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ANTICANCER RESEARCH 31: 1367-1372 (2011)

Effect of or on Sulfatase Activity and Serum Concentrations in Postmenopausal Women with

S.J. STANWAY1, C. PALMIERI2, F.Z. STANCZYK3, E.J. FOLKERD4, M. DOWSETT4, R. WARD2, R.C. COOMBES2, M.J. REED1† and A. PUROHIT1

1Oncology Drug Discovery Group, Section of Investigative Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, U.K.; 2Cancer Research UK Laboratories, Department of Oncology, Hammersmith Hospital, London W12 0NN, U.K.; 3Reproductive Endocrine Research Laboratory, University of Southern California, Keck School of Medicine, Women’s and Children’s Hospital, Los Angeles, CA, U.S.A.; 4Department of Biochemistry, Royal Marsden Hospital, London, SW3 6JJ, U.K.

Abstract. Background: In postmenopausal women and levels. Results: Neither can be formed by the pathway, which anastrozole nor tamoxifen had any significant effect on STS gives rise to , and the (STS) route activity as measured in PBLs. Anastrozole did not affect which can result in the formation of estrogens and serum concentrations. Conclusion: androstenediol, a steroid with potent estrogenic properties. Anastrozole and tamoxifen did not inhibit STS activity and Aromatase inhibitors, such as anastrozole, are now in serum androstenediol concentrations were not reduced by clinical use whereas STS inhibitors, such as STX64, are still aromatase inhibition. As androstenediol has estrogenic undergoing clinical evaluation. STX64 was recently shown properties, it is possible that the combination of an to block STS activity and reduce serum androstenediol and STS inhibitor may give a therapeutic concentrations in postmenopausal women with . advantage over the use of either agent alone. In contrast, little is known about the effects of aromatase inhibitors or anti-estrogens on STS activity or serum Anti-estrogens and aromatase inhibitors are now widely used androgen levels. Patients and Methods: Study 1: Blood was for the treatment of postmenopausal women with hormone- collected from ten postmenopausal women with breast dependent breast cancer (1, 2). While anti-estrogens, such as cancer before and after two-week treatment with anastrozole tamoxifen, block the interaction of (E2) with the and serum concentrations of androstenediol and other receptor (ER), aromatase inhibitors such as and estrogens were assessed. Study 2: Blood anastrozole, and inhibit the conversion samples were collected from 15 breast cancer patients before of to estrone (E1), the major source of and after four-week treatment with anastrozole and 10 estrogen in postmenopausal women (3). Recent clinical studies patients before and after four-week treatment with tamoxifen. have revealed that third-generation aromatase inhibitors are of Blood was used to assess STS activity in peripheral blood greater clinical benefit compared with tamoxifen in lymphocytes (PBLs) and serum dehydroepiandrosterone postmenopausal women with breast cancer (4, 5). In addition to the aromatase pathway of estrogen synthesis, the steroid sulfatase (STS) route is also thought to contribute to the in situ synthesis of estrogens in breast †Deceased. tumors (6). STS is responsible for the hydrolysis of (E1S) and dehydroepiandrosterone sulfate (DHEAS) Correspondence to: Dr. A. Purohit, Oncology Drug Discovery to E1 and dehydroepiandrosterone (DHEA) respectively. E1 Group, Section of Investigative Medicine, Imperial College London, and DHEA can be reduced by 17β-hydroxysteroid 6th Floor, Commonwealth Building (6N2B) Hammersmith Hospital, dehydrogenase type I (17β-HSD1) to with potent Du Cane Road, London W12 0NN, U.K. Tel: +44 2083834287, i.e. Fax: +44 2083838320, e-mail: [email protected] estrogenic properties, E2 and androstenediol. 17β-HSD1 is expressed in many tissues in the body including breast Key Words: Aromatase inhibitors, anti-estrogens, breast cancer, tumors (7, 8). Several potent STS inhibitors have now been steroid sulfatase, tamoxifen, anastrozole, postmenopausal women. developed including STX64 (also known as 667 Coumate,

0250-7005/2011 $2.00+.40 1367 ANTICANCER RESEARCH 31: 1367-1372 (2011)

