| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Article |
Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois
Abstract
Abstract I. Introduction A. The Aromatase Enzyme B. The Aromatase (CYP19) Gene C. Physiologic Regulation of Aromatase Expression in Human Tissues D. Pathology Related to Aromatase Overexpression II. Aromatase and Breast Cancer A. Estrogen Formation in Breast Cancer 1. Aromatase in Adipose Tissue and Skin: Endocrine Effect. 2. Local Aromatase in Breast Cancer Tissue: Paracrine/Intracrine Effect. B. Cellular Mechanisms That Regulate Aromatase Expression in Normal and Malignant Breast Tissue 1. Cellular Localization of Aromatase in Breast Cancer. 2. Inhibition of Adipogenic Differentiation in Breast Cancer: Link to Aromatase Overexpression. C. Molecular Mechanisms Responsible for Elevated Aromatase Expression in Breast Cancer 1. Up-Regulation of Promoters I.3 and II. 2. Regulation of Promoters II and I.3 in MCF7 Cells. 3. Up-Regulation of Promoter I.7 in Breast Cancer. D. Summary of Regulation of Aromatase Expression in Breast Cancer E. Aromatase Inhibitors in the Treatment of Breast Cancer III. Aromatase and Endometriosis A. Mechanisms of Growth and Inflammation in Endometriosis B. Definitions of Experimental Models and Abnormal Tissues in Women with Endometriosis C. Estrogen Formation in Endometriosis D. Cellular Mechanisms That Regulate Aromatase Expression in Eutopic Endometrium and Endometriosis E. Prostaglandin E2 Biosynthesis and Action in Endometriosis 1. Prostaglandin E2 Biosynthesis in Endometrial Disease. 2. Prostaglandin E2 Action on Endometriotic and Endometrial Stromal Cells. F. Molecular Mechanisms Responsible for Increased Expression of Steroidogenic Genes in Endometriosis 1. Up-Regulation of Promoter II for Increased Aromatase Expression. 2. Transcriptional Mechanisms Responsible for Increased Expression of the Aromatase Gene in Endometriosis. G. Activation of Multiple Steroidogenic Promoters in Endometriosis H. Summary of Regulation of Promoter II in Endometriotic Stromal Cells and Breast Adipose Fibroblasts I. Aromatase Inhibitors in the Treatment of Endometriosis IV. Aromatase and Endometrial Cancer V. Aromatase and Uterine Fibroids (Leiomyomata) A. Molecular Basis for Estrogen Dependence of Uterine Leiomyomata for Growth B. Regulation of Estrogen Biosynthesis in Leiomyoma Smooth Muscle Cells C. Aromatase Expression in Uterine Leiomyoma Tissues D. Regulation of Aromatase Expression in Leiomyoma Tissues and Smooth Muscle Cells E. Treatment of Uterine Leiomyomata with Aromatase Inhibitors VI. Aromatase Excess Syndrome A. Background and Clinical Manifestations B. Genetic Basis for Familial Aromatase Excess Syndrome C. Discussion on Gain-of-Function Mutations That Affect the Aromatase Gene VII. Conclusions Regarding Aromatase Overexpression in Estrogen-Dependent Human Disease
A single gene encodes the key enzyme for estrogen biosynthesis termed aromatase, inhibition of which effectively eliminates estrogen production. Aromatase inhibitors successfully treat breast cancer and endometriosis, whereas their roles in endometrial cancer, uterine fibroids, and aromatase excess syndrome are less clear. Ovary, testis, adipose tissue, skin, hypothalamus, and placenta express aromatase normally, whereas breast and endometrial cancers, endometriosis, and uterine fibroids overexpress aromatase and produce local estrogen that exerts paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kilobase regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. Three mechanisms are responsible for aromatase overexpression in a pathologic tissue versus its normal counterpart. First, cellular composition is altered to increase aromatase-expressing cell types that use distinct promoters (breast cancer). Second, molecular alterations in stromal cells favor binding of transcriptional enhancers versus inhibitors to a normally quiescent aromatase promoter and initiate transcription (breast/endometrial cancer, endometriosis, and uterine fibroids). Third, heterozygous mutations, which cause the aromatase coding region to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, result in excessive estrogen formation owing to the overexpression of aromatase in many tissues.
The initial entry of cytosolic cholesterol into the mitochondrion, which is facilitated by steroidogenic acute regulatory protein (StAR1), represents a major step for steroidogenesis. Six enzymes encoded by at least five specific genes then catalyze the conversion of cholesterol to the biologically active estrogen estradiol. The aromatase enzyme catalyzes the final and key step, i.e., the conversion of C19 steroids to estrogens (Fig. 1).
|
The aromatase enzyme is localized in the endoplasmic reticulum of estrogen-producing cells (Sebastian and Bulun, 2001
; Simpson et al., 2002). Aromatase enzyme complex is comprised of two polypeptides. The first of these is a specific cytochrome P450 (P450), namely aromatase cytochrome P450 (P450arom) (the product of the CYP19 gene) (Simpson et al., 2002). The second is a flavoprotein, NADPH-cytochrome P450 reductase and is ubiquitously distributed in most cells. Thus, cell-specific expression of P450arom determines the presence or absence of aromatase activity. For practical purposes, we will refer to "P450arom" as "aromatase" throughout this text. Since only a single gene (CYP19) encodes aromatase in mice and humans, targeted disruption of this gene or inhibition of its product effectively eliminates estrogen biosynthesis in these species (Simpson et al., 2002).
