.
[Cancer Research 61, 2547-2551, March 15, 2001]
© 2001 American Association for Cancer Research

Epidemiology and Prevention

Reciprocal Expression of ER{alpha} and ERß Is Associated with Estrogen-mediated Modulation of Intestinal Tumorigenesis1

Michael J. Weyant, Adelaide M. Carothers, Najjia N. Mahmoud, H. Leon Bradlow, Helen Remotti, Robyn T. Bilinski and Monica M. Bertagnolli2

Departments of Surgery and Pathology, Weill College of Medicine of Cornell University, New York, New York [M. J. W., A. M. C., N. N. M., H. R., R. T. B.]; Division of Surgical Oncology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 [M. M. B.]; and The Strang Cancer Prevention Center, New York, New York 10021 [M. J. W., A. M. C., N. N. M., H. L. B., M. M. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Menopausal hormone replacement therapy has been widely used to alleviate the symptoms of menopause and to decrease the detrimental effects of ovarian hormone loss on bone density and cardiovascular health. Multiple studies of colorectal cancer epidemiology also support a role for hormone replacement therapy in prevention of colorectal cancer. We studied the effect of ovariectomy and estrogen replacement on tumor formation in C57BL/6J-Min/+ (Min/+) mice, animals that bear a germ-line mutation in murine Apc. These mice develop multiple intestinal tumors that show loss of wild-type Apc protein. After ovariectomy, intestinal adenomas in Min/+ mice increased by 77% (P = 0.0004). Ovariectomized Min/+ mice that were treated with a replacement dose of 17ß-estradiol had the same number of tumors as Min/+ mice that were neither castrated nor treated with estrogen replacement (P = 0.85). Examination of estrogen receptor (ER) levels in intestinal tissue by immunoblot showed changes in relative expression levels of ER{alpha} and ERß, with highest ER{alpha} and lowest ERß expression in the normal-appearing intestine of Min/+ mice, and lowest ER{alpha} and highest ERß expression in the enterocytes of animals that received 17ß-estradiol. These results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17ß-estradiol is associated with an increase in ERß and a decrease in ER{alpha} expression in the target tissue.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although males and females develop CRC3 with approximately the same frequency, women in developed countries who are normally at high risk for CRC have recently experienced a substantial decline in mortality from this disease (1 , 2) . This decline has been attributed to the use of menopausal HRT, a practice that began in the 1970s (3) . Numerous epidemiological studies of CRC incidence show a protective effect of menopausal HRT use. A recent meta-analysis of studies published up to December 1996 suggests as much as a 25% reduction with HRT use, with a summary relative risk of 0.85 (95% CI, 0.73–0.99). Studies identifying the duration of HRT use also suggest a dose response, with a relative risk among current or recent HRT users of 0.69 (95% CI, 0.52–0.91) compared to 0.88 (95% CI, 0.64–1.21) for short-term users (4) .

The mechanism responsible for HRT-associated prevention of CRC is unclear. Estrogens undergo metabolism to a variety of compounds that have different half-lives and receptor affinities, and they produce varying effects on cell growth. For example, the endogenous estrogen 17ß-estradiol is metabolized by cytochrome P450 enzymes to form monohydroxy metabolites, and some of these metabolites may play important roles in the induction of estrogen-induced cancers (5) . In addition, some of these metabolites (2-hydroxyestradiol and 4-hydroxyestradiol) can be further metabolized by catechol-O-methyltransferase to methylated estrogens. One of these methylated metabolites (2-methosyestradiol) may protect against tumor formation or inhibit tumor growth (5, 6, 7) . A tissue-specific distribution of estrogen-metabolizing enzymes influences the effect of a systemically administered estrogen, such as those used for HRT, on cell proliferation in target tissues.

The activity of estrogens and antiestrogens in both males and females is mediated through binding of these compounds to ERs, which are ligand-activated transcription factors. In the absence of ligand, ER resides in the nucleus, where it is bound to an inhibitory heat shock protein complex (8) . Upon ligand binding, the ER forms a stable dimer that then interacts with specific estrogen response elements to initiate the transcription of target genes. There are at least two major ERs, known as ER{alpha} and ERß. These two ER isoforms appear to have a differential tissue distribution: ER{alpha} is the predominant isoform in breast and uterine tissue (9 , 10) ; and ERß is expressed in significant quantities in the urogenital tract, the central nervous system, and endothelial cells (10, 11, 12) . The two ER isoforms also exhibit differences in binding affinity, potency, and efficacy after interaction with various estrogenic compounds (13 , 14) . To add yet another level of complexity, multiple ERß isoforms have been identified (15 , 16) .

Several studies have examined the relative expression of ER{alpha} and ERß in the colon. ER{alpha} is present at very low levels in the colon, with no difference in mRNA or protein expression between normal colon, adenomas, and colon cancers and no differences between males and females (17 , 18) . ERß protein is the predominant isoform present in normal colon, and expression of ERß protein may be decreased in colon cancers (18) , although ERß mRNA levels remain unchanged, suggesting that this decrease is due to a posttranscriptional mechanism. The ER{alpha}:ERß protein ratio observed in normal colon is the reverse of that found in human endometrium (18) .