BN83495 or Irosustat) (9, 10). In a recent phase I trial of be measured in PBLs. In addition, serum concentrations of STX64 in postmenopausal women with advanced breast androstenediol were assessed by radioimmunoassay, together cancer, treated with either 5 or 20 mg/day, STS activity in with the levels of androstenedione, , E1, E2 and peripheral blood lymphocytes (PBLs) and tumor tissues was E1S, which were measured using gas chromatographic- almost completely blocked (>90%) (11). Inhibition of STS tandem mass spectroscopy (GC-MS/MS). activity in these patients resulted in a significant decrease in serum concentration of E1 and E2. Serum concentrations of Patients and Methods androstenediol, which in postmenopausal women is mainly derived from the peripheral conversion of DHEAS, also fell Study 1. Blood was collected for the measurement of serum steroid by up to 90% (12). Unexpectedly, serum androstenedione concentrations from ten postmenopausal women with breast cancer concentration also decreased, as a result of STS inhibitor before and two weeks after starting treatment with anastrozole (1 mg/d). Three of the patients in this group achieved an objective therapy, indicating that in postmenopausal women the major response. For these patients, serum concentrations of androstenediol, part of this steroid is derived from the peripheral conversion androstenedione, testosterone, E1, E2 and E1S were measured. of DHEAS. In the phase I STX64 inhibitor trial, all patients selected Study 2. Blood samples were also collected from a further 15 for entry into the trial had previously progressed while on postmenopausal women with breast cancer before and four weeks anti-estrogen or aromatase inhibitor therapy. However, 5 out after treatment with anastrozole (1 mg/d) and ten patients before and of 14 patients still showed evidence of stable disease after four-week treatment with tamoxifen (20 mg/d). In addition, blood samples were collected from these patients before and after according to RECIST criteria. This finding suggests that STS four-week treatment for the assessment of STS activity in PBLs. For inhibitor therapy may have a role in the treatment of this blood was collected into CPT vacutainers (Beckton Dickinson, postmenopausal women with hormone-dependent breast Franklin Lakes, NJ, USA). After centrifugation (1,500 ×g at 22˚C for cancer and, in particular, for those patients progressing on 30 min), isolated PBLs were washed with phosphate-buffered saline anti-estrogen or aromatase inhibitor therapy. (PBS, 5 ml ×2) and stored at –20˚C until assayed. The STS in PBLs Although aromatase inhibitors have now been in clinical was solubilised before assaying using Triton-X 100/PBS (0.2%). STS 3 use for a number of years there is still no convincing evidence activity was assayed using [6,7- H] E1S (2-3 nmol/l, 46-57 Ci/mmol; Perkin-Elmer Life Sciences, Wellesley, MA, USA) over a 20 h period that the response to this form of endocrine therapy is related using [4-14C] E1 to monitor procedural losses (11, 22). to baseline levels of E2 or to the extent that E2 levels are The protocols for these studies were approved by the suppressed (13, 14). It is also not known why patients become Hammersmith Hospital’s Ethics Committees and all patients gave resistant to this form of therapy. Androstenediol, which is written informed consent for their participation in the study. derived from DHEA, has been shown to have potent estrogenic properties, stimulating the proliferation of ER- Steroid concentrations. For serum steroid concentration positive breast cancer cells in vitro and the growth of induced measurements in samples obtained from patients in the first study, androstenediol concentrations were measured by radioimmunoassay mammary tumors in vivo in rodents (15-17). One possible (RIA) after organic solvent extraction and Celite column partition reason why disease might progress whilst patients are treated chromatography (23). Concentrations of androstenedione, with aromatase inhibitors may be that over time tumor cells testosterone, E1, E2 and E1S were measured using a GC-MS/MS become more sensitive to the proliferative effects, not only of method by SBFC Taylor (Princeton, NJ, USA) (24, 25). The limits the low residual levels of E2, but also to steroids with potent of quantitation of E1, E2 and E1S in this assay were 5.8, 2.3 and 8.9 estrogenic properties, such as androstenediol (18). As yet, pmol/l, respectively. For samples of serum obtained from the second little is known about the effects of aromatase inhibitors on study, concentrations of DHEAS and DHEA were assayed using kits obtained from DSL (Webster, TX, USA) according to the serum concentrations of androstenediol, although in a manufacturer’s instructions. Intra-and inter-assay coefficients of previous study no effect on serum concentrations of DHEAS, variation for these assays were <15%. DHEA or androstenedione was detected (14). There is also a paucity of information about the effects of tamoxifen on Statistics. Mean and standard error of the mean (sem) levels of STS serum androgen concentrations. There are, however, a number activity and serum steroid concentrations were calculated using the of reports suggesting that tamoxifen may either inhibit or Instat 3 programme (GraphPad Software Inc., La Jolla, CA, USA). stimulate in vitro STS activity (19-21). To date, no studies The significance of differences in mean concentrations of serum steroids and STS activity in PBLs was assessed using the paired appear to have been carried out to assess the effects of Student’s t-test. Data are presented as mean±sem. tamoxifen on STS in postmenopausal women. In the present study, the effects of anastrozole or Results tamoxifen on STS activity were assessed in postmenopausal women with breast cancer. In contrast to the difficulty of Study 1. In the first study, the effect of two-week therapy measuring peripheral aromatase activity, which involves a with anastrozole on serum androgen and estrogen double isotopic infusion technique, STS activity can readily concentrations was assessed (Figure 1). Basal levels of