In the human, aromatase is expressed in a number of cells, including the ovarian granulosa cell, the placental syncytiotrophoblast, the testicular Leydig cell, and various extraglandular sites, including the brain and skin fibroblasts (Simpson et al., 1994). The principal product of the ovarian granulosa cells during the follicular phase is estradiol (-17
). Additionally, aromatase is expressed in human adipose tissue. Whereas the highest levels of aromatase are in the ovarian granulosa cells in premenopausal women, the adipose tissue becomes the major aromatase-expressing body site after menopause (Fig. 2) (Grodin et al., 1973; Bulun and Simpson, 1994). Although aromatase level per adipose tissue fibroblast may be small, the sum of estrogen arising from billions of adipose tissue fibroblasts in the entire body makes a physiologic impact. The principal product of the ovary is the potent estrogen estradiol. In adipose tissue, estrogenically weak estrone is produced from androstenedione of adrenal origin in relatively large quantities. However, at least half of this peripherally produced estrone is eventually converted to estradiol in extraovarian tissues (Fig. 2) (MacDonald et al., 1979).
|
The recent availability of the Human Genome Project on the Internet permitted us to reconstruct the entire aromatase gene by computer-assisted analysis (Sebastian and Bulun, 2001) (Fig. 3A). Two overlapping bacterial artificial chromosome clones contained the entire gene, which spans approximately 123 kb (Sebastian and Bulun, 2001) (Fig. 3A). The gene is transcribed from the telomere to the centromere, and the region encoding the aromatase protein spans 30 kb of the 3'-end and contains nine exons (II-X) (Shozu et al., 2003b). The ATG translation start site is located in coding exon II. The upstream (telomeric) 93 kb of the gene contains a number of promoters (Simpson et al., 1994; Sebastian and Bulun, 2001). The most proximal gonad-specific promoter II and two other proximal promoters, I.3 (expressed in adipose tissue and breast cancer) and I.6 (expressed in bone), are found within the 1-kb region upstream of the ATG translation start site in exon II, as expected (Fig. 3A). Promoter I.2, the minor placenta-specific promoter, is located approximately 13 kb upstream of the ATG site in exon II. The promoters specific for the brain (I.f), endothelial cells (I.7), fetal tissues (I.5), adipose tissue (I.4), and placenta (2a and I.1) are localized in tandem order at 
33, 36, 43, 73, 78, and 93 kb, respectively, upstream of the first coding exon, the exon II (Fig. 3A) (Mahendroo et al., 1993; Sebastian and Bulun, 2001). In addition to promoter II-specific sequences, transcripts containing two other unique sequences, untranslated exons I.3 and I.4, are present in adipose tissue and in adipose tissue fibroblasts maintained in culture (Mahendroo et al., 1993). Transcription initiated by use of each promoter gives rise to a transcript with a unique 5'-untranslated end that contains the sequence encoded in the first exon immediately downstream of this particular promoter (Fig. 3B). Therefore, the 5'-untranslated region of aromatase mRNA is promoter-specific and may be viewed as a signature of the particular promoter used. It should be emphasized again that all of these 5'-ends are spliced onto a common junction 38 bp upstream of the ATG translation start site (Mahendroo et al., 1993). Consequently, the sequence encoding the open reading frame is identical in each case. Thus, the expressed protein is the same regardless of the splicing pattern (Fig. 3B).
|
Aromatase expression in fish, birds, and lower mammals (rodents) is expressed in the brain and gonads via highly conserved promoters named I.f and II, respectively (Simpson et al., 1994). Aromatase is expressed in placenta and other extragonadal tissues such as the skin, adipose tissue, and bone of higher mammals via recruitment of additional promoters throughout the evolution. To our knowledge, the human is unique in that it has the largest number of tissues with aromatase expression and the highest levels of aromatase expression per tissue among all species.
The placental promoter I.1 is the most distally located promoter (93 kb), which gave rise to splicing of a 103-bp first exon onto the common splice junction immediately upstream of the ATG translational start site (Kamat et al., 1998; Jiang et al., 2000; Jiang and Mendelson, 2003) (Fig. 3A). The activity of promoter I.1 is the basis for 100 to 1000 times elevated levels of circulating estrogen in pregnant women (Kamat et al., 1998; Jiang et al., 2000; Jiang and Mendelson, 2003). Thus, recruitment of this promoter may have an evolutionary impact, since, of all species, humans are unique to acquire and maintain extraordinarily high levels of aromatase expression in placenta.
A transgene containing the human genomic region upstream of the placental exon I.1 was found to exhibit readily detectable promoter activity in the mouse placenta (Kamat et al., 2002). Thus, although mouse placental tissue does not express aromatase endogenously, it may contain the necessary transcriptional factors for human promoter I.1 expression. This suggests that the recruitment of aromatase expression in human placenta occurred via alterations in the mammalian genome throughout the evolution. One of the key mechanisms that permits the recruitment of such a large number of promoters seems to be the extremely promiscuous nature of the common splice acceptor site, since activation of each promoter gives rise splicing of an untranslated first exon onto this common junction immediately upstream of the translation start site in the coding region.
