1 ,25-Dihydroxyvitamin D3 Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells1

Vol. 6, 3371–3379, August 2000 Clinical Cancer Research 3371

␣ 1 ,25-Dihydroxyvitamin D3 Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells1

Srilatha Swami, Aruna V. Krishnan, and increase in breast cancer susceptibility gene (BRCA1) pro- 2 David Feldman tein is reduced by 1,25(OH)2D3 treatment. Overall, these Department of Medicine, Stanford University School of Medicine, results suggest that the antiproliferative effects of Stanford, California 94305 1,25(OH)2D3 and its analogues on MCF-7 cells could partially be mediated through their action to down-regulate ER levels and thereby attenuate estrogenic bioresponses, includ- ABSTRACT ing breast cancer cell growth. ␣ 1 ,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Because the estrogen receptor (ER) plays INTRODUCTION a key role in breast cancer progression, we have studied the Breast cancer is the most commonly diagnosed cancer and effects of 1,25(OH)2D3 on the regulation of ER in the estro- the second leading cause of cancer-related deaths among women gen-responsive MCF-7 human breast cancer cell line, which in the United States (1). Because breast cancer is generally ␣ is known to predominantly express ER . 1,25(OH)2D3 characterized by estrogen-dependent growth, the abundance of 3 causes significant inhibition of MCF-7 cell growth, and it ERs in these cells assumes critical importance (2). E2 acts via also decreases the growth-stimulatory effect of 17␤-estradiol the ER, a member of the steroid/thyroid/retinoid receptor super-

(E2). Treatment of MCF-7 cells with 1,25(OH)2D3 reduces family (3). The factors and mechanisms that control the level of ER levels in a dose-dependent manner, as shown by ligand ER expression are important in determining the amplitude of binding assays and Western blot analysis. The 1,25(OH)2D3 E2-mediated actions on the breast cancer cells (2). analogues EB-1089, KH-1060, Ro 27-0574, and Ro 23-7553 1,25(OH)2D3, the biologically active form of vitamin D, is are more potent than 1,25(OH)2D3 in both their antiprolif- a major regulator of calcium and phosphate homeostasis in the erative actions as well as ER down-regulation. There is a body (4, 5). The regulatory effects of 1,25(OH)2D3 are mediated /between the growth-inhibi- via the VDR, which is also a member of the steroid/thyroid (0.98 ؍ striking correlation (R2 tory actions of 1,25(OH)2D3 or analogues and their ability to retinoid receptor superfamily (4–6). In addition to its effects on down-regulate ER levels. Treatment with 1,25(OH)2D3 calcium and phosphate homeostasis, 1,25(OH)2D3 is an impor- shows that the reduction in ER is accompanied by a signif- tant modulator of cellular proliferation and differentiation in a icant decrease in the steady-state levels of ER mRNA. The number of normal and malignant cells (4, 7–9). In breast cancer decrease in ER mRNA is not abolished by the protein syn- cells, 1,25(OH)2D3 has potent growth-inhibitory actions (10– thesis inhibitor cycloheximide. Inhibition of mRNA synthe- 13). Although the growth-inhibitory effects of 1,25(OH)2D3 on sis with actinomycin D reveals no significant differences breast cancer cells have been well established, the effects of between ER mRNA half-life in control and 1,25(OH)2D3- 1,25(OH)2D3 on ER expression are less well documented. Stud- treated cells. Nuclear run-on experiments demonstrate sig- ies on ER␣ in human breast cancer cell lines have reported nificant decreases in ER gene transcription at the end of 17 h minor decreases (14) or no change (15) in ER expression with of treatment with 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 treatment. A more recent study reported signifi- 1,25(OH)2D3 exerts a direct negative effect on ER gene cant decreases in ER protein levels in the MCF-7 cells treated transcription. Coincident with the decrease in ER levels with EB-1089, a potent analogue of 1,25(OH)2D3 (13). Studies there is an attenuation of E2-mediated bioresponses after have also been conducted to indicate that the E2-mediated 1,25(OH)2D3 treatment. Induction of progesterone receptor bioresponses are attenuated by 1,25(OH)2D3 treatment (16, 17). by E2 is suppressed by 1,25(OH)2D3, and the E2-mediated Although the above-mentioned reports have suggested potential cross-talk between 1,25(OH)2D3 and estrogen signaling pathways, the extent of the interaction and the mechanism by which

1,25(OH)2D3 causes down-regulation of ER are still not clarified. Received 2/14/00; revised 5/5/00; accepted 5/11/00. The purpose of the present investigation is to study the The costs of publication of this article were defrayed in part by the effects of 1,25(OH)2D3 on ER and E2-mediated effects in payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant DK42482 and Department of the Army Grant DAMD 17-98-8556. 2 3 ␤ To whom requests for reprints should be addressed, at Division of The abbreviations used are: ER, estrogen receptor; E2,17 -estradiol; ␣ Endocrinology, SUMC, Room S-005, Stanford, CA 94305-5103. Phone: 1,25(OH)2D3,1 ,25-dihydroxyvitamin D3; VDR, vitamin D receptor; (650) 725-2910; Fax: (650) 725-7085. E-mail: feldman@cmgm. nVDRE, negative vitamin D response element; ERE, estrogen response stanford.edu. element; CSS, charcoal-stripped serum; PR, progesterone receptor.