1368 Stanway et al: Steroid Sulfatase Activity in Breast Cancer

Figure 1. Serum concentrations of androstenediol, androstenedione, testosterone and estrone sulfate in samples obtained from patients with breast cancer before (pre) and after (post) two-week treatment with anastrozole (1 mg/d). Data are presented as mean±SEM. NS, Not significant. Figure 2. Steroid sulfatase activity as measured in peripheral blood lymphocytes prepared from blood samples from patients before and after four-week treatment with either anastrozole (1 mg/day) or tamoxifen (20 mg/d). Data are presented as mean±SEM. NS, Not significant. androstenediol, androstenedione, testosterone, E1, E2 and E1S were similar to those previously reported by this group in postmenopausal women with breast cancer (11). It is evident from this study that daily treatment with anastrozole collected from postmenopausal women participating in the had no significant effect on serum concentrations of STS inhibitor trial (11). It was clear that treatment with androstenediol, androstenedione or testosterone. Pre- either tamoxifen or anastrozole for the four-week period had treatment levels of E1, E2 and E1S in this study were 60±25, no significant effect (p>0.05) on STS activity in PBLs from 17.7±8.3 and 980±233 pmol/l, respectively. As shown in these groups of patients (Figure 2). Similarly, serum Figure 1, E1S concentrations were significantly lower concentrations of DHEAS and DHEA were not affected by (p<0.01) at the end of the two-week period, but detectable in treatment with either tamoxifen or anastrozole (Figure 3). all the samples assayed at the end of this period (range, 26- There is no obvious explanation for the differences in the 210 pmol/l). The mean level of aromatase suppression pre- and post-treatment levels of DHEAS and DHEA for calculated from the change in serum E1S concentrations was patients receiving tamoxifen or anastrozole. Interestingly, 94%. There was no significant difference in the serum E1S differences in basal levels of DHEAS and DHEA were concentrations for three patients who subsequently showed previously seen by this group in different cohorts of patients a response to aromatase inhibitor therapy (52±9 pmol/l) and receiving either the 5 or 20 mg of STX64 (11). that of non-responders (72±35 pmol/l). For all patients, serum concentrations of E1 after two-week treatment with Discussion anastrozole were below the limits of quantitation for this assay (5.8 pmol/l). For E2, significant concentrations (3.1 The main findings from the two studies presented here are and 2.9 pmol/l) were still detectable after treatment with that neither tamoxifen nor anastrozole has any effect on STS anastrozole for two weeks in two of the patients who did not activity, assayed in PBLs and that serum concentrations of subsequently respond to aromatase inhibitor therapy. androgens, including androstenediol, are not altered by anastrozole therapy. This is in contrast to the results obtained Study 2. As previous reports had suggested that tamoxifen with the specific STS inhibitor, STX64, where STS activity may influence STS activity and possible effects of aromatase was almost completely inhibited in PBLs and a significant inhibitors on STS are not yet known, PBLs were collected reduction in serum androstenediol concentrations was from a second group of patients receiving either tamoxifen detected. Although it was not anticipated that anastrozole or anastrozole over a four-week period. STS activity in these would modulate STS activity, a number of previous reports PBLs was similar to the range detected in basal samples had suggested that tamoxifen may affect STS activity.

1369 ANTICANCER RESEARCH 31: 1367-1372 (2011)

Figure 3. Serum concentrations of DHEAS and DHEA in samples obtained from patients before and after four-week treatment with either anastrozole (1 mg/d) or tamoxifen (20 mg/d). Data are presented as mean±SEM. NS, Not significant.

