Control of aromatase activity in breast cancer cells: The role of cytokines and growth factors

The aromatase complex has a key role in regulating oestrogen formation in normal and malignant breast tissues. Using dexamethasone-treated fibroblasts, derived from breast tumours, breast tumour cytosol and breast tumour-derived conditioned medium (CM) markedly stimulate aromatase activity. The cytokine, interleukin-6 (IL-6) has been identified as a factor present in CM which is capable of stimulating aromatase activity. To examine whether IL-6 may have a role in vivo in regulating breast tissue aromatase activity, IL-6 production and aromatase activity in breast tumour and adipose tissue from breast quadrants were examined. In 5/6 breasts examined so far, aromatase activity was highest in adipose tissue in the breast quadrant containing the tumour or on which the tumour impinged. There was a significant correlation (P < 0.05, Kendall's rank correlation) between IL-6 production and aromatase activity in these breast tissues. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast tissues.     

Factors influencing aromatase activity in the breast

Particularly in postmenopausal women, peripheral aromatase appears to be the major source of oestrogens which may encourage the growth of hormone-dependent tumours. Studies have therefore been undertaken to determine factors which influence biosynthesis of oestrogens within breast tissues. Aromatase activity was measured in (i) breast cancers by incubating tumour homogenates with [7³H]testosterone and characterizing the production of radioactively-labelled oestradiol and (ii) breast fat by incubation of sub-cellular fractions of fibroblast cell lines with [1ß³H]androstenedione and monitoring the formation of ³H₂O. Evidence has been presented that (i) certain treatment regimes for cancer profoundly influence aromatase activity in breast tumours, (ii) aromatase activity is elevated in breast fat associated with malignancy and (iii) breast-derived fluids and extracts can markedly affect aromatase activity in cultured fibroblasts of breast fat.     

Role of human adipose tissue in the production and metabolism of steroid hormones

Radioimmunological methods have been employed for the simultaneous determination of dehydroepiandrosterone, androstenedione, testosterone, oestrogens (oestradiol + oestrone), progesterone, 17 alpha-hydroxyprogesterone and cortisol in human adipose tissue and peripheral blood to compare the hormone pool of adipose tissue with that in the general circulation. Extremely high steroid concentrations in the adipose tissue and hormone pool in the fat of obese subjects were observed. For adipose tissue/serum steroid ratios, the highest values were obtained for dehydroepiandrosterone and the lowest ones for cortisol. A preliminary study showed a great accumulation of steroids in adjacent adipose tissue of breast tumors. Striking differences were observed in the adipose tissue steroid concentrations between benign and malignant mammary tumors. The present findings revealed that blood hormone determinations may be insufficient to consider the steroid hormone availability in various endocrinopathies or steroid responsive tumors, especially when the endocrine state of extremely obese subjects is observed.     

The importance of local synthesis of estrogen within the breast

Tumor aromatase has been correlated with clinical response to treatment with aminoglutethimide in patients with estrogen receptor-positive advanced breast cancer. There was a significant positive relationship between aromatase status and likelihood of response to therapy, none of five patients with aromatase-negative tumors responding compared with 11 of 18 having aromatase-positive cancers. Measurement of aromatase in sequential biopsies of large primary tumors before and during treatment with aminoglutethimide-hydrocortisone showed a marked but paradoxical rise in activity following therapy. Assays of aromatase in adipose tissue from the different quadrants of mastectomy specimens from patients with breast cancer indicate that activity was always higher in quadrants associated with tumor as compared with non-involved quadrants. These results emphasize the importance of local estrogen synthesis within the breast in terms of both the natural history and behavior of breast cancers.     

On the significance of in situ production of oestrogens in human breast cancer tissue

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

Aminoglutethimide--a new endocrine therapy in breast cancer. A cancer research review

Aminoglutethimide is an effective treatment for advanced postmenopausal breast cancer, acting in a novel way. It inhibits peripheral and tumour aromatase, which converts androgens to oestrogens. Marked suppression of oestrone and oestradiol is produced. Aminoglutethimide was used as an inhibitor of adrenal steroid biosynthesis, but the concentrations required to inhibit aromatase in vitro are 10 times lower. This has led to its clinical evaluation in much lower doses. Doses of 125 mg twice daily produce oestrogen suppression equivalent to conventional doses of 1 g daily, and also similar tumour responses. Toxicity is much reduced by the lower dose.     

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.     

Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.     

Studies to confirm the source of 11ß-hydroxyandrostenedione

In a longitudinal study of 82 children we found a gradual rise in median plasma concentrations of 11ß-hydroxyandrostenedione (11ß-OH-A4) from 2.5 to 6.4 nmol/1 during childhood which was similar in both sexes. This could reflect changes in adrenal function during the adrenarche and sexual maturation. Plasma concentrations of 11ß-OH-A4 in adults follow the patterns of cortisol secretion. In patients with diseases of the adrenal cortex, the plasma concentrations of 11ß-OH-A4 were consistent with the pathology of each condition. In women with polycystic ovaries (PCO) undergoing gonadotrophic stimulation for in vitro fertilization and embryo transfer, 11ß-OH-A4 (median = 3.8 nmol/l), testosterone and androstenedione, were raised when compared to women with normal ovaries (11ß-OH-A4 median = 2.6 nmol/l). Follicular fluid has concentrations of 11ß-OH-A4 six to twelve times greater than plasma levels and in women with PCO, 11ß-OH-A4 concentrations were lower than in women with normal ovaries, which is consistent with an inhibition of ovarian 11ß-hydroxylase. Granulosa cells in vitro demonstrated the production of 11ß-OH-A4 by side chain cleavage of cortisol. These data support an adrenal source for 11ß-OH-A4 but the raised plasma concentrations in women with polycystic ovary syndrome (PCOS) may reflect the excess androgen output from the ovary. 11ß-OH-A4 may therefore be an additional marker for ovarian dysfunction.     

