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[Cancer Research 66, 3495-3503, April 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Distinct Tumor Stage–Specific Inhibitory Effects of 2-Methoxyestradiol in a Breast Cancer Mouse Model Associated with Id-1 Expression

Jung-Im Huh, Alfonso Calvo, Rhonda Charles and Jeffrey E. Green

Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: Jeffrey E. Green, Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, NIH, Room C629, Building 41, 41 Medlars Drive, Bethesda, MD 20892. Phone: 301-435-5193; Fax: 301-496-8395; E-mail: jegreen{at}nih.gov.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
2-Methoxyestradiol (2ME2), a metabolite of 17-ß-estradiol, inhibits angiogenesis and has additional antitumor activities. We have analyzed the tumor stage–specific effects of 2ME2 in the C3(1)/Tag transgenic mouse model for breast cancer, which spontaneously develops estrogen receptor–negative mammary tumors following a predictable progression of lesion formation. When given either as a therapeutic agent in established tumors (late intervention study) or in mice with pre-invasive mammary lesions (early intervention study), tumor growth was reduced by 60% compared with untreated controls and was associated with an induction of apoptosis. In a prevention study, a significant reduction in mammary intraepithelial neoplasia (MIN) lesions was observed in animals beginning treatment at 6 weeks of age, before the appearance of histopathologic abnormalities. However, although 2ME2 reduced the number of MIN lesions in the prevention study, a paradoxical increase in tumor multiplicity and growth rate was observed. This was associated with unusual cystic tumor formation, in which significant central necrosis was observed, surrounded by an outer region of proliferative tumor cell growth. The characteristics of the cystic tumor formation in mice treated with 2ME2 at early ages are consistent with an impaired angiogenic response as observed in mice deficient for inhibitor of differentiation (Id-1). We further show that Id-1 expression is negatively regulated by 2ME2, which may be an additional mechanism for the antiangiogenic effect of 2ME2. Although 2ME2 significantly reduced tumor growth at late stages, these results also suggest that altered tumor morphology and accelerated tumor growth may occur if 2ME2 is administered in a prevention setting for prolonged periods. (Cancer Res 2006; 66(7): 3495-503)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is estimated that one in nine women in the United States will develop breast cancer, resulting in >40,000 deaths each year (1). Both environmental and genetic factors seem to contribute to the risk of developing breast cancer (2). The prognosis is better for patients with hormone-responsive disease, where antiestrogens, such as tamoxifen, are used as adjunct therapy for estrogen receptor (ER)–positive tumors (3). However, treatment of ER-negative breast cancer remains quite problematic without widely effective therapies.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of 17-ß-estradiol, is produced following hydroxylation and methylation reactions catalyzed primarily in the liver (4). 2ME2 has been shown to be an effective antitumor agent for reducing tumor volume and neovascularization in several xenograft models and in a transgenic model for prostate cancer (5, 6), but tumor stage specificity has not been investigated, and the mechanisms through which 2ME2 inhibits tumorigenesis are not fully understood.

Although a metabolite of estradiol, 2ME2 has low affinities for ER{alpha} and ERß (7, 8), and its antitumor activity is independent of the presence of ERs (8, 9). 2ME2 is a potent inhibitor of angiogenesis and has been shown to reduce endothelial cell proliferation and migration (10). 2ME2 induces changes in the levels and activities of many proteins involved in the regulation of the cell cycle, including stress kinases, cell division kinases, cyclin B, and regulators of cell cycle arrest and apoptosis (6). It also increases the expression of p53 and death receptor 5 and activates downstream caspases in human tumor and endothelial cells (11), leading to increased apoptosis. A recent study showed that 2ME2 depolymerizes microtubules and down-regulates hypoxia-inducible factor-1{alpha} protein levels and transcriptional activity in an oxygen-independent and proteasome-independent manner (12). Additional studies have shown that 2ME2 inhibits the activity of superoxide dismutase, leading to free radical–mediated damage to mitochondrial membranes and the release of cytochrome c, inducing apoptosis (13). Importantly, 2ME2 has been well tolerated both clinically and in preclinical studies, and neither dose-limiting toxicity nor a maximum tolerated dose has been established (14).

