Regulation of steroid sulphatase expression and activity in breast cancer

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Abstract

Steroid sulphatase (STS) catalysis 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 α (TNFα) 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 TNFα 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. TNFα 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 TNFα and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.

Introduction

The hydrolysis of oestrone sulphate (E1S) to oestrone (E1) by steroid sulphatase (STS) is thought to make a major contribution to the production of oestrogens within breast tumours [1], [2]. The activity of this enzyme in breast tumours is considerably higher than that of the aromatase enzyme complex, which can also contribute to tumour oestrogen synthesis [3]. STS activity is also higher in malignant and benign breast tumours than in adjacent non-involved tissues [4], [5]. Evidence for the potential importance of STS in regulating oestrogen synthesis within breast tumours has recently been obtained. Patients with tumours containing high levels of STS mRNA were found to have a significantly shorter disease free survival as compared with those with low levels of STS mRNA [6]. Furthermore, STS mRNA levels are significantly higher in malignant than non-malignant breast tissues [7].

The highest incidence of breast cancer occurs in post-menopausal women. There is evidence that STS activity is increased in breast tumours from post-menopausal women compared with the activity in tumours from pre-menopausal women [8]. It is well established that peripheral aromatase activity is related to age and body weight [9]. Cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor α (TNFα) are thought to be responsible for the increases in peripheral aromatase activity associated with ageing and obesity [10], [11], [12].

In contrast to our understanding of the regulation of aromatase expression and activity, little is known about the control of STS expression and activity. In breast cancer cells IL-6 and TNFα are able to stimulate STS activity and act synergistically to increase enzyme activity [13]. There is also evidence that progestins may inhibit the expression and activity of E1-STS in breast cancer cells [14]. Whether this regulation occurs at the transcriptional level or post transcriptionally is not known. Studies on the promoter region of the STS gene are limited [15]. While no specific transcription binding sites in the promoter region of this gene have so far been identified the basal promoter and 5′ enhancer element activities did show tissue specificity in transient reporter gene expression studies. Both were only active in cells of placental origin where STS activity is known to be high but showed no activity in COS-1, HeLa or B82 cells. Post translationally a novel protein modification is required at the active site for full enzyme activity. An α-formylglycine residue is formed by oxidation of a cysteine residue at position 75 within the active site [16]. The regulation of, and modifying enzymes involved in this process have not so far been elucidated. It is possible that this process represents another level of regulation of STS activity.

In the present study we have employed a semi-quantitative RT-PCR technique to examine basal and cytokine stimulated STS expression in fibroblasts derived from normal and malignant breast tissues. In addition, we have also examined the ability of IL-6 and TNFα to regulate STS expression in MCF-7 breast cancer cells. We have also explored the possibility that compounds, such as 2-methoxyoestrone-3-O-sulphamate (2-MeOEMATE) may block the ability of cells to respond to cytokines such as TNFα. 2-MeOEMATE, while a potent inhibitor of STS activity [17], can also induce cells to undergo apoptosis and has been found to reduce the ability of TNFα to stimulate aromatase activity in fibroblasts [18], [19].

Section snippets

Cell culture

MCF-7 breast cancer cells and COS-1 cells were obtained from the European Collection of Cell Cultures, (Salisbury, Wilts, UK). Cells were routinely maintained in Dulbeco's minimal essential medium (DMEM) with 5% fetal calf serum (FCS) and other essential nutrients [13]. Cells were cultured at 37°C under 5% CO2 and used when 50–60% confluent. For experiments cells were cultured in DMEM with 5% charcoal stripped FCS.

Culture of breast fibroblasts

Fibroblasts were cultured from breast tissues of women undergoing lumpectomy for

Steroid sulphatase expression in breast tumour tissue-derived fibroblasts

Having developed a RT-PCR method to examine STS mRNA expression it was used to investigate the expression of this enzyme in fibroblasts derived from samples of normal and malignant breast tissue (Fig. 1). Three matched pairs of samples of breast tumour and tissue proximal to the tumour were used to culture fibroblasts and to subsequently determine their relative levels of STS mRNA expression. For subjects 1 and 3, who were pre-menopausal, no significant difference in basal STS mRNA expression

Discussion

The formation of E1 from E1S by the action of STS is a major pathway for in situ oestrogen production in breast tumours. In the present investigation a semi-quantitative RT-PCR method has been used to start to explore the regulation of STS mRNA expression. As previously reported by Evans et al. [21], using an RT-PCR technique it was possible to detect STS mRNA expression in breast tissue-derived fibroblasts and MCF-7 breast cancer cells.

Using cultured fibroblasts derived from breast tumour and

Acknowledgements

We would like to thank B. Malini for technical assistance with the sulphatase assay. This research was supported by Sterix Ltd.

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