Mediated Activation of Estrogen Sulfotransferase

Research Article

Glucocorticoids Antagonize Estrogens by Glucocorticoid Receptor–Mediated Activation of Estrogen Sulfotransferase

Haibiao Gong,1 Michael J. Jarzynka,2 Timothy J. Cole,4 Jung Hoon Lee,1 Taira Wada,1 Bin Zhang,1 Jie Gao,1 Wen-Chao Song,5 Donald B. DeFranco,3 Shi-Yuan Cheng,2 and Wen Xie1,3

1Center for Pharmacogenetics and Department of Pharmaceutical Sciences, 2University of Pittsburgh Cancer Institute and Department of Pathology, and 3Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania; 4Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and 5Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Abstract superfamily. In the absence of a ligand, GR resides in the cytoplasm Glucocorticoids and estrogens are two classes of steroid associating with the heat shock protein hsp90 and several other hormones that have essential but distinct physiologic proteins. Upon ligand binding, GR is dissociated with hsp90 and functions. Estrogens also represent a risk factor for breast translocated into the nucleus, where it activates gene expression by cancer. It has been suggested that glucocorticoids can binding to glucocorticoid-responsive elements (GRE) located in the attenuate estrogen responses, but the mechanism by which promoter regions of target genes (4–6). glucocorticoids inhibit estrogenic activity is unknown. In this Estrogens are sex hormones essential for mammals’ reproduc- tion. Although produced primarily in the ovary, estrogens circulate study, we show that activation of glucocorticoid receptor (GR) by dexamethasone (DEX) induced the expression and systemically and exert their effects on various reproductive and activity of estrogen sulfotransferase (SULT1E1 or EST), nonreproductive tissues (7, 8). The estrogen receptor (ER), also a an enzyme important for the metabolic deactivation of nuclear hormone receptor, plays an important role in mediating estrogens, because sulfonated estrogens fail to activate the estrogenic effects. The uterus is one of the organs highly estrogen receptor. Treatment with DEX lowered circulating responsive to estrogens. In mice, uterine estrogen responses estrogens, compromised uterine estrogen responses, and include water imbibition and increased uterine weight, as well as inhibited estrogen-dependent breast cancer growth in vitro transactivation of estrogen-responsive genes, such as c-fos and and in a xenograft model. We further showed that the mouse lactoferrin (Ltf; ref. 9). In addition to their physiologic functions, and human SULT1E1 genes are transcriptional targets of estrogens also play an important role in the initiation and progression of human breast cancers (10–12). Accordingly, GR and deletion of Sult1e1/Est in mice abolished the DEX effect on estrogen responses. These findings have revealed a strategies to down-regulate estrogen activities have been effective novel nuclear receptor–mediated and metabolism-based mech- to treat and prevent breast cancers (13, 14). anism of estrogen deprivation, which may have implications An important metabolic deactivation pathway for estrogens is in therapeutic development for breast cancers. Because the sulfotransferase (SULT)–mediated sulfation, because sulfonated glucocorticoids and estrogens are widely prescribed drugs, estrogens cannot bind to and activate ER, thus losing their our results also urge caution in avoiding glucocorticoid- estrogenic activities (15). Sulfotransferases belong to a family of estrogen interactions in patients. [Cancer Res 2008;68(18):7386–93] phase II drug metabolizing enzymes. Although several SULT isoforms have been shown to sulfonate estrogens in vitro, the estrogen sulfotransferase (SULT1E1 or EST) is believed to be the Introduction major SULT isoform responsible for estrogen sulfation at Glucocorticoids and estrogens are two classes of steroid physiologic concentrations due to its high affinity at nanomolar hormones essential to mammals. Glucocorticoids have been concentrations of estrogens (16, 17). The importance of SULT1E1 in implicated in a variety of cellular processes, ranging from estrogen homeostasis and reproductive function was manifested in development to metabolism, immune response, and apoptosis. the Sult1e1/Est null mice (18). SULT1E1 expression has also been Endogenous glucocorticoids, such as cortisol in humans and implicated in human breast cancer. SULT1E1 is highly expressed in corticosterone in rodents, are synthesized in the adrenal cortex normal human mammary epithelial cells, but its expression is under the control of the hypothalamic-pituitary-adrenal axis (1, 2). diminished in breast cancer cells, including the ER-positive and In addition to their physiologic functions, glucocorticoids are estrogen-responsive MCF-7 cells (19). The differential expression of among the most commonly prescribed drugs for their antiin- SULT1E1 in normal human mammary epithelial cells and breast flammatory and immunosuppressive effect (1, 3). Glucocorticoids cancer cells suggests that down-regulation of SULT1E1 may have exert most of their functions by binding to the glucocorticoid led to unchecked estrogen stimulation and cancerous transforma- receptor (GR; NR3C1), a member of the nuclear hormone receptor tion of the breast epithelium. An ectopic expression of SULT1E1 in MCF-7 cells suppressed the estrogen response (20, 21), suggesting that reactivation of endogenous SULT1E1 gene expression may represent a novel therapeutic strategy to inhibit estrogen- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). dependent growth of breast cancer cells. Requests for reprints: Wen Xie, Center for Pharmacogenetics, University of It has been recognized that glucocorticoids inhibited estrogen Pittsburgh, Pittsburgh, PA 15261. Phone: 412-648-9941; Fax: 412-648-1664; E-mail: responses (22–24). Treatment with dexamethasone (DEX), a [email protected]. I2008 American Association for Cancer Research. synthetic glucocorticoid, attenuated the estrogen-induced uterine doi:10.1158/0008-5472.CAN-08-1545 expression of insulin-like growth factor-I (IGF-I; ref. 25). DEX also