The Min/+ mouse bears a germ-line mutation in the murine Apc gene and is therefore a genetic model for both familial adenomatous polyposis and sporadic human CRC. Loss of APC protein function produces increased intracellular levels of the oncoprotein ß-catenin (19) . In the nucleus, ß-catenin combines with the protein Tcf-4, providing a DNA-binding domain that allows transcription of genes modulating cell proliferation and apoptosis (20) . Min/+ mice develop multiple intestinal adenomas and frequently succumb to adenoma-related anemia or intestinal obstruction by 18–20 weeks of age (21) . The development of adenomas in Min/+ mice is sensitive to a variety of compounds administered in the diet, making these animals an excellent screen for promising chemopreventive agents. Adenoma number in Min/+ mice is decreased by a variety of agents including aspirin (22) , sulindac (23) , piroxicam (24) , and plant phenolics (25) .

We investigated the role of estrogens in intestinal tumorigenesis in Min/+ mice by examining the effect of ovariectomy with and without estrogen replacement on tumor number and intestinal ER expression. The results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17ß-estradiol is associated with a relative increase in ERß and a decrease in ER{alpha} in the target tissue.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Min/+ mice and their wild-type littermates (+/+) were purchased from The Jackson Laboratory (Bar Harbor, ME). AIN-76A pelleted diet was obtained from Research Diets (New Brunswick, NJ). Controlled release 90-day pellets containing 17ß-estradiol (NE-121) or control vehicle were obtained from Innovative Research of America (Sarasota, FL). Primary antibodies and protein standards were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; ER{alpha}, MC-20 rabbit polyclonal antibody sc-542) and Affinity Bioreagents (Golden, CO; ER{alpha} standard, rp-310; ERß, PA1–310A rabbit polyclonal antibody; ERß standard, RP-311). Western blot analyses used Optitran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Electrotransfer of proteins used the electroblot buffers of Owl Separation Systems (Woburn, MA). Western blotting used the enhanced chemiluminescence detection reagents of Amersham Pharmacia Biotech (Piscataway, NJ).

Animal Treatments and Tissue Harvesting.
Female Min/+ mice and their wild-type littermates (+/+) were obtained at 5 weeks of age. Ovariectomy was performed at The Jackson Laboratory on Min/+ mice at 4 weeks of age. Animals receiving estrogen replacement underwent implantation of a 90-day controlled release pellet containing 1.7 mg/pellet 17ß-estradiol at 5–6 weeks of age. Control animals underwent placement of an inert pellet containing identical substances but lacking the hormone. Beginning at 5–6 weeks of age, all mice were fed AIN-76A chow diet. The animals were checked daily for signs of distress or anemia, and animals and their food were weighed weekly. During the course of the experiment, there were no differences in body weight or food consumption among the various study groups. Hence, the typical approximate intake of the animals included 2.5 grams/day AIN-76A and 3.0 ml/day water. At 15 weeks of age, all mice were euthanized by CO2 inhalation, and their intestinal tracts were removed from esophagus to distal rectum, opened, flushed with saline, and examined under x3 magnification to obtain tumor counts. The tumors were counted by an individual blinded to the animal’s genetic status and treatment. Successful ovariectomy was documented by histological comparison of uterine mucosa from ovariectomized animals, animals treated with estrogen replacement, and control animals. Analyses of the different animal tissues were performed by an observer blinded to the animal’s genetic status and treatment group.

Enterocyte Preparation and Western Blotting.
Several tumors from all regions of the small intestine (duodenum, jejunum, and ileum) were excised from each animal, as well as segments of tumor-free duodenum, small bowel, and colon. These tissues were snap frozen and stored separately in liquid nitrogen. Enterocyte samples for Western blotting were obtained by mechanical dissociation using the edge of a glass microscope slide (26) and washed twice in cold PBS before storage at -70°C. For consistency, all samples of enterocytes were collected from 4-cm segments of the proximal small intestines. These were deemed macroscopically "normal" because they were collected after removal of all adenomas seen under x3 magnification. Samples from two mice of the same treatment group were pooled to prepare the protein extracts. Procedures and buffers for cell lysis, protein determination, and Western blot analyses were performed exactly as described previously (27) . Western blots used 50 µg protein/lane (500 ng for standards), 10% SDS-PAGE, and the following primary antibodies: (a) ER{alpha}, MC-20 rabbit polyclonal antibody sc-542; (b) ER{alpha} standard, rp-310; (c) ERß, PA1–310A rabbit polyclonal antibody; and (d) ERß standard, RP-311. All antibodies were used at a concentration of 1:1000. The Western blot determinations were repeated separately at least two times.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ovariectomy Increased Tumor Production in Min/+ Mice.
To simulate the hormonal environment of menopause, ovariectomy was performed on healthy female Min/+ mice at 4–5 weeks of age. At this age, Min/+ mice have not yet developed tumors measurable by our counting method. Both ovariectomized Min/+ mice and control Min/+ mice with intact ovarian function were fed a standard AIN-76A diet. At 15 weeks of age, the animals were euthanized, and the entire intestinal tract was examined under x3 magnification.

The average tumor number in the control Min/+ animals was 23.9 ± 11.9, a value consistent with that seen in previous studies using the tumor counting technique used here (Fig. 1)Citation . Ovariectomized Min/+ mice contained an average of 42.3 ± 13.9 tumors, a 77% increase in total tumor number compared to the control animals (P = 0.0004). Consistent with previous studies, 93% of the tumors were found in the small intestine, with 5% in the duodenum, and approximately 2% in the colon (Table 1)Citation . The distribution of tumors throughout the intestinal tract was not altered by ovariectomy (Table 1)Citation .