MCF-7 breast cancer cells. As discussed below, the MCF-7 cells Lincoln Park, NJ) at a density of 50,000 cells/well in 3 ml of used in this study do not express ER␤, as measured by reverse RPMI 1640 containing 10% calf serum. Twenty-four h later, transcription-PCR. Therefore, in these studies the effect is lim- fresh medium was added. Cells were grown in RPMI 1640 ited to ER␣. For simplicity, we refer to the ER␣ in these cells as medium with 10% calf serum or CSS and were treated with

ER. To achieve our goal of investigating the effect of various doses of 1,25(OH)2D3 or its analogues in the presence or M 1,25(OH)2D3 on breast cancer cells, we have studied the rela- absence of 10 n E2. Fresh medium and hormones were added tionship between changes in MCF-7 growth rate, levels of ER every other day. At the end of 6 days, cell monolayers were protein, steady-state ER mRNA, and gene transcription in cells processed as described earlier (19), and DNA contents were determined by the method of Burton (20). treated with 1,25(OH)2D3 or its analogues. We have also as- Ligand Binding Assays. MCF-7 cells growing in CSS- sessed the correlation between the effects of 1,25(OH)2D3 and its analogues KH-1060, EB-1089, Ro 27-0574, and Ro 23-7553 containing medium were treated with either E2 (10 nM)or on the growth of MCF-7 cells and the changes elicited in ER 1,25(OH)2D3 or analogues (1, 10, or 100 nM) for 2 days. Cells levels. Furthermore, we have investigated how changes in ER were then harvested, and high salt cell extracts were made as described previously (21). The protein concentration of the abundance induced by 1,25(OH)2D3 and its analogues alter the extracts was measured by the method of Bradford (22). Aliquots functional responses to E2 in MCF-7 cells. We have established of the extracts were incubated overnight at 4°C with either 10 that the 1,25(OH)2D3-mediated effect on ER gene expression is 3 3 at the transcriptional level. nM [ H]E2 or 10 nM [ H]progesterone for ER and PR measurements, respectively. Two hundred-fold excess of nonradioactive hormone was used to correct for nonspecific binding. Bound and MATERIALS AND METHODS free hormones were separated using hydroxylapatite, and spe- 3 Materials. [ H]Estradiol-17␤-D-glucuronide (specific cific binding was calculated as described earlier (21). activity, 40 Ci/mmol) and [3H]progesterone (specific activity, Western Blot Analysis. Aliquots of cell extracts pre- 54.1 Ci/mmol) were purchased from DuPont NEN (Wilmington, pared as described above were mixed with 3ϫ SDS sample DE). Radioinert steroids were obtained from Steraloids, Inc. buffer, boiled for 5 min, and subjected to 10% SDS-PAGE.

(Wilton, NH). Nonradioactive 1,25(OH)2D3 and its analogues After transfer to nitrocellulose membranes, immunoblotting 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (Ro 23-7553) with either the antimouse monoclonal antibody to human ER and 1,25-dihydroxy-23-yne-26,27-hexafluoro-20-cyclopropyl- (H222; 1:500 dilution in 1% Carnation nonfat milk; a gift from 19-nor-cholecalciferol (Ro 27-0574) were generous gifts Abbott laboratories) or antimouse monoclonal antibody to the from Dr. M. Uskokovic (Hoffmann La-Roche Co., Nutley, human BRCA1 protein (C-20; 2 ␮g/ml in 1% Carnation nonfat NJ). 1␣,25-Dihydroxy-22,24-diene-24,26,27-trihomovitamin milk; from Santa Cruz Biotechnology) was carried out as de- ␣ D3 (EB-1089) and 1 ,25-dihydroxy-20-epi-22-ene-24,26,27- scribed previously (19). The blots were then probed with a trihomovitamin D3 (KH-1060) were generous gifts from Dr. horseradish peroxidase-conjugated antimouse secondary anti- L. Binderup (Leo Pharmaceuticals, Ballerup, Denmark). body, and the immunoreactive bands were detected using an MCF-7 cells were obtained from American Type Culture enhanced chemiluminescence (ECL) kit obtained from Amer- Collection (Rockville, MD). Culture media and other supple- sham (Arlington Heights, IL). High molecular weight markers ments were purchased from Mediatech (Herndon, VA). All from Life Technologies, Inc. (Grand Island, NY) were used to other chemicals and reagents, including anti-actin antibody, estimate the sizes of the immunoreactive bands. were obtained from Sigma Chemical Co. (St. Louis, MO). Northern Blot Analysis. Total RNA was isolated from