In this study, we have analyzed the tumor stage–specific effects of 2ME2 in the C3(1)/Tag transgenic mouse model of mammary cancer. This model recapitulates important histopathologic and molecular alterations in mammary cancer development over a highly predictable time course (15). Low-grade mammary intraepithelial neoplasia (MIN) lesions develop at about 8 weeks of age and progress to high-grade MIN (similar to human ductal carcinoma in situ) by 12 to 14 weeks of age. ER{alpha}-negative invasive carcinomas are observed at about 15 to 16 weeks of age (15, 16).

We show that 2ME2 can affect tumorigenesis at both early and late stages, via different mechanisms. When 2ME2 is administered in late stages to animals with lesions in the C3(1)/Tag mouse model, it inhibits mammary tumor growth and multiplicity by significantly increasing apoptosis in mammary tumor epithelial cells. In early stages, when 2ME2 is administered at 6 weeks of age before significant lesion formation, an unusually high rate of cystic tumor formation is observed, consistent with a strong inhibition of tumor angiogenesis. However, a paradoxical increase in tumor multiplicity and tumor size occurs in mice in the early treatment group, where rates of apoptosis are not increased. The cystic tumor formation observed with 2ME2 treatment is similar to the cystic changes reported in a mammary breast cancer model deficient for the transcription factor Id-1 (17). Id-1 has been shown to inhibit differentiation, and its expression has been correlated with increased angiogenesis and endothelial growth (18). In this study, we show for the first time that 2ME2 down-regulates Id-1 expression in mouse and human mammary tumor and endothelial cell lines.

These observations suggest that different biological responses to 2ME2 may predominate (antiangiogenic or increased apoptosis), depending upon the stage at which 2ME2 is administered during mammary tumor development. Although 2ME2 may be a useful adjunct in established ER– tumors, caution may be warranted for its prolonged use as an agent to prevent breast cancer or breast cancer recurrence.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The mammary tumor epithelial cell line M6 was previously derived from C3(1)/Tag mammary gland tumors (19, 20). M6 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 50 units/mL of penicillin G sodium/50 µg/mL of streptomycin sulfate. SV40 T-antigen transformed mouse endothelial cells, SVEC-4, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES, 2 mmol/L glutamine, 50 units/mL of penicillin G sodium, and 50 µg/mL of streptomycin sulfate. MDA MB 231 cells were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum. Human microvascular endothelial cells-1 (HMEC-1) were grown in MCDB 131 supplemented with 15% FBS, 10 mmol/L L-glutamine, 50 units/mL of penicillin G sodium, and 50 µg/mL of streptomycin sulfate (21). All supplements were purchased from Invitrogen.

In vitro proliferation assay. Detection of DNA synthesis was done using the bromodeoxyuridine (BrdUrd) cell proliferation colorimetric ELISA kit (Roche, Indianapolis, IN) according to the manufacturer's instructions. For BrdUrd assays, 2,500 cells per well were seeded in a 96-well plate, allowed to attach overnight, and were then incubated with the compound in the growth medium for 48 hours.

In vitro apoptosis assay. Apoptosis was measured by examination of the presence of cytoplasmic histone-associated DNA fragments according to the manufacturer's instructions (Roche, Indianapolis, IN). Briefly, 10,000 cells per well were seeded in a 24-well plate and after 24 hours were exposed to 2ME2 at concentrations ranging from 0 to 10 µmol/L for 48 hours. The detection of cytoplasmic histone associated DNA fragments was quantitated as per manufacturer's protocol.

Measurement of caspase-3 enzymatic activity. Caspase activity was measured using a caspase fluorometric protease assay kit (BioVision Research Products, Palo Alto, CA). Briefly, after treatment of cells with 5 µmol/L 2ME2 for the indicated periods of time, cells were harvested, pelleted, and frozen on dry ice. This concentration was selected based on our previous results studying the effects of 2ME2 on apoptosis (8). Cell lysis buffer was added to the cell pellets, and the protein concentration was determined using the Bradford assay. Two hundred micrograms of each cell lysate was incubated with 5 µL of 1 mmol/L stock of fluorescently labeled tetrapeptide caspase substrate at 37°C for 1 hour. The release of 7-amino-4-trifluoromethyl coumarin was measured with a fluorometer (Wallac Victor, Perkin-Elmer Life Sciences, Boston, MA) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Caspase activity was assayed in duplicate samples.

