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2-Methoxyestradiol Induces Mammary Gland Differentiation through Amphiregulin-Epithelial Growth Factor Receptor-Mediated Signaling: Molecular Distinctions from the Mammary Gland of Pregnant Mice

Laboratories of Cell Regulation and Carcinogenesis (J.-I.H., T.H.Q., R.C., J.E.G.), Biosystems and Cancer (G.V.R.C.), and Receptor Biology and Gene Expression (M.W., G.L.H.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; Division of Oncology (R.C., A.C.), Center for Applied Medical Research (CIMA), and Department of Histology and Pathology, University of Navarra, Pamplona, 31080 Spain; EntreMed, Inc. (T.M.L.), Rockville, Maryland 20850; and California Pacific Medical Center (P.-Y.D.), Cancer Research Institute, San Francisco, California 94143

Address all correspondence and requests for reprints to: Jeffrey E. Green, Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Building 41, Room C629, 41 Medlars Drive, Bethesda, Maryland 20892. E-mail: jegreen{at}nih.gov.

	Abstract

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Levels of 2-methoxyestradiol (2ME₂), an endogenous metabolite of estradiol, are highly elevated during late stages of pregnancy when mammary glands have differentiated with the formation of alveolar structures producing milk proteins. Based upon our previous demonstration that 2ME₂ induces mammary ductal dilation associated with expression of mammary differentiation markers when administered to transgenic mice that spontaneously develop mammary cancer, we studied the effects of 2ME₂ on normal mammary gland development. The results of this study demonstrate that 2ME₂ can induce a partial differentiation of normal mammary glands in virgin mice, as evidenced by the appearance of limited numbers of alveolar cells and significantly increased expression of the differentiation markers ß-casein and whey acidic protein. 2ME₂-induced differentiation is associated with inhibition of expression of inhibitor of differentiation 1 (Id-1) in normal mammary epithelial cells through elements in the 5'-flanking region of the Id-1 gene. Microarray analysis revealed that 2ME₂-induced differentiation of the mammary gland shares some significant similarities in gene expression with that of mammary glands from late-stage pregnancy, including elevated expression of many milk protein differentiation markers. However, several genes are differentially regulated between 2ME₂-treated mammary glands and differentiated mammary glands through pregnancy. Significantly, amphiregulin, ATF3, serpine2, and SOX6 were up-regulated in 2ME₂-treated mammary glands but not in mammary glands from pregnant mice. Using the SCp2 differentiation cell line system, we demonstrate that 2ME₂ induces differentiation through the down-regulation of Id-1 and up-regulation of amphiregulin. Administration of amphiregulin to SCp2 cells induced differentiation, whereas inhibition of 2ME₂-induced expression of amphiregulin by small interfering RNA blocked differentiation. Estrogen receptor-negative SCp2 cells differentiate in response to 2ME₂, but not estradiol, suggesting that 2ME₂ operates through an estrogen receptor-independent mechanism. These data demonstrate that 2ME₂ can induce a partial differentiation of the mammary gland through mechanisms that differ from those normally used during pregnancy.

	Introduction

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DIFFERENTIATION OF THE mammary gland is a complex process that involves the coordinated response to several hormone signals during pregnancy, lactation, and subsequent involution (1). Pregnancy has paradoxical effects on breast cancer risk, suggesting that the state of mammary gland differentiation and history of parity can influence the development of cancer (2, 3). It has been proposed that pregnancy early in life provides a protective effect against the delayed development of breast cancer by permanently altering the state of differentiation of the mammary gland and rendering it less susceptible to transformation (1). However, the risk of breast cancer is actually increased during and immediately after pregnancy, which may be influenced by the extensive remodeling of the mammary gland during this period (3).

Relatively modest increases in maternal serum estradiol (E₂) (100 nM) and estrone (75 nM) occur during pregnancy compared with estriol, which may reach approximately 900 nM, produced primarily through the fetal-placental unit (4). However, levels of 2-methoxyestradiol (2ME₂), an endogenous metabolite of 17ß-E₂ with low affinity to estrogen receptors (ER) and ß, dramatically increase during pregnancy. 2ME₂ is formed by the sequential hydroxylation of 17ß-E₂ by cytochrome P450 enzymes followed by O-methylation by catechol-O-methyltransferase. Significant levels of 2ME₂ can be measured in both the blood and urine of humans, with highly elevated levels during pregnancy. Serum levels of 2-methoxyestrogens between the 11th and 16th weeks of pregnancy range between 2 and 15 nM; between the 37th and 40th weeks of pregnancy, the serum level of 2ME₂ reaches 18–96.21 nM; and during labor, levels are 12.15–90 nM (5). Although the levels of 2ME₂ during pregnancy are quite high, the normal physiological roles for 2ME₂ have not been extensively explored.

During the course of our previous study to determine the effects of 2ME₂ on mammary tumor development, it was observed that 2ME₂ induced limited differentiation of the mammary glands resulting in ductal dilatation and production of milk proteins (6), although no direct role for 2ME₂ on normal mammary differentiation has been previously reported. This intriguing observation suggested that 2ME₂ could exert some differentiating effects upon the mammary gland, through a non-prolactin-dependent manner and without signaling directly through the ER.