C. Physiologic Regulation of Aromatase Expression in Human Tissues
The primary site of aromatase expression in premenopausal women is the ovarian follicle, where FSH induces aromatase and thus estradiol production in a cyclic fashion (Simpson et al., 1994). Ovarian aromatase expression is mediated primarily by FSH receptors, cAMP production and activation of the proximal promoter II (Simpson et al., 1994) (Fig. 4). Men and postmenopausal also produce estrogen by aromatase that resides in extragonadal tissues such as adipose tissue and skin (Simpson et al., 1994) (Fig. 4). Estrogen produced in these extragonadal tissues are of paramount importance for the closure of bone plates and bone mineralization in both men and postmenopausal women, since the phenotype of men with defective genes of aromatase or estrogen receptor (ER)-
include severe osteoporosis and extremely tall stature with growth into adulthood (Bulun, 2000). A distal promoter (I.4) located 70 kb upstream of the coding region directs aromatase expression in adipose tissue and skin fibroblasts. Promoter I.4 in these tissues is regulated by combined action of a glucocorticoid and a member of the class I cytokine family [e.g., interleukin (IL)-6, IL-11, leukemia inhibitory factor, and oncostatin-M] (Fig. 4) (Zhao et al., 1995).
|
; Jiang et al., 2000). On the other hand, extremely low baseline levels of promoter II in the ovary are stimulated strikingly by FSH via a cAMP-dependent pathway in the developing follicle (Simpson et al., 1994) (Fig. 4). Serum, cytokines, and growth factors are inhibitory to promoter II. In case of adipose and skin fibroblasts, promoter I.4 is used in vivo and activated coordinately by a glucocorticoid in the presence of a cytokine (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M). Glucocorticoid receptors and the Janus kinase-1/signal transducer and activator of transcription (STAT)-3 pathway mediate this induction (Zhao et al., 1995).
Promoter use in cultured adipose tissue fibroblasts is a function of hormonal treatments. For example, in vitro studies showed that prostaglandin (PG) E2 or cAMP analogs stimulate aromatase expression strikingly via proximally located promoters II and I.3, whereas treatment with a glucocorticoid plus a member of the class I cytokine family switches promoter use to I.4 (Zhao et al., 1995, 1996a).
D. Pathology Related to Aromatase Overexpression
Thus far, aromatase overexpression in four estrogen-responsive human diseases has been shown to be critical, since the use of aromatase inhibitors has been therapeutic in 1) breast cancer, 2) endometriosis, 3) endometrial cancer, 4)uterine fibroids, and 5) hypogonadism in aromatase excess syndrome. We will initially discuss the cellular and mechanisms responsible for aromatase overexpression in each disorder in detail. Then we will point out common mechanisms that mediate aromatase overexpression in these disorders.
Breast cancer, endometriosis, and uterine leiomyomata are highly responsive to estrogen for growth, as evidenced by high concentrations of estrogen receptors in these tissues (Bulun et al., 1997). Thus, increased estrogen formation as a consequence of aromatase expression in these tissues has primarily local (paracrine and intracrine) effects (O'Neill and Miller, 1987, 1988; James et al., 1990; Bulun et al., 1993, 1994a,b; Noble et al., 1996; Shozu et al., 2001). On the other hand, aromatase may be overexpressed in many human tissues ubiquitously in aromatase excess syndrome (Shozu et al., 2003b). This gives rise to increased circulating estrogen and both local and systemic (endocrine) effects of estrogen such as gynecomastia and hypogonadism in males and premature breast development and anovulation in females.
II. Aromatase and Breast Cancer
Paracrine interactions between malignant breast epithelial cells, proximal adipose fibroblasts, and vascular endothelial cells are responsible for estrogen biosynthesis and the lack of adipogenic differentiation in breast cancer tissue. It seems that malignant epithelial cells secrete factors that inhibit the differentiation of surrounding adipose fibroblasts to mature adipocytes and stimulate aromatase expression in these undifferentiated adipose fibroblasts (Meng et al., 2001). The in vivo presence of malignant epithelial cells also enhances aromatase expression in endothelial cells in breast tissue (Zhou et al., 2001). We developed a model in breast cancer, which reconciles the inhibition of adipogenic differentiation and estrogen biosynthesis in a positive feedback cycle (Fig. 5).
|
). This consistency comes from the tightly packed undifferentiated adipose fibroblasts around malignant epithelial cells. Malignant epithelial cells achieve this by secreting large quantities of TNF and IL-11 that inhibit the differentiation of fibroblasts to mature adipocytes. Thus, large numbers of these estrogen-producing cells are maintained proximal to malignant cells (Meng et al., 2001; Zhou et al., 2001). At the same time, a separate set of factors secreted by malignant epithelial cells activates aromatase expression in surrounding adipose fibroblasts (Meng et al., 2001; Zhou et al., 2001) (Fig. 5).
Malignant epithelial cells induce aromatase via activation of aberrant promoters in breast cancer tissue and adipose fibroblasts proximal to tumor (Fig. 6). The breast adipose tissue in disease-free women maintains low levels of aromatase expression primarily via promoter I.4 that lies 73 kb upstream of the common coding region (Mahendroo et al., 1993) (Fig. 6). The proximal promoters I.3 and II are used only minimally in normal breast adipose tissue (Mahendroo et al., 1993). Transcription via activity of promoters II and I.3 in the breast tumor fibroblasts and malignant epithelial cells, however, is strikingly increased (Harada et al., 1993; Agarwal et al., 1996; Utsumi et al., 1996; Zhou et al., 1996) (Fig. 6). Additionally, the endothelial-type promoter I.7 is also up-regulated in breast tumor tissue (Sebastian et al., 2002) (Fig. 6). Thus, it seems that the prototype estrogen-dependent malignancy breast cancer takes advantage of four promoters used in various cell types for aromatase expression (Fig. 6). The sum of aromatase mRNA species arising from these four promoters markedly increases total aromatase mRNA levels in breast cancer compared with the normal breast that uses almost exclusively promoter I.4 (Fig. 6).
|
A. Estrogen Formation in Breast Cancer
There are two sources of estrogen for breast cancer. First, estrogen that arises from extraovarian body sites such as subcutaneous adipose tissue and skin reaches breast cancer by way of circulation in an endocrine manner. Second, estrogen locally produced in breast cancer tissue makes an impact via paracrine or intracrine mechanisms.