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. The Antagonistic Effect of Glucocorticoid on Estrogen blocked the stimulatory effect of estrogen on MCF-7 cell maintained in the hepatocyte maintenance medium from Cambrex -7 proliferation (26). However, the mechanism by which glucocorti- BioScience supplemented with 10 mol/L insulin, 50 Ag/mL gentamicin, coids inhibit estrogenic activity is unknown. and 50 ng/mL amphotericin and incubated overnight to allow the cells to In this study, we show that activation of GR by DEX induced the attach. Cells were then treated with appropriate drugs for 24h before cell harvesting. expression and activity of SULT1E1, which in turn facilitated Xenograft model. 1 Â 107 of MCF-7/vascular endothelial growth factor estrogen deactivation and inhibited estrogen-dependent breast (VEGF) cells were inoculated into the mammary fat pads of 7-wk-old cancer growth in cell culture and in vivo. Our results have revealed ovariectomized female nude mice that were implanted with E2 pellets a unique GR-mediated and metabolism-based hormone-hormone (0.72 mg/60-day release) or placebo pellets from the Innovative Research of interaction. Moreover, the GR-mediated SULT1E1 gene activation America. The E2-treated mice were randomly divided into three groups, may represent a novel strategy for estrogen deprivation that can be with two groups receiving a daily treatment of DEX at different doses used to treat and prevent breast cancers. (2 or 6 mg/kg) and the other group receiving vehicle. The volumes of the tumors were measured using a caliper every 5 d. Mice were labeled with BrdUrd for 30 min before sacrifice. Serum, tumor, and liver tissues were Materials and Methods collected for analysis. Mouse models. The GR null (27), Sult1e1/Est null (28), and PXR null (29) Electrophoretic mobility shift assay and chromatin immunoprecip- mice have been described previously. The wild-type C57BL/6J mice were itation analysis. Electrophoretic mobility shift assay (EMSA) were purchased from JAX Mice and Services. The ovariectomized nude mice used performed using receptor proteins prepared with the TNT in vitro in the xenograft model were purchased from Taconic. The use of mice in transcription and translation system and [32P]-labeled double-stranded this study has complied with all relevant federal guidelines and institutional DNA fragments, as described previously (30). DMSO-treated or DEX-treated policies. primary hepatocytes were used for chromatin immunoprecipitation (ChIP) Uterotropic bioassay. Three-week-old virgin female mice were sub- assays using a GR antibody (sc8992) from Santa Cruz, as we described A ¶ jected to daily s.c. injections of vehicle (5% ethanol in PBS) or E2 (5 g/kg/d) previously (30). The PCR primers for mouse Sult1e1/GRE are 5 - for 3 d, as previously described. Mice were then sacrificed 24h after the last GGTCCTGTTTGTGTAGCCAGTC-3¶ and 5¶-CCACAAATCCAGCCCTT- ¶ ¶ E2 dose, and the uteri were dissected and weighed. In the DEX-treated CAAAGG-3 . The primers for human SULT1E1/GRE are 5 -TAAACTT- group, mice received daily i.p. injection of DEX (20 mg/kg) starting 3 d GTGTCCACACAAA-3¶ and 5¶-TTTGTGTGGACACAAGTTTA-3¶. The primers ¶ before the E2 treatment. Injections were continued until the completion of for Sult1e1/liver X receptor (LXR) response element (LXRE) are 5 - the experiments. CCAAAGGGGAGAAACAGCTG-3¶ and 5¶-GAGAAGGAGGCAGAGACTAAC- Uterine estrogen responses. Five-week-old mice were ovariectomized 3¶.TheprimersforCyp7b1/ROR response element (RORE) are 5¶- and recovered for 1 wk before treatment with DMSO or DEX (20 mg/kg in AACCTTAGGAGGAAGGAGCCCATGAA-3¶and 5¶-TGATGAATACTC- A ¶ DMSO) for 3 d. Mice were then given a single s.c. injection of E2 (20 g/kg) CATGTGCAATGAGA-3 (34). The PCR products of Sult1e1/GRE, SULT1E1/ and sacrificed 20 h after. Mice also received an i.p. injection of 5-bromo 2- GRE, Sult1e1/LXRE, and Cyp7b1/RORE are 252, 174, 142, and 125 bp, deoxyuridine (BrdUrd; 60 mg/kg) 2 h before sacrifice. For each mouse, one respectively. uterine horn was processed for RNA extraction and real-time PCR analysis, Statistical analysis. When applicable, results are presented as means and the other for paraffin section and immunostaining using an anti- F SD. Animal numbers are labeled or shown in figure legends. The BrdUrd antibody, as we have previously described (30). Student’s t test was used to calculate P values. P < 0.05 was considered Northern blot analysis and real-time reverse transcription–PCR. statistically significant. Total RNA was extracted using the TRIZOL Reagent from the Invitrogen. Northern hybridization was carried out as described (31). Total RNA was Results treated with RNase-free DNase I and reverse transcribed into single- Treatment with DEX induced the hepatic expression of stranded cDNA. Real-time PCR using the SYBR Green–based assay was Sult1e1 performed with the ABI 7300 Real-Time PCR System (30). in a GR-dependent manner and decreased the Estrogen sulfotransferase activity assay. Liver cytosols were prepared circulating level of estrogens. In our effort to identify nuclear by homogenizing tissues in 5 mmol/L KPO4 buffer (pH 6.5) containing receptors and their ligands that control the expression of Sult1e1 in 0.25 mol/L sucrose. The cytosols were then used for sulfotransferase assay, mice, we found that treatment with DEX in wild-type mice induced which was carried out using [35S]phosphoadenosine phosphosulfate (PAPS; the mRNA expression of Sult1e1 in the liver (Fig. 1A). The basal Perkin-Elmer) as the sulfate donor (30). In brief, 20 Ag/mL of total liver expression of Sult1e1 mRNA in the liver is low. The hepatic Sult1e1 cytosolic extract was incubated with 1 Amol/L of estrone substrate. After 35 mRNA induction in DEX-treated mice was so dramatic that its the reaction, free [ S]PAPS was removed by extracting with ethyl acetate. A expression reached a level similar to that of the testis (Fig. 1A), the 300-AL aliquot of the aqueous phase was then counted in the Beckman tissue known to have the highest constitutive expression of Sult1e1 LS6500 scintillation counter for radioactivity. (15). DEX is known to activate both GR and the pregnane X Measurement of serum bioactive E2 level. The serum concentrations receptor (PXR; ref. 35). The DEX effect on Sult1e1 mRNA expression of active E2 were measured using the Ultra-Sensitive Estradiol RIA kit (DSL-4800) from Diagnostic Systems Laboratories, Inc., as previously was PXR-independent, because the Sult1e1 mRNA induction was described (30). intact in PXR null mice (29) and the PXR agonist PCN failed to Cell transient transfection and luciferase activity assay. HepG2 cells increase Sult1e1 mRNA level in wild-type mice (Fig. 1A). The lack of were transfected with the reporter constructs and hGRa expression vector the PXR effect was also supported by the lack of Sult1e1 mRNA in 48-well plates, as previously described (30). When necessary, cells were induction in transgenic mice that expressed a constitutively A treated with DEX (1 mol/L) in media containing 10% charcoal-stripped activated PXR in the liver (data not shown). Instead, by using GR serum for 24h before the luciferase assay. The transfection efficiency was null mice (27), we showed that the DEX effect on Sult1e1 mRNA normalized against the h-gal activities from the cotransfected CMX–h-gal expression was GR-dependent, because the Sult1e1 mRNA vector. Human and mouse primary hepatocyte preparation and treatment. induction by DEX was completely abolished in GR null mice Human livers were obtained through the Liver Tissue Procurement and (Fig. 1B). Moreover, the DEX-induced Sult1e1 mRNA expression Distribution System (LTPADS), and hepatocytes were isolated by three-step was suppressed by the coadministration of RU486, a known GR collagenase perfusion (32). Mouse hepatocytes were also prepared by antagonist (Supplementary Fig. S1). The DEX effect on Sult1e1 collagenase perfusion (33). Cells were plated on gelatin-coated T25 flask and mRNA expression seemed to be tissue specific. DEX had little effect www.aacrjournals.org 7387 Cancer Res 2008;68: (18). September 15, 2008