Animals and experimental schedule. Transgenic female mice heterozygous for the C3(1)/Tag transgene in the FVB/N background were generated as previously described (15). Three sets of experiments were done: early and late intervention studies and a prevention study. The scheme of the study is depicted in Fig. 2.


Figure 2
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  		Figure 2. Stage-specific experimental design using 2ME2 in vivo using C3(1)/Tag transgenic mice. Three different studies were performed. In the late intervention study, 2ME2 treatment was begun when the tumor size reached 0.5 cm and maintained until tumor reached 2 cm in diameter. In the early intervention study, 2ME2 treatment was initiated at 12 weeks of age, continued for 6 weeks, and the mice were sacrificed when tumor size reached 2 cm. In the prevention study, 2ME2 was given from 6 weeks of age until the tumor reached 2 cm.

 
As shown in previous studies, 2ME2 causes very low signs of toxicity when administered in vivo. Doses of 75 mg/kg body weight did not cause any clinical toxicity in mice (22, 23). Based on previous studies using several mouse models (14) that showed that doses of 2ME2 ranging from 50 to 150 mg/kg/d are effective but not toxic against various cancer types, such as melanoma (5), neuroblastoma (24), and lung metastasis (23), we used the highest dose, 150 mg/kg body weight for our study in the C3(1)/Tag transgenic breast cancer model.

For the intervention experiments, mice were randomly selected for a control group (n = 12) and an experimental group (n = 12). Animals in the experimental group received 150 mg/kg of 2ME2 daily in 0.2 mL 0.5 % methylcellulose by oral gavage. For the late intervention group, treatment was initiated when mice developed tumors 0.5 cm in diameter and was continued for 6 weeks or until the tumors reached a maximum diameter of 2 cm.

For the early intervention experiments, designed to show inhibition from MIN to invasive tumor formation (typically occurring at 16-20 weeks of age), another cohort of mice was randomly separated into a control (n = 10) and an experimental group (n = 10). The same dosing regimen was used as above, but treatment was initiated at 12 weeks of age and continued for 6 weeks.

The effect of 2ME2 on preneoplastic mammary glands was analyzed in a prevention experiment. Six-week-old mice were divided into two groups with treatment continuing until the mice were euthanized or the tumors reached 2 cm in diameter: (a) a control group that received vehicle (0.2 mL of 1% hydroxypropylcellulose in sterile water, n = 22) and (b) a group that received 150 mg/kg/d 2ME2 (formulated with 1% hydroxypropylcellulose in sterile water, 0.2 mL, n = 24). From this group, 10 mice (5 controls and 5 mice treated with 2ME2) were sacrificed at 12 and 15 weeks for mammary gland analysis. The formulation of 2ME2 used in the prevention experiment (done after the intervention studies) was altered based upon data, indicating improved pharmacokinetics and activity with this formulation in other tumor models (25).

Body weights were checked weekly for all mice. Mammary tumor size was measured by caliper, and tumor volume was calculated using the formula: the largest diameter x (the smallest diameter)2 x 0.4 (26). All mice were treated in accordance with the guidelines of the Animal Care and Use of Laboratory Animals (NIH publication no. 86-23, 1985) under an approved animal protocol.

Antibodies. The following primary antibodies were used: SV40 Tag (for Western blot: 1:80, Ab-2; Oncogene, Boston, MA; for immunohistochemistry: 1:25, PAB101; PharMingen, San Diego, CA), mouse monoclonal anti-Ki-67 (DAKO, Carpinteria, CA; 1:10,000), and mouse monoclonal anti-proliferating cell nuclear antigen (anti-PCNA; DAKO; 1:1,000). Mouse monoclonal antibody (1:12,000) to SV40 Tag and Id-1 polyclonal antibody (1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Histology, immunohistochemistry, and terminal deoxynucleotidyl transferase–mediated nick end labeling analysis. Mammary tissues and tumors were excised, fixed in 4% paraformaldehyde, embedded in paraffin, cut at a thickness of 4 µm, and stained with H&E.

