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Estrogen—the Good, the Bad, and the Unexpected

Prince Henry’s Institute of Medical Research, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3168, Australia

Correspondence: Address all correspondence and requests for reprints to: Evan R. Simpson, Ph.D., Prince Henry’s Institute of Medical Research, Monash Medical Center, 246 Clayton Road, Clayton, Melbourne, Victoria 3168, Australia. E-mail: evan.simpson{at}phimr.monash.edu.au

I. Introduction

II. The Concept of Local Estrogen Biosynthesis

III. Aromatase and Its Gene

IV. The ArKO Mouse

V. The ArKO Mouse and the Metabolic Syndrome

VI. The Metabolic Syndrome in Humans with Natural Mutations in Aromatase

VII. Summary of the Metabolic Effects of Estrogens

VIII. Local Aromatase Expression in the Breast and Breast Cancer

IX. Role of LRH-1 in Aromatase Expression in the Breast

	I. Introduction

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
IN THE LAST DECADE or so, our knowledge of the roles of the steroids referred to as "sex hormones," namely testosterone and estradiol, has undergone a quiet revolution. In the first place, models of androgen and estrogen insufficiency, whether they be natural or engineered, have revealed new and unexpected roles for sex steroids, some of which have nothing to do with reproduction. Furthermore, both categories of steroid have roles in both sexes, which blunts the definition of the terms "androgen" and "estrogen." Secondly, the gradual acceptance of the role of local steroid hormone action, particularly as it applies in postmenopausal women and in men, provides new insights into the significance of paracrine and intracrine actions and requires a reevaluation of the importance of circulating steroid hormone levels. This article will attempt to review both of these developments, particularly in the context of the work of this laboratory on the aromatase knockout (ArKO) mouse and the role of local aromatase expression within the breast and breast cancer.

	II. The Concept of Local Estrogen Biosynthesis

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
Models of estrogen insufficiency have revealed new and unexpected roles for estrogens in both males and females. These models include natural mutations in the aromatase gene, as well as mouse knockouts of aromatase and the estrogen receptors (1, 2, 3, 4, 5, 6). In addition there is one man described with a natural mutation in the ER (7). Some of the roles of estrogens apply equally to males and females and do not relate to reproduction, for example the bone, vascular, and metabolic syndrome phenotypes.

In postmenopausal women and in men, estradiol does not appear to function as a circulating hormone; instead, it is synthesized in a number of extragonadal sites such as breast, brain, and bone where its actions are mainly at the local level as a paracrine or intracrine factor. Thus, in postmenopausal women and in men, circulating estrogens are not the drivers of estrogen action; instead, they reflect the metabolism of estrogens formed in these extragonadal sites, and they are reactive rather than proactive (8). Importantly, estrogen biosynthesis in these sites depends on a circulating source of androgenic precursors such as testosterone.

Table 1 shows the plasma steroid levels in postmenopausal women and in men. As can be seen, the levels of estrone and estradiol in the plasma of postmenopausal women are extremely low, lower in fact than those in the circulation of men; and moreover, the levels of circulating testosterone are an order of magnitude greater than those of estrogens in postmenopausal women. This in itself would suggest that circulating testosterone is better placed to serve as a precursor of estradiol in target tissues than is circulating estradiol. On the other hand, the levels of testosterone in the blood of men are an order of magnitude higher than those of women. Significantly, levels of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) in the blood of both men and women are orders of magnitude higher than those of the circulating active steroids. In postmenopausal women, the ovaries secrete 25–35% of the circulating testosterone. The remainder is formed peripherally from androstenedione and DHEA produced in the ovaries and from androstenedione, DHEA, and DHEAS secreted by the adrenals. However, the secretion of these steroids and their plasma concentrations decrease markedly with advancing age (9).

