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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.
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Introduction |
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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.
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Materials and Methods |
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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.
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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.
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, 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).
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, addition of 2ME2 resulted in the growth arrest of SCp2 cells, which became larger and more rounded than untreated SCp2 cells whose morphology remained flattened (Fig. 2A, upper panel). Staining for F-actin revealed that 2ME2 induced significant intracellular cytoskeleton reorganization with larger nuclei associated with the differentiation of the SCp2 cells (Fig. 2A, lower panel).
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). Because thrombospondin-1 (TSP-1) is a downstream target and negatively inhibited by Id-1 (14), we evaluated TSP-1 expression. Consistent with the down-regulation of Id-1, TSP-1 expression was increased in response to 2ME2 treatment. These results suggest that the induction of mammary epithelial cell differentiation is strongly associated with the down-regulation of Id-1 expression (Fig. 2B).
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.
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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.
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, C and D. Most milk proteins were up-regulated in both 2ME2-treated and pregnant glands, whereas Per1, Per2, and oxysterol binding protein-like 9 were similarly down-regulated in 2ME2-treated glands and glands from pregnant mice (Fig. 4A). Clusters of genes that are differentially expressed between 2ME2 treatment in vivo and pregnant mammary glands were selected in Fig. 4, C and D. Serpine2, AREG, Sox6, and ATF3 were up-regulated by 2ME2 treatment but were down-regulated in pregnant glands (Fig. 4C). Catenin 
2, endothelin 2, Mesp1, and Gjbp4 were down-regulated by 2ME2 but up-regulated in glands during pregnancy (Fig. 4D).
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.
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), with the maximal effect observed at 75 mg/kg·d.
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.
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). E2 at 10–2 µM activated ERE-dependent transcription in SCp2 cells, but even at concentrations of 10 µM 2ME2 or 0.1 µM tamoxifen, no effect on the transcriptional activity through the ERE was observed, consistent with previous reports that suggest that 2ME2 may operate through non-ER-ERE mechanisms (Fig. 7C).
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.
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, B and D) and down-regulated AREG expression in SCp2 cells. Changes in cell morphology induced by 2ME2 differentiation were also inhibited by Tarceva (Fig. 8C). ATF3 was significantly up-regulated by exogenous amphiregulin or 2ME2 treatment for 24 h, and Id-1 expression was down-regulated under the same conditions, suggesting these genes are involved in 2ME2-induced differentiation (Fig. 8D). |
Discussion |
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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.
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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.
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reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471–486
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