The potential role of estrogen in aromatase regulation in the breast

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3–4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.     

The role of cytokines and sulphatase inhibitors in regulating oestrogen synthesis in breast tumours

Synthesis of oestrogens within breast tissues makes an important contribution to the high concentrations of oestradiol which are found in breast tumours. The activities of the enzymes involved in oestrogen synthesis, i.e. the aromatase, oestradiol dehydrogenase (E2DH) and oestrone sulphatase (E1-STS), can be stimulated by several growth factors and cytokines. As it is possible that some of these factors may be derived from cells of the immune system (macrophages and lymphocytes), the effects of basic fibroblast growth factor (bFGF) and interleukin-2 (IL-2), which are produced by these cells, on E2DH activity was examined in MCF-7 cells. Treatment of these cells with bFGF resulted in a dose-dependent increase in E2DH reductive activity whereas IL-2 was inactive at the concentration tested. To obtain further evidence that factors produced by macrophages and lymphocytes can modulate the activities of enzymes involved in oestrogen synthesis, conditioned medium was collected from these cells and found to stimulate both E1-STS and E2DH activities. In addition to understanding the control of oestrogen synthesis in breast tumours an inhibitor to block the synthesis of oestrone via the oestrone sulphatase pathway was developed. Oestrone-3-O-sulphamate (EMATE) is a potent, irreversible, inhibitor of E1-STS. A single dose of EMATE (10 mg/kg) inhibited tissue E1-STS activity in rats by more than 95% for up to 7 days, indicating that this compound may have considerable therapeutic potential for the treatment of breast cancer. Evidence is also reviewed that another steroid sulphatase, dehydroepiandrosterone sulphate sulphatase, may have a crucial role in regulating cytokine production and that this may indirectly control tumour oestrogen synthesis.     

Aromatase activity in adipose tissue from breast quadrants: a link with tumour site

To determine the importance of local oestrogen biosynthesis within the breast, aromatase activity was measured in adipose tissue from the breast quadrants of 12 consecutive mastectomies from patients with breast cancer. Activity was detected in all samples (range 3·6-35·0 fmol oestrogen/mg protein/h) but varied considerably not only among different patients but also among the quadrants of individual breasts. The highest activity in a breast was always found in a quadrant that contained tumour, whereas quadrants with the lowest activity were never associated with the presence of tumour.These results provide evidence of a significant relation between breast adipose tissue and breast cancer. Whether such an association occurs because breast tumours are more likely to develop in areas with enhanced oestrogen biosynthesis or because they secrete into their local environment factors capable of stimulating oestrogen biosynthesis remains to be determined.     

Local endocrine effects of aromatase inhibitors within the breast

To determine the effects of aromatase inhibitors on oestrogen uptake, in situ aromatase activity and endogenous oestrogens in the breast, postmenopausal women with large primary ER-rich breast cancers have been treated neoadjuvantly for 3 months with either letrozole (2.5 or 10 mg daily) or anastrozole (1 or 10 mg daily) or exemestane (25 mg daily). Patients were given an infusion of ³H-androstenedione and ¹⁴C-oestrone for 18 h before and at the end of the study period. Blood, tumour and non-malignant breast were taken immediately after each infusion; oestrogens were extracted and purified. Tumour volume was measured before and during treatment at monthly intervals so that endocrinological changes could be related to clinical response. Treatment with each of the aromatase inhibitors was associated with a profound reduction in peripheral aromatase (as monitored by the level of plasma ³H-oestrone). There was no consistent effect on uptake of radioactively labelled oestrogen into breast tumours but a tendency for levels to increase after treatment in non-malignant breast. Conversely, therapy was associated with a marked inhibition of in situ oestrogen synthesis in both tumour and non-malignant breast (in occasional tissues, inhibitors appeared to be less effective but the effect was not related to clinical or pathological responses). Similar decreases were apparent in endogenous levels of oestrone and oestradiol. The absence of in situ aromatase activity tended to be associated with lack of clinical response to aromatase inhibition but the relationship was not absolute, limiting the utility of measurements of tumour aromatase as a predictive indices. Ex vivo studies of tissue aromatase indicated that such measurements consistently underestimate the inhibitory potential of reversible non-steroidal agents (and occasionally paradoxical in vitro increases in aromatase activity were seen with treatment). However, in situ assays demonstrate that new aromatase inhibitors such as anastrozole, exemestane and letrozole have profound effects on the local endocrinology within the postmenopausal breast, these being compatible with the clinico-pathological changes which occur with treatment.     

Inhibitory effect of a new androstenedione derivative, 14 alpha-hydroxy-4-androstene-3,6,17-trione (14 alpha-OHAT) on aromatase activity of human uterine tumors

The development of human uterine estrogen-dependent tumors is considered to be closely related to estrogen biosynthesis. This study examined whether or not 14 alpha-hydroxy-4-androstene-3,6,17-trione (14 alpha-OHAT), a new 4-androstene-3, 17-dione derivative synthesized microbiologically, inhibits estrogen biosynthetase (aromatase) activities of human uterine tumors (i.e. uterine endometrial cancer, uterine leiomyoma and uterine adenomyosis tissues). 14 alpha-OHAT inhibited aromatase activity in all uterine tumors, dose-dependently (0.1-10 microM). Moreover, 14 alpha-OHAT did not show the binding affinity to rabbit uterine cytosol-sex steroids, and it was not converted to estrogen in human placental preparations. Thus, 14 alpha-OHAT, an aromatase inhibitor, may be useful clinically as an endocrine chemotherapy for peri- or post-menopausal women with uterine estrogen-dependent tumors.