View larger version (33K): [in this window] [in a new window]   FIG. 1. Pathways of metabolism of testosterone and estradiol in target tissues. HSD, Hydroxysteroid dehydrogenase; 5-diol, 5/ß-androstane-diol; 4-dione, androstenedione; testo, testosterone; E1, estrone; E2, estradiol; DHT, dihydrotestosterone; UGT, UDP-glucuronyl transferase; G, glucuronate. [Reproduced with permission from Labrie et al.: Endocr Rev 24:152–182, 2003 (8 ). © The Endocrine Society.]

2. However, in premenopausal women, most of the testosterone is formed, acts, and is metabolized in specific target tissues. Testosterone is a paracrine and intracrine factor, whereas in men it circulates as a hormone and acts globally.

3. On the other hand, in men most of the estradiol is formed, acts, and is metabolized in specific target tissues, whereas in women it circulates as a hormone and acts globally.

4. Finally, in postmenopausal women, neither testosterone nor estradiol functions to any extent as a circulating hormone. Both are mainly formed locally in target tissues and act and are metabolized therein.

The power of local estrogen biosynthesis is illustrated in the case of postmenopausal breast cancer (10). It has been determined that the concentration of estradiol present in breast tumors of postmenopausal women is at least 20-fold greater than that present in the plasma. With aromatase inhibitor therapy, there is a precipitous drop in the intratumoral concentrations of estradiol and estrone together with a corresponding loss of intratumoral aromatase activity, consistent with this activity within the tumor and the surrounding breast adipose tissue being responsible for these high tissue concentrations (11).

In bone, aromatase is expressed primarily in osteoblasts and chondrocytes (12), and aromatase activity in cultured osteoblasts is comparable to that present in adipose stromal cells (13). Thus, it appears that in bone also, local aromatase expression is a major source of estrogen responsible for the maintenance of mineralization, although this is extremely difficult to prove due to sampling problems. Hence, for both breast tumors and for bone, it is likely that circulating estrogen levels are minimally responsible for the relatively high endogenous tissue estrogen levels; rather, the circulating levels reflect the sum of local formation in its various sites. This is a fundamental concept for the interpretation of relationships between circulating estrogen levels in postmenopausal women and estrogen insufficiency or excess in specific tissues.

The second important point is that estrogen production in these extragonadal sites is dependent on an external source of C19 androgenic precursors, because these extragonadal tissues are incapable of converting cholesterol to the C19 steroids (9, 14). As a consequence, circulating levels of testosterone and androstenedione as well as DHEA and DHEAS become extremely important in terms of providing adequate substrate for estrogen biosynthesis in these sites, and therefore differences in the levels of circulating androgens are likely to be important determinants for the maintenance of local estrogen levels in extragonadal sites.

In this context, it is appropriate to consider why osteoporosis is more common in women than in men and why it affects women at a younger age in terms of fracture incidence. We have suggested that uninterrupted sufficiency of circulating testosterone in men throughout life supports the local production of estradiol by aromatization of testosterone in estrogen-dependent tissues, and thus affords ongoing protection against the so-called estrogen deficiency diseases. This appears to be important in terms of protecting the bones of men against mineral loss and may also contribute to the maintenance of cognitive function and prevention of Alzheimer’s disease (15).

	III. Aromatase and Its Gene

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
Estrogen biosynthesis is catalyzed by a microsomal member of the cytochrome P450 superfamily, namely aromatase cytochrome P450 (P450arom, the product of the CYP19 gene). The P450 gene superfamily is a very large one, containing over 3000 members in some 350 families, of which cytochrome P450arom is the sole member of family 19 (website of D. Nelson, http://drnelson.utmem.edu/cytochromeP450.html). This heme protein is responsible for binding of the C19 androgenic steroid substrate and catalyzing the series of reactions leading to formation of the phenolic A ring characteristic of estrogens.