In this study, we have confirmed that 2ME₂ can cause differentiation of mammary epithelial cells in vitro as well as normal mammary glands in vivo with milk protein expression but that the mechanisms for the induction of mammary differentiation is different from that which occurs during normal pregnancy. We demonstrate that 2ME₂-induced differentiation involves the transcriptional down-regulation of the inhibitor of differentiation-1 (Id-1) and Id-3 and induction of amphiregulin signaling through the epithelial growth factor receptor (EGFR). Additionally, 2ME₂ induces some changes in the expression of extracellular matrix (ECM) and immune-related genes similarly altered during pregnancy. These results provide new insights into 2ME₂-induced mammary differentiation independent of prolactin or ER signaling.

	Materials and Methods

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Cell culture and reagents
SCp2 cells (7) were generously provided by Dr. Mina Bissell (Lawrence Berkeley National Laboratory, Livermore, CA). SCp2 cells were maintained in DMEM/nutrient mixture F-12 (Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS) (Invitrogen), 5 µg/ml insulin (Invitrogen), and 50 µg/ml gentamycin (Sigma Chemical Co., St. Louis, MO). SCp2 cells were incubated with 0, 5, 10, and 50 µM 2ME₂ for indicated time points, and RNA was extracted for RT-PCR. Amphiregulin (R&D Systems, Inc., Minneapolis, MN) was added to SCp2 cells at a concentration of 6 ng/ml. Tarceva, obtained from OSI Pharmaceuticals (Melville, NY), was used at a 10 µM concentration (100 mM stock in dimethylsulfoxide). A stock solution of 100 mM 2ME₂ (kindly provided by EntreMed, Inc., Rockville, MD) in dimethylsulfoxide was used and diluted as indicated.

Animals and experimental schedule
To determine the effects of 2ME₂ on normal mammary glands, four groups of wild-type FVB mice were studied using the same experimental schedule for 6 wk. Seven mice per group received 25, 75, or 150 mg/kg of 2ME₂ daily by gavage formulated in 1% hydroxypropylcellulose beginning at 6 wk of age for 6 wk. Control mice received 0.2 ml of 1% hydroxypropylcellulose by gavage daily, following the same schedule as the experimental group. All of the mice were killed at diestrus to exclude variations due to the estrous cycle. The stage of estrus was determined by measuring the electrical impedance using an MK-10 rat estrous cycle monitor (Fine Science Tools, Inc., Foster City, CA). Mammary tissues were immediately collected in liquid nitrogen for RT-PCR or fixed in 4% paraformaldehyde for histological analyses. All mice were treated in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 86-23, 1985) under an approval animal protocol.

Whole-mount analysis and immunohistochemistry
For whole-mount analysis, four mammary glands from 12-wk-old normal FVB mice were removed and mounted on slides, fixed overnight in 10% formalin, placed overnight in acetone to remove fat, dehydrated, stained with hematoxylin, rehydrated, cleared with xylene, and coverslipped (Daigger, Wheeling, IL) with VectaMount (Vector Laboratories, Burlingame, CA) for morphological analysis.

For immunohistochemical analysis, sections were heated in a microwave oven in 0.1 M sodium citrate for antigen retrieval, hybridized with casein antisheep antibody (1:250, kindly provided by Dr. Barbara K. Vonderhaar, National Cancer Institute, National Institutes of Health), and processed using the avidin-biotin complex method (Vectastain ABC Elite kit; Vector Laboratories). Secondary sheep polyclonal antibody (1:12,000) to casein was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Promoter assays
Id-1 promoter luciferase constructs (Id-1 Luc 2.2 kb, 5'-del 1, 5'-del 2, 5'-del 3, and 5'-del 6) (8) and pRL-CMV were cotransfected into SCp2 cells over 4 h using Lipofectamine 2000 (Invitrogen) with 2% FBS following the manufacturer’s protocol. 2ME₂ was added for 24 h at which time cells were lysed and promoter activity was measured using the dual-luciferase reporter assay system kit (Promega, Madison, WI) and Wallac Victor 2 1420 multilabel counter (Perkin Elmer, Shelton, CT). Transfection efficiency was normalized using the pRL-CMV vector as an internal control (Promega). For the estrogen response element (ERE) reporter assay, SCp2 cells were plated either on 24-well plates (5 x 10⁴ cells per well) or 96-well plates (0.75 x 10⁴ cells per well) and transfected 2 d later. Two hours before transfection, the medium was changed to contain 5% charcoal-stripped serum. Cells were cotransfected with the green fluorescent protein-ER expression plasmid pCL-nGL1-HEGO and pERE-Luc plasmid (27) with pRL-CMV as an internal control. Two days after transfection, cells were treated with 10 µM 2ME₂, 10^–2 µM E₂ (Equitech-Bio, Inc., Kerrville, TX) or 0.1 µM tamoxifen (Tocris Bioscience, Ellisville, MO) for 8 h and subjected to the dual-luciferase assay with the Dual-Glo Luciferase assay system (Promega) by means of a POLARstar OPTIMA (BMG Labtech, Durham, NC). All experiments were conducted in triplicate.