Cell Culture. MCF-7 cells were routinely cultured in cells treated with either ethanol vehicle or 1,25(OH)2D3 as T-75 flasks at 37°C under an atmosphere of 5% CO2. They were described previously (23, 24). Changes in ER mRNA levels maintained in RPMI 1640 supplemented with 10% calf serum, attributable to 1,25(OH)2D3 treatment were detected by North- 100 units/ml penicillin, and 100 ␮g/ml streptomycin. At con- ern blot analysis. A 2.1-kb EcoRI fragment of the human ER fluence, the flasks contained approximately 1.5–2 ϫ 107 cells cDNA was subjected to random prime labeling using [32P]dCTP and were routinely subcultured every 7–10 days. For experi- and the Rediprime labeling kit (Amersham), and the labeled ments, the growth medium was replaced with phenol red-free fragment was used to probe the blots. To control for differences RPMI 1640 supplemented with 10% calf serum (CSS), twice in RNA sample loading and transfer, the blots were also hybrid- stripped of endogenous hormones using charcoal and Dextran ized with a 32P-labeled, 0.9-kb EcoRI fragment of the gene T-70. We used medium containing CSS in most of our studies encoding the human L7 ribosomal protein. The membranes were

to minimize the effects of E2, which is known to down-regulate exposed to X-ray films (Hyperfilm MP) for about 17 h at its own receptor (18). The medium containing CSS (treatment Ϫ80°C. Autoradiograms were scanned using a Molecular Dy- medium) and hormones, as indicated, was introduced to the cells namics Computing densitometer (model 300A; Molecular De- 24 h after subculture, and fresh medium and hormones were vices, Menlo Park, CA), and the ER mRNA levels were indexed replenished every 2 days. Stock solutions of steroid hormones to the corresponding L7 mRNA levels. were made in 100% ethanol and added to the treatment medium. Measurement of ER mRNA Half-Life. To determine All controls received ethanol vehicle at a concentration equal to the half-life of ER mRNA, MCF-7 cells were grown as de- that in the hormone-treated cells (0.1% v/v). scribed earlier and treated with ethanol (control) or 100 nM

Cell Proliferation Assay. DNA content or attained cell 1,25(OH)2D3 for 24 h. At the end of 24 h, transcription was mass was used as a measure of cell proliferation. MCF-7 cells terminated by the addition of 4 ␮M actinomycin D. Because the were seeded in six-well tissue culture plates (Becton Dickinson, reported half-life of ER mRNA is ϳ4 h, total RNA was ex-

Table 1 Comparison of IC50 of 1,25(OH)2D3 and its analogues

Cell proliferation and E2 binding were determined in MCF-7 cells as described in “Materials and Methods.” IC50 was calculated as the concentration required to achieve 50% of the maximal inhibition.

IC50 (nM)

Analogue Proliferation E2 binding

1,25(OH)2D3 4.70 7.50 Ro 23-7553 1.50 3.90 EB 1089 0.30 0.59 KH 1060 0.26 0.50 Ro 27-0574 0.22 0.48

RESULTS

Effect of 1,25(OH)2D3 and Its Analogues on MCF-7 Cell Proliferation. Fig. 1 demonstrates the effect of 1,25(OH)2D3 and its analogues on the growth of MCF-7 cells cultured in Fig. 1 Effect of 1,25(OH) D and analogues on the proliferation of 2 3 medium containing 10% calf serum. 1,25(OH)2D3 and each of MCF-7 cells. MCF-7 cells were grown in six-well dishes for 6 days in the analogues tested caused a dose-dependent decrease in the RPMI 1640 containing 10% calf serum and treated with various con- cell growth. The analogues were more potent inhibitors of centrations of 1,25(OH)2D3 or analogues, whereas controls received ethanol vehicle. Fresh medium and hormones were replenished every growth than 1,25(OH)2D3, with the order of potency being other day. DNA levels are expressed as percentages of control, which Ro 27-0574 Ͼ KH-1060 Ͼ EB-1089 Ͼ Ro 23-7553 Ͼ was 43 Ϯ 0.42 ␮g DNA/well. Experiments were conducted in triplicate, 1,25(OH)2D3. The calculated IC50 values are shown in Table 1. and values were a mean of at least three individual experiments; bars, Fig. 2 shows the effect of 1,25(OH) D and its analogues SD. All groups were significantly different from the control with P Ͻ 2 3 0.05. on growth of MCF-7 cells cultured in phenol red-free RPMI containing 10% CSS in the absence or presence of 10 nM E2. The total DNA content in these controls was slightly less than that observed with the controls grown in medium containing tracted at regular time intervals up to 6 h after actinomycin 10% calf serum (31 Ϯ 6.6 versus 43 Ϯ 0.42 ␮g DNA/well). ␮ treatment. Twenty g of total RNA were used for Northern blot 1,25(OH)2D3 and its analogues demonstrated a higher degree of analysis as described earlier. growth inhibition in medium containing serum (Fig. 1) than