The human CYP19 gene was cloned some years ago (16, 17, 18) when it was shown that the coding region spans nine exons beginning with exon II. Upstream of exon II are a number of alternative first exons that are spliced into the 5'-untranslated region of the transcript in a tissue-specific fashion (Fig. 2). For example, placental transcripts contain at their 5'-end a distal exon, I.1. This is because placental expression is driven by a powerful distal promoter upstream of exon I.1 (19). Examination of the Human Genome Project data reveals that exon I.1 is 89 kb upstream of exon II (20). On the other hand, transcripts in ovary and testes contain sequence at their 5'-end which is immediately upstream of the translational start site. This is because expression of the gene in the gonads utilizes a proximal promoter, promoter II. By contrast, transcripts in cells of mesenchymal origin such as adipose stromal cells and osteoblasts, contain yet another distal exon (I.4) located 20 kb downstream of exon I.1 (21). Adipose tissue transcripts also contain promoter II-specific exonic sequences, but these are undetectable in bone (13).

View larger version (13K): [in this window] [in a new window]   FIG. 2. Diagram of the human aromatase (CYP19) gene showing tissue-specific promoter usage. The coding region comprises exons II–X. Upstream of the translational start site (ATG) are a number of untranslated exons I that are spliced into the coding region at a common 3'-splice junction in a tissue-specific fashion due to use of the promoters I.1–I.4. The promoters are regulated by the factors indicated. Because this splice junction is upstream of the start of translation, the coding region is always the same, regardless of the tissue of expression. HBR, Heme-binding region.

	IV. The ArKO Mouse

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
To investigate the phenotypes resulting from lack of estrogen, some years ago we and others generated the ArKO mouse (5, 6, 23, 24). This was done by replacing most of exon 9 with the neomycin resistance cassette. Because exon 9 contains many of the amino acids involved in substrate binding, and many of the natural point mutations that result in a complete loss of aromatase activity are located in exon 9, deletion of this exon results in a complete abrogation of aromatase activity. The main features of the phenotype of the ArKO mouse can be summarized as follows: infertility and lack of sexual behavior in both males and females; progressive defects in folliculogenesis and spermatogenesis; elevated gonadotropins and testosterone levels; loss of bone mass in both sexes; and a metabolic syndrome with insulin resistance, truncal obesity, and hepatic steatosis. Many, but not all aspects of this phenotype are also present in the ERKO and ER/ßKO mice (reviewed in Ref. 25). The requirements of estrogen for male sexual behavior and for maintenance of male bone mineralization were quite unexpected at the time, but space does not permit discussion of these aspects, which can be found in Refs. 26, 27, 28 . Instead, we will focus here on the role of estrogen in energy homeostasis.

	V. The ArKO Mouse and the Metabolic Syndrome

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
From the age of 12–14 wk onward, ArKO mice develop a progressive phenotype of truncal obesity with increased adiposity in the gonadal and visceral fat pads (6). MRI data show that ArKO females have three to four times as much adipose as wild types, whereas males have twice as much, so this phenotype of increased adiposity is more marked in the females than in the males. As might be expected then, serum leptin levels are also elevated, as shown in Table 2; by 1 yr of age, ArKO females have three times as much circulating leptin as the wild types, whereas males have twice as much, consistent with the degree of adiposity in the males and females.

While conducting these experiments, we noticed that the livers of the male ArKO mice were paler in color than those of the wild types or of the females. Microscopic examination revealed that the livers of the male ArKO mice were engorged in lipid, whereas those of the females were not (30) (Fig. 3). Analysis of the lipid content revealed that this was primarily due to a 4- to 5-fold increase in the triglyceride content of the male ArKO livers. Treatment with estradiol for 6 wk effectively blocked this increase in hepatic lipid accumulation. Thus, the phenotype of the ArKO mice is characterized by a markedly sexually dimorphic lipid partitioning, with the increase in lipid in the case of the females occurring primarily in the visceral adipose depots, whereas in the males there is a shift in lipid deposition such that an increased proportion is deposited in the liver, resulting in marked hepatic steatosis. We also examined the expression of enzymes involved in fatty acid synthesis in the livers of these mice and found that in the males there was a 3- to 4-fold increase in the expression of fatty acid synthase and of acetyl coenzyme A carboxylase-. There was a similar increase in the levels of adipose differentiation-related protein, a fatty acid transporter. Again, these increases were normalized by estradiol replacement (30).

