Joanna M. Day

Design and validation of specific inhibitors of 17β-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17beta-HSDs have been identified to date, and with one exception, 17beta-HSD type 5 (17beta-HSD5), an aldo-keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17beta-HSDs, reflecting the major redox activity at the 17beta-position of the steroid, the activities of these 15 enzymes vary, with several of the 17beta-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17beta-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17beta-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.

17β‐hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone‐dependent breast cancer

Oestradiol (E2) stimulates the growth of hormone‐dependent breast cancer. 17β‐hydroxysteroid dehydrogenases (17β‐HSDs) catalyse the pre‐receptor activation/inactivation of hormones and other substrates. 17β‐HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17β‐HSD, 17β‐HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17β‐HSD1 which is important for E2 production and report the inhibition of E1‐stimulated breast tumor growth by STX1040, a non‐oestrogenic selective inhibitor of 17β‐HSD1, using a novel murine model. 17β‐HSD1 and 17β‐HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17β‐HSD12 was highly expressed in breast cancer cell lines, only 17β‐HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1‐stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 μg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1‐stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17β‐HSD, 17β‐HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17β‐HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone‐dependent breast cancer.

The role of 17β-hydroxysteroid dehydrogenases in modulating the activity of 2-methoxyestradiol in breast cancer cells

The bis-sulfamoylated derivative of 2-methoxyestradiol (2-MeOE2), 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE), has shown potent antiproliferative and antiangiogenic activity in vitro and inhibits tumor growth in vivo. 2-MeOE2bisMATE is bioavailable, in contrast to 2-MeOE2 that has poor bioavailability. In this study, we have examined the role of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 in the metabolism of 2-MeOE2. In MDA-MB-231 cells, which express high levels of 17beta-HSD type 2, and in MCF-7 cells transfected with 17beta-HSD type 2, high-performance liquid chromatography analysis showed that a significant proportion of 2-MeOE2 was metabolized to inactive 2-methoxyestrone. Furthermore, MCF-7 cells transfected with 17beta-HSD type 2 were protected from the cytotoxic effects of 2-MeOE2. In contrast, no significant metabolism of 2-MeOE2bisMATE was detected in transfected cells and 17beta-HSD type 2 transfection did not offer protection against 2-MeOE2bisMATE cytotoxicity. This study may go some way to explaining the poor bioavailability of 2-MeOE2, as the gastrointestinal mucosa expresses high levels of 17beta-HSD type 2. In addition, this study shows the value of synthesizing sulfamoylated derivatives of 2-MeOE2 with C17-position modifications as these compounds have improved bioavailability and potency both in vitro and in vivo.

STX140 Is Efficacious In vitro and In vivo in Taxane-Resistant Breast Carcinoma Cells

Purpose: The aim of these studies was to characterize the action of STX140 in a P-glycoprotein–overexpressing tumor cell line both in vitro and in vivo. In addition, its efficacy was determined against xenografts derived from patients who failed docetaxel therapy. Experimental Design: The effects of STX140, Taxol, and 2-methoxyestradiol (2-MeOE2) on cell proliferation, cell cycle, and apoptosis were assessed in vitro in drug-resistant cells (MCF-7DOX) and the parental cell line (MCF-7WT). Mice bearing an MCF-7DOX tumor on one flank and an MCF-7WT tumor on the other flank were used to assess the in vivo efficacy. Furthermore, the responses to STX140 of three xenografts, derived from drug-resistant patients, were assessed. Results: In this study, STX140 caused cell cycle arrest, cyclin B1 induction, and subsequent apoptosis of both MCF-7DOX and MCF-7WT cells. Taxol and 2-MeOE2 were only active in the MCF-7WT parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7WT cells, only STX140 inhibited the growth of tumors derived from MCF-7DOX cells. 2-MeOE2 was ineffective at the dose tested against both tumor types. Two out of the three newly derived docetaxel-resistant xenografts, including a metastatic triple-negative tumor, responded to STX140 but not to docetaxel treatment. Conclusions: STX140 shows excellent efficacy in both MCF-7WT and MCF-7DOX breast cancer xenograft models, in contrast to Taxol and 2-MeOE2. The clinical potential of STX140 was further highlighted by the efficacy seen in xenografts recently derived from patients who had failed on taxane therapy.

The effects of 2-substituted oestrogen sulphamates on the growth of prostate and ovarian cancer cells.