Transcriptional Run-On Assay. Nuclei were isolated medium containing CSS (Fig. 2). From Fig. 2, E2 treatment Ͻ from MCF-7 cells treated with 100 nM 1,25(OH)2D3 for various caused a 2-fold increase in growth (P 0.001) when compared time intervals according to the procedure described by Stott with controls. Significant decreases in DNA content were seen

(25). Briefly, MCF-7 cells treated with 1,25(OH)2D3 or ethanol after cotreatment with E2 and 1,25(OH)2D3 or its analogues vehicle were harvested at various time intervals and resus- when compared with E2 treatment alone. 1,25(OH)2D3 and its pended in ice-cold nuclei isolation buffer [10 mM Tris-HCl (pH analogues were able to partially or completely counteract the

7.4), 10 mM NaCl, 5 mM MgCl2,and1mM DTT] containing E2-mediated increase in growth, with the analogues being more 0.5% NP40. The intact nuclei were then pelleted by centrifuga- potent than 1,25(OH)2D3. tion at 2000 rpm for 5 min. The pellets were resuspended in Effect of 1,25(OH)2D3 on ER Abundance. To deter- nuclei freezing buffer [50 nM Tris-HCl (pH 8.5), 50% w/v mine the effects of 1,25(OH)2D3 and its analogues on ER 8 3 glycerol, 5 mM MgCl2, and 0.1 mM EDTA] in aliquots of 10 abundance, we used [ H]E2 ligand binding assays in cells cul- nuclei/ml and frozen as aliquots of 210 ␮latϪ70°C until tured in medium containing CSS treated with graded concen-

needed. RNA elongation was carried out as described earlier trations of 1,25(OH)2D3 or its analogues for 2 days. As shown (25). Frozen aliquots of nuclei were thawed on ice and incubated in Fig. 3, 1,25(OH)2D3 induced a dose-dependent decrease in 32 with [ P]UTP and unlabeled ATP, CTP, and GTP at 30°C for ER levels, which was modest at 1 and 10 nM and higher (ϳ50%) 45 min. The radiolabeled nascent RNA transcripts were isolated at 100 nM (P Ͻ 0.001). As in the proliferation experiments, the

using TRIzol reagent, followed by chloroform extraction and analogues were more potent than 1,25(OH)2D3, with the order ethanol precipitation. Isolated RNA was then hybridized to of potency being Ro 27-0574 Ͼ KH-1060 Ͼ EB-1089 Ͼ Ro Ͼ nitrocellulose filters containing cDNAs for ER and L7 for 72 h 23-7553 1,25(OH)2D3.IC50 values for ER down-regulation at 65°C. Filters were then washed and exposed to X-ray film. are shown in Table 1. Data in Table 1 demonstrate that the

Autoradiographs were scanned, and results were normalized by antiproliferative effect of 1,25(OH)2D3 or a given analogue comparison to the transcriptional level of L7. correlated very well (R2 ϭ 0.98) with ER suppression. Statistical Analysis. Data are presented as mean Ϯ SD In the next set of experiments, MCF-7 cells grown in of three to four individual measurements. Statistical analysis medium containing 10% CSS were treated with 100 nM

was done by Student’s t test or ANOVA using the Statview 4.5 1,25(OH)2D3, and the time course of the 1,25(OH)2D3 effect on software (Abacus Concepts, Berkeley, CA). P Ͻ0.05 is consid- ER levels was investigated. Although decreases in ER levels

ered significant. could be seen as early as 6 h after treatment with 1,25(OH)2D3,

Fig. 2 Effect of 1,25(OH)2D3 and analogues on MCF-7 cell proliferation in the presence and absence of E2. MCF-7 cells cultured in phenol Fig. 3 Effect of 1,25(OH) D and analogues on ER abundance. MCF-7 red-free RPMI 1640 containing 10% CSS were treated with 10 nM E2 2 3 cells grown in medium containing 10% CSS were treated with graded and/or 10 nM 1,25(OH)2D3 or analogues. Controls received ethanol vehicle. Fresh medium and hormones were replenished every other day. concentrations of 1,25(OH)2D3 or analogues as indicated. After 2 days, 3 After 6 days, cells were collected, and DNA content in each well was cells were collected, and [ H]E2 binding assays performed. Values are measured. Experiments were conducted in triplicate and are expressed expressed as a percentage of control levels, which was 430 Ϯ 49 as a percentage of vehicle-treated controls (31 Ϯ 6.6 ␮g DNA/well). fmol/mg protein. All values represent means of at least three separate Values represent the means from at least three individual experiments; experiments conducted in duplicate; bars, SD. All groups, with the Ͻ ϩ exception of cells treated with 1 nM 1,25(OH) D , were significantly ءءء Ͻ ءء Ͻ ء bars, SD. , P 0.01; , P 0.001; , P 0.0001 when E2 2 3 ϩ Ͻ ϩϩ Ͻ different when compared with control (P Ͻ 0.05–0.001). 1,25(OH)2D3/analogues were compared with E2. , P 0.01; , P ϩϩϩ Ͻ 0.001; , P 0.0001 when 1,25(OH)2D3 or analogues were compared with controls in the absence of E2.