Figure 1. Treatment with DEX induced the hepatic expression of Sult1e1 in a GR-dependent manner and decreased the circulating estrogens. A, up-regulation of hepatic Sult1e1 in the liver of wild-type mice treated with a single i.p. dose of DEX (20mg/kg) for 24 h, as shown by Northern blot analysis. Membranes were first probed with Sult1e1 and subsequently stripped and reprobed with Gapdh as a loading control. Lanes represent individual mice. B, the effect of DEX on Sult1e1 mRNA expression is GR-dependent, as shown by Northern blot analysis. DEX treatment was the same as in A. Lanes represent individual mice. C, treatment with DEX increased estrogen sulfation. Cytosolic liver extracts from vehicle (DMSO)–treated or DEX-treated wild-type mice were subjected to sulfation assay using estrone as the substrate. [35S]PAPS was used as the sulfate donor. Radioactivity was measured by scintillation counting after separation and removal of free [35S]PAPS. D, 4-wk-old virgin wild-type female mice treated with vehicle or DEX were measured for serum estradiol levels. Mice in C and D received three daily i.p. doses of DEX (20mg/kg) before being sacrificed. Lanes in C and D represent four to six mice per group, and results are presented as averages and SD. *, P < 0.05; **, P <0.01, compared with the vehicle controls.