The human endogenous metabolite 2-methoxyoestradiol (2-MeOE2) has been shown to inhibit the proliferation of breast cancer cells. We have previously shown that sulphamoylation of a series of 2-substituted oestrogens greatly enhances their ability to inhibit breast cancer cell proliferation and induce apoptosis. In this study, we have investigated the ability of a number of 2-substituted oestrogens and their sulphamoylated derivatives to inhibit the proliferation of two prostate cancer cell lines, an ovarian cancer cell line and its drug-resistant derivatives. 2-Methoxyoestrone, 2-ethyloestrone and 2-ethyloestradiol had little effect on the growth of the cell lines tested (IC(50)>10 microM). 2-MeOE2 did inhibit the growth of the cells (IC(50)<10 microM), but to a lesser extent than any of the sulphamoylated derivatives tested (IC(50)<1.0 microM). Cells treated with the sulphamoylated derivatives became detached and rounded, displaying a characteristic apoptotic appearance. FACS analysis revealed induced G(2)/M cell cycle arrest. Treatment of cells and subsequent drug removal indicated that the effects of the drugs on the cells were irreversible. Immunoblot analysis indicated that apoptosis may be induced by phosphorylation of BCL-2. From these studies, 2-substituted oestrogen sulphamates are emerging as a potent new class of drug that may be effective against AR+/AR- prostate and ovarian tumours, and against tumours that are resistant to conventional chemotherapeutic regimens.

Overexpression of 17β-hydroxysteroid dehydrogenase type 1 increases the exposure of endometrial cancer to 17β-estradiol.

CONTEXT The local interconversions between estrone (low activity) and 17β-estradiol (potent compound) by 17β-hydroxysteroid dehydrogenases (17β-HSDs) can lead to high 17β-estradiol generation in endometrial cancer (EC). OBJECTIVE Examine the balance between the 17β-HSDs reducing estrone to 17β-estradiol (types 1, 5, 12, and 7) and those oxidizing 17β-estradiol to estrone (2, 4, and 8), in EC. PATIENTS AND METHODS Reducing and oxidizing 17β-HSD activities (HPLC) and mRNA level (RT-PCR) were assessed in normal post-menopausal (n = 16), peritumoral endometrium (normal tissue beside cancer, n = 13), and 58 EC (29 grade 1, 18 grade 2, 11 grade 3). RESULTS Grade 1 EC displayed a shifted estrone reduction/17β-estradiol oxidation balance in favor of 17β-estradiol compared with controls. This was more pronounced among estrogen receptor-α (ER-α)-positive biopsies. Type 1 17β-HSD mRNA (HSD17B1 gene expression, real time PCR) and protein levels (immunohistochemistry) were higher in ER-α-positive grade 1 EC than controls. The mRNA coding for types 4, 5, 7, 8, and 12 17β-HSD did not vary, whereas that coding for type 2 17β-HSD was increased in high-grade lesions compared with controls. Three-dimensional ex vivo EC explant cultures demonstrated that 17β-HSD type 1 generated 17β-estradiol from estrone and increased tumor cell proliferation. Additional in vitro studies using EC cells confirmed that in the presence of 17β-HSD type 1, estrone induced estrogen signaling activation similarly to 17β-estradiol. Therefore, estrone was reduced to 17β-estradiol. CONCLUSIONS Type 1 17β-HSD increases 17β-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

17β-Hydroxysteroid dehydrogenase Type 1 and Type 2: Association between mRNA expression and activity in cell lines

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) are a family of enzymes that regulate steroid availability within a tissue by catalysing the interconversion of active and inactive forms. Type 1 is up-regulated in many breast tumours, and is responsible for the reduction of oestrone to active oestradiol which stimulates cell proliferation within the tumour. Type 2 oxidises many active steroids to their inactive forms, including oestradiol to oestrone. In this study, we have compared the mRNA expression and enzyme activities of Type 1 and Type 2 in MCF-7, MDA-MB-231, T47D, JEG3 and 293-EBNA cell lines. Also studied were two cell lines stably expressing transfected Type 1 cDNA. RT-PCR indicated that little Type 1 mRNA is expressed in two of the breast cancer cell lines, MCF-7 and MDA-MB-231, and in 293-EBNA cells, but that expression is much higher in the T47D breast cancer cell line, and in the choriocarcinoma cell line, JEG3. However, a higher level of expression of Type 1 is seen in the transfected cell lines MCF-7.8H and 293-EBNA[His617beta-HSD1]. Activity assays show that there is high association between mRNA expression and enzyme activity. Assays indicate that, with the exception of MDA-MB-231 cells, Type 2 activity is low in these lines. The study of the basal activities of these enzymes will be used in future studies investigating the regulation of the enzymes by endogenous and exogenous factors. An understanding of their regulation in both healthy and malignant tissues may lead to future therapeutic intervention at the regulatory level.