Small interfering RNA (siRNA) transfection
SCp2 or M6 cells (0.5 x 10⁵) were plated in six plates with DMEM/F12 containing 2% FBS or DMEM containing 5% FBS without antibiotics the day before transfection. siRNA was transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Ten micromolar 2ME₂ was added for the indicated time periods. Protein was extracted from M6 cells for Western blotting, and RNA was extracted from SCp2 cells for RT-PCR. All experiments were repeated at least twice. Two different sequences of siRNA for Id-1 were designed, but only one of them could knock down Id-1 expression, and we used this sequence for our experiment. The target sequence of siRNA for Id-1 used in this study is GGCTACGTCCAGGAGCGCACC. To knock down amphiregulin expression, three different siRNA entities for AREG were transfected into SCp2 cells at the same time (each at a concentration of 30 nM) following the same method as described for Id-1 siRNA transfection. 2ME₂ (10 µM) was added 15 h after transfection, and RNA was extracted 12 h after treatment with 2ME₂. Target sequences of siRNA for AREG were CCACAAATATCCGGCTATA (no. 1), ACAAGGACCTATCCAAGAT (no. 2), and AGAGAGGTTTCCACCATAA (no. 3). siRNA for the green fluorescent protein duplex was used as a control for the siRNA experiments. All the target sequences for siRNA for Id-1 and AREG were designed using the Dharmacon siRNA design center (http://www.dharmacon.com/sidesign) and synthesized by Dharmacon, Inc (Lafayette, CO).

Western blotting
Protein was extracted from M6 cells using RIPA lysis buffer [1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM Na₃VO₄] with protease inhibitor cocktail tablets (Roche, Penzberg, Germany) for protein analysis. Thirty micrograms of protein were resolved on 16% Tris-glycine gradient gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), blocked with 5% nonfat milk, and incubated with the Id-1 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horseradish-peroxidase-conjugated antirabbit secondary antibody (Santa Cruz Biotechnology). Signal was visualized using an ECL detection kit (Amersham Biosciences, Piscataway, NJ). -Tubulin (Sigma-Aldrich Inc., St. Louis, MO) was used as an internal control.

RNA extraction and semiquantitative RT-PCR and real-time RT-PCR
Total RNA was isolated from frozen tissues and cells using Trizol reagent (Invitrogen) following the manufacturer’s instructions, and RT-PCR was performed as follows: RT reactions were performed using the Superscript first-strand synthesis system for RT-PCR (Invitrogen) following the manufacturer’s protocol. The PCR mixture included 1 µl of the cDNA generated from the RT reaction, 1 µl of 10x PCR buffer, 1.5 µl of 50 mM MgCl₂, 1 µl of 10 mM dNTP, 0.4 µl of Taq polymerase (5 U/µl), 0.5 µl of 20 µM sense primer, and 0.5 µl of 20 µM antisense primer in a total volume of 50 µl. PCR conditions were as follows: 94 C for 5 min, 94 C for 30 sec, annealing temperature as indicated in Table 1 for 30 sec, 72 C for 1 min, 72 C for 7 min, and 4 C at the completion of the reactions. Primer sequences with annealing temperature and cycles of PCR are listed in Table 1. Quantitative real-time PCR for amphiregulin in 2ME₂-treated glands and mammary glands during pregnancy was done using the Brilliant SYBR Green QPCR kit (Stratagene, La Jolla, CA) and an iCycler IQ real-time detection system (Bio-Rad Laboratories, Hercules, CA). The result was normalized by GAPDH expression. The same primer sets and cDNA for amphiregulin and GAPDH used for RT-PCR were used for real-time PCR.

Oligonucleotide microarray hybridization and data analysis
Four normal controls and four mammary gland samples from individual mice treated with 75 and 150 mg/kg 2ME₂ were collected for Affymetrix (Santa Clara, CA) microarray analyses. Additionally, three mammary gland samples were collected from FVB/N mice at each of following time points: 12-wk-old virgin (12W), d 16 of pregnancy (P16), d 2 of lactation (L2), and five samples each of d 30 post lactation (W30) and age-matched control animals for W30. SCp2 cells treated with 10 µM 2ME₂ and the corresponding untreated controls were collected at 6, 24, and 48 h after treatment. Total RNA was extracted from frozen tissues or cells using TRIzol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s protocol. Ten micrograms of total RNA were reverse transcribed using a T7 (dT)₂₄ primer to synthesize the cDNA, followed by the incorporation of biotinylated ribonucleotides by in vitro transcription using T7 RNA polymerase. The biotinylated RNA was fragmented and hybridized to an oligonucleotide microarray murine genome 430 2.0 chip containing 45,100 features (for normal controls and 2ME₂-treated samples) and a murine genome U74Av2 chip containing 12,500 features (for the pregnancy samples) (Affymetrix) according to the manufacturer’s protocol. Gene expression analysis was performed using the GeneChip Operating Software (GCOS) version 1.1 software (Affymetrix). Signal intensities of each chip were normalized to an average target value for 500 excluding the lowest 2% and highest 2% signals and transformed to a logarithmic scale for statistical calculations.