1. Aromatase in Adipose Tissue and Skin: Endocrine Effect.
The potential roles of extraovarian aromatase in human physiology and pathology were also recognized initially in the 1960s (MacDonald et al., 1968). These studies demonstrated that the conversion rate of plasma androstenedione to estrone in humans increased as a function of obesity and aging (Grodin et al., 1973; Hemsell et al., 1974) (Fig. 2). These studies also revealed the importance of extraovarian tissues (primarily adipose tissue and skin) as the origin of estrogen in postmenopausal women (Figs. 2 and 4). Extraovarian estrogen formation was shown to be correlated positively with excess body weight in both pre- and postmenopausal women and may be increased as much as 10-fold in morbidly obese postmenopausal women (Grodin et al., 1973; Hemsell et al., 1974). This elevation in association with both obesity and aging bears a striking relationship to the incidence of endometrial hyperplasia and cancer, which are more commonly observed in elderly obese women (MacDonald et al., 1978). It is now recognized that the continuous production of estrogen by the adipose tissue in these women is one of the risk factors of endometrial hyperplasia and cancer.
Evidence also suggests a role of estrogens produced by adipose tissue in the pathogenesis of the breast cancer. For example, breast cancer incidence correlates positively with the body fat content or serum estradiol levels in postmenopausal women, suggesting that estrogen collectively produced in all extraovarian sites reach the breast tissue by circulation in an endocrine fashion and stimulate tumor growth (Huang et al., 1997; Hankinson et al., 1999). A role for adipose tissue estrogen biosynthesis in promoting the growth of breast cancer is implied because of the palliative effects of adrenalectomy in the past. Because estrone production by adipose tissue depends on plasma androstenedione secreted by the adrenal cortex as substrate, the role of adrenalectomy is explicable in terms of the denial of substrate precursor for adipose tissue estrogen biosynthesis. Today, reduction of estrogen biosynthesis in the adipose tissue is accomplished by the use of aromatase inhibitors in the treatment of breast cancer (Brodie et al., 1999a,b) (Fig. 7).
|
; Chetrite et al., 2000; Geisler et al., 2000). These studies showed strikingly increased local levels of estrone, estrone sulfate, and eastradiol in breast tumor tissue compared with circulating estrogen levels (Van Landeghem et al., 1985; Chetrite et al., 2000; Geisler et al., 2000).
A series of paracrine interactions between malignant breast epithelial cells and surrounding adipose stroma were uncovered and explained increased local estrogen levels in the breast bearing a cancer. For example, independent studies from at least six different laboratories indicated striking increases in aromatase enzyme activity and mRNA levels in breast fat adjacent to cancer compared with those in distal fat or disease-free breast adipose tissue (O'Neill et al., 1988; Bulun et al., 1993; Harada et al., 1993; Reed et al., 1993; Sasano et al., 1994; Utsumi et al., 1996; Zhou et al., 1996) (Fig. 7). We also found that an elevation in aromatase expression in adipose stroma surrounding malignant epithelial cells is regulated by complex cellular, molecular, and genomic mechanisms (Bulun et al., 1993; Agarwal et al., 1996; Zhou et al., 2001). Interestingly, the overall aromatase expression in breast adipose tissue in mastectomy specimens bearing a breast tumor was significantly higher than that in benign breast tissue removed for reduction mammoplasty (Agarwal et al., 1995, 1996, 1997).
Estrogens can act both directly or indirectly on human breast cancer cells to promote proliferation. Breast cancer cells in culture elaborate a number of growth stimulants in response to estrogen, which can act in an autocrine and paracrine manner to stimulate their growth; however, there is also evidence that estrogens can directly induce proliferation of breast cancer cells. The pathologic significance of local aromatase activity in breast cancer was recognized based on the following in vitro data. MCF7 breast cancer cells, which were stably transfected to express an mouse mammary tumor virus-promoter-driven human aromatase cDNA and inoculated into oophorectomized nude mice, remained dependent on circulating androstenedione for their rapid growth (Yue et al., 1994). Further evidence for the importance of local aromatase expression in the breast tissue came from an in vivo mouse model demonstrating that aromatase overexpression in breast tissue is sufficient for maintaining hyperplasia in the absence of circulating estrogen and that aromatase inhibitors abrogated hyperplasia (Tekmal et al., 1999). These transgenic mice with mouse mammary tumor virus-promoter-driven local aromatase in breast tissue are more prone for breast cancer development (Kovacic et al., 2004).
B. Cellular Mechanisms That Regulate Aromatase Expression in Normal and Malignant Breast Tissue
1. Cellular Localization of Aromatase in Breast Cancer.
Breast adipose tissue is primarily composed of mature lipid-containing cells and other stromal elements. This latter group of cells in the breast adipose tissue was characterized using immunohistochemical methods (Price et al., 1992). Ninety percent of these resident cells of adipose tissue are fibroblasts, i.e., the potential precursors of mature adipocytes, and another 7% represented endothelial cells. Most (80-90%) aromatase transcripts in adipose tissue was demonstrated to reside in fibroblasts compared with mature adipocytes (Price et al., 1992). Moreover, aromatase enzyme activity was found to reside primarily in the fibroblast component of the adipose tissue in a previous study from the same laboratory (Ackerman et al., 1981).