on Sult1e1 mRNA expression in the testis, a tissue known to gene expression was evaluated. As shown in Fig. 2C, compared with express GR (Fig. 1B; ref. 36). Consistent with Sult1e1 mRNA vehicle control, treatment with E2 increased the mRNA expression induction, increased estrone sulfation was observed in liver of progesterone receptor, Ltf, and Igf-1, and suppressed Txnip cytosols prepared from DEX-treated wild-type mice (Fig. 1C). expression in wild-type mice, as expected (37–40). Pretreatment Moreover, treatment of 4-wk-old intact virgin female mice with with DEX abolished E2-responsive expression of all four genes in DEX for 3 days resulted in a significantly reduced circulating wild-type mice, but this inhibition was abrogated in Sult1e1/Est estradiol level (Fig. 1D). null mice. DEX inhibited mouse uterine estrogen responses in a DEX induced SULT1E1 mRNA expression in human hepato- Sult1e1/Est-dependent manner. The uterus is highly responsive cytes and MCF-7 cells and inhibited E2-stimulated MCF-7 cell to estrogen stimulation. Therefore, we used uterine estrogen proliferation. The effect of DEX on human SULT1E1 mRNA responses to examine the biological consequence of DEX-induced expression was investigated in primary human hepatocytes and Sult1e1 expression. Treatment with 17h-estradiol (E2)almost breast carcinoma MCF-7 cells. DEX treatment up-regulated doubled uterine weight in intact wild-type mice (Fig. 2A). The SULT1E1 mRNA expression in both cell types (Fig. 3A). Over- uterotropic effect of E2 was largely abolished when the mice were expression of GR in MCF-7 cells by transfection further enhanced simultaneously treated with DEX, whereas DEX alone had little DEX-induced SULT1E1 mRNA expression (Fig. 3A, right). MCF-7 effect (Fig. 2A). The uterine epithelial proliferation and gene cells are ER-positive and estrogen responsive. As expected, E2 expression were examined in ovariectomized virgin female mice by treatment induced f2.5-fold increase in MCF-7 cell proliferation, BrdUrd labeling and real-time PCR, respectively. As shown in as measured by BrdUrd labeling assay. Although DEX alone had Fig. 2B, treatment with E2 caused a dramatic increase of BrdUrd little effect, it inhibited E2-stimulated MCF-7 cell proliferation in a labeling index (Fig. 2B, b), but this effect was abolished in mice dose-dependent manner (Fig. 3B). The inhibition was specific for pretreated with DEX (Fig. 2B, d). The inhibitory effect of DEX was E2-dependent breast cancer cell growth, because DEX had little abolished in Sult1e1/Est null mice (Fig. 2B, f ), suggesting that effect on the proliferation of MDA-MB-231, an ER-negative and Sult1e1 played an essential role in this inhibition. Similar effects of E2-independent breast cancer cell line (Fig. 3B). The induction DEX and Sult1e1 null background were observed when uterine of SULT1E1 mRNA expression by DEX in MCF-7 cells was