Santner and Santen examined the effect of tamoxifen on STS induced mammary tumors in vivo (15-17). The finding that activity in preparations of rat mammary tumors and human serum androstenediol concentrations are not affected by breast tumors and concluded that it was a potent STS treatment by anastrozole may be one possible reason why inhibitor (19). Similarly, Gelly and Pasqualini found that patients on this form of therapy do eventually progress. using R-27 cells, a tamoxifen-resistant cell line derived from However, three of the patients in the first cohort studied did MCF-7 cells, tamoxifen was able to inhibit STS activity (20). subsequently respond to aromatase inhibitor therapy, In contrast, in other studies employing MCF-7 cells, breast demonstrating that responses can occur in the presence of tumor preparations or in vivo studies in rodents, tamoxifen relatively high levels of androstenediol. Further studies, was found to have either no effect or a small, but significant, involving the combination of an aromatase inhibitor with an stimulatory effect on STS activity (21, 26, 27). To the best STS inhibitor will be required to examine whether reduction of the Authors’ knowledge, this is the first study to of serum androstenediol levels improves the response or investigate the possible effects of tamoxifen on STS activity extends the duration of endocrine therapy for breast cancer. in humans and clearly demonstrated that tamoxifen is devoid In the present study serum concentrations of E1, E2 and of any inhibitory effects on STS activity. The lack of an E1S were measured using a GC-MS/MS method. There has affect of aromatase inhibition on serum concentrations of been considerable debate recently about the merits of RIA DHEAS, DHEA and androstenedione is concordant with the when tying to measure estrogen levels in postmenopausal results obtained in a previous study (14). This contrasts with women and, in particular, in serum from women receiving the effects of STS inhibition where serum concentrations of aromatase inhibitor therapy. It is well documented that the DHEA were reduced (11). use of RIA to measure low levels of estrogen in With regard to androstenediol, as discussed previously, postmenopausal women is markedly difficult, mainly due to this steroid has potent estrogenic properties as shown by its cross-reacting materials in the serum. Such problems led ability to stimulate breast cancer cell growth in vitro and Santen to conclude that measurements of E2 by RIA may not

1370 Stanway et al: Steroid Sulfatase Activity in Breast Cancer be sufficiently sensitive or precise to use for serum samples Stuart N, Snowdon CF, Carpentieri M, Massimini G, Bliss JM from postmenopausal women (28). The measurements using and van de Velde C; Intergroup Exemestane Study: A GC-MS/MS in this study showed a good correlation (r=0.84) randomised trial of exemestane after two to three years of tamoxifen therapy in postmenopausal women with primary with those using a recombinant ultrasensitive bioassay (24). breast cancer. N Engl J Med 350: 1081-1092, 2004. In the present study, the level of aromatase inhibition 6 Reed MJ, Purohit A, Woo LW, Newman SP and Potter BVL: calculated from the suppression of serum E1S concentrations Steroid sulfatase: molecular biology, regulation and inhibition. was 94%, which is in good agreement with the level of Endocr Rev 26: 171-202, 2005. suppression derived from measurements of E1S using an RIA 7 Bonney RC, Reed MJ, Davidson K, Beranek PA and James in association with an extensive purification procedure (29, VHT: The relationship between 17β-hydroxysteroid dehydrogenase activity and estrogen concentrations in human 30). While the levels of E1S were, therefore, effectively suppressed by anastrozole, levels above the limit of breast tumor and in normal breast tissue. Clin Endocrinol 19: 727-739, 1983. quantitation for this steroid conjugate were detected in all the 8 Gunnarsson C, Hellqvist E and Stal O: 17β-Hydroxysteroid samples analysed. Again this would suggest that, although dehydrogenases involved in local estrogen synthesis have these levels of E1S do not preclude an initial response to prognostic significance in breast cancer. Br J Cancer 92: 547- anastrozole, they may contribute to disease progression in 552, 2005. the longer term. 9 Woo LWL, Purohit A, Malini B, Reed MJ and Potter BVL: In summary, the results from the present investigations Potent -directed inhibitors of steroid sulfatase by revealed that neither tamoxifen nor anastrozole has any effect tricyclic -based sulphamates. Chem Biol 7: 773-791, 2000. on STS activity in PBLs. Anastrozole was also found not to 10 Purohit A, Williams GJ, Howarth NM, Potter BVL and Reed affect serum concentrations of androstenediol, a steroid with MJ: Inactivation of steroid sulfatase by an active site-directed potent estrogenic properties. Serum concentrations of E1, E2 inhibitor, estrone-3-O-sulfamate. Biochem 34: 11508-11514, and E1S were effectively suppressed by anastrozole treatment 1995. for two weeks but significant concentrations of E1S were still 11 Stanway SJ, Purohit A, Woo LW, Sufi S, Vigushin D, Ward R, detectable. Given the findings that reduced concentrations of Wilson RH, Stanczyk FZ, Dobbs N, Kulinskaya E, Elliott M, Potter BVL, Reed MJ and Coombes RC: Phase I study of E1S are still detectable after aromatase inhibitor treatment and that serum concentrations of androstenediol are not STX64 (667 Coumate) in breast cancer patients: the first study of a steroid sulfatase inhibitor. Clin Cancer Res 12: 1585-1592, affected by this form of therapy, it will be important to test 2005. whether the combination of an aromatase inhibitor with a 12 Poortman J, Andriesse R, Agema A, Donker GH, Schwarz F and sulfatase inhibitor may give a therapeutic advantage over the Thijssen JHH: Adrenal androgen secretion and in use of either agent alone. postmenopausal women. 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Lancet 365: 60-62, piandrosterone sulfate: inhibition by novel non-steroidal 2005. sulfatase inhibitors. J Steroid Biochem Mol Biol 73: 225-235, 5 Coombes RC, Hall E, Gibson LJ, Paridaens R, Jassem J, 2003. Delozier T, Jones SE, Alvarez I, Bertelli G, Ortmann O, Coates 18 Masamura S, Santner SJ, Heitjan DF and Santen RJ: Estrogen AS, Bajetta E, Dodwell D, Coleman RE, Fallowfield LJ, deprivation causes estradiol hypersensitivity in human breast Mickiewicz E, Andersen J, Lønning PE, Cocconi G, Stewart A, cancer cells. J Clin Endocrinol Metab 80: 2918-2925, 1995.