For immunohistochemical analysis, sections were heated in a microwave oven in 0.1 mol/L sodium citrate for antigen retrieval, hybridized with primary antibody, and processed using the avidin-biotin complex method (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA). Using a x10 objective, the number of MIN lesions counted in five fields of the #4 mammary gland from each of five mice in each group at 15 weeks of age were determined in the intermediate prevention study and reported as the average number of MIN lesions per mouse. Apoptotic cells were identified using the TUNEL in situ end-labeling technique (Apoptag, Oncor, Gaithersburg, MD). Cells staining positively for Ki-67, PCNA, and terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assay were quantified using a Zeiss Axioplan microscope and ImagePro software (Media Cybernetics, L.P., Silver Spring, MD). At least 1,000 cell nuclei per section of mammary gland were counted in randomly selected fields in non-necrotic areas. Final data were expressed as a mean percentage of positively stained epithelial cells with respect to the total number of epithelial cells examined. Mammary glands from five animals in the 2ME2-treated and control groups from the intervention experiment were used for quantification.

RNA extraction and semiquantitative reverse transcription-PCR. Total RNA was isolated using Trizol reagent (Invitrogen) from mammary tissues or cells following the manufacturer's instruction from frozen tissues and cells, and reverse transcription-PCR (RT-PCR) was done for Id-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): Id-1 sense primer sequence for mouse samples was 5'-GTACAACCTTTCTCCAACTT-3', and antisense primer sequence was 5'-ATCTGGTCCCTCAGTGCGCC-3'. GAPDH sense primer sequence for mouse samples was 5'-ATCACCATCTTCCAGGAGCGAGAC-3' and antisense primer sequence was 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'. Id-1 sense primer sequence for human samples was 5'-GTCTGTCTGAGCAGAGCGTG-3', and antisense primer sequence was 5'-TAGTCGATGACGTGCTGGAG-3'. GAPDH sense primer sequence for human samples was 5'-GGAAATCCCATCACCATCTTC-3', and antisense primer sequence was 5'-GTCTTCTGGGTGGCAGTGAT-3'.

Reverse transcription reactions were done using the Superscript first-strand synthesis system for RT-PCR (Invitrogen) following manufacturer's protocol: 1 µg total RNA, 1 µL of 10 mmol/L deoxynucleotide triphosphate (dNTP) mix, and 1 µL of oligo(dT)12-18 (0.5 µg/µL) in a total volume of 10 µL at 65°C for 5 minutes followed by the addition of 2 µL of 10x RT buffer, 4 µL of 25 mmol/L MgCl2, 2 µL of 0.1 mol/L DTT, 1 µL of RNaseOUT (40 units/µL) with incubation at 42°C for 2 minutes. Fifty units of reverse transcriptase were added and incubated at 42°C for 50 minutes. The reaction was terminated at 70°C for 15 minutes and then chilled on the ice. One microliter of RNase H (2 units/µL) was added to each tube and incubated for 20 minutes at 37°C.

The PCR mixture included 1 µL of the cDNA generated from the reverse transcription reaction, 1 µL of x10 PCR buffer, 1.5 µL of 50 mmol/L MgCl2, 1 µL of 10 mmol/L dNTP, 0.4 µL of Taq polymerase (5 units/µL), 0.5 µL of 20 µmol/L sense primer, and 0.5 µL of 20 µmol/L antisense primer in a total volume 50 µL. PCR condition for Id-1: 94°C for 5 minutes/94°C for 30 seconds; 58°C for 30 seconds; 72°C for 1 minute/72°C for 7 minutes, 4°C for 30 cycles. PCR condition for GAPDH: 94°C for 5 minutes/94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 minute/72°C for 7 minutes, 4°C for 25 cycles.

Western blotting. Mammary tissues or cells were homogenized using radioimmunoprecipitation assay lysis buffer [1% Triton X-100, 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 10 mmol/L Na3VO4] with protease inhibitor cocktail tablets (Roche, Penzberg, Germany) for protein analysis. Thirty micrograms of protein were resolved on 4% to 20% Tris-glycine gradient gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), blocked with 5% nonfat milk, and incubated with the indicated primary antibodies followed by horseradish peroxidase–conjugated secondary antibody. Signal was visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ). For sequential blotting with additional antibodies, the membranes were stripped using a Re-Blot western recycling kit (Chemicon International, Inc., Temecula, CA) and reprobed with the indicated antibodies.