View larger version (96K):
[in this window]
[in a new window]
  		Fig. 1. Ovariectomy increases tumors in Min/+ mice. Female Min/+ mice underwent ovariectomy at 4–5 weeks of age. Mice receiving estrogen replacement underwent s.c. implantation of a controlled release estrogen pellet at 5–6 weeks of age. Tumor counts from the entire intestine were obtained at 15 weeks of age. Values represent total tumor number/mouse ± SD, where n = 32 (Min/+, Experiment A), n = 9 (Min/+ sham, Experiment B), n = 15 (Min/+ ovariectomy, Experiment A), and n = 9, Min/+ E2 + ovariectomy, Experiment B). *, P = 0.0004 compared to Min/+; **, P = 0.85 compared to Min/+ animals treated with sham implant.

 

View this table:
[in this window]
[in a new window]
  		Table 1 Distribution of intestinal tumors

 
Administration of 17ß-Estradiol Decreased Intestinal Tumor Number in Ovariectomized Min/+ mice.
To simulate the effect of postmenopausal HRT, 17ß-estradiol was given to ovariectomized Min/+ mice in the form of controlled release pellets implanted in the s.c. tissue of 5–6-week-old animals. Control Min/+ mice were given a sham operation, with implantation of a pellet containing the only inert vehicle and lacking the hormone. As in the previous experiment, the mice were allowed to reach 15 weeks of age before tumor counts were obtained. The Min/+ control mice that were implanted with an inert pellet each contained 28.3 ± 7.5 tumors, an amount unchanged from that seen in previous Min/+ control animals (P = 0.19). The ovariectomized animals that also received estrogen replacement, however, contained 29.2 ± 12.4 tumors each and were therefore equal in tumor number to the sham vehicle-treated Min/+ controls (P = 0.85). When the distribution of tumors throughout the gastrointestinal tract was examined, the number of small intestinal tumors in the animals receiving 17ß-estradiol was no different from that of animals receiving a sham operation (P = 0.36; Table 2Citation ). In the duodenum, treatment with 17ß-estradiol decreased the number of tumors per mouse from 6.7 ± 1.2 to 3.7 ± 3.2 (P = 0.04). In the colon, the opposite effect was noted, with an increase in tumor number from 0.22 ± 0.44 tumor/animal in the sham operation group to 0.77 ± 0.66 tumor/animal in the 17ß-estradiol-treated mice (P = 0.05). Although statistically significant, these differences are unlikely to be important because of the very small numbers of tumors found in the duodenum and colon of Min/+ mice.


View this table:
[in this window]
[in a new window]
  		Table 2 Effect of 17ß-estradiol supplementation on tumor distribution

 
Ovariectomy and Estrogen Replacement Altered the Relative Expression of ER{alpha} and ERß in Enterocytes from Min/+ Mice.
ERs effect the biological responses to estrogens at the tissue level. We therefore performed Western blotting to determine the relative expression of ER{alpha} and ERß in enterocytes isolated from Min/+ mice and their wild-type littermates, as well as in pooled preparations of adenomas arising in the intestines of Min/+ mice.

The small intestine of the Min/+ mouse is the region of the gastrointestinal tract most prone to tumor formation. By immunoblot, the tumor-prone small intestine of Min/+ mice showed elevated ER{alpha} and lowered ERß levels when compared to comparable tissues from wild-type animals (Fig. 2)Citation . The intestinal mucosa that was used for the Min/+ small intestine immunoblots appeared normal under x3 magnification, although it is possible that a small fraction of this preparation contained adenoma cells. When pooled samples of adenomas from Min/+ small intestine were examined, however, Western blots showed slightly more ERß and slightly less ER{alpha} than the normal-appearing Min/+ mucosa. Thus, potential adenoma contamination is unlikely to explain the different ER{alpha}:ERß ratios observed between Min/+ and wild-type animals.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 2. ER{alpha} and ERß are differentially expressed in Min/+ intestine. Samples of intestinal mucosa from wild-type (WT), control Min/+ (Min/+), Min/+ mice after ovariectomy (Min/+/OVX), and ovariectomized Min/+ mice receiving 17ß-estradiol replacement (Min/+/OVX/17ßE2) and pooled adenomas from control Min/+ mice (Min/+ adenoma) were examined by immunoblotting using antibodies to ER{alpha} and ERß. The blots shown are representative of two or more using separately prepared cell lysates and show 50 µg protein/lane and 500 ng of protein for the standards.

 
To assess the consequences of hormone deficiency resulting from ovariectomy, as well as hormone replacement, Western blotting for ER{alpha} and ERß was performed on enterocyte lysates prepared as described above. When compared to the control Min/+ animals, ovariectomized mice had lower ER{alpha} expression and higher ERß expression in the tumor-prone small intestine. When ovariectomized animals were given 17ß-estradiol, ER{alpha} expression was reduced further, in conjunction with a significant rise in ERß levels.