1,25(OH)2D3 and its analogues on ER levels in the presence of medium containing 10% calf serum. A significant fall in ER

changes were significant only at the end of 2 days (Fig. 4A). At levels could be seen with 1,25(OH)2D3 and all of the analogues this time point, the ER levels declined by ϳ50% (from 479 Ϯ tested. Expression of actin, which was used as a control, did not Ϯ 41 to 213 37 fmol/mg protein). This decrease was transient, change. Thus, ER down-regulation by 1,25(OH)2D3 or its ana- because by day 4, the decrease in ER was less pronounced, and logues could be seen in medium containing CSS as well as by day 6, ER levels were back to that seen in control cells. regular serum. These results correlate with the observations 3 Scatchard analysis (data not shown) of [ H]E2 binding revealed from ligand binding studies (Fig. 4A) and growth assays (Fig. 2), that 1,25(OH)2D3 treatment for 2 days caused a decrease in ER confirming that the analogues were more potent than

abundance (Nmax) from 492 fmol/mg protein to 251 fmol/mg 1,25(OH)2D3 in both assays (Table 1). ϭ protein, with no change in the affinity for E2 (Kd 0.70 nM in Effect of 1,25(OH)2D3 on ER mRNA. To determine controls versus 0.62 nM in 1,25(OH)2D3-treated cells). whether the changes in ER attributable to 1,25(OH)2D3 treat- Changes in ER protein levels were also assessed by West- ment occurred at the mRNA level, we examined the steady-state ern blot analysis. Cell extracts from MCF-7 cells, grown in levels of ER mRNA by Northern blot analysis. Fig. 5A is a

medium containing CSS, were treated with 1,25(OH)2D3 for representative Northern blot and Fig. 5B its corresponding den- various time intervals and probed with the H222 anti-ER mono- sitometric scan. All experiments (n ϭ 4) were conducted in the

clonal antibody. Western blot analysis (Fig. 4B) revealed a presence of CSS to minimize the effects of E2 present in the ϳ Ͻ pattern of changes similar to that observed with ligand binding medium. 1,25(OH)2D3 decreased ER mRNA ( 60%; P studies (Fig. 4A). A 66-kDa band representing the immunore- 0.001) only at 24 h. The decreased levels of ER mRNA were active ER protein could be detected in both treated and untreated transient, with ER mRNA returning to near normal levels by groups. A significant decrease (ϳ50%) in the ER protein levels 48 h. Data from Northern blot analysis correlate with the ob-

was seen at the end of 2 days in the 1,25(OH)2D3-treated cells. servations from Western blot analysis and ligand binding stud- The ER protein levels remained suppressed (ϳ30%) at day 4 ies, which showed an ϳ50% drop in the ER protein levels after and gradually returned to control levels by the end of 6 days. 48 h of treatment. The experiment was repeated three times to confirm the pattern Effects of Cycloheximide on ER mRNA Levels. To

of changes and to rule out possible differences attributable to determine whether the effect of 1,25(OH)2D3 on ER required de loading. novo protein synthesis, we assessed the ability of 1,25(OH)2D3 Because the growth-inhibitory effects of 1,25(OH)2D3 and to regulate ER mRNA in the presence and absence of cyclohex- its analogues were more evident in medium containing serum imide, a potent inhibitor of protein synthesis. MCF-7 cells were

(Fig. 1), it was important to establish the down-regulation of ER treated with 100 nM 1,25(OH)2D3 in the presence of cyclohex- in serum containing medium. Fig. 4C demonstrates the effect of imide (10 ␮g/ml of culture medium) for 24 h. Controls received

Fig. 5 Northern blot analysis of ER mRNA after 1,25(OH)2D3 treatment. A, a representative Northern blot. MCF-7 cells grown to 60% confluence in medium containing 10% CSS were treated with 100 nM ␮ 1,25(OH)2D3 for various time intervals. Total RNA (10 g) from each sample was subjected to Northern blot analysis and probed for ER

mRNA and L7. Lanes C, control; Lanes D, 1,25(OH)2D3 treated. The numbers in subscript denote the number of hours of treatment with

1,25(OH)2D3. B, densitometric scan of the Northern blot from A. Values are the ratio of ER mRNA indexed to the corresponding L7 mRNA. Lanes 7 and 8, representing time points C48 and D48, were taken from another experiment. The results are representative of four individual experiments.