Significant differences in gene expression between classes were identified by ANOVA calculation. Two-class comparisons were made of 2ME₂-treated vs. respective controls at each of the doses and time points, and pregnancy and post-pregnancy days vs. corresponding controls. Genes altered by 2-fold at P < 0.05 and having one of the geometric average signal values above 100 with at least one "Present" call were selected for further examination. Hierarchical clustering of both genes and samples was performed using the Eisen Cluster program (9) with 1 – as the distance metric where is the Pearson correlation coefficient and the complete linkage option of the software for agglomerative clustering. Matching of probes present on the 45 K mouse 430 2.0 chip with the probes present on the 12.5 K murine genome array U74Av2 was done using the good match file provided by Affymetrix (http://www.affymetrix.com).

Multiple matches were unified by best overlap of GenBank sequences.

	Results

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2ME₂ induces morphological changes associated with expression of milk proteins in mammary glands
In our previous study demonstrating that 2ME₂ suppressed mammary tumor formation in C3(1)/Tag transgenic mice (6), we observed that 2ME₂ caused significant dilation of mammary gland ducts with accumulation of secretory materials including casein. This suggested that 2ME₂ might induce partial differentiation of the mammary glands. To further investigate the role of 2ME₂ on mammary differentiation, 6-wk-old wild-type virgin female FVB/N mice were administered 25, 75, or 150 mg 2ME₂/kg body weight per day by gavage for 6 wk in this study. Whole-mount analysis of the mammary glands revealed morphological changes in ductal terminal lobular units with small numbers of alveolar-type cells and dilated ductal termini (Fig. 1A, right) compared with the untreated virgin controls (Fig. 1A, left). No significant difference in the number of terminal lobular units was observed between the 2ME₂-treated and control animals as determined by computer-assisted quantification (data not shown). The dilated ductal terminal lobular units in the 2ME₂-treated mammary glands exhibited increased cellularity (Fig. 1B, right) compared with mammary glands from control mice (Fig. 1B, left). The morphological changes were associated with increased expression of milk protein ß-casein as determined by immunohistochemical analysis (Fig. 1C). A milder differentiating effect was observed at 75 mg/kg·d but not at 25 mg/kg·d, demonstrating a dose response. Increased expression of whey acidic protein (WAP), which is usually detected at a late stage of pregnancy, was demonstrated by RT-PCR at the higher dose of 2ME₂, whereas increased levels of ß-casein, which is usually detected at early stages of pregnancy, was observed at both the 75- and 150-mg/kg doses of 2ME₂ (Fig. 1D).

View larger version (93K): [in this window] [in a new window]   FIG. 1. Wild-type FVB mice were treated with 2ME2 from 6 wk of age for 6 wk. A, Mammary whole-mount analysis demonstrates that morphological changes were induced by 2ME2 in mammary glands. In the 150-mg/kg 2ME2-treated mice, alveolar side branching and budding was observed compared with the control groups (magnification, x100). B, Red boxes in A were enlarged. C, Immunohistochemical analysis of casein shows that casein expression was dramatically secreted inside of lumen of mammary glands by 2ME2. D, RT-PCR was performed to show the expression of milk proteins. ß-Casein was expressed in a dose-dependent manner, whereas WAP expression was highly detected in the highest dose (150 mg/kg·d).

View larger version (47K): [in this window] [in a new window]   FIG. 2. A, Morphological changes and F-actin staining (green) of SCp2 cells by 2ME2. Actin cytoskeleton images were visualized using confocal microscopy in normal mammary epithelial cells. Nuclei are visualized by 4',6-diamidino-2-phenylindole staining (blue). SCp2 cells were treated with 10 µM 2ME2 for 48 h. B, SCp2 cells were starved for 3 d before treatment and then maintained in 2% FBS medium treated with 2ME2 in 0, 5, 10, or 50 µM concentration for 24 and 48 h. To detect mRNA expression, RT-PCR for ß-casein, Id-1, Id-2, Id-3, and TSP-1 was performed in SCp2 cells. GAPDH was used as an internal control. C, siRNA for Id-1 (50 and 100 nM) was transfected with Lipofectamine 2000 for 24 h following the manufacturer’s instruction in M6 cells for Western blotting to detect Id-1 protein expression. D, Knocking down of Id-1 alone by siRNA could not produce ß-casein. However, 2ME2 had a synergic effect when it was used with siRNA for Id-1. The cells were treated with siId-1 or 2ME2 for 6 and 48 h.

To examine whether inhibition of Id-1 expression alone might directly lead to mammary differentiation, we used Id-1 siRNA to inhibit expression. The efficacy of the siRNA in reducing Id-1 protein levels was demonstrated by Western blot analysis. Id-1 protein levels in M6 tumor cells, which express high basal protein levels of Id-1, significantly decreased using Id-1 siRNA (Fig. 2C). SCp2 cells (whose basal level of Id-1 protein was below the sensitivity threshold for our Western blot) transfected with Id-1 siRNA (Fig. 2D) did not differentiate because ß-casein expression did not increase, even though Id-1 expression decreased (Fig. 2D). However, reducing the level of Id-1 expression in combination with administration of 2ME₂ augmented the level of ß-casein expression compared with 2ME₂ treatment alone (Fig. 2D). This suggests that Id-1 may play a cooperative role in mammary differentiation with other factors induced by 2ME₂, because reduction of Id-1 alone is not sufficient to induce differentiation of SCp2 cells.