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GR-dependent. Knocking-down of GR by small interfering RNA Consistent with the tumor inhibition phenotype, DEX treatment (siRNA), as confirmed by real-time PCR (Fig. 3C) and Western blot decreased the serum level of E2 in a dose-dependent manner analysis (data not shown), compromised DEX-induced SULT1E1 (Fig. 4C), which was associated with an increased SULT1E1 mRNA expression (Fig. 3C). DEX failed to inhibit E2-stimulated cell expression in the liver and xenograft tumor tissues (Fig. 4D). In the proliferation in GR siRNA-transfected MCF-7 cells (Fig. 3D). same animals, the mRNA expression of several other hepatic DEX inhibited estrogen-dependent, but not estrogen- estrogen metabolizing enzymes, including Cyp1a2, Cyp3a11, and independent, human breast cancer cell tumorigenicity in Ugt1a1 (42), was not induced by DEX (Fig. 4D). Although high nude mice. The induction of SULT1E1 and inhibition of estrogenic doses of DEX (20–50 mg/kg) can activate PXR in vivo (29), the DEX activity prompted us to determine whether DEX can inhibit doses (2–6 mg/kg) we used in the xenograft model had little effect estrogen-dependent breast cancer tumorigenicity in vivo by using on PXR activation and, thus, failed to activate PXR target genes, the MCF-7 cell xenograft model. Due to the low tumorigenicity of such as Cyp3a11 and Ugt1a1 (43). These results suggested that both the parent MCF-7 cells, we chose the MCF-7/VEGF cells that local (in the xenograft tumor) and systemic (in the liver) activation overexpress VEGF. The MCF-7/VEGF cells exhibited a higher of SULT1E1 may have contributed to E2 deprivation and tumor penetrance of estrogen-independent growth and robust estrogen- inhibition. dependent promotion (41). Ovariectomized mice were used to SULT1E1 is a transcriptional target of GR. To understand the minimize the effect of endogenous E2. The DEX effect was molecular mechanism by which GR regulates SULT1E1 gene evaluated in the presence of E2 supplied by implanted E2 pellets. expression, we have cloned and analyzed the mouse and human Consistent with our previous report (41), the MCF-7/VEGF cells SULT1E1 gene promoters. Putative GREs were found in both the were highly tumorigenic in response to E2 stimulation (Fig. 4A). mouse and human promoters (Supplementary Fig. S2A). EMSA Compared with the vehicle treatment group, daily treatment with showed that GR can bind to both the mouse Sult1e1/GRE DEX (2 or 6 mg/kg) significantly decreased tumor volume. The (Supplementary Fig. S2B) and human SULT1E1/GRE (Supplemen- difference was noticeable as early as 10 days after tumor cell tary Fig. S2C). In both cases, the bindings can be efficiently inoculation, and the inhibition was sustained thereafter. The DEX competed by excess unlabeled wild-type Sult1e1 (SULT1E1)/GRE or effect was dose-dependent, with a more dramatic tumor inhibition MTV/GRE (a prototypical GRE), but not by Sult1e1 (SULT1E1)/GRE observed when the higher dose (6 mg/kg) was used (Fig. 4A). mutants (Supplementary Fig. S2B and C). ChIP assay using a Shown in the insert of Fig. 4A are representative tumors from GR-specific antibody showed that GR protein can be recruited to vehicle-treated and DEX-treated mice. The reduced tumor size in the GRE in the mouse Sult1e1 promoter, and DEX treatment DEX-treated mice was associated with a decreased cell proliferation enhanced the recruitment (Supplementary Fig. S2D). The recruit- rate, as shown by BrdUrd labeling and immunostaining (Fig. 4B). ment of GR was GRE-specific, because no binding was detected