Fig. 4 Time course of 1,25(OH)2D3 regulation of ER levels in MCF-7 3 cells by [ H]E2 binding assays and Western blot analysis. MCF-7 cells in phenol red-free RPMI 1640 containing 10% CSS were treated with 100 nM 1,25(OH)2D3. Cells were collected at the time points indicated the 1,25(OH)2D3-mediated decrease in steady-state levels of ER 3 and processed. A, [ H]E2 binding data. ER levels are expressed as mRNA compared with either the ethanol-treated cells or the fmol/mg protein. Values represent means of three to six individual cells treated with only cycloheximide. An ϳ75% decrease in ER P Ͻ ,ءء ;P Ͻ 0.05 ,ء .experiments conducted in duplicate; bars, SD 0.001 compared with corresponding control. B, Western blot analysis of mRNA was seen 24 h after treatment with 1,25(OH)2D3 (Fig. 6, MCF-7 cells cultured in RPMI 1640 containing 10% CSS. ER protein Lanes 2 and 4), suggesting that ongoing protein synthesis is not was visualized as a Mr 66,000 immunoreactive band using the ECL necessary for the down-regulation of ER mediated by detection system. Lanes C, control; Lanes D, 1,25(OH)2D3 treated. The 1,25(OH) D . numbers in subscript denote the number of days of treatment with 2 3 Effect of 1,25(OH)2D3 on the Half-Life of ER mRNA. 1,25(OH)2D3. Similar results were obtained in three individual experiments. C, Western blot analysis of MCF-7 cells grown in RPMI 1640 To measure the half-life of ER mRNA, MCF-7 cells were containing 10% calf serum. Cells grown to 60% confluence were treated pretreated with 100 nM 1,25(OH)2D3 or vehicle, and transcrip- with 100 nM 1,25(OH)2D3 or analogues for a period of 2 days. ER tion was terminated at the end of 24 h by the addition of 4 ␮M protein was visualized as a M 66,000 immunoreactive band. Actin (M r r actinomycin D. RNA was isolated at various times after the 46,000) was used as a control to correct for loading differences. Lane 1, addition of actinomycin D and subjected to Northern blot anal- control; Lane 2, 1,25(OH)2D3; Lane 3, EB-1089; Lane 4, Ro 23-7553; Lane 5, KH-1060; and Lane 6, Ro 27-0574. ysis of ER and L7. ER/L7 mRNA at the time of actinomycin D addition (zero time) was represented as 100% for both control

and 1,25(OH)2D3 treated cells. It is clear from Fig. 7 that the half-life of ER mRNA did not decrease with 1,25(OH)2D3 either ethanol vehicle or cycloheximide alone. At the end of treatment; rather, a small increase was seen. The half-life was ϳ ϳ 24 h, total RNA was extracted from each treatment group and 4.5 h in the controls versus 6 h in the 1,25(OH)2D3-treated used for Northern blot analysis. As can be seen from Fig. 6, cells, a difference that was not statistically significant, suggest-

inhibition of protein synthesis by cycloheximide did not prevent ing that 1,25(OH)2D3 had no effect on ER mRNA turnover.

Fig. 7 Effect of 1,25(OH)2D3 on the stability of ER mRNA. MCF-7 cells cultured in medium containing 10% CSS were treated with 100 nM

1,25(OH)2D3 for 24 h. Controls received ethanol vehicle for the same period. RNA synthesis was terminated at the end of 24 h by the addition of actinomycin D (4 ␮M). Total RNA was isolated at the indicated time Fig. 6 Effect of cycloheximide on 1,25(OH)2D3-mediated suppression intervals after the addition of actinomycin D, and Northern blot analysis of ER mRNA. Cells at 60% confluence grown in medium containing of ER and L7 mRNA was carried out. Zero time is the ER:L7 mRNA ␮ CSS were treated with a single dose of cycloheximide (10 g/ml) in the ratio at the time at which actinomycin D was added to both groups of presence or absence of 100 nM 1,25(OH)2D3 for 24 h. Total RNA was cells and is represented as 100%. Half-life is calculated as the time taken isolated and processed for Northern blot analysis. C, control; D, for ER:L7 mRNA to fall by 50%. Bars, SD. 1,25(OH)2D3; CY, cycloheximide. The Northern blot shown here is a representative of three individual experiments.