Id-1 promoter assay. Id-1 promoter constructs (kindly provided by Dr. Pierre-Yves Desprez, California Pacific Medical Center, Cancer Research Institute, San Francisco, CA; 5'del-1, 5'-del-3, 5'del-6) were transfected to the cells for 4 hours with 2% (for M6 cells) or 10% (for MCF 7 and MDA MB 231 cells) serum condition using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. Cells were incubated for 4 hours, and then 2ME2 was added for 24 hours. Promoter activity was measured using the Dual-luciferase reporter assay system (Promega, Madison, WI) and Wallac Victor-2 1420 multilabel counter (Perkin-Elmer, Shelton, CT) and was normalized for transfection efficiency using the pRL-CMV vector (Promega).

Statistical analyses. The two-sided Student's t test was used to evaluate whether significant differences existed between the mean values of the groups analyzed for the apoptosis assay. The Mann-Whitney test was used for the analysis of tumor volumes. The log-rank test was used to determine the differences in survival curves.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
2ME2 Inhibits Proliferation and Induces Apoptosis in M6 and SVEC-4 Cells In vitro
We first evaluated the effects of 2ME2 on proliferation and apoptosis in M6 and SVEC-4 cells in vitro. M6 is a tumorigenic cell line isolated from C3(1)/Tag mammary tumors and previously characterized (19, 20). SVEC-4 cells are SV40 T-antigen transformed mouse endothelial cells that have been previously described (27).

2ME2 caused a dose-dependent inhibition of cell proliferation for both M6 and SVEC-4 cells (Fig. 1A ). The IC50 value was ~4 µmol/L. Levels of apoptosis were assessed by detection of cytoplasmic histone-associated DNA fragments and caspase-3 activation. 2ME2 induced apoptosis in M6 and SVEC-4 cells as evidenced by the induction of DNA fragmentation at ≥1 µmol/L concentrations (Fig. 1B). To investigate the kinetics of apoptosis in response to treatment with 2ME2 in these cell lines, we evaluated caspase-3 activation (Fig. 1C). Caspase-3 activation was detectable in both M6 and SVEC-4 cells treated with 5 µM 2ME2 beginning after about 16 hours of treatment and markedly increased by 24 hours of treatment (Fig. 1C). These data show that 2ME2 inhibits cell proliferation and increases apoptosis of M6 and SVEC-4 cells in vitro.


Figure 1
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  		Figure 1. Effect of 2ME2 on proliferation and apoptosis in vitro in M6 ({circ}) and SVEC-4 cells (bullet). A and B, antiproliferative (A) and apoptotic (B) activities of increasing concentrations of 2ME2 for 48 hours. C, kinetics of 2ME2-induced caspase-3 activation in M6 and SVEC-4 cells. Increased caspase-3 activation was observed after 16 hours of treatment with 5 µmol/L 2ME2. Points, average of three independent experiments; bars, SD. Proliferation, apoptosis, and caspase assays were done as described in Materials and Methods.

 
Stage-Specific Effects of 2ME2 on Mammary Tumor Growth, Tumor Multiplicity, and Survival in C3(1)/Tag Transgenic Mice
To evaluate the tumor stage–specific effects of 2ME2 in C3(1)/Tag mice, we did three independent in vivo experiments: (a) a late intervention study, (b) an early intervention study, and (c) a prevention study. The experimental scheme is depicted in Fig. 2 .

Late intervention study. The late intervention experiment was designed to determine whether 2ME2 could retard tumor growth or result in tumor regression of established mammary tumors in C3(1)/Tag mice. For this purpose, we treated mice bearing 0.5 cm tumors with 150 mg/kg/d 2ME2 for 6 weeks (or until tumor size reached 2 cm, requiring euthanasia of the animal). Three weeks after the initiation of treatment, a significant decrease (*, P < 0.05) in tumor burden was observed in the 2ME2-treated mice compared with the control mice (Fig. 3A ). After 6 weeks of treatment, tumor burden was reduced by ~60% (**, P < 0.01) in animals treated with 2ME2 compared with the control mice.


Figure 3
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  		Figure 3. A and B, tumor stage–specific effects of 2ME2 on mammary tumor burden in the late intervention and early intervention studies. A, tumor burden was significantly (**, P < 0.01) inhibited by ~60% after 6 weeks of treatment in the late intervention study compared with the control. B, tumor burden was significantly (**, P < 0.01) decreased in mice that received 2ME2 in the early intervention study. C and D, tumor stage–specific effects of 2ME2 on mammary tumor multiplicity. C, mean number of tumors was significantly decreased (***, P < 0.001) after 6 weeks of 2ME2 treatment in the late intervention study compared to control. D, mean number of tumors is significantly lower (*, P < 0.05) in the 2ME2-treated mice compared with the control group in the early intervention study. E, a modest but significant (P = 0.0072) increase in survival was observed in 2ME2-treated mice compared to controls in the late intervention study. F, 2ME2 induced apoptosis in mammary tumors in the late intervention study. Quantification of apoptosis in tumors showed increased apoptosis in the 2ME2-treated group compared with the control group (*, P < 0.05). Arrows, termination of treatment in (B) and (D).