Immunoreactive aromatase was localized to both the malignant epithelial cells and surrounding fibroblasts in breast tumor tissues (Santen et al., 1994; Sasano et al., 1996; Brodie et al., 1998). Different antibodies, however, showed variable affinity to malignant epithelial cells versus fibroblasts (Sasano et al., 2003). Immunoreactive aromatase was also observed in endothelial cells in normal breast tissue and breast tumors. Recently published data using RNA from laser-captured breast tumor cells showed aromatase mRNA in both stromal and malignant epithelial cells in tumor tissues from three patients (Sasano et al., 2003). Markedly high levels of aromatase enzyme activity have been consistently detected in breast adipose fibroblasts freshly isolated from breast tissue with or without cancer (Ackerman et al., 1981; Price et al., 1992). Aromatase enzyme activity in malignant breast epithelial cells, on the other hand, is either undetectable or extremely low (Pauley et al., 2000).
Adjacent adipose tissue including the dense fibroblast layer seems to account for most aromatase expression in breast tumors for the following reasons. First, the quantity of adjacent adipose tissue surrounding a clinically detectable breast tumor is comparatively very large; e.g., the volume of adipose tissue within a 1-inch radius of a 1-ml breast tumor is 129 ml. Second, the most intense aromatase immunostaining was observed in the adipose tissue fibroblasts located in and around the fibrous capsule (i.e., desmoplastic reaction) surrounding malignant cells (Sasano et al., 1994). Third, levels of aromatase expression and activity in fibroblasts isolated from breast adipose tissue or tumor are 10 to 15 times higher than those found in malignant epithelial cells or cell lines (Pauley et al., 2000).
2. Inhibition of Adipogenic Differentiation in Breast Cancer: Link to Aromatase Overexpression.
Extraordinarily large quantities of TNF and IL-11 are produced and secreted by malignant breast epithelial cells (Meng et al., 2001) (Fig. 8). These two cytokines mediate one of the most commonly observed biological phenomena in breast tumors, the desmoplastic reaction. Desmoplastic reaction or accumulation of fibroblasts around malignant epithelial cells serves to maintain the strikingly hard consistency in many of these tumors (i.e., the traditional macroscopic description of malignant breast tumors as "scirrhous cancer") and increased local concentrations of estrogen via aromatase overexpression localized to these undifferentiated fibroblasts. The inhibition of differentiation of fibroblasts to mature adipocytes mediated by TNF and IL-11 is the key event responsible for desmoplastic reaction, because neither malignant cell-conditioned media nor these cytokines caused the proliferation of adipose tissue fibroblasts (Meng et al., 2001). Moreover, blocking both TNF and IL-11 in cancer cell-conditioned media using neutralizing antibodies is sufficient to reverse this antidifferentiative effect of cancer cells completely (Fig. 8) (Meng et al., 2001). In summary, desmoplastic reaction primarily occurs via the action of cytokines (TNF and IL-11) secreted by the malignant epithelial cells to inhibit the differentiation of adipose tissue fibroblasts to mature adipocytes. This tumor-induced block in adipocyte differentiation is mediated by the selective inhibition of expression of the essential adipogenic transcription factors C/EBP and PPAR
(Meng et al., 2001) (Fig. 8).
|
Estrogen per se seems to potentiate this antiadipogenic action via indirect mechanisms. For example, treatment of T47D breast cancer cells with estradiol increased the mRNA levels of IL-11 by 3-fold (Crichton et al., 1996). Moreover, the cellular actions of TNF are mediated by two distinct receptors: TNF receptor type 1 (TNFR1) (also known as p60 in humans) and TNFR2 (p80). TNFR1 but not TNFR2 was found to be responsible for the inhibition of adipocyte differentiation using mutants of TNF specific for the stimulation of either receptor type (Hube and Hauner, 2000). We recently demonstrated that TNFR1 is responsible for inhibition of adipocyte differentiation in breast cancer (Deb et al., 2004). Interestingly, estradiol enhances this antiadipogenic effect by inducing TNFR1 levels in adipose fibroblasts (Deb et al., 2004) (Fig. 8).
Thus, large amounts of antiadipogenic cytokines (e.g., TNF and IL-11) secreted by malignant epithelial cells serve to maintain increased numbers of the aromatase-expressing cell type, i.e., undifferentiated adipose fibroblast, in breast tumor tissue. This is further enhanced by stimulatory effects of estrogen on IL-11 production in cancer cells and on the TNF receptor type that mediates adipogenic inhibition (Fig. 8).
C. Molecular Mechanisms Responsible for Elevated Aromatase Expression in Breast Cancer
Alternative promoter use is a major mechanism that mediates increased aromatase expression in breast cancer. The normal breast adipose tissue maintains low levels of aromatase expression primarily via promoter I.4 that lies 73 kb upstream of the common coding region (Fig. 6). The proximally located promoters I.3 and II are used only minimally in normal breast adipose tissue. Promoter II and I.3 activities in the breast cancer, however, are strikingly increased (Harada et al., 1993; Agarwal et al., 1996; Utsumi et al., 1996; Zhou et al., 1996). Additionally, the endothelial-type promoter I.7 is also up-regulated in breast cancer (Sebastian et al., 2002). Thus, it seems that the prototype estrogen-dependent malignancy breast cancer takes advantage of four promoters (II, I.3, I.7, and I.4) for aromatase expression (Fig. 6). The sum of aromatase mRNA species arising from these four promoters markedly increases total aromatase mRNA levels in breast cancer compared with the normal breast that uses almost exclusively promoter I.4 (Fig. 6).