2ME₂ represses Id-1 transcriptional activity
To investigate 5' genomic regions of the Id-1 gene potentially regulating transcription in response to 2ME₂, Id-1 promoter luciferase assays were performed using five different mutant reporter constructs (8): Id-1 Luc 2.2 kb (–2212 to 0 bp), 5' del-1(–1572 to 0 bp), 5' del-2 (–1361 to 0 bp), 5' del-3 (–1200 to 0 bp), and 5' del-6 (–272 to 0 bp) (Fig. 3A). Binding sites for transcription factors YY1, EGR-1, CREB/ATF, AP2, and E-box are contained between –1156 and –946 bp, whereas sites for C/EBP, NF1, SP1, CP1, and Zeste are located between –272 and –145 bp. 2ME₂ had a significant dose-dependent effect on Id-1 transcriptional levels in all of the constructs used, including the longest promoter available (2.2 kb). An element downstream from –2.2 kb appears to suppress general transcriptional activity (Fig. 3B). 2ME₂ also decreased 75% of the promoter activity in the 5' del-3 construct at the 50 µM concentration. This region has been shown to be crucial for constitutive Id-1 expression in metastatic cells as well as serum responsiveness in nonaggressive breast cancer cells (8). Interestingly, the basal promoter activity increased with loss of the –2212 to –1572 sequences, suggesting that a suppressor element may be contained within this portion of the 5'-flanking region of the Id-1 gene.

View larger version (20K): [in this window] [in a new window]   FIG. 3. A, Id-1 promoter constructs; B, Id-1 promoter luciferase vectors (Id-1 Luc 2.2 kb, 5'del-1, 5'del-2, 5'del-3, and 5'del-6) were transfected into SCp2 cells to investigate transcriptional regulation. Four hours after Id-1 promoter luciferase vectors, 2ME2 was added with 0, 10, 50, or 100 µM concentration in 2% FBS. Id-1 promoter activity was down-regulated by 2ME2 in a dose-dependent manner. The result was normalized with pCMV luciferase activity. The experiments were done in triplicate.

Compared with the control virgin mammary gland, we found 100 up-regulated and 141 down-regulated genes in mice treated with 75 mg 2ME₂/kg·d, whereas 60 up-regulated and 202 down-regulated genes were observed in the 150-mg 2ME₂/kg·d group. A comparison between 2ME₂ treatment and P16 groups demonstrated 27 genes with similar expression patterns including the up-regulation of many milk proteins (2-fold change at P < 0.05) (Table 2). Several genes significantly altered by 2ME₂ treatment in vivo (P < 0.05 and 2-fold) were similarly regulated in mammary glands during pregnancy (Fig. 4A) including the caseins , ß, and , WAP, lactalbumin , and lactotransferrin.

View larger version (45K): [in this window] [in a new window]   FIG. 4. For microarray studies, mammary glands of wild-type FVB mice from 6 wk of age were treated with 2ME2 for 6 wk for in vivo. For in vitro samples, SCp2 cells treated with 10 µM 2ME2 for 6, 24, or 48 h were used. Pregnancy mammary tissue samples were collected at P16, L2, and W30. Shown is hierarchical clustering of 243 genes altered on 2ME2 treatment in vitro by at least 2-fold at P < 0.05 and comparison with the expressions in pregnancy and 2ME2 treatment in vivo. A and B, Genes regulated in the same direction in 2ME2-treated and pregnancy mammary glands. C and D, Differentially expressed genes between 2ME2 treatment and pregnancy were clustered.

To identify whether any changes in gene expression induced by 2ME₂ are also seen in fully involuted mammary glands, we compared 2ME₂-treated samples with mammary glands from animals 30 d after the cessation of lactation (W30). Genes whose expression was similarly altered between W30 and 2ME₂ treatment are depicted in the cluster in Fig. 5. Immune mediators (IgM, Igk-V8, Igh-1a, Igj, Ighg, Igh-4, and Igk-C) and milk proteins were up-regulated in both 2ME₂-treated glands and mammary glands during pregnancy. Several genes that modify the ECM were identified in 2ME₂-treated mammary glands. Spondin 1, elastin, cathepsin R, and fibulin1 were up-regulated in 2ME₂-treated mammary gland, whereas MMP-9 and MMP-12 were up-regulated in both 2ME₂-treated glands and mammary glands during pregnancy.

View larger version (35K): [in this window] [in a new window]   FIG. 5. Hierachical clustering of gene expression profiles compared between mammary glands from 2ME2 treatment and post lactation (W30) altered by 2-fold changes at P < 0.05.

View larger version (33K): [in this window] [in a new window]   FIG. 6. A, Validation of microarray data by RT-PCR. AREG, MMP-12, Foxf2, Pax4, and ATF3 were up-regulated and Id-1 was down-regulated in mammary glands from normal virgin mice by 0, 25, 75, and 150 mg/kg·d of 2ME2 treatment for 6 wk beginning at 6 wk of age. B, Relative mRNA expression of AREG in 2ME2-treated glands and mammary glands during pregnancy was quantified by real-time RT-PCR. The result was normalized by GAPDH. N, 12-wk-old virgin; W, d 30 post lactation. *, P < 0.05 for control vs. 75 or 150 mg/kg·d. Bar, SD.