View larger version (62K): [in this window] [in a new window]   FIG. 3. Hepatic phenotype of the male ArKO mouse and the effect of estradiol replacement. The photomicrographs are representative sections of livers from wild-type (WT) and ArKO (KO) male mice and ArKO mice treated with estradiol (KO + E2). The right panel shows the corresponding triglyceride levels.

	VI. The Metabolic Syndrome in Humans with Natural Mutations in Aromatase

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
Currently, about a dozen individuals have been characterized with natural aromatase mutations, of whom five are men (34, 35, 36, 37, 38). The women so far described have been diagnosed at the time of puberty and placed on estrogen replacement, so it has not been possible to study their lipid and carbohydrate profiles. Consequently, these studies have been confined to men with aromatase mutations. The most recent study is of an Argentinian male whose phenotype was characterized by Dr. Laura Maffei and her colleagues in Buenos Aires and by Dr. Cesare Carani and his colleagues in Modena, Italy (38). The metabolic parameters of this subject are presented in Table 4. As can be seen, his glucose and insulin levels are markedly elevated, and these levels are decreased after estradiol replacement. He also has acanthosis nigricans. Based on this, the subject was diagnosed as having type 2 diabetes at the age of 29 yr. Estradiol replacement also caused a decrease in total circulating and low-density lipoprotein cholesterol and an increase in high-density lipoprotein cholesterol. His liver function parameters were also profoundly elevated, as indicated in Table 4, and once again these were markedly reduced upon estrogen replacement. A liver biopsy revealed substantial macro- and microsteatosis as well as portal vein fibrosis and steatosis. He also had carotid plaques. which are unusual in a man of his relative youth, and once again these disappeared after estrogen treatment. Thus, this man with a natural mutation in aromatase has a metabolic syndrome phenotype that is similar in many ways to that of the male ArKO mice.

	VII. Summary of the Metabolic Effects of Estrogens

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

	VIII. Local Aromatase Expression in the Breast and Breast Cancer

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
As indicated previously, aromatase expression in the breast is implicated as the main source of estrogen driving breast cancer development. Studies to examine aromatase activity and expression in breast cancer quadrants have indicated that this activity is highest in quadrants of the breast containing a tumor (39, 40). Indeed, there is a gradient of aromatase expression extending from a tumor, such that expression in the tumor-containing quadrant is equal to that in the tumor itself, but double that in a quadrant of the same breast that does not contain a tumor, which in turn is double again the expression present in a cancer-free breast (41). These results suggest that the tumor is elaborating a factor or factors that stimulate aromatase expression within the tumor and in the surrounding adipose tissue.

To understand which factor or factors might be responsible, we and others have examined not only total aromatase transcript expression but also expression of promoter-dependent transcripts (41, 42, 43) (Fig. 4). In adipose tissue of healthy breast, as indicated previously, aromatase expression is low and is driven primarily by a distal promoter I.4 that is regulated by class 1 cytokines and TNF produced locally in the tissue and acting in a paracrine and autocrine fashion. On the other hand, in the presence of a tumor, the increase in aromatase expression is due primarily to an increase in expression driven off the proximal gonadal promoter, promoter II. This promoter is regulated by factors that stimulate adenylate cyclase. We reasoned that a likely candidate produced by tumors would be PGE2, and indeed it turned out that PGE2 is a most powerful stimulator of aromatase expression in human breast adipose stromal cells (44, 45) (Fig. 5). Moreover, recent work has indicated that estrogen has a role itself in up-regulating PGE2 synthesis and aromatase in estrogen-receptor-positive breast cancer cells (46). Moreover, as is well known, cyclooxygenase-2 is expressed in many breast carcinomas where it correlates with tumor size, high grade, HER-2/neu positivity as well as a worse disease-free interval (53). We would anticipate then that factors that inhibit COX2 activity and thus PGE2 synthesis would inhibit aromatase expression within the breast. Moreover, because this pathway of regulation of aromatase within the breast is unique and does not occur within the bone or in the ovaries (because the ovaries of postmenopausal women cease to synthesize estrogens), such inhibition would specifically inhibit estrogen formation within the breast but would leave other sites of estrogen formation where it serves an important function, such as bone, brain, and the cardiovascular system, protected. Such COX2 inhibitors are common analgesic drugs such as aspirin and ibuprofen, so the question arises, are such compounds beneficial in terms of breast cancer therapy.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Promoter-specific aromatase transcript expression in cancer-free breast tissue and in proximity to a tumor. The left panel shows the situation in healthy breast tissue where promoter I.4 predominates, regulated by cytokines produced by the adipose tissue in a paracrine or autocrine fashion. The right panel shows the situation in a tumor-containing breast in which PGE2 produced by the tumorous epithelium causes switching from promoter I.4 to promoter II and increased aromatase expression.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Stimulation of aromatase activity by PGE2 in human breast adipose stromal cells. The left panel shows the dependence on PGE2 concentration; the right panel shows a time course.