Figure 2. DEX inhibited mouse uterine estrogen responses in a Sult1e1/Est-dependent manner. A, quantitation of estrogen uterotropic bioassay results. Three-week-old wild-type virgin female mice were treated with vehicle or DEX (20mg/kg) for 3 d before being mock treated or treated with a single dose of E2 (5 Ag/kg). Each group contained five mice. *, P < 0.05; **, P <0.01, compared with the vehicle control. B, BrdUrd immunostaining on uterine paraffin sections from mice of different genotypes and drug treatments. All mice were ovariectomized 7 d before estrogen treatment. Mice received a single s.c. injection of vehicle or E2 (20mg/kg) 20h before being sacrificed. Mice were labeled for BrdUrd (60mg/kg) for 2 h before sacrifice. Percentages of BrdUrd-positive nuclei are quantitated and labeled. When applicable, mice were pretreated with DEX for 3 d before E2 treatment. The original magnification is 200Â for all panels. C, regulation of uterine gene expression as measured by real-time PCR. Pgr, progesterone receptor; Txnip, thioredoxin interacting protein. **, P < 0.01, compared with the wild-type vehicle control. Three mice were used for each group in B and C.

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Figure 3. DEX induced SULT1E1 mRNA expression in human hepatocytes and MCF-7 cells and inhibited E2-stimulated MCF-7 cell proliferation. A, expression of SULT1E1 in primary human hepatocytes and MCF-7 cells treated with DEX (10nmol/L) for 24 h, as shown by real-time PCR. The MCF-7 cell experiment was performed in the absence or presence of GR cotransfection. Results of the human hepatocytes represent the averages and SD from three cases of primary hepatocytes (case IDs: HH1139, 1280, 1281). **, P <0.01.B, cotreatment with DEX inhibited E2-stimulated MCF-7 cell proliferation but had little effect on the proliferation of MDA-MB-231 cells. Cells were treated with E2 (0.1 nmol/L) in serum-free condition for 48 h before being measured for cell proliferation by BrdUrd labeling. **, P < 0.01. C, transfection of GR siRNA down-regulated GR mRNA expression and inhibited DEX-induced SULT1E1 mRNA expression in MCF-7 cells. **, P <0.01. D, the inhibitory effect of DEX on E2-dependent MCF-7 cell proliferation was compromised in GR siRNA-transfected cells. Cell proliferation was measured by BrdUrd labeling. *, P < 0.05; **, P <0.01; #, P > 0.05 (not significant), compared with the Veh control or the comparison is labeled. At least three replicate experiments were conducted to generate data for statistical analysis.

when PCR primers specific for LXRE on the Sult1e1 promoter (30) Discussion or RORE on the Cyp7b1 promoter (34) were used (Supplementary It has been recognized that glucocorticoids inhibited the Fig. S2D). ChIP assay showed that GR was also recruited to the uterine responses stimulated by estrogens (22, 44). DEX treatment human SULT1E1/GRE encompassing promoter region, but not to a has also been shown to attenuate the estrogen-induced uterine control promoter region that was distal from the SULT1E1/GRE Igf-1 and hepatic ER expression in ovariectomized rodents (25). site (Supplementary Fig. S2E). Consistent with EMSA and ChIP However, the molecular basis for the antagonistic effect of results, synthetic thymidine kinase (tk) reporter genes that contain glucocorticoids on estrogens has been elusive. Our results 700-bp promoter sequences encompassing Sult1e1/GRE (Sult1e1, suggest that transcriptional activation of SULT1E1 by the DEX- 700 bp) or three synthetic copies of Sult1e1/GRE (GRE*3), but not GR pathway plays an important role in DEX-mediated estrogen its mutant variant (Sult1e1/GRE-M*3), were activated by GR in the deprivation. Our conclusions are supported by the observations presence of DEX (Supplementary Fig. S2F). The GR-mediated that (a) DEX induces SULT1E1 gene expression in a GR- Sult1e1/GRE*3 activation was comparable with that of a synthetic dependent manner (Fig. 1), (b) SULT1E1 is necessary for the tk reporter gene containing three copies of the GRE derived from estrogen deprivation effect of DEX (Fig. 2), (c) SULT1E1 is the rat tyrosine aminotransferase gene (Supplementary Fig. S2F), a a transcriptional target of GR (Supplementary Fig. S2), and (d) known GR target gene. LXR had no effect on this GRE*3 reporter a forced expression of SULT1E1 is sufficient to inhibit E2 activity gene (data not shown). A synthetic tk reporter gene that contains in transient transfection and reporter gene assay (data not three copies of the human SULT1E1/GRE, but not its mutant shown). variant, was also activated by GR in a ligand-dependent manner The current study has revealed an important role of SULT1E1 in (Supplementary Fig. S2G). glucocorticoid-induced estrogen deprivation. As the major SULT