Quantitative analysis of amphiregulin was performed using quantitative real-time PCR. A representative result is demonstrated in Fig. 6B. Mammary glands from 2ME₂-treated mice demonstrated increased expression of amphiregulin in a dose-dependent manner, which was not detectable in mammary glands from pregnant mice (Fig. 6B). Results were normalized using GAPDH expression as an internal control.

2ME₂ but not E₂ induces differentiation
To determine whether mammary differentiation induced by 2ME₂ can be similarly induced by E₂, induction of ß-casein by SCp2 cells was assessed upon stimulation by 2ME₂ or E₂. Exposure to 2ME₂, but not E₂, resulted in a small but detectable level of ß-casein production in SCp2 cells (Fig. 7A). To further confirm the specific effects of 2ME₂, SCp2 cells were exposed to a wide range of concentrations (0, 10^–1, 10^–2, 1, and 10 µM) of 2ME₂ and E₂. AREG and Id-1 expression were determined by RT-PCR after 24 h or 48 h treatment, respectively (Fig. 7B). AREG expression was up-regulated by 2ME₂, but E₂ did not alter AREG expression. Conversely, Id-1 expression was down-regulated by 2ME₂ but was up-regulated by E₂, suggesting 2ME₂ and E₂ operate through different mechanisms to alter gene transcription and differentiate mammary glands.

View larger version (20K): [in this window] [in a new window]   FIG. 7. A, Casein expression by RT-PCR. ß-Casein was expressed mildly by 2ME2 (10 µM) for 24 h in 2% FBS medium but not by E2 (10–2 µM) in SCp2 cells. B, Various concentrations of 2ME2 and E2 (0, 10–2, 10–1, 1, and 10 µM) were treated in SCp2 cells. Amphiregulin and Id-1 expression were compared by RT-PCR for 24 or 48 h treatment, respectively. Amphiregulin expression was up-regulated, whereas Id-1 expression was down-regulated by 2ME2. E2 treatment did not change AREG expression but up-regulated Id-1 expression. GAPDH was used as an internal control. C, ER promoter assay after treatment with 2ME2, E2, tamoxifen (Tam, 0.1 µM) in SCp2 cells. E2 increased ER promoter activity; however, no effect was observed with 2ME2 or tamoxifen. *, P < 0.05, 2ME2 vs. E2.

Amphiregulin is involved in 2ME₂-induced mammary differentiation
Because AREG was significantly induced by 2ME₂ both in vitro and in vivo, we investigated whether AREG plays a role in 2ME₂-induced differentiation. AREG levels were altered in SCp2 cells either through the knockdown of expression by siRNA or through the exogenous administration of amphiregulin to SCp2 cells. SCp2 cells were transfected with AREG siRNA followed 15 h later with the supplementation of 2ME₂ for 12 h. Inhibition of AREG was associated with a complete lack of ß-casein expression, suggesting that AREG is involved in 2ME₂-induced mammary differentiation (Fig. 8A). Additionally, knockdown of AREG expression was associated with the up-regulation of Id-1 and the down-regulation of ATF3, whose expression was shown by microarray analysis to be up-regulated by 2ME₂ treatment.

View larger version (62K): [in this window] [in a new window]   FIG. 8. A, SCp2 cells were transfected with siRNA for AREG (100 nM). 2ME2 (10 µM) was added 15 h after transfection for 12 h. RT-PCR was performed to detect AREG, ATF3, Id-1, and ß-casein. GAPDH was used as an internal control for in vivo and in vitro samples. siRNA for AREG induced Id-1 expression resulting in blocking ß-casein expression, whereas it had a minor effect on ATF3 expression induced by 2ME2. B, Tarceva (10 µM) inhibited expression of ß-casein induced by 2ME2; however, Tarceva alone showed no effects on Id-1 expression. C, Tarceva blocked morphological changes caused by 2ME2 in SCp2 cells. Cells were treated with Tarceva for 1 h, and then 2ME2 was added for 24 h before cells were photographed. D, Overexpression of AREG induced expression of ß-casein. SCp2 cells were treated with 2ME2 (10 µM), AREG (6 ng/ml), or Tarceva (10 µM) for 24 h in the culture condition mentioned in Materials and Methods. Both 2ME2-induced AREG and exogenous AREG treatment induced ß-casein expression. Tarceva blocked AREG expression, which is induced by 2ME2 or exogenous AREG treatment. ATF3 expression was up-regulated by up-regulation of AREG, whereas Id-1 expression was inversed.

	Discussion

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Differentiation of the mammary gland is a complex process that requires coordinated hormone signaling and stromal-epithelial interactions (15) leading to tremendous expansion of alveolar cell numbers with the ability to secrete significant quantities of milk. Aside from the critical role of mammary differentiation for lactation, it has been proposed that pregnancy and mammary differentiation may strongly influence risk factors related to the development of mammary cancer. Early pregnancy is associated with a reduced risk of breast cancer years later, whereas the periparous period is associated with an increased risk of aggressive breast cancer development (3).