However, there are drawbacks to the use of these compounds. This stems from the fact that because these are highly specific and high-affinity inhibitors of the catalytic activity of aromatase, they inhibit aromatase activity in every site of expression, not only in breast but also in bone, brain, and other sites. Not surprisingly therefore, their use is associated with an increase in bone loss and fracture risk. Interestingly, there is also an increase in arthralgia or inflammatory joint pain (50), and based on the studies discussed earlier in this chapter, it might be anticipated that there is a potential for a poorer lipid profile as well as perhaps development of a metabolic syndrome with long-term use, although as yet there is no evidence for this.

For these reasons therefore, there will clearly be a benefit if one could specifically inhibit aromatase in the breast but leave other sites of expression such as bone protected. The only way to do this is to specifically inhibit aromatase expression within the breast. The fact that there is a unique pathway of aromatase expression within the breast due to the promoter switching described previously allows the possibility for this in principle. This leads to the concept of selective aromatase modulators (28), which are to estrogen synthesis what selective estrogen receptor modulators are to estrogen action, and their tissue site specificity is based on the fact that: 1) the role of estradiol is as a paracrine and intracrine factor in postmenopausal women and in men; 2) the tissue-specific regulation of the aromatase gene is based on the use of tissue-specific promoters; and 3) these promoters employ different stimulatory and inhibitory factors in the various tissue-specific sites of expression. Thus, inhibitors of COX2 would serve as the first generation of such selective aromatase modulators. However, these compounds inhibit the COX enzymes in a ubiquitous fashion, and it would clearly be of benefit to specifically inhibit the pathway of aromatase expression within the breast.

	IX. Role of LRH-1 in Aromatase Expression in the Breast

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 
Aromatase expression from promoter II in the ovary requires the presence of activated CREB, which binds to cAMP response element (CRE) in the promoter II sequence (51). In the ovary, CREB is activated via the signaling pathway that originates when FSH binds to its receptor and activates adenylyl cyclase. In addition to CREB binding to its CRE, activation of the promoter requires the presence of a monomeric orphan member of the nuclear receptor family to bind to a nuclear receptor half site downstream of the CRE. In the ovary, this factor is SF1. In the case of adipose, no SF1 is present (52), so although PGE2 can substitute for FSH in terms of the cAMP signaling pathway, the question arises as to what factor occupies the nuclear receptor half site to activate promoter 2 in breast adipose. We tested a number of monomeric orphan nuclear receptors known to bind to such a half site including ERR, Nurr1, Nor1, NGF1B and liver-receptor homolog-1 (LRH-1) (52). The only factor that could substitute for SF1 in terms of promoter II activation was LRH-1. SF1 and LRH-1 share a high degree of homology, and both belong to the NR5A subfamily of nuclear receptors. In contrast to SF1, LRH-1 is expressed in human adipose tissue as well as in human breast tumors, whereas SF1 is not. Using real time PCR, it was found that in adipose tissue, LRH-1 is expressed in the mesenchymal preadipocytes rather than in the adipocytes themselves, a similar distribution to that of aromatase. Moreover, upon differentiation of human preadipocytes to the lipid-laden phenotype, LRH-1 expression drops precipitously preceding the loss of aromatase expression, suggesting that aromatase expression is dependent on LRH-1. LRH-1 and cAMP activate promoter II synergistically in 3T3L1 preadipocytes, and mutation of the nuclear receptor half site completely abrogates this action of LRH-1 (52).