Similar analyses were performed using enterocytes isolated from the colon (Fig. 2)Citation . In the colonic mucosa, there is apparently little difference in ER expression between wild-type, Min/+, and ovariectomized Min/+ mice. The overall expression of ER{alpha} appeared higher than that of ERß in these three tissue types, although this experiment was not performed to quantitate exact protein levels. Just as in the small intestine, however, supplementation of ovariectomized Min/+ mice with 17ß-estradiol produced a relative decrease in ER{alpha} and an increase in ERß expression. These qualitative results also suggest that augmented ERß expression relative to ER{alpha} correlates with suppression of tumors in Min/+ intestine.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although it is clear that estrogens modulate tumor initiation in hormone-sensitive tissues such as the breast (5, 6, 7 , 28) , the role of estrogens in colorectal carcinogenesis is unclear. Data from observational studies suggest that some women may have a reduced CRC incidence because HRT replacement and multiparity may be protective (2) . Despite these data, there are no clear gender differences in CRC incidence. The peak incidence of CRC for both men and women comes approximately 10 years after the onset of age-related decline in hormone production, an appropriate time span for completion of carcinogenesis once a protective effect has been removed (29 , 30) . Male Min/+ mice do not have a higher tumor number than female Min/+ mice (21) . Despite this, our data show that tumor formation is increased in female Min/+ mice after ovarian hormone withdrawal. Taken together, these observations suggest that both male and female hormones may protect against intestinal tumors. Studies of the effect of castration on tumor formation in Min/+ mice are in progress and will help resolve this question.

Several studies suggest that ER{alpha} and ERß are differentially regulated in estrogen-sensitive tissues. In blood vessels, removal of the endothelial cell layer to simulate vascular injury causes a dramatic increase in ERß expression in the underlying vascular smooth muscle, with little change in the levels of ER{alpha} (31) . This study suggests that estrogens act through ERß to protect against arteriosclerotic vascular lesions by inhibiting smooth muscle proliferation. Another example of reciprocal regulation of ER{alpha} and ERß is seen after neonatal estrogenization of male rats (32) . This treatment produces increased susceptibility to estrogen-induced carcinogenesis of the urogenital tract and is associated with increased expression of ER{alpha} and decreased expression of ERß in the prostate (32) .

The ER{alpha}:ERß ratio may be a determinant of the susceptibility of a tissue to estrogen-induced carcinogenesis. Estrogens induce gene transcription both by binding to the classic estrogen response element and by signaling through an AP1 enhancer element requiring the products of c-fos and c-jun (33) . ER{alpha} and ERß may have similar effects on gene transcription mediated via the estrogen response element but opposite effects on promoters containing AP1 (34) . Estradiol activates AP1-mediated gene transcription when bound to ER{alpha} but inhibits promoter activity when bound to ERß (34) . The converse is true for the antiestrogens, such as tamoxifen, raloxifene, and ICI 164384, which are AP1 transcriptional suppressors via ER{alpha} and activators via ERß (34) . In addition, tumor-prone tissues responsive to hormones, such as breast and endometrium, have overall higher expression levels of ER{alpha} than ERß (9 , 10) .

These observations led to the theory that binding of estrogens to ER{alpha} induces a cancer-promoting response, whereas ERß binding is protective. The results of our study support a reciprocal role for ER{alpha} and ERß expression in the promotion of intestinal tumor formation in vivo. In animals with intact ovaries, the tumor-prone Min/+ small intestine showed increased ER{alpha} and decreased ERß when compared to wild-type tissue. The effect of estradiol replacement after ovariectomy was also informative. The tumor-reducing effect of estradiol replacement in the ovariectomized mice was accompanied by increased ERß and decreased ER{alpha} in the target tissue. It is not known precisely what metabolites of estrogen are present in the intestinal mucosa, nor it is known whether ER{alpha} and ERß compete for ligands in this tissue. The apparent tumor-promoting effects of increased ER{alpha} expression in Min/+ enterocytes are unlikely to be due to differential ligand binding, however, because the affinity of 17ß-estradiol for ER{alpha} and ERß is essentially equivalent (12 , 35 , 36) .

By in situ hybridization, ERß is expressed in the mucosa of the human duodenum, colon, and rectum (10) . In the colonic tissue, expression of ERß protein is significantly higher than that of ER{alpha} (18) . We found the opposite result for the colonic tissue of both wild-type and Min/+ mice, although site-specific comparisons between human and mouse intestinal mucosa tend not to be useful. For example, the phenotype of Min/+ mice is different from that of humans with APC-associated tumors because most Min/+ tumors are located in the small intestine, with very few found in the mouse colon. When we examined noninvasive adenomas in Min/+ mice, we found less ER{alpha} and slightly more ERß than in the normal-appearing Min/+ mucosa. This result is again opposite that observed in humans because Western blot of human CRCs showed a marked decrease in ERß compared to normal tissue, whereas ER{alpha} levels remained the same (18) . It is possible that a shift from ERß to ER{alpha} predominance occurs when a tumor becomes invasive, although this is not supported by previous studies of ER expression in human colonic mucosa, adenomas, and carcinomas (17 , 18) . Unfortunately, invasive cancers cannot be studied in our model because Min/+ mice die from adenoma-related anemia or bowel obstruction before developing a significant number of invasive tumors. Studies of ER distribution and activity in human CRC cell lines show variable results. ER{alpha} is present in HT29 colon cancer cells (37) , although these cells did not show increased proliferation in response to in vitro culture with estradiol. Additional studies showed that ERß mRNA and protein were present in HCT8, HCT116, DLD-1, and LoVo cells (38) , whereas ER{alpha} mRNA and protein were absent. Binding studies determined that all four of these cell lines contained a high affinity receptor for 17ß-estradiol. Cell proliferation studies, however, yielded mixed results because 17ß-estradiol induced the growth of HCT8 cells but inhibited the growth of HCT116, DLD-1, and LoVo cultures (38) .