Given the fact that endogenous levels of 2ME₂ are extremely high during later stages of pregnancy and that our previous study suggested that 2ME₂ may have a differentiating effect on the mammary gland, we further evaluated the differentiating effects and mechanisms by which 2ME₂ influences mammary epithelial cell differentiation.

An extensive analysis evaluating the pharmacokinetic and pharmacodynamic relationship of 2ME₂ has been done. Mice orally administered 150 mg/kg 2ME₂ achieve a plasma maximal concentration of 1967 nM and area under the curve of 3971 nM-h (data not shown). In the mice at 150 mg/kg, drug levels in the range of 9–60 nM can be achieved (data not shown). At these plasma levels in mice, which are considerably lower than those needed in vitro, we see effects on tubulin and an antitumor response (16). In vitro effects of 2ME₂ on tubulin and HIF-1 were demonstrable at 10–100 µM concentrations, whereas in vivo activity was seen at 30–150 mg 2ME₂/kg·d (16). This could occur for several reasons: 1) plasma drug levels do not reflect tumor drug levels or 2) in vitro experiments are done with constant exposure to drug, whereas once-daily administration of 2ME₂ to mice does not result in significant drug levels for the 24-h period. Indeed, in vitro experiments looking at transient exposure to drug demonstrated that cells had to be exposed to drug for greater than 6 h in a 24-h period to approach the activity of constant drug exposure. Furthermore, in vivo experiments have shown that fractionated dosing of 2ME₂ results in better activity, such that 2 mg/d given as 0.5 mg four times a day gives better antitumor activity than 2 mg given once a day. In a previously published study from our lab, we demonstrated that 2ME₂ has antiangiogenic activity when administered to mice at 150 mg/kg·d (6).

The results of the present study demonstrate that 2ME₂ alters the state of mammary epithelial differentiation both in vitro and in vivo. However, although 2ME₂ can stimulate the expression of mammary differentiation markers such as ß-casein and WAP, it appears to do so through mechanisms that are distinct from pregnancy-induced differentiation. This is exemplified by the significant differences in gene expression observed in 2ME₂-induced differentiation in normal FVB/N mice compared with gene expression changes that occur during pregnancy. Mice treated with 2ME₂ did not develop an extensive lobuloalveolar system as seen in pregnancy, and prolactin serum levels were not changed in the 2ME₂-treated mice compared with the control mice (data not shown).

Previous studies using SCp2 cells have also demonstrated that there is an inverse association between the expression of Id-1 and cellular differentiation (13). Inhibitor of differentiation (Id) proteins are key regulators of myogenesis, neurogenesis, angiogenesis, and differentiation of many cell lineages (17, 18). We have previously demonstrated that 2ME₂ inhibits Id-1 expression in endothelial cells and breast cancer cells, which is related to antiangiogenic and antitumorigenic effects (6). In this study, we observed that expression of Id-1 and Id-3, but not Id-2, are down-regulated in normal mammary epithelial cells by 2ME₂. Id-3 is often coexpressed temporally and spatially with Id-1 during murine neurogenesis and angiogenesis (18), whereas little is known about the role of Id-3 in association with mammary differentiation. The results of this study are in agreement with these previous reports and suggest that Id-3 expression may also be important for regulating mammary gland differentiation.

Additionally, we have demonstrated that 2ME₂ is able to inhibit Id-1 expression, at least in part, through repression of transcription in normal mammary epithelial cells. We demonstrated that 2ME₂ inhibits Id-1 transcriptional activity in several breast cancer cell lines and proposed that this may, in part, account for the antitumorigenic activity of 2ME2 observed in our previous study (6). A major effect of 2ME₂ transcriptional repression in normal mammary epithelial cells appears to be through a cis-acting region within approximately 270 bp upstream of the transcriptional start site and another region extending up to –1200 bp. In the breast cancer cell lines studied, 2ME₂ has less of an inhibitory effect on Id-1 promoter activity in the 5' del-6 construct, containing the –270 to 0 bp region, compared with the other promoter sequences contained in the 5' del-1 and 5' del-3 constructs (6). Although several transcriptional binding sites are contained in these regions, more studies will be required to determine exactly through which cis-acting elements 2ME₂ results in transcriptional repression.

A major difference between 2ME₂-induced differentiation and pregnancy was noted in the expression of AREG. Mice lacking functional AREG exhibit severely stunted ductal outgrowth during puberty, consistent with an important role for AREG during normal development of mammary ducts and terminal end buds (19). AREG has been shown to influence early stages of mammary differentiation, but its expression during pregnancy is dramatically down-regulated. This is in contrast to both our in vitro and in vivo demonstration that 2ME₂ induces the overexpression of AREG, which is known to signal through EGFR.