1. Up-Regulation of Promoters I.3 and II.
Using an in vivo approach, we and two other groups demonstrated by quantitative exon-specific RT-PCR that the use of the proximal promoters II/I.3 are strikingly up-regulated in adipose tissue adjacent to breast cancer and in breast cancer tissue per se (Agarwal et al., 1996; Utsumi et al., 1996; Zhou et al., 1996). As noted earlier, promoters II and I.3 are located within 215 bp from each other and are coordinately induced by cAMP-dependent or -independent mechanisms in adipose fibroblasts in breast tumors. These promoters possibly share common regulatory DNA motifs.
Increased promoter I.3/II activity is, in part, the basis for increased aromatase expression in peri- and intratumoral adipose tissue fibroblasts (Fig. 6) (Agarwal et al., 1996). Over the past several years, we and others sought to elucidate the mechanisms underlying this cancer-induced increase in promoters I.3/II activity in adipose tissue fibroblasts.
There are two experimental models, which are not mutually exclusive, for the regulation of promoters I.3 and II in breast adipose fibroblasts in tumor tissue. In the first model, PGE2 induces aromatase via promoters I.3 and II employing both cAMP/protein kinase A- and protein kinase C-dependent pathways (Zhao et al., 1996a). In this model, PGE2 stimulates binding activity of an orphan nuclear receptor, LRH-1, to a nuclear receptor half-site (-136/-124 bp) upstream of promoter II (Clyne et al., 2002; Karuppu et al., 2002). Treatment with PGE2 strikingly increased both LRH-1 expression and its binding activity to the aromatase promoter II in cultured adipose fibroblasts. LRH-1 overexpression significantly increased aromatase promoter II activity and aromatase enzyme activity in cultured adipose fibroblasts (Fig. 9). From an in vivo perspective, LRH-1 was up-regulated in undifferentiated fibroblasts in breast tumor tissue compared with those in disease-free breast tissue (C. Clyne, personal communication). It was suggested that a corepressor of LRH-1, short heterodimer partner (SHP), may inhibit aromatase expression in fibroblasts of normal breast tissue (Kovacic et al., 2004) (Fig. 9). The recently reported increases in cyclooxygenase (COX)-2 expression and beneficial effects of nonsteroidal anti-inflammatory in breast cancer support this model (Brueggemeier et al., 1999; Richards et al., 2002). In vivo evidence for increased PGE2 formation in breast cancer, however, is still lacking at this time.
|
) (Fig. 9). We hypothesize that a hormonal cocktail secreted from malignant epithelial cells induces aromatase in undifferentiated adipose fibroblasts via redundant pathways. For example, the addition of a COX-2 inhibitor or an adenylyl cyclase inhibitor or mutation of the LRH-1 binding site of the aromatase promoter II does not reverse induction of aromatase expression by malignant epithelial cell-conditioned medium. We isolated two cis-acting elements that stimulated promoter I.3/II use in response to cancer cell-conditioned medium (Zhou et al., 2001). Two critical elements were determined as a C/EBP site (-317/-304) and a CRE (-211/-197), since mutation of either element abolished conditioned medium-induced promoter activity (Fig. 9). Malignant epithelial cell-conditioned medium strikingly induced the expression of C/EBP, which binds to the C/EBP site in promoter I.3/II region and increases its activity (Zhou et al., 2001) (Fig. 9). In contrast, promoter I.3 and II are inhibited by transcription factors C/EBP and 
that bind to the same site in adipose fibroblasts treated with benign epithelial cell conditioned medium.
Malignant epithelial cell-conditioned medium also induced phosphorylation of ATF-2 and its binding to the CRE (Fig. 9). This CRE is occupied by nonphosphorylated ATF-2 in fibroblasts treated with benign epithelial cell-conditioned medium associated with inactivation of promoters I.3 and II. Moreover, chromatin immunoprecipitation PCR showed that the activator transcriptional complex in a malignant environment contains C/EBP, phosphorylated ATF-2, and p300/cAMP response element-binding protein-binding protein (CBP), whereas the inactivator complex (benign environment) contained nonphosphorylated ATF-2 and histone deacetylase-1 (HDAC1) (Fig. 9). Although this second model reflects in vivo conditions, the individual factors in malignant cell-conditioned medium, which contribute to up-regulation of aromatase, are not currently known. Cytokines in malignant cell-conditioned medium probably do not account for this up-regulation, since the TNF and IL-6 group of cytokines selectively induce only promoter I.4, which is not up-regulated either in vivo or in vitro in breast tumors (Zhao et al., 1995, 1996b; Agarwal et al., 1996; Zhou et al., 2001).
A unified model for promoter II/I.3 activation in breast cancer therefore predicts that malignant epithelial cells secrete a number of factors including PGE2 (Fig. 9). These factors induce a number of signaling pathways in a redundant fashion to activate the transcription of the aromatase gene via promoter I.3/II in adipose fibroblasts. PGE2 possibly arising from malignant epithelial cells is a candidate factor for activation of promoters I.3 and II in breast cancer. This, however, has not been demonstrated in vivo. Neither PGE2 nor its downstream regulators cAMP or LRH-1-binding site in promoter II were found to be essential for activation of promoters II in adipose fibroblasts treated with malignant cell-conditioned medium (Zhou et al., 2001). In disease-free breast tissue, the incorporation of a number of transcriptional repressors into the multimeric complex that occupies promoter I.3/II region is associated with inhibition of transcription. Malignant epithelial cell conditioned medium, on the other hand, gives rise to replacement of this inhibitory complex by an activator transcriptional complex composed of distinct factors such as phosphorylated ATF-2, C/EBP, p300/CBP, and possibly LRH-1 (our unpublished observations) (Fig. 9).