The effect of ovariectomy on ER expression in Min/+ intestine was interesting. The withdrawal of ligand generally produces up-regulation of its receptor. In this case, loss of endogenous estrogen production by ovariectomy increased ERß and decreased ER{alpha}. This supports a role for ERß as the natural receptor in the intestinal mucosa. This result also suggests that the response to ovariectomy may be due to more than just loss of estradiol stimulation of the intestinal mucosa. ER undergoes a conformational change after ligand binding, releasing inhibitory proteins and leading to dimerization followed by DNA binding and transcriptional activation (39 , 40) . ER{alpha} and ERß can either homo- or heterodimerize. ER{alpha} and ERß differ significantly in their NH2-terminal domains and are therefore likely to interact with different sets of proteins. The various ER ligands may also produce distinct conformational changes in these receptors (41) . Subtle changes in the ER{alpha}:ERß ratio, such as those produced here by ovariectomy, may therefore result in significant changes in the transcriptional activity and/or gene promoter specificity of these receptors.

ER ligands other than endogenous hormones may alter intestinal tumor formation. Diet is known to influence the development of CRC, with high consumption of fruits and vegetables conferring a protective effect. These food categories contain a variety of phytoestrogens that are capable of modulating ER activity. Phytoestrogens appear to have a greater affinity for ERß than they do for ER{alpha} (42) . In the brain, where estrogen may play a protective role against the progression of neurodegenerative conditions such as Alzheimer’s disease, ERß mRNA expression in the paraventricular nucleus of ovariectomized rats is decreased by 17ß-estradiol and up-regulated by the phytoestrogen coumestrol, a substance found in legumes (43) . Our results, which show that ovariectomy followed by estrogen replacement markedly increased expression of ERß in a susceptible tissue, suggest that phytoestrogens may also decrease tumor formation when endogenous hormones are lacking.

One of the possible mechanisms of estrogen-induced carcinogenesis is induction of peroxisome proliferation. Estrogen-induced peroxisome proliferation may be mediated through regulation of members of the PPAR family of nuclear transcription factors (44, 45, 46) . PPAR activation is associated with carcinogenesis in the liver and may also be important in the development of APC-associated colorectal tumors (47) . Interestingly, ligands of PPAR{gamma}, an isoform expressed in human colonic mucosa (48) , stimulate tumorigenesis in Min/+ mice (49) as well as diet-induced carcinogenesis in mice (50) , although they can induce differentiation and suppress the growth of certain human colon cancer cell lines (51) . Thus, estrogens may modify tumorigenesis under conditions of APC deficiency via the regulation of PPAR activity. The possible effects of differential ER{alpha} and ERß expression on PPAR activity in the intestinal tissue remain to be tested.

In conclusion, hormone-associated carcinogenesis is a complex process with species- and tissue-specific differences in receptor expression, receptor isoform distribution, and ligand metabolism as well as significant cross-talk between the different signaling pathways that govern cell fate. This study demonstrates a role for estrogen in modulating Apc-associated intestinal tumors and indicates that tumorigenesis is associated with reciprocal modulation of ER isoform expression in adult female Min/+ mice. Further study is needed to address how ER-estrogen interactions affect enterocyte proliferation and/or differentiation in vivo.


   FOOTNOTES
 
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.

1 Supported by National Cancer Institute Grant NCI-1R29CA74162 (to M. M. B.), NIH Surgical Oncology Research Training Grant T32-CA-68971 (to M. J. W.), and the Irving Weinstein Foundation (A. M. C.). Back

2 To whom requests for reprints should be addressed, at Department of Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. Phone: (617) 732-8910; Fax: (617) 582-6177; E-mail: mbertagnolli{at}partners.org Back

3 The abbreviations used are: CRC, colorectal cancer; HRT, hormone replacement therapy; ER, estrogen receptor; Min/+, C57BL/6J-Min/+; CI, confidence interval; PPAR, peroxisome proliferator-activated receptor. Back

