Regulation of steroid sulphatase expression and activity in breast cancer

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.     

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.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts

The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1-DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal D-amino acid (instead of L form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1-DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner.     

Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Regulation of estrogen synthesis in postmenopausal women

The decrease in ovarian estrogen production that occurs at the menopause may lead to an increase in peripheral aromatase activity. While estrogens can have beneficial effects on some body tissues, such as bone and the cardiovascular system, they also have a crucial role in supporting the growth and development of breast tumors. A number of factors, including interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and prostaglandin E(2) (PGE(2)), which can stimulate aromatase activity, have now been identified. As plasma concentrations of some cytokines increase at the menopause, this may account for the increased peripheral aromatase activity that is detected in older women. Macrophages and lymphocytes which infiltrate breast tissue are now thought to be an important source of cytokines that can stimulate aromatase activity in this tissue. Studies, we have recently carried out, have suggested that the endogenous estrogen metabolite, 2-methoxy-estradiol, may be able to modulate the ability of cytokines and PGE(2) to stimulate aromatase activity. Understanding the role of endogenous estrogen metabolites in regulating estrogen synthesis may give rise to new strategies for the prevention or treatment of breast cancer.     

Relationship between tumour aromatase activity, tumour characteristics and response to therapy

Aromatase activity has been measured in human breast cancers by incubating tumour minces with [7-³H]testosterone and characterizing purified oestradiol (E₂) fractions by chemical derivative formation. Of 247 primary tumours, 178 showed evidence of oestrogen biosynthesis, levels varying between 0.5 and 12.5 fmol E₂ produced/h/g tissue. These values were quantitatively small but at least comparable with those in other peripheral tissues. There was no correlation between presence or level of aromatase activity and the histopathology of the tumours although oestrogen biosynthesis was more likely to be present in more cellular tumours. Aromatase activity was also unrelated to age, menopausal status, lymph node status and T stage of the patient from which the tumour was derived. In a subgroup of patients presenting without clinical evidence of distant metastatic disease, no significant relation was detected between tumour aromatase and disease-free interval, but tumours without aromatase activity were associated with increased survival at 36 months after primary treatment. A statistically significant correlation was also detected between the presence of tumour aromatase and oestrogen receptors. Furthermore, in small subgroups of patients with “advanced” breast cancer tumour aromatase was related to response to aminoglutethimide but not tamoxifen therapy. Whilst these results do not conclusively define a role for local synthesis of oestrogen in the progression of breast cancer, this possibility still exists and further studies on tumour aromatase are warranted.     

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.     

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E2 (PGE2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity (p<0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 microM) and the aromatase inhibitor, formestane (10nM) (p<0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 microM celecoxib and 10nM formestane but not with 10 microM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 microM. Celecoxib (25 microM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE2 and estradiol.     

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.     

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.     

Functional heterogeneity of breast fibroblasts is defined by a prostaglandin secretory phenotype that promotes expansion of cancer-stem like cells

Fibroblasts are important in orchestrating various functions necessary for maintaining normal tissue homeostasis as well as promoting malignant tumor growth. Significant evidence indicates that fibroblasts are functionally heterogeneous with respect to their ability to promote tumor growth, but markers that can be used to distinguish growth promoting from growth suppressing fibroblasts remain ill-defined. Here we show that human breast fibroblasts are functionally heterogeneous with respect to tumor-promoting activity regardless of whether they were isolated from normal or cancerous breast tissues. Rather than significant differences in fibroblast marker expression, we show that fibroblasts secreting abundant levels of prostaglandin (PGE2), when isolated from either reduction mammoplasty or carcinoma tissues, were both capable of enhancing tumor growth in vivo and could increase the number of cancer stem-like cells. PGE2 further enhanced the tumor promoting properties of fibroblasts by increasing secretion of IL-6, which was necessary, but not sufficient, for expansion of breast cancer stem-like cells. These findings identify a population of fibroblasts which both produce and respond to PGE2, and that are functionally distinct from other fibroblasts. Identifying markers of these cells could allow for the targeted ablation of tumor-promoting and inflammatory fibroblasts in human breast cancers.     

Correlation of interleukin 6 with interleukin 1alpha in human mammary tumours, but not with oestrogen receptor expression

The authors hypothesized that IL-6 production by breast tumour tissues would correlate with OR-positivity, as only those tumours that contain oestrogen receptors (OR) and use oestrogen as a mitogen would benefit from locally increased oestrogen. IL-6 increases the activity of the 17beta-oxidoreductase, which converts oestrone to oestradiol, a process that may contribute to the increased concentration of oestrogen around breast tumours. IL-1alpha upregulates IL-6 production; therefore, the correlation between IL-1alpha and IL-6 immunoreactivity and OR-positivity in paraffin-embedded human breast tumours was further investigated.The results indicate IL-6 immunoreactivity in 40 of 66 paraffin embedded breast tumour specimens, a finding which did not correlate with the clinical evaluation of oestrogen receptor positivity (P=0.32 by Fisher's exact test). However, there was a correlation between IL-6 and IL-1alpha immunoreactivity (P<0.05). To study an in vitro model for this phenomenon, the IL-6 immunoreactivity in available cell lines was tested. Surprisingly, no production of IL-6 protein or mRNA could be detected in any of the cell lines, and this did not change with IL-1alpha stimulation. Therefore, none of the cell lines apparently reflected the immunological potential observed in the majority of surgical specimens.     

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.     

Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status

Elevated serum IL-6 concentrations have been associated with poor prognosis in a variety of cancers, and decreases in serum IL-6 concentrations have been reported after chemotherapy. We have demonstrated that serum IL-6 concentrations are elevated in breast cancer patients [normal women 0.7 +/- 2.5 pg/ml (n=36), breast cancer patients 38.3 +/- 138.7 pg/ml (n = 111)]. After vaccination of breast cancer patients with a combination of tumour-associated antigens and biological adjuvants (IL-2 and GM-CSF), the concentration of IL-6 decreased significantly (P<0.05) to 8.1 +/- 14.6 pg/ml (n=85). Other studies have shown that oestrogen suppresses IL-6 production in oestrogen receptor positive breast cancer cells. We have demonstrated that the decrease in IL-6 associated with vaccination is related to the oestrogen receptor status of the tumours from breast cancer patients, as a decrease in IL-6 from 124.0 +/- 267.5 pg/ml (n=26) to 6.2 +/- 11.0 pg/ml (n=34) only occurs in patients with oestrogen receptor negative tumours. The IL-6 concentration in breast cancer patients with oestrogen receptor positive tumours remained unchanged (9.5 pg/ml before vaccination, and 9.3 pg/ml after vaccination). These results suggest that postmenopausal women with oestrogen receptor negative breast cancers, who do not respond well to either hormonal therapy with tamoxifen or adjuvant chemotherapy, may have a significant response to vaccination with autologous tumour-associated antigens.     

Tibolone and its delta-4, 7alpha-methyl norethisterone metabolite are reversible inhibitors of human aromatase

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3alpha- and 3beta-hydroxy derivatives and the delta-4, 7alpha-methylnorethisterone (7alpha-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) activities but also stimulate steroid sulphotransferase and 17beta-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7alpha-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7alpha-MeNET, but not the 3alpha- or 3beta-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10muM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7alpha-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17beta-HSD1, tibolone and 7alpha-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.