In summary, the proximal promoters II and I.3 clustered within a 215-bp region are coordinately regulated. They remain quiescent in fibroblasts of normal breast tissue via redundant binding of multiple transcriptional inhibitors (Fig. 9). In a malignant breast environment, however, these promoter regions are occupied by multiple transcriptional enhancers as a result of activation of multiple signaling pathways in a fail-safe fashion to increase aromatase expression in breast fibroblasts (Fig. 9).
2. Regulation of Promoters II and I.3 in MCF7 Cells.
Chen et al. (2002) studied the regulation of a number of gene reporter constructs of the promoter II/I.3 region. They found that estrogen receptor-related -1 and CREB1 up-regulate and v-erbA-related factor-2, chicken ovalbumin upstream transcription factor (COUP-TF) inhibitor, retinoic acid receptor , Snail, and Slug proteins down-regulate this promoter region (Chen et al., 2002). The in vivo relevance of these findings will become clearer in the future once the relative significance of aromatase enzyme activity and estrogen biosynthesis is demonstrated in malignant epithelial cells.
3. Up-Regulation of Promoter I.7 in Breast Cancer.
Studies summarized above employed exon-specific RT-PCR analysis of 5'-untranslated ends of aromatase mRNA in breast cancer tissues. This limited strategy permitted only the detection of promoters previously identified from healthy tissues (Agarwal et al., 1996; Utsumi et al., 1996; Zhou et al., 1996). Discovery-driven approaches designed to identify novel promoter regions in breast cancer or adjacent adipose tissues, however, have not been published until recently. To identify novel promoter regions in cancer tissues and proximal fat, we employed the 5'-rapid amplification of cDNA ends (RACE) procedure using total RNA isolated from breast cancer and proximal adipose tissue samples. We cloned a novel 101-bp untranslated first exon (I.7) that comprises the 5'-end of 29 to 54% of aromatase mRNA isolated from breast cancer tissues (Sebastian et al., 2002) (Fig. 10). The levels of aromatase mRNA with exon I.7 were significantly increased in breast cancer tissues and adipose tissue adjacent to tumors (Fig. 10). We identified a promoter immediately upstream of exon I.7 and mapped this to about 36 kb upstream of the ATG translation start site of the aromatase gene (Sebastian et al., 2002) (Fig. 10). Promoter I.7 is a TATA-less promoter containing cis-regulatory elements found in megakaryocytic and endothelial type promoters (Fig. 10). Maximal promoter activity could be demonstrated in human microvascular endothelial cells (Fig. 10). Binding of the transcription factor GATA-2 to a specific GATA cis-regulatory element in this promoter was critical for its regulation in endothelial cells (Sebastian et al., 2002). In conclusion, promoter I.7 is a GATA-2-regulated endothelial-type promoter of the human aromatase gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessel physiology (Fig. 10).
|
D. Summary of Regulation of Aromatase Expression in Breast Cancer
Several alternative cellular and molecular mechanisms serve to maintain excessive levels of aromatase activity in breast stroma proximal to malignant epithelial cells. First, malignant epithelial cell-derived factors induce aromatase overexpression via the transcription factors LRH-1, C/EBP, and phosphorylated ATF-2 (Fig. 5). These factors are incorporated into a multimeric transcriptional complex that occupies the aromatase promoter I.3/II region in adipose tissue fibroblasts adjacent to epithelial cells. Second, aromatase is overexpressed in vascular endothelial cells of tumor tissue via binding of GATA-2 and other endothelial-type transcription factors to promoter I.7. These factors may also mediate angiogenesis in tumor tissue. Moreover, estrogen is known to induce the angiogenic factor VEGF in cancer cells (Nakamura et al., 1996, 1999; Ruohola et al., 1999; Shekhar et al., 2000) (Fig. 5). Third, we demonstrated recently that antiadipogenic cytokines IL-11 and TNF secreted by malignant epithelial cells block the differentiation of the aromatase-expressing cells (fibroblasts) to mature adipocytes that do not express aromatase (Fig. 5). These cytokines thereby secreted abundantly by malignant epithelial cells serve to maintain a dense layer of aromatase-expressing fibroblasts proximal to malignant epithelial cells to provide structural and hormonal support. Fourth, the expression of IL-11 in malignant epithelial cells and antiadipogenic-type TNF receptors in adjacent adipose tissue fibroblasts are up-regulated by estrogen produced as a consequence of elevated aromatase activity in breast tumors. This positive feedback involving complex epithelial-stromal interactions favor higher numbers of undifferentiated fibroblasts, angiogenesis, and increased local estrogen concentrations in breast tumors (Fig. 5). These four mechanisms interact to maintain high levels of estrogen production in a breast tumor.
E. Aromatase Inhibitors in the Treatment of Breast Cancer
Today, aromatase inhibitors are the most effective endocrine treatment of estrogen-responsive breast cancer (Santen, 2002) (Fig. 7). Six recent head-to-head randomized clinical trials published since 2000 demonstrated the superiority of aromatase inhibitors to tamoxifen in the treatment of breast cancer (Bonneterre et al., 2000; Mouridsen et al., 2001; Baum et al., 2002, 2003; Goss et al., 2003; Milla-Santos et al., 2003; Paridaens et al., 2003; Buzdar et al., 2004). Long-term side effect profiles of these agents will determine whether aromatase inhibitors will replace tamoxifen or other selective estrogen receptor modulators in the long run.
There are two intriguing implications of these results. First, it is pharmacologically more efficacious to block estrogen formation rather than its action at least by currently approved estrogen antagonists or selective estrogen receptor modulators. Second, the local effect of aromatase inhibitors at the target tissue level to block local estrogen formation possibly represents the most critical mechanism for the superior therapeutic potential of aromatase inhibitors (Fig. 7).