 
Tumor multiplicity was significantly decreased in the mice treated with 2ME2 compared with the controls (P < 0.001 after 3 weeks). The mean number of tumors per mouse (MNT) in control mice was 2.1 after 2 weeks of treatment, whereas in the 2ME2-treated mice, the MNT was 1.2, a 45 % reduction. After 6 weeks of treatment, the MNT for the control group was 5.8 compared with 2.5 for the mice treated with 2ME2, a 57% reduction in the 2ME2-treated group (Fig. 3C). Animals receiving 2ME2 showed a modest but statistically significant 2-week increase in survival compared with the control mice (P = 0.0072; Fig. 3E). To determine the mechanism of tumor inhibition and increased survival, we evaluated proliferation and apoptosis. In the late intervention group, the apoptotic index was significantly increased by ~2-fold in tumors from the 2ME2-treated group compared with the control group (Fig. 3F). The proliferation index, as determined by Ki67 and PCNA staining, was not statistically different between the control and treated mice (data not shown) in any of the intervention experimental groups.

Early intervention study. To evaluate whether 2ME2 inhibits progression from pre-invasive MIN (ductal carcinoma in situ like) lesions to palpable, invasive tumors in C3(1)/Tag mice, animals were treated beginning at 12 weeks of age for a total of 6 weeks. We have previously shown that at 12 weeks of age, MIN lesions are found in the mammary glands of C3(1)/Tag mice, but few invasive tumors and no palpable tumors are seen. Although onset of palpable tumors was not significantly delayed in mice treated with 2ME2 compared with controls in the early intervention study (data not shown), tumor burden (cumulative tumor volume) was significantly decreased in animals that received 2ME2 compared with the control mice (Fig. 3B). The degree of tumor reduction was similar to the late intervention experiment (50-60%). The MNT was also statistically lower in mice treated with 2ME2 compared with controls (Fig. 3D). The effect in the late intervention study was more pronounced than in the early intervention experiment (compare Fig. 3B and D). However, in the early intervention study, survival was not significantly increased in the 2ME2-treated animals compared with the control mice (data not shown). Unlike the late intervention study, the apoptotic index was not changed in MIN lesions of 15-week-old mice in the early intervention group.

Prevention study. Animals received 2ME2 beginning at 6 weeks of age until mice were euthanized either from tumor burden (diameter of 2 cm) or because animals seemed moribund. Surprisingly, tumor burden seemed to be increased in animals that received 2ME2 compared with the control mice in this experiment (Fig. 4A ; *, P < 0.05), but this seemed due to the cystic nature of the tumors that developed, as discussed below. At 20 weeks of age, tumor size in the 2ME2-treated group was 2.3-fold larger than the control group.


Figure 4
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  		Figure 4. In the prevention study, 2ME2 increases cystic tumor formation. A, tumor growth. B, tumor multiplicity. C, histologic features of mammary glands and tumor progression in the 2ME2-treated and control C3(1)/Tag mice in the prevention study. Mammary glands and mammary tumors were taken at 13 weeks (left) and 20 to 21 weeks (middle) of age from both control and 2ME2-treated groups. Large central necrosis (arrows) was observed in most tumors treated with 2ME2 (bottom right) but not in tumors from the control mice (top right). Magnification, x100. Bars, 1 mm.

 
The MNT seemed to be higher in mice treated with 2ME2 compared with controls (Fig. 4B; *, P < 0.05). At 20 weeks of age, the MNT in the control group was 0.5, whereas it was 2.2 in the 2ME2-treated group, an almost 4.4-fold induction of MNT compared with the control group.

Apoptosis was not increased in the preinvasive lesions, and the proliferation index was not decreased in the prevention study, similar to the early intervention study.