treatment when compared with controls. E2 failed to induce a measurable increase in PR in the presence of 1,25(OH)2D3, suggesting that this functional response of ER was suppressed in Effect of 1,25(OH)2D3 on ER Gene Transcription. To the presence 1,25(OH) D . determine whether the 1,25(OH)2D3 induced decrease in ER 2 3 mRNA was a transcriptional event, ER gene transcription run-on BRCA1 expression was assessed by Western blot analysis assays were performed using nuclei isolated from MCF-7 cells in MCF-7 cells treated with 10 nM E2 and/or 100 nM 1,25(OH) D . The immunoreactive protein representing treated with either 1,25(OH)2D3 or ethanol vehicle for various 2 3 time intervals. L7 gene transcription was used as an internal BRCA1 could be seen as a single Mr 210,000 species using the control. ER:L7 mRNA ratio was calculated for vehicle-treated C-20 anti-BRCA1 monoclonal antibody (Fig. 9B). Detectable controls for each time point and was represented as 100%. increases in BRCA1 levels were seen at the end of 2 days of E2 treatment (Lane 3). 1,25(OH) D , on its own, did not have any ER:L7 mRNA for 1,25(OH)2D3-treated cells were calculated 2 3 and represented as a percentage of its corresponding vehicle- effect on the levels of BRCA1 protein (Lane 2). However, treated control. Fig. 8 demonstrates a decrease in the level of ER 1,25(OH)2D3 suppressed the estrogen-mediated rise in BRCA1 levels (Lane 4). No changes could be seen in the levels of actin gene transcription with 1,25(OH)2D3 treatment. ER transcription decreased at 6 h (50% inhibition) and reached a nadir at used to control for loading differences. 17 h (80% inhibition) before rising back to control levels at 48 h. The decrease was specific for ER transcription because L7 did DISCUSSION

not change with 1,25(OH)2D3 treatment. These data suggest that The growth-stimulatory effects of E2 on breast cancer cells 1,25(OH)2D3 down-regulates ER gene expression at the tran- have been well established (26, 27). MCF-7, the most studied scriptional level. and well-characterized breast cancer cells, have been shown to ␣ Effect of 1,25(OH)2D3 on the ER-mediated Functional express high levels of ER (28). Although there have been Responses. This set of experiments evaluated the impact of reports indicating that ER␤ may also be expressed in MCF-7 ER regulation on estrogen-dependent responses, i.e., the induc- cells (29, 30), we could not detect any ER␤ mRNA by reverse

tion of PR and expression of BRCA1, which is stimulated by E2. transcription-PCR in our cultures of MCF-7 cells. Hence, we Fig. 9A shows the changes in PR induction by E2 in have interpreted our observations on the assumption that the ␣ response to 1,25(OH)2D3 treatment. Cell extracts were made at MCF-7 cells used in this study express only ER . Further the end of 2 days of treatment with E2, 1,25(OH)2D3,ora investigations are needed to ascertain whether these observa- combination of both; and PR levels were determined. tions can be extrapolated to other breast cancer cell lines. It will

1,25(OH)2D3 by itself did not have any effect on PR levels. A also be of great interest to study the effects of 1,25(OH)2D3 on ␣ ␤ 6-fold increase in PR levels was seen at the end of 2 days of E2 ER regulation in cells that express both the and isoforms.

Fig. 8 Effect of 1,25(OH)2D3 on ER gene transcription. MCF-7 cells grown to 50% confluence in RPMI 1640 containing 10% CSS were treated with 100 nM 1,25(OH)2D3. Nuclei were isolated at various time points, and nuclear run-on assays were performed. Autoradiographs were scanned, and the level of transcription was computed as a ratio of ER:L7 for each time point. Vehicle-treated controls were represented as

100%. ER:L7 mRNA ratios for 1,25(OH)2D3 treated cells were calculated and represented as a percentage of its corresponding vehicle- treated control. Values are means of two experiments; bars, SD.