Received 8/25/00. Accepted 1/12/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Potter J. D., Slattery M. L., Bostick R. M., Gapstur S. M. Colon cancer: a review of the epidemiology. Epidemiol. Rev., 15: 499-545, 1993.[Free Full Text]
  2. Potter J. D. Hormones and colon cancer. J. Natl. Cancer Inst. (Bethesda), 87: 1039-1040, 1995.[Free Full Text]
  3. McMichael A. J., Potter J. D. Reproduction, endogenous and exogenous sex hormones, and colon cancer: a review and hypothesis. J. Natl. Cancer Inst. (Bethesda), 65: 1201-1207, 1980.
  4. Hebert-Croteau N. A meta-analysis of hormone replacement therapy and colon cancer in women. Cancer Epidemiol. Biomark. Prev., 7: 653-659, 1998.[Abstract]
  5. Zhu B. T., Conney A. H. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis?. Cancer Res., 58: 2269-2277, 1998.[Abstract/Free Full Text]
  6. Telang N. T., Sato A., Wong G. Y., Osborne M. P., Bradlow H. L. Induction by estrogen metabolite 16{alpha}-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells. J. Natl. Cancer Inst. (Bethesda), 84: 634-638, 1992.[Abstract/Free Full Text]
  7. Klauber N., Paragni S., Flynn E., Hamel E., D’Amato R. J. Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and Taxol. Cancer Res., 57: 81-86, 1997.[Abstract/Free Full Text]
  8. Smith D. F., Toft D. O. Steroid receptors and their associated proteins. Mol. Endocrinol., 7: 4-11, 1993.[Medline]
  9. Gustafsson J. A. Estrogen receptor ß: a new dimension in estrogen mechanism of action. J. Endocrinol., 163: 379-383, 1999.[Medline]
  10. Enmark E., Pelto-Huikko M., Grandien K., Lagercrantz S., Lagercrantz J., Fried G., Nordenskjold M., Gustafsson J-A. Human estrogen receptor ß-gene structure, chromosomal localization, and expression pattern. J. Clin. Endocrinol. Metab., 82: 4258-4265, 1997.[Abstract/Free Full Text]
  11. Hess R. A., Gist D. H., Bunick D., Lubahn D. B., Farrell A., Bahr J., Cooke P. S., Greene G. L. Estrogen receptor ({alpha} and ß) expression in the excurrent ducts of the adult male rat reproductive tract. J. Androl., 18: 602-611, 1997.[Abstract/Free Full Text]
  12. Mosselman S., Polman J., Dijkema R. ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett., 392: 49-52, 1996.[Medline]
  13. Kuipfer G. G. J., Carlsson B., Grandien K., Enmark E., Haggblad J., Nilsson S., Gustafsson J-A. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology, 138: 863-870, 1997.[Abstract/Free Full Text]
  14. Pike A. C. W., Brozowski A. M., Hubberd R. E., Bonn T., Thorsell A-G., Engstrom O., Ljunggren J., Gustafsson J-A., Carlquist M. Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full antagonist. EMBO J., 18: 4608-4618, 1999.[Medline]
  15. Hanstein B., Liu H., Yancisin M. C., Brown M. Functional analysis of a novel estrogen receptor-ß isoform. Mol. Endocrinol., 13: 129-137, 1999.[Abstract/Free Full Text]
  16. Moore J. T., McKee D. D., Slenz-Kesler K., Moore L. B., Jones S. A., Horne E. L., Su J., Kliewer S. A., Lehmann J. M., Willson T. M. Cloning and characterization of human estrogen receptor ß isoforms. Biochem. Biophys. Res. Commun., 247: 75-78, 1998.[Medline]
  17. Singh S., Sheppard M. C., Langman M. J. S. Sex differences in the incidence of colorectal cancer: an exploration of oestrogen and progesterone receptors. Gut, 34: 611-615, 1993.[Abstract/Free Full Text]
  18. Foley E. F., Jazaeri A. A., Shupnik M. A., Jasaeri O., Rice L. W. Selective loss of estrogen receptor ß in malignant human colon. Cancer Res., 60: 245-248, 2000.[Abstract/Free Full Text]
  19. Morin P. J., Sparks A. B., Korinek V., Barker N., Clevers H., Vogelstein B., Kinzler K. W. Activation of ß-catenin-Tcf signaling in colon cancer by mutations in ß-catenin or APC. Science (Washington DC), 275: 1787-1790, 1997.[Abstract/Free Full Text]
  20. Korinek V., Barker N., Morin P. J., van Wichen D., de Weger R., Kinzler K. W., Vogelstein B., Clevers H. Constituitive transcriptional activation by a ß-catenin-Tcf complex in APC-/- colon carcinoma. Science (Washington DC), 275: 1784-1787, 1997.[Abstract/Free Full Text]
  21. Su L-K., Kinzler K., Vogelstein B., Preisinger A. C., Moser A. R., Luongo C., Gould K., Dove W. F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science (Washington DC), 256: 668-670, 1992.[Abstract/Free Full Text]
  22. Mahmoud N. N., Dannenberg A. J., Mestre J., Bilinski R. T., Churchill M. R., Martucci C., Newmark H., Bertagnolli M. M. Aspirin prevents tumors in a murine model of familial adenomatous polyposis. Surgery (St. Louis), 124: 225-231, 1998.[Medline]
  23. Mahmoud N. N., Boolbol S. K., Dannenberg A. J., Mestre J. R., Bilinski R. T., Newmark H. L., Chadburn A., Bertagnolli M. M. The sulfide metabolite of sulindac prevents tumors and restores enterocyte apoptosis in a murine model of familial adenomatous polyposis. Carcinogenesis (Lond.), 19: 87-91, 1998.[Abstract/Free Full Text]