To more fully assess the role of AREG in 2ME₂-induced differentiation of mammary epithelial cells, we used the previously well-characterized SCp2 cell system that was derived from normal mammary epithelial cells of a pregnant mouse. SCp2 cells have been shown to differentiate into casein-producing cells upon stimulation with prolactin and laminin. In the present study, we demonstrate that SCp2 cells can undergo differentiation by 2ME₂ alone, as evidenced by ß-casein expression independent of prolactin. This differentiation effect is blocked by inhibition of AREG expression using siRNA, whereas the addition of amphiregulin to the media results in the expression of the differentiation markers. Consistent with these findings is the observation that Tarceva, an inhibitor of EGFR signaling, also blocks differentiation induced by both 2ME₂ and amphiregulin.

ATF3 (a gene involved in stress response, wound healing, cell cycle, sexual differentiation, and estrogen signaling) (20) is a component of the ATF/SMAD3 complex that mediates the repression of Id-1 by TGF-ß (21). Based on these observations, we hypothesized that 2ME₂ might suppress Id-1 expression through a mechanism involving ATF3, leading to mammary epithelial differentiation, and therefore, we also measured levels of ATF3. The expression level of ATF3 was the highest in mice that received 75 mg 2ME₂/kg·d, not 150 mg/kg·d. Such a U-shaped dose-response curve has been observed in other systems. A biphasic dose-response has been observed for endostatin, an endogenous antiangiogenic factor (22). Our results similarly suggest that there may be an optimal concentration of 2ME₂ to alter Id-1 and ATF expression.

Our current study suggests that 2ME₂ does not require ER to induce its effects and does not stimulate transcription through an ERE. 17ß-E₂ does not induce ß-casein in SCp2 cells, further suggesting that 2ME₂ uses a non-ER signaling pathway to differentiate mammary epithelial cells. However, additional studies are required to more definitively exclude the role of ER in mediating 2ME₂ effects. Interestingly, 2ME₂ could increase casein expression in SCp2 cells without stimulation by lactogenic hormones or ECM components, whereas E₂ did not have this effect. Signaling through EREs by E₂, however, was demonstrated in SCp2 cells using an ERE-luciferase reporter system cotransfected with ER expression plasmid, but much higher concentrations of 2ME₂ did not induce transactivation of the ERE reporter. The expression level of casein induced by 2ME₂ was quite low, however, compared with prolactin stimulation, indicating that prolactin is a much stronger inducer of differentiation than 2ME₂. Additionally, a previous study reported that estradiol induced AREG expression in ER-positive human breast cancer cells (9). However, in ER-negative SCp2 cells, AREG expression was induced by 2ME₂ but not by E₂. This result further suggests that 2ME₂ and E₂ might use different mechanisms to induce differentiation in mammary epithelial cells based on their ER status.

Changes in the expression of genes influencing the microenvironment and immune response during involution have been proposed as factors that may promote tumorigenesis in the periparous period (3). Degradation of the ECM is an important element of tissue remodeling during mammary gland involution and also for tumor invasion and progression. Several ECM proteins such as spondin1, MMP-9, elastin, and cathepsin R were found to be up-regulated by 2ME₂ in this study and may lead to partial remodeling of the ECM. In this regard, it is possible that the increased cystic tumor formation we previously observed when 2ME₂ was administered in a cancer prevention setting (6) could also be due in part to changes induced in the mammary microenvironment in addition to the inhibitory effect of 2ME₂ on endothelial cell growth. Some of these extracellular components, such as MMP-9 and elastin, are potentially involved in promoting cancer progression (23). Previous microarray studies identified alterations in the expression of numerous immune-related genes during mammary involution (24, 25). Immune mediators, primarily Ig, were also identified in 2ME₂-treated mammary glands in our present study.

Multiple signaling pathways have also been known to be involved in mammary budding, branching, and differentiation, such as stimulation by progesterone, Wnt, prolactin, EGF family members, and Stat5a signaling (26). However, the results of this study demonstrate that 2ME₂ can induce a partial differentiation of the mammary gland involving the amphiregulin-EGFR pathway. Additionally, 2ME₂ led to changes in expression of immune- and ECM-related genes.

Although differentiation therapy has been proposed as a potential method to reduce breast cancer risk beyond antiangiogenic activity of 2ME₂ in cancer therapy, caution would be warranted in using 2ME₂ for this purpose because it does not induce identical changes as observed in pregnancy and because our previous studies have demonstrated that it may increase cystic tumor formation in a prevention setting.

	Acknowledgments

 
We are grateful to Lisa Riffle for animal care, Dr. Barbara Vonderhaar for generously providing casein antibody, Dr. Mina Bissell for kindly providing SCp2 cells, and Dr. Christina Bennett for her critical review of this manuscript. We also appreciate assistance with confocal imaging from Drs. James McNally and Tatiana Karpova in the Fluorescence Imaging Facility, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health.

	Footnotes

 
First Published Online December 7, 2006

Abbreviations: E₂, Estradiol; ECM, extracellular matrix; EGFR, epithelial growth factor receptor; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; Id, inhibitor of differentiation; L2, d 2 of lactation; 2ME₂, 2-methoxyestradiol; P16, d 16 of pregnancy; si RNA, small interfering RNA; TSP-1, thrombospondin-1; W30, d 30 post lactation; WAP, whey acidic protein.

Received July 19, 2006.

Accepted for publication November 29, 2006.

	References

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