Aromatase excess in cancers of breast, endometrium and ovary

Abstract

Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb 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. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.

Figure 1. Tissue sites of estrogen production in women

The biologically active estrogen, estradiol (E₂) is produced at least in three major sites: 1- direct secretion from the ovary in reproductive-age women; 2- by conversion of circulating androstenedione (A) of adrenal and/or ovarian origins to estrone (E₁) in peripheral tissues; and 3- by conversion of A to E₁ in estrogen-target tissues. In the latter two instances, estrogenically weak E₁ is further converted to E₂ within the same tissue. The presence of the enzyme aromatase and 17β-hydroxysteroid dehydrogenase (17β-HSD) is critical for E₂ formation at these sites. E₂ formation by peripheral and local conversion is particularly important for postmenopausal women and for estrogen-dependent diseases such as breast cancer, endometriosis and endometrial cancer.

Figure 2. CYP19 (aromatase) gene

Expression of the aromatase gene is regulated by the tissue-specific activation of a number of promoters via alternative splicing.

Figure 3. Physiological regulation of aromatase expression

FSH induces aromatase expression via a cAMP-dependent pathway in ovarian granulosa cells via promoter II. Steroidogenic factor-I (SF-1) mediates this action of FSH. On the other hand, a combination of a glucocorticoid and a member of the class I cytokine family induces aromatase expression in skin and adipose tissue fibroblasts via promoter I.4 located 70 kb upstream of the coding region. Binding of signal transducers and activators of transcription (STAT)-3 and glucocorticoid receptor (GR) upstream of promoter I.4 mediate regulation of aromatase expression in these fibroblasts.

Figure 4. Promoter use for aromatase expression in normal and malignant breast tissues

The levels of total aromatase mRNA levels in breast cancer tissue are strikingly higher than normal breast tissue. The normal breast adipose tissue maintains low levels of aromatase expression primarily via promoter I.4. 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. Additionally, the endothelial-type promoter I.7 is also upregulated in breast cancer. Thus, it appears that the prototype estrogen-dependent malignancy breast cancer takes advantage of four promoters (II, I.3, I.7 and I.4) for aromatase expression. The sum of aromatase mRNA species arising from these four promoters markedly increase total aromatase mRNA levels in breast cancer compared with the normal breast.

Figure 5. Origins of estrogen in postmenopausal breast cancer

This figure exemplifies the important pathologic roles of extraovarian (peripheral) and local estrogen biosynthesis in an estrogen-dependent disease in postmenopausal women. The estrogen precursor androstenedione (A) originates primarily from the adrenal in the postmenopausal woman. Aromatase expression and enzyme activity in extraovarian tissues such as fat increases with advancing age. The aromatase activity in skin and subcutaneous adipose fibroblasts gives rise to formation of systemically available estrone (E₁) and to a smaller extent estradiol (E₂). The conversion of circulating A to E₁ in undifferentiated breast adipose fibroblasts compacted around malignant epithelial cells and subsequent conversion of E₁ to E₂ in malignant epithelial cells provide high tissue concentrations of E₂ for tumor growth. The clinical relevance of these findings is exemplified by the successful use of aromatase inhibitors to treat breast cancer.

Figure 6. Detail of epithelial-stromal interaction via estrogen and cytokines in breast cancer

Estradiol (E₂) increases secretion of antiadipogenic cytokines (IL-11) from malignant epithelial cells and upregulates their antiadipogenic-type receptors (TNF-receptor type 1, TNFR1) in undifferentiated fibroblasts. These redundant mechanisms give rise to accumulation of undifferentiated fibroblasts around malignant epithelial cells (desmoplastic reaction), which express aromatase and form E₂.

Figure 7. Effects of PGE₂, PKA, PKC and sodium butyrate (NaBu) on aromatase expression in breast adipose fibroblasts

We recently found that sodium-butyrate (NaBu) profoundly decreased promoter I.3/II-specific aromatase mRNA expression induced by PGE₂ or a surrogate hormomonal cocktail made of dibutyryl cAMP (Bt₂cAMP) plus the PKC activator phorbol diacetae (PDA). MCM, Bt₂cAMP+PDA or NaBu regulated aromatase mRNA levels or enzyme activity only specifically via promoters I.3/II but not other promoters. Recruitment of phosphorylated ATF-2 by a CRE (-211/-199) in the promoter I.3/II region conferred the response to malignant epithelial cells conditioned medium, PGE₂ or Bt₂cAMP+PDA. Malignant cell-conditioned medium, PGE₂ or Bt₂cAMP+PDA stabilized a complex comprised of phosphorylated ATF-2, C/EBPβ and CBP in the common regulatory region of promoters I.3/II. The inhibitory effect of NaBu on transcription was not accompanied by comparable changes in overall histone acetylation patterns of promoters I.3/II. NaBu treatment, however, consistently decreased ATF-2 phosphorylation and disrupted the activating complex. Taken together, these findings represent a novel mechanism of NaBu action and provide evidence that aromatase activity can be attenuated in a signaling pathway- and tissue-specific fashion. Our data also suggested that malignant cells secreted substances other than PGE2. These unknown substances were associated with signaling pathways other than cAMP-PKA in the activation of aromatase promoters I.3 and II.