Targeting aromatase in breast cancer as a therapeutic strategy was first conceptualized in the 1960s (Santen, 2002). Aminoglutethimide was the first aromatase inhibitor tested for this purpose. Although the first-generation aromatase inhibitor aminoglutethimide was as efficacious as tamoxifen in the treatment of breast cancer, its adverse side effects precluded widespread use (Santen, 2002). Tamoxifen was introduced in the 1970s and became the gold standard for hormonal treatment of breast cancer (Santen, 2002). Second-generation aromatase inhibitors were tested in Europe in the 1980s and were found to be as efficacious as tamoxifen (Santen, 2002). Finally, the third-generation aromatase inhibitors were approved in the United States to treat postmenopausal breast cancer in the 1990s and proven to be superior to tamoxifen (Lu et al., 1998; Bonneterre et al., 2000; Dixon et al., 2000; Mouridsen et al., 2001; Baum et al., 2002, 2003; Goss et al., 2003; Milla-Santos et al., 2003; Paridaens et al., 2003; Buzdar et al., 2004). These new inhibitors have a benign side-effect profile and suppress estrogen production in extraovarian tissues and within the breast cancer tissue itself. This effectively blocks estrogenic action, reduces recurrences, and prolongs disease-free survival in postmenopausal women with breast cancer (Baum et al., 2002, 2003). Aromatase inhibitors are also effective in the treatment of breast cancer that becomes resistant to treatment with tamoxifen (Goss et al., 2003).
In these studies, tumors that express ER were more responsive to aromatase inhibitors compared with the tumors with an unknown receptor status (Baum et al., 2002, 2003). Future studies are required to determine whether aromatase inhibitors might be beneficial in ER-negative tumors via ER-independent mechanisms.
III. Aromatase and Endometriosis
Endometriosis is an estrogen-dependent disease that affects 6 to 10% of American women of reproductive age (approximately 4-6 million) and is the most common cause of chronic pelvic pain (Giudice and Kao, 2004). Endometriosis is a systemic disorder that is characterized by the presence of endometrium-like tissue in ectopic sites outside the uterus, primarily on pelvic peritoneum and ovaries, and is linked to chronic pelvic pain, pain during sex, and infertility (Giudice and Kao, 2004). In the United States, endometriosis is the third most common gynecologic disorder that requires hospitalization and is a leading cause of hysterectomy. Only 50% of women with endometriosis achieve pain relief in response to existing hormonal treatments or conservative surgery (Vercellini et al., 1997). Thus, there is a clear need to develop novel and effective therapies for endometriosis.
The clinical significance of estrogen biosynthesis in endometriosis is exemplified by the clinical observations that estrogen is essential for growth of endometriosis. We and others demonstrated abundant aromatase expression and local estrogen production in endometriotic tissue (Kitawaki et al., 1997; Zeitoun et al., 1999; Bulun et al., 2001; Fang et al., 2002; Gurates et al., 2002; Yang et al., 2002). The subsequent introduction of aromatase inhibitors in the treatment of endometriosis successfully underscored the presence of aromatase in endometriotic tissue (Takayama et al., 1998; Ailawadi et al., 2004). These recent results suggested that aromatase inhibitors might treat endometriosis more effectively than GnRH analogs via suppression of local estrogen formation in endometriotic tissue. Thus, as in breast cancer, aromatase is a critical therapeutic target in endometriosis.
A. Mechanisms of Growth and Inflammation in Endometriosis
Two basic pathologic processes, growth and inflammation, are responsible for chronic pelvic pain and infertility, which are the primary devastating symptoms of endometriosis. Estrogen, growth factors, and metalloproteinases enhance the growth and invasion of endometriotic tissue, whereas prostaglandins and cytokines mediate pain, inflammation, and infertility (Bruner et al., 1997; Ryan and Taylor, 1997). Research work from our laboratory and other investigators over the past 10 years uncovered a molecular link between inflammation and estrogen production in endometriosis (Bulun et al., 2001). This is mediated by a positive-feedback cycle that favors expression of key steroidogenic genes, most notably StAR and aromatase, expression of COX-2, and continuous local production of estradiol and PGE2 in endometriotic tissue (Noble et al., 1997; Tsai et al., 2001; Sun et al., 2003) (Fig. 11). We find that the aberrantly expressed transcription factor steroidogenic factor-1 (SF-1) mediates PGE2-cAMP-dependent coactivation of multiple steroidogenic genes, most notably StAR and aromatase, in endometriosis (our unpublished observations) (Zeitoun et al., 1999).
|
We and others demonstrated a number of molecular abnormalities in endometriosis (Fig. 12). The prototype abnormality was the presence of significant levels of StAR and aromatase activity and expression of protein and mRNA in the stromal cell component of endometriosis, whereas StAR or aromatase expression was either absent or barely detectable in the eutopic endometrium of disease-free women (Noble et al., 1996; Noble et al., 1997; Tsai et al., 2001; Gurates et al., 2002; Sun et al., 2003) (Fig. 12). The eutopic endometrium of women with endometriosis contains low but significant levels of aromatase mRNA and enzyme activity and represents an intermediate state of this disease. It seems that upon retrograde menstruation and implantation of this inherently abnormal tissue on pelvic peritoneal surfaces, aromatase expression and enzyme activity are amplified by up to 400 times (Noble et al., 1996, 1997). COX-2 expression, which is important for PGE2 synthesis, is increased markedly in both eutopic endometrium and endometriotic tissue of women with endometriosis (Ota et al., 2001; Wu et al., 2004) (Fig. 12).
|