Based on these studies, therefore, we can conclude that LRH-1 substitutes for SF1 in human breast preadipocytes to activate aromatase promoter II expression (Fig. 6). Thus, inhibition of LRH-1 would result in loss of aromatase activity in the breast and hence of estrogen biosynthesis. Therefore LRH-1 is a potential target for new breast-specific breast cancer therapies, because inhibitors of LRH-1 would specifically inhibit aromatase in breast and thus spare estrogen formation in other tissues. Thus, they would serve as selective aromatase modulators and thus could find utility as the next generation of breast cancer therapeutic agents.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Role of LRH-1 in activation of aromatase promoter II expression in human breast adipose stromal cells. EPIIR, The isoform of the PGE2 receptor that activates adenylyl cyclase; TGA(A)CGTCA, cAMP response element; (CCA)AGGTCA, nuclear receptor half site binding element.

	Footnotes

 
The work from this laboratory described in this review was supported by United States Public Health Service Grant R37AG08174 and by the Victorian Breast Cancer Consortium Inc.

First Published Online April 7, 2005

Abbreviations: ArKO, Aromatase knockout; CRE, cAMP-response element; CREB, CRE binding protein; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; LRH, liver receptor homolog; PGE2, prostaglandin E2; SF1, steroidogenic factor 1.

	References

Top I. Introduction II. The Concept of... III. Aromatase and Its... IV. The ArKO Mouse V. The ArKO Mouse... VI. The Metabolic Syndrome... VII. Summary of the... VIII. Local Aromatase Expression... IX. Role of LRH-1... References

 

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36. Bilezikian JP, Morishima A, Bell J, Grumbach MM 1998 Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. N Engl J Med 339:599–603[Free Full Text]
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42. Harada N, Utsume T, Takagi Y 1993 Tissue-specific expression of the human aromatase cytochrome P450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc Natl Acad Sci USA 90:11312–11316[Abstract/Free Full Text]
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45. Richards JA, Brueggemeier RW 2003 Prostaglandin E2 regulates aromatase activity and expression in human adipose stromal cells via two distinct receptor subtypes. J Clin Endocrinol Metab 88:2810–2816[Abstract/Free Full Text]
46. Frasor J, Danes JM, Komm B, Chang K, Lyttle CR, Katzenellenbogen BS 2003 Profiling of estrogen up-and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144:4562–4574[CrossRef][Medline]
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48. Terry MB, Gammon MD, Zhang FF, Tawfik H, Teitelbaum SL, Britton JA, Subboramaiah K, Dannenberg AJ, Neugut AL 2004 Association of frequency and duration of aspirin use and hormone receptor status with breast cancer risk. JAMA 291:2433–2440[Abstract/Free Full Text]
49. Arun B, Goss P 2004 The role of COX-2 inhibition in breast cancer treatment and prevention. Semin Oncol 31(2 Suppl 7):22–29
50. Howell A, Dowsett M 2004 Endocrinology and hormone therapy in breast cancer: aromatase inhibitors versus antiestrogens. Breast Cancer Res 6:269–274[CrossRef][Medline]
51. Michael MD, Kilgore MW, Morokashi K, Simpson ER 1995 Ad4BB/SF1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566[Abstract/Free Full Text]
52. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 277:20591–20597[Abstract/Free Full Text]
53. Brueggemeier RW, Hackett JC, Diaz-Cruz ES 2005 Aromatase inhibitors in the treatment of breast cancer. Endocr Rev 26:331–345[Abstract/Free Full Text]

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