Novel inhibitors of 17beta-hydroxysteroid dehydrogenase type 1: templates for design.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the interconversion between the oxidized and reduced forms of androgens and estrogens at the 17 position. The 17beta-HSD type 1 enzyme (17beta-HSD1) catalyzes the reduction of estrone (E1) to estradiol and is expressed in malignant breast cells. Inhibitors of this enzyme thus have potential as treatments for hormone dependent breast cancer. Syntheses and biological evaluation of novel non-steroidal inhibitors designed to mimic the E1 template are reported using information from potent steroidal inhibitors. Of the templates investigated biphenyl ethanone was promising and led to inhibitors with IC(50) values in the low micromolar range.

Focused libraries of 16-substituted estrone derivatives and modified E-ring steroids : Inhibitors of 17ß-hydroxysteroid dehydrogenase type 1

17β‐Hydroxysteroid dehydrogenase type 1 (17β‐HSD1), an oxidoreductase which has a preferential reductive activity using NADPH as cofactor, converts estrone to estradiol and is expressed in many steroidogenic tissues including breast and in malignant breast cells. As estradiol stimulates the growth and development of hormone‐dependent breast cancer, inhibition of the final step of its synthesis is an attractive target for the treatment of this disease. The parallel synthesis of novel focused libraries of 16‐substituted estrone derivatives and modified E‐ring pyrazole steroids as new potent 17β‐HSD1 inhibitors is described. Substituted 3‐O‐sulfamoylated estrone derivatives were used as templates and were immobilised on 2‐chlorotrityl chloride resin to give resin‐bound scaffolds with a multi‐detachable linker. Novel focused libraries of 16‐substituted estrone derivatives and new modified E‐ring steroids were assembled from these immobilised templates using solid‐phase organic synthesis and solution‐phase methodologies. Among the derivatives synthesised, the most potent 17β‐HSD1 inhibitors were 25 and 26 with IC50 values in T‐47D human breast cancer cells of 27 and 165 nm, respectively. Parallel synthesis resulting in a library of C5′‐linked amides from the pyrazole E‐ring led to the identification of 62 with an IC50 value of 700 nM. These potent inhibitors of 17β‐HSD1 have a 2‐ethyl substituent which will decrease their estrogenic potential. Several novel 17β‐HSD1 inhibitors emerged from these libraries and these provide direction for further template exploration in this area. A new efficient diastereoselective synthesis of 25 has also been developed to facilitate supply for in vivo evaluation, and an X‐ray crystal structure of this inhibitor is presented.

The development of steroid sulfatase inhibitors for hormone-dependent cancer therapy.

Steroid sulfatase (STS) regulates the hydrolysis of steroid sulfates to their unconjugated forms. Estrone sulfate and dehydroepiandrosterone sulfate can be hydrolyzed by STS to estrone and dehydroepiandrosterone, respectively, with these steroids being the precursors for the synthesis of more biologically active estrogens or androgens. A number of potent STS inhibitors have now been developed including STX64, which entered a phase I trial for the treatment of postmenopausal women with advanced metastatic hormone‐dependent breast cancer. The results from this phase I trial were encouraging, suggesting that STS inhibitors may also have a role in the treatment of other hormone‐dependent cancers including those of the endometrium, ovary, and prostate. In this paper the potential use of STS inhibitors to treat these hormone‐dependent cancers is reviewed. In addition, results from in vitro studies show that Ishikawa endometrial cancer cells, OVCAR‐3 ovarian cancer cells, and LNCaP prostate cancer cells all possess significant STS activity. Furthermore, STS activity in these cells can be almost completely inhibited by STX64 or the second‐generation STS inhibitor, STX213. Results from these investigations therefore suggest that STS inhibitors could have therapeutic potential for the treatment of a range of hormone‐dependent cancers.