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. The Antagonistic Effect of Glucocorticoid on Estrogen isoform to sulfonate estrogens under physiologic conditions, study, we showed that the expression of SULT1E1 is also subjected SULT1E1 has been recognized for its role in estrogen deprivation. to the transcriptional regulation by GR. The regulation of Sult1e1 The critical role of SULT1E1 in estrogen homeostasis was shown by by GR and LXR in the mouse liver seemed to be independent. The the disruption of the Sult1e1 gene in mice. Sult1e1/Est null males DEX and LXR agonists had an additive effect on activating hepatic exhibited structural and functional lesions in their reproductive Sult1e1 mRNA expression (data not shown). There are several system, a phenotype resulting from chronic estrogen stimulation notable differences between GR and LXR in their regulation of (28). In female Sult1e1/Est null mice, the defect in estrogen SULT1E1. First, the DEX effect on SULT1E1 mRNA expression can deprivation caused estrogen excess, leading to placental thrombosis be seen in the liver and MCF-7 cells; whereas the LXR effect was and spontaneous fetal loss (18). In humans, ectopic expression of limited to the liver (30). Second, the DEX effect can be seen in both SULT1E1 in MCF-7 cells reduced the response to physiologic con- mouse and human cells, including human hepatocytes and breast centrations of estradiol and inhibited estrogen-stimulated DNA cancer cells; whereas the LXR effect seemed to be mouse-specific synthesis and cell proliferation (19, 20). (30), reminiscent of the rodent-specific Cyp7a1 regulation by LXR In spite of the extensive biochemical characterization and (46). The DEX/GR-mediated SULT1E1 induction was also consis- functional studies of SULT1E1 (18, 19), until recently, little has been tent with the observation that the hypercorticosteronemia in db/db known about the transcriptional regulation of this SULT isoform. C57BL/KsJ mice was associated with a marked increase in Sult1e1 We have recently shown that LXR, a nuclear receptor known for its mRNA expression in the liver (47). It has also been reported that function in lipid homeostasis and inflammation (45), activated DEX treatment in mice induced hepatic Sult1e1 gene expression Sult1e1/Est gene expression in the mouse liver (30). In the current (48, 49). However, neither the molecular mechanism nor the

Figure 4. DEX inhibited estrogen-dependent human breast cancer cell tumorigenicity in nude mice. A, growth kinetics of the MCF-7/VEGF tumors in the presence of indicated hormone and drug treatments. The DEX treatment groups received daily treatment of DEX at indicated doses by i.p. injection. Tumor volumes were measured at the indicated times. Results are presented as means F SD. Each group contains at least nine mice. *, P < 0.05; **, P < 0.01, compared with the DEX treatment group. Shown in the insert are representative E2-induced tumors in vehicle-treated or DEX-treated mice with the DEX doses labeled. B, BrdUrd immunostaining in E2-induced tumors 25 d postinoculation. Quantitation of BrdUrd-positive nuclei is labeled. Three mice of each group from A were selected for BrdUrd immunostaining. C, serum levels of E2 from mice in A at the time of sacrifice (35 d postinoculation). *, P < 0.05. At least five mice of each group were used for E2 measurement. D, DEX treatment increased hepatic and MCF-7/VEGF tumor expression of Sult1e1/SULT1E1, but not for Cyp1a2, Cyp3a11, and Ugt1a1. **, P < 0.01. Three mice of each group were used for gene expression analysis. www.aacrjournals.org 7391 Cancer Res 2008;68: (18). September 15, 2008