  24. Jacoby R. F., Marshall D. J., Newton M. A., Novakovic K., Tutsch K., Cole C. E., Lubet R. A., Kelloff G. J., Verma A., Moser A. R., Dove W. F. Chemoprevention of spontaneous intestinal adenomas in the ApcMin mouse model by the nonsteroidal anti-inflammatory drug piroxicam. Cancer Res., 56: 710-714, 1996.[Abstract/Free Full Text]
  25. Mahmoud N. N., Carothers A. M., Grunberger D., Bilinski R. T., Churchill M. R., Bertagnolli M. M. Phenolic antioxidants prevent tumors in an animal model of familial adenomatous polyposis. Carcinogenesis (Lond.), 21: 921-927, 2000.[Abstract/Free Full Text]
  26. Moyer M. P. Culture of human gastrointestinal epithelial cells. Proc. Soc. Exp. Biol. Med., 174: 12-15, 1983.[Abstract]
  27. Weyant M. J., Carothers A. M., Bertagnolli M., Bertagnolli M. M. Colon cancer chemopreventive drugs modulate integrin-mediated signaling pathways. Clin. Cancer Res., 6: 949-956, 2000.[Abstract/Free Full Text]
  28. Guzman R. C., Yang J., Rajkumar L., Thordarson G., Chen X., Nandi S. Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy. Proc. Natl. Acad. Sci. USA, 96: 2520-2525, 1999.[Abstract/Free Full Text]
  29. Ilyas M., Straub J., Tomlinson I. P. Genetic pathways in colorectal and other cancers. Eur. J. Cancer, 35: 335-351, 1999.
  30. Kelloff G. J., Hawke E. T., Crowell J. A., Boone C. W., Nayfield S. G., Perloff M., Steele V. E., Lubet R. Strategies for identification and clinical evaluation of promising chemopreventive agents. Oncology (Basel), 10: 1471-1484, 1996.[Medline]
  31. Lindner V., Kim S. K., Karas R. H., Kuiper G. G. J., Gustafsson J-A., Mendelsohn M. E. Increased expression of estrogen receptor-ß mRNA in male blood vessels after vascular injury. Circ. Res., 83: 224-229, 1998.[Abstract/Free Full Text]
  32. Prins G. S., Marmer M., Woodham C., Chang W., Kuiper G., Gustafsson J-A., Birch L. Estrogen receptor ß mRNA ontogeny in the rate prostate and effects of neonatal estrogen exposure on the expression pattern. Endocrinology, 139: 874-883, 1998.[Abstract/Free Full Text]
  33. Gaub M-P., Bellard M., Scheuer I., Chambon P., Sassone-Corse P. Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex. Cell, 63: 1267-1276, 1990.[Medline]
  34. Paech K., Webb P., Kuiper G. G. J. M., Nilsson S., Gustafsson J., Kushner P. J., Scanlan T. S. Differential ligand activation of estrogen receptors ER-{alpha} and ER-ß at AP-1 sites. Science (Washington DC), 277: 1508-1510, 1997.[Abstract/Free Full Text]
  35. Kuipfer G. G. J., Enmark E., Pelto-Huikko M., Nilsson S., Gustafsson J. Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA, 93: 5925-5930, 1996.[Abstract/Free Full Text]
  36. Tremblay G, B., Tremblay A., Copeland N. G., Gilbert D. J., Jenkins N. A., Labrie F., Giguere V. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol. Endocrinol., 11: 353-365, 1997.[Abstract/Free Full Text]
  37. Singh S., Paraskeva C., Gallimore P. H., Sheppard M. C., Langman M. J. S. Differential growth response to oestrogen of premalignant and malignant colonic cell lines. Anticancer Res., 14: 1037-1042, 1994.[Medline]
  38. Fiorelli G., Picariello L., Martineti V., Tonelli F., Brandi M. L. Functional estrogen receptor ß in colon cancer cells. Biochem. Biophys. Res. Commun., 261: 521-527, 1999.[Medline]
  39. Beekman J. M., Allan G. F., Tsai S. Y., Tsai M-J., O’Malley B. W. Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol. Endocrinol., 7: 1266-1274, 1993.[Abstract]
  40. Cowley S. M., Hoare S., Mosselman S., Parker M. G. Estrogen receptors {alpha} and ß form heterodimers on DNA. J. Biol. Chem., 272: 19858-19862, 1997.[Abstract/Free Full Text]
  41. Paige L. A., Christensen D. J., Gron H., Norris J. D., Gottlin E. B., Padilla K. M., Chang C-Y., Ballas L. M., Hamilton P. T., McDonnell D. P., Fowlkes D. M. Estrogen receptor (ER) modulators each induce distinct conformational changes in ER {alpha} and ER ß. Proc. Natl. Acad. Sci. USA, 96: 3999-4004, 1999.[Abstract/Free Full Text]
  42. Kuiper G. G. J. M., Lemmen J. G., Carlsson B., Corton J. C., Safe S. H., van der Saag P. T., van der Burg B., Gustafsson J-A. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology, 139: 4252-4263, 1998.[Abstract/Free Full Text]
  43. Patisaul H. B., Whitten P. L., Young L. J. Regulation of estrogen receptor ß mRNA in the brain: opposite effects of 17ß estradiol and the phytoestrogen, coumestrol. Mol. Brain Res., 67: 165-171, 1999.[Medline]
  44. Ma H., Sprecher H. W., Kolattukudy P. E. Estrogen-induced production of a peroxisome proliferator-activated receptor (PPAR) ligand in a PPAR{gamma}-expressing tissue. J. Biol. Chem., 273: 30131-10138, 1998.[Abstract/Free Full Text]
  45. Llopis J., Westin S., Ricote M., Wang J., Cho C. Y., Kurokawa R., Mullen T-M., Rose D. W., Rosenfeld M. G., Tsien R. Y., Glass C. K. Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc. Natl. Acad. Sci. USA, 97: 4363-4368, 2000.[Abstract/Free Full Text]
  46. Tcherepanova I. L., Puigserver P., Norris J. D., Spiegelman B. M., McDonnell D. P. Modulation of estrogen receptor-{alpha} transcriptional activity by the coactivator PGC-1. J. Biol. Chem., 275: 16302-16308, 2000.[Abstract/Free Full Text]