The overall survival in the 2ME2-treated group compared with the control group was reduced due to the accelerated cystic tumor expansion requiring earlier euthanasia (data not shown).

These results suggest that increased survival in the intervention study is likely due to increased apoptosis that was not observed in the 2ME2-treated mice in the prevention group.

2ME2 Altered the Phenotype of Tumors during Mammary Cancer Progression in the Prevention Study
To further understand the nature of the cystic tumor formation induced by 2ME2 in the prevention study, several histologic analyses were done using five mice from each group (Fig. 4C). Tissues were analyzed histologically, and the number of MIN lesions was quantitated.

At all ages, dilated ducts filled with proteinaceous material were noted in all the treated mice, but only in one of the mice from the control group (Fig. 4C, left). At early stages, small lesions with evidence of early central necrosis were observed within the lumens of ducts in the 2ME2-treated mice (Fig. 4C, middle). By 20 to 21 weeks of age, when palpable mammary tumors were developing, marked necrosis in the central regions of many of the tumors were observed in the 2ME2-treated group (Fig. 4C, bottom right), but a similar degree of central necrosis was not observed in the control group (Fig. 4C, top right). Eventually, most 2ME2-treated mice developed multiple cystic tumors filled with secreted fluid with significantly dilated mammal ductal architecture (Fig. 4C, bottom right), whereas tumors in the control mice remained primarily solid (Fig. 4C, top right). Overall, cystic tumors were observed in 71 % of the mice treated with 2ME2 but rarely seen in the mice in the control group, where only an occasional tumor with small cysts was observed.

The average number of MIN lesions per mouse was 15.25 ± 3.3 in the control group compared with 6.6 ± 2.9 in the 2ME2-treated group (P < 0.01). This result indicates that overall, 2ME2 inhibited MIN lesion development, although large cystic tumors still developed. It is likely that the cystic nature of the tumors led to larger tumor sizes compared with the controls, and detection of these smaller tumors was more apparent as they expanded from the cystic fluid that would not have been palpable in the untreated mice.

To exclude the possibility that these effects mediated by 2ME2 were a consequence of changes in transgene expression, we did immunohistochemical staining for T-antigen. The distribution of expression and the intensity of staining of T-antigen in the 2ME2-treated animals were similar to those found in the control mice (data not shown).

2ME2 Down-regulates Id-1 Expression in Mammary Tumor and Endothelial Cells in a Dose-Dependent Manner
Id-1 expression has been correlated with aggressive breast cancer and poor prognosis (28, 29). Interestingly, the tumor phenotype from our prevention study seemed very similar to that observed for MMTV-neu mice deficient for Id-1, in which tumors exhibited central necrosis and were quite cystic (17). The lack of Id-1 in that setting resulted in impaired angiogenesis. Therefore, we investigated the expression of Id-1 in response to 2ME2 treatment. Id-1 was down-regulated by 2ME2 in M6 cells [which are derived from C3(1)/Tag mammary tumors] and HMEC-1 in a dose-dependent manner at the RNA levels (Fig. 5A ). Similar results in protein levels were observed using M6 cells, MDA MB 231, and SVEC-4 (Fig. 5B). Tumors from vehicle-treated C3(1)/Tag mice showed inconsistent levels of Id-1 expression, perhaps due to tumor heterogeneity (data not shown), and thus did not allow for the adequate evaluation of this in vivo.


Figure 5
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  		Figure 5. M6 cells (10% serum), SVEC-4 (10% serum), and HMEC-1 (15% serum) were treated with 2ME2 for 24 hours, and then RNA or protein was extracted as described in Materials and Methods. 2ME2 down-regulated Id-1 RNA expression in mammary tumor M6 cells and HMEC-1 in a dose-dependent manner (A). GAPDH was used as an internal control for RNA. B, Id-1 protein levels were down-regulated by 2ME2 treatment in a dose-dependent manner in M6 and SVEC-4 cells. MDA MB 231 human breast cancer cells were grown to 80% confluence and treated with 2ME2 overnight (0.5 and 5 µmol/L). 2ME2 also down-regulated Id-1 expression in MDA MB 231. ß-Actin was used as an internal control for Western blotting.