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Cancer Research physiologic implication of this regulation has been systemically with DEX (4mg/kg) for 4days decreased the mRNA expression evaluated in previous studies. of Sult1a1, whereas treatment of nude mice with DEX (6 mg/kg) The stimulatory role of estrogens in breast cancer initiation and for 5 weeks had little effect on the mRNA expression of Sult1a1 progression has been well recognized. Accordingly, strategies to (Supplementary Fig. S3). These results suggest that induction of down-regulate estrogen activities have been proved effective to Sult1a1 may not play a role in our mouse models. treat and prevent breast cancers (13, 14). These include the use of Other than their implications in breast development and breast antiestrogens to prevent estrogen binding and use of aromatase cancer, estrogens are known to have diverse roles in many other inhibitors to inhibit estrogen synthesis. Activation of estrogen- tissues, including bone, liver, the central nervous system, and the metabolizing enzymes represents an independent strategy to vascular system (7, 8). Most, if not all, hormone replacement down-regulate estrogenic activity. The latter notion was supported therapy regimens contain estrogens (55). Meanwhile, glucocorti- by the observation that SULT1E1 expression is decreased in many coids are among the most frequently prescribed antiinflammatory human breast cancer cell lines (19) and an ectopic expression of and immunosuppressive drugs. The inhibition of estrogenic activity SULT1E1 in MCF-7 cells was sufficient to inhibit estrogen- by glucocorticoids raises the concern of hormone-hormone dependent growth response. A more recent study showed that interactions in clinical practice. For example, it may be desirable SULT1E1 levels were inversely correlated with the tumor size or to limit or be cautious about using glucocorticoids in patients who lymph node status in breast cancer patients (50). Our results clearly have undergone hormone replacement therapies. In conclusion, showed that pharmacologic activation of GR was sufficient to GR-mediated estrogen deprivation may have broad implications in restore SULT1E1 expression and inhibit MCF-7 cell growth in cell estrogen homeostasis, endocrine therapies, and tumorigenesis, culture and in vivo. We propose that GR and SULT1E1 may including a potential use in breast cancer prevention and represent novel therapeutic targets to down-regulate estrogen treatment. activity. Indeed, the inhibition of estrogen-dependent MCF-7/VEGF tumor growth by a GR agonist supports the practicality of this therapeutic strategy. Disclosure of Potential Conflicts of Interest DEX is known to trigger insulin resistance and diabetes after a No potential conflicts of interest were disclosed. prolonged (2–5 months) drug treatment (51). The longest DEX treatment in our xenograft model was 35 days; the phenotype was Acknowledgments obvious by day 10 of treatment (Fig. 4). In short-term experiments, we found that treatment with DEX (4mg/kg) for 4days modestly, Received 4/24/2008; revised 7/5/2008; accepted 7/10/2008. but significantly, lowered serum glucose levels (data not shown). Grant support: NIH grants ES014626 and CA107011 (W. Xie) and Department of Therefore, we do not feel that the in vivo DEX treatments used in Defense Breast Cancer Program grants DAMD17-01-1-0375 and DAMD-17-02-1-0584 our study produced diabetic conditions. The aryl sulfotransferase/ (S.Y. Cheng). H. Gong is supported by a Postdoctoral Fellowship (PDF0503458) from the Susan G. Komen Breast Cancer Foundation. SULT1A1 can also sulfonate estrogens. The rat liver SULT1A1 gene The costs of publication of this article were defrayed in part by the payment of page expression has been shown to be glucocorticoid-inducible (52, 53). charges. This article must therefore be hereby marked advertisement in accordance Interestingly, the DEX effect on SULT1A1 expression seemed to be with 18 U.S.C. Section 1734solely to indicate this fact. Normal human hepatocytes were obtained through the Liver Tissue Procurement species specific. Alnouti and Klaassen showed that DEX (75 mg/kg) and Distribution System, Pittsburgh, Pennsylvania, which was funded by NIH contract had little effect on the expression of Sult1a1 in mice (49). The N01-DK-7-0004/HHSN267200700004C. We thank Yonggong Zhai, Shaheen ‘‘Sean’’ Khadem, and Diana Xie for technical expression of human SULT1A1 was also not affected by DEX (54). assistance, and Dr. Stephen Strom (University of Pittsburgh Department of Pathology) In our experiments, treatment of female wild-type C57BL/6J mice for providing human hepatocytes.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Glucocorticoids Antagonize Estrogens by Glucocorticoid Receptor−Mediated Activation of Estrogen Sulfotransferase

Haibiao Gong, Michael J. Jarzynka, Timothy J. Cole, et al.

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