  47. He T-C., Chan T. A., Vogelstein B., Kinzler K. W. PPAR{delta} is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell, 99: 335-345, 1999.[Medline]
  48. DuBois R. N., Gupta R., Brockman J., Reddy B. S., Krakow S. L., Lazar M. A. The nuclear eicosanoid receptor, PPAR{gamma}, is aberrantly expressed in colonic cancers. Carcinogenesis (Lond.), 19: 49-53, 1998.[Abstract/Free Full Text]
  49. Lefebvre A-M., Chen I., Desreumaux P., Najib J., Fruchart J-C., Geboes K., Briggs M., Heyman R., Auwerx J. Activation of the peroxisome proliferator-activated receptor {gamma} promotes the development of colon tumors in C57BL/6J-APC Min/+ mice. Nat. Med., 4: 1053-1057, 1998.[Medline]
  50. Saez E., Tontonoz P., Nelson M. C., Alvarez J. G., Ming U. T., Baird S. M., Thomazy V. A., Evans R. M. Activators of the nuclear receptor PPAR{gamma} enhance colon polyp formation. Nat. Med., 4: 1058-1061, 1998.[Medline]
  51. Sarraf P., Mueller E., Jones D., King F. J., DeAngelo D. J., Partridge J. B., Holden S. A., Chen L. B., Singer S., Fletcher C., Spiegelman B. M. Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat. Med., 4: 1046-1052, 1998.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. J. Gunter, D. R. Hoover, H. Yu, S. Wassertheil-Smoller, T. E. Rohan, J. E. Manson, B. V. Howard, J. Wylie-Rosett, G. L. Anderson, G. Y.F. Ho, et al.
Insulin, Insulin-like Growth Factor-I, Endogenous Estradiol, and Risk of Colorectal Cancer in Postmenopausal Women
Cancer Res., January 1, 2008; 68(1): 329 - 337.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
V. Martineti, L. Picariello, I. Tognarini, S. C. Sala, A. Gozzini, C. Azzari, C. Mavilia, A. Tanini, A. Falchetti, G. Fiorelli, et al.
ER{beta} is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components
Endocr. Relat. Cancer, June 1, 2005; 12(2): 455 - 469.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
K. Woodson, D. J. Weisenberger, M. Campan, P. W. Laird, J. Tangrea, L. L. Johnson, A. Schatzkin, and E. Lanza
Gene-Specific Methylation and Subsequent Risk of Colorectal Adenomas among Participants of the Polyp Prevention Trial
Cancer Epidemiol. Biomarkers Prev., May 1, 2005; 14(5): 1219 - 1223.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S. H. Javid, A. E. Moran, A. M. Carothers, M. Redston, and M. M. Bertagnolli
Modulation of tumor formation and intestinal cell migration by estrogens in the ApcMin/+ mouse model of colorectal cancer
Carcinogenesis, March 1, 2005; 26(3): 587 - 595.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. E. Moran, D. H. Hunt, S. H. Javid, M. Redston, A. M. Carothers, and M. M. Bertagnolli
Apc Deficiency Is Associated with Increased Egfr Activity in the Intestinal Enterocytes and Adenomas of C57BL/6J-Min/+ Mice
J. Biol. Chem., October 8, 2004; 279(41): 43261 - 43272.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
A Bardin, N Boulle, G Lazennec, F Vignon, and P Pujol
Loss of ER{beta} expression as a common step in estrogen-dependent tumor progression
Endocr. Relat. Cancer, September 1, 2004; 11(3): 537 - 551.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
H. Xu, J. K. Uno, M. Inouye, L. Xu, J. B. Drees, J. F. Collins, and F. K. Ghishan
Regulation of intestinal NaPi-IIb cotransporter gene expression by estrogen
Am J Physiol Gastrointest Liver Physiol, December 1, 2003; 285(6): G1317 - G1324.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
V. Pais, I. Leav, K.-M. Lau, Z. Jiang, and S.-M. Ho
Estrogen Receptor-{beta} Expression in Human Testicular Germ Cell Tumors
Clin. Cancer Res., October 1, 2003; 9(12): 4475 - 4482.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. L. Kettunen, A. S. L. Kettunen, and N. E. Rautonen
Intestinal Immune Responses in Wild-Type and ApcMin/+ Mouse, a Model for Colon Cancer
Cancer Res., August 15, 2003; 63(16): 5136 - 5142.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
G. Akiri, E. Sabo, H. Dafni, Z. Vadasz, Y. Kartvelishvily, N. Gan, O. Kessler, T. Cohen, M. Resnick, M. Neeman, et al.
Lysyl Oxidase-related Protein-1 Promotes Tumor Fibrosis and Tumor Progression in Vivo
Cancer Res., April 1, 2003; 63(7): 1657 - 1666.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. T. Kim, B. C. Kim, I. Y. Kim, M. Mamura, D. H. Seong, J.-J. Jang, and S.-J. Kim
Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apoptosis through Cleavage of BAD in TSU-PR1 Human Cancer Cells
J. Biol. Chem., August 30, 2002; 277(36): 32510 - 32515.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. Signoretti and M. Loda
Estrogen Receptor {beta} in Prostate Cancer : Brake Pedal or Accelerator?
Am. J. Pathol., July 1, 2001; 159(1): 13 - 16.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Weyant, M. J.
Right arrow Articles by Bertagnolli, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weyant, M. J.
Right arrow Articles by Bertagnolli, M. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online