In our studies with MCF-7 cells, 1,25(OH)2D3 and its structural analogues clearly demonstrate dose-dependent antiproliferative properties (Fig. 2). These results confirm earlier observations (10, 11, 13, 14, 31–35) and show that the analogues, in addition to being less calcemic (36, 37), are more Fig. 9 Effect of 1,25(OH)2D3 to attenuate E2 actions in MCF-7 cells. A, PR induction. Cells grown in medium containing 10% CSS were potent than 1,25(OH)2D3 in their ability to inhibit growth of treated with 10 nM E2 or 100 nM 1,25(OH)2D3 or both for 2 days, and MCF-7 cells. The growth-inhibitory effects are more evident in PR levels were assessed by [3H]progesterone binding. Controls received serum containing medium than medium containing CSS. Fur- ethanol vehicle. Values represent the means from at least four experi- P Ͻ 0.001 compared with ,ء .ments conducted in duplicate; bars, SD thermore, 1,25(OH)2D3 and its analogues are antiproliferative, even in the presence of added E . Although 1,25(OH) D and controls. B, Western blot analysis of BRCA1 expression. Experimental 2 2 3 conditions are the same as in A. Western blot analysis revealed BRCA1 Ro 23-7553 could only partially counteract the E -mediated 2 protein as a Mr 210,000 immunoreactive band. Actin (Mr 46,000) was growth of MCF-7 cells, the other analogues are clearly more used as a control to correct for loading differences. C, control; D, ϩ ϩ potent because they are able to completely abolish the stimula- 1,25(OH)2D3; E, E2; E D,E2 1,25(OH)2D3. tory effects of E2. We next investigated whether the antiproliferative effects of 1,25(OH)2D3 on the MCF-7 cells could be attributable to its action to down-regulate ER levels. Although previous studies of ER mRNA and ER gene transcription. The steady-state levels have shown that 1,25(OH)2D3 down-regulates ER protein, the of ER mRNA are decreased 60–80% (Figs. 5A and 6) by results in different studies have been variable (13, 14). Using 1,25(OH)2D3, indicating that the regulation occurs at the mRNA ligand binding studies and Western blot analysis, we have level. 1,25(OH)2D3 does not significantly alter the half-life of demonstrated significant down-regulation of ER levels in me- ER mRNA, and the decrease in ER mRNA is not dependent on dium containing CSS as well as in the presence of complete new protein synthesis, suggesting that the 1,25(OH)2D3 effects serum. There is a high degree of correlation (R2 ϭ 0.98) be- on ER are transcriptional rather than posttranscriptional. This tween the ability of the different 1,25(OH)2D3 analogues to hypothesis is confirmed by nuclear run-on assays, which dem- inhibit cell growth and their ability to decrease ER levels. These onstrate significant decreases in ER gene transcription after results suggest that the actions of 1,25(OH)2D3 and its ana- 1,25(OH)2D3 treatment. The magnitude of changes in transcrip- logues to inhibit cell growth might be, in part, attributable to tion rate is more pronounced, and they seem to occur earlier than their ability to down-regulate ER levels. However, because those observed with the changes in steady-state ER mRNA

1,25(OH)2D3 has also been shown to inhibit the growth of levels. The trends, however, are similar. Taken together, these

ER-negative breast cancer cell lines (35), we emphasize that ER data suggest that the predominant mechanism of 1,25(OH)2D3- down-regulation is only one of several pathways through which mediated down-regulation of ER gene expression is at the tran-

1,25(OH)2D3 and its analogues act to inhibit breast cancer cell scriptional level. growth. Evidence from our study supports the hypothesis that the

In an attempt to elucidate the mechanism of ER regulation, 1,25(OH)2D3-bound VDR interacts directly with a nVDRE in we studied the effect of 1,25(OH)2D3 on the steady-state levels the ER gene promoter inhibiting its transcription. Further sup-

port for this hypothesis will require the identification of an along the estrogen response pathway, affecting the levels of ER nVDRE in the ER gene promoter. nVDRE sequences have been as well as their ability to function as enhancers of transactiva- described in other genes (38, 39). tion. This study adds down-regulation of ER to the many pos-

Coincident with decreases in ER levels, the functional tulated mechanisms by which 1,25(OH)2D3 inhibits breast can- responses to E2 are also attenuated because of 1,25(OH)2D3 cer cell growth. treatment (Fig. 9). It was demonstrated earlier (17) that EB- 1089, a potent analogue of vitamin D, down-regulates ER ex- Note Added in Proof pression in MCF-7 cells and limits E2 responsiveness measured as the induction of PR protein and pS2 mRNA. In our study, the A potential nVDRE has recently been identified in the ER promoter (Stoica et al., J. Cell Biochem., 75: 640–651, 1999). E2 induction of PR protein is completely inhibited by 1,25(OH)2D3, although there is only a 50% decrease in ER levels. Thus, the attenuation of the ER functional response is ACKNOWLEDGMENTS greater than that of ER down-regulation. One possible explana- We thank Dr. M. Uskokovic (Hoffmann La-Roche Co., Nutley, NJ)

tion for this finding is that 1,25(OH)2D3 might act at multiple for providing the 1,25(OH)2D3 Ro 27-0574 and Ro 23-7553; Dr. L. sites in the E2-mediated pathway. Demirpence et al. (16) have Binderup (Leo Pharmaceuticals, Ballerup, Denmark) for providing us demonstrated that 1,25(OH)2D3 decreases ER binding to an with the EB-1089 and KH-1060; and Dr. P. Chambon (University of ERE element, suggesting that in addition to ER regulation, Louis Pasteur, Strasbourg, France) for providing the human ER cDNA. The authors thank Drs. Xiao Yan Zhao and Stephen Sarabia for assist- 1,25(OH)2D3 has other sites of action in the ER pathway to ance in the preparation of the manuscript. inhibit E2-mediated transactivation of target genes. We also studied the effect of 1,25(OH)2D3 on BRCA1 expression, which has been implicated in the development REFERENCES and/or progression of hereditary breast cancer (40). In addition 1. Greenlee, R. T., Murray, T., Bolden, S., and Wingo, P. A. Cancer to its role as a tumor suppression gene (41, 42), BRCA1 has been statistics, 2000. CA Cancer J. Clin., 50: 7–33, 2000.

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Srilatha Swami, Aruna V. Krishnan and David Feldman

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