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19 Santner SJ and Santen RJ: Inhibition of estrone sulfatase and 26 Evans TRJ, Rowlands MG, Jarman M and Coombes RC: 17β-hydroxysteroid dehydrogenase by anti-estrogens. J Steroid Inhibition of oestrone sulfatase in human placenta and Biochem Mol Biol 45: 383-390, 1993. breast carcinoma. J Steroid Biochem Mol Biol 39: 493-499, 20 Gelly C and Pasqualini JR: Effect of tamoxifen and tamoxifen 1991. derivatives on the conversion of estrone sulfate and estradiol in 27 Purohit A, Williams GJ, Roberts CJ, Potter BVL and Reed MJ: R-27 cells, a tamoxifen resistant cell line derived from MCF-7 In vivo inhibition of oestrone sulfatase and dehydroe- human breast cancer cells. J Steroid Biochem 30: 321-324, 1988. piandrosterone sulfatase by oestrone-3-O-sulphamate. Int J 21 Purohit A and Reed MJ: Estrogen sulfatase activity in hormone- Cancer 63: 106-111, 1995. dependent breast cancer cells: modulation by steroidal and non- 28 Santen RJ, Lee JS, Wang S, Demers LM, Mauras N, Wang H steroidal therapeutic agents. Int J Cancer 50: 901-905, 1992. and Singh R: Potential role of ultra-sensitive estradiol assays in 22 Purohit A, Froome VA, Wang DY, Potter BVL and Reed MJ: estimating the risk of breast cancer and fractures. Steroids 73: Measurement of estrone sulfatase activity in white blood cells to 1318-1321, 2008. monitor in vivo inhibition of steroid sulfatase activity by 29 Lonning P, Geisler J, Johannessen DC and Ekse D: Plasma oestrone-3-O-sulphamate. J Steroid Biochem Mol Biol 62: 45- estrogen suppression with aromatase inhibitors elevated by a 51, 1997. novel, sensitive assay for estrone sulfate. J Steroid Biochem Mol 23 Dorgan JF, Stanczyk FZ, Longcope C, Stephenson HE Jr, Chang L, Biol 61: 255-260, 1997. Miller R, Franz C, Falk RT and Kahle L: Relationship of serum 30 Geisler J, Helle H, Ekse D, Duong NK, Evans DB, Nordbø Y, dehydroepiandrosterone (DHEA), DHEA sulfate and 5-androstene- Aas T and Lønning PE: Letrozole is superior to anastrozole in 3β,17β-diol to risk of breast cancer in postmenopausal women. suppressing breast cancer tissue and estrogen levels. Clin Cancer Cancer Epidemiol Biomarkers Prev 6: 177-181, 1997. Res 14: 6330-6335, 2008. 24 Wang S, Paris F, Sultan CS, Song RX, Demers LM, Sundaram B, Settlage J, Ohorodnik S and Santen RJ: Recombinant cell ultra- sensitive bioassay for measurement of estrogens in postmenopausal women. J Clin Endocrinol Metab 90: 1407-1413, 2005. 25 Sundaram B, Settlage JA, Ohorodnik SK and Taylor PA: A combined GC-MS/MS and LC-MS/MS bioanalytical method for the quantification of estradiol, estrone, estrone sulfate, testosterone Received January 20, 2011 and androstenedione. 51st ASMS Conference on Mass Revised February 23, 2011 Spectrometry and allied topics. Montreal, Canada, June 2003. Accepted February 24, 2011

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