 
The transcriptional regulation of Id-1 has been well characterized in breast cancer cell lines by other groups (30, 31). Based upon the results above, we studied the transcriptional regulation of Id-1 by 2ME2 using three different cell lines, in which luciferase reporter constructs containing various lengths of the Id-1 5' regulatory region were assayed with or without 2ME2. All these cell lines showed a consistent reduction (maximum 50%) in transcriptional activity of Id-1 in a dose-dependent fashion (Fig. 6, A and B ).


Figure 6
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  		Figure 6. Id-1 promoter-luciferase assay. A, Id-1 promoter constructs (5'del-1, 5'del-3, and 5'del-6). Numbers indicate bp from transcriptional start site. B, Id-1 promoter activity was assayed in three different breast cancer cells. Id-1 promoter activity was down-regulated in the constructs, 5'-del-1 and 5'del-3 in MCF7, MDA-MB-231, and M6 cells in a dose-dependent manner. The experiments were repeated at least twice in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Columns, mean; bars, SD.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report evaluating the effects of 2ME2 on several stages of tumor development in a transgenic breast cancer mouse model. An important feature of the C3(1)/Tag model used in this study is that mammary tumor progression can be monitored and tumor stage–specific responses can be evaluated. Several studies have shown tumor stage–specific effects of antiangiogenic agents using different mouse models (32, 33). We took advantage of the C3(1)/Tag model in designing experiments to address how 2ME2 might affect different stages of mammary tumorigenesis in a model that develops ER-negative mammary tumors. Different phenotypic responses were observed depending upon the stage of tumor development when 2ME2 was administered, and these results may be due to different mechanisms of action.

Transgenic mice that were treated with 2ME2 following the development of MIN (lesions similar to human ductal in situ carcinoma) in the early intervention study, or once palpable tumors of ~0.5 cm were observed in the late intervention study, showed a significant reduction in tumor growth and tumor multiplicity associated with increased apoptosis in vivo. Our in vitro studies using M6 mammary tumor cells from C3(1)/Tag transgenic mice and mouse endothelial SVEC-4 cells showed that 2ME2 could result in a significant reduction in proliferation and increased apoptosis associated with increased DNA fragmentation and elevated caspase-3 activity. However, when similar variables were studied in tumors arising spontaneously in vivo and treated with 2ME2, no reduction in cell proliferation was observed, but a 2-fold increase in the rate of apoptosis of tumor cells was observed. This suggests that at the late stage of tumorigenesis studied in vivo, 2ME2 exerted an inhibitory effect, at least in part through increased apoptosis. The tumor inhibitory effects we observed with 2ME2 in the early intervention study were more modest and showed a reduction in tumor growth but not complete eradication of tumor development. No significant difference in tumor cell proliferation or apoptosis was observed in the early lesions from either the early intervention or prevention studies (data not shown). These results suggest that 2ME2 is able to significantly decrease mammary tumor size and number in established yet relatively small invasive tumors.

The majority of tumors exposed to 2ME2 before the development of MIN lesions were remarkably cystic with a relatively thin rim of viable tumor cells in the periphery. The tumors seemed to originate as nodular lesions within the lumens of mammary ducts. As these lesions enlarge, central necrosis develops, ultimately resulting in large, fluid-filled cystic tumors. This type of cystic morphology was not observed in the tumors that arose in the control group of transgenic mice. Although cystic mammary tumors have occasionally been observed in C3(1)/Tag mice, in our long experience with this model system, we have not observed this striking morphologic characteristic of tumors in an experimental cohort of mice.

To further define the effects of 2ME2 on lesion formation, we quantitated the average number of MIN lesions per mouse after 7 weeks of treatment in the prevention cohorts (at 13 weeks of age). The significant reduction in the number of MIN lesions in the 2ME2-treated group suggests that 2ME2 inhibits this type of early tumor progression in addition to altering histologic characteristics of the invasive tumors that do develop. Previous studies have also shown that 2ME2 has potent antiangiogenic activities (5, 12, 34). In the case of the present prevention study, it seems that once the tumor has grown to a critical size, vascularization becomes limiting, and the central tumor region undergoes necrosis. However, the peripheral region of the tumor receives sufficient oxygen and nutrients and is able to continue to proliferate and survive as had similarly been observed in MMTV-neu;Id-1–/– mice.

Our observations in the prevention study are remarkably similar to those reported by de Candia et al., where it was shown that deficiency of Id-1 and Id-3, which play an important role in tumor angiogenesis, did not prevent or delay tumor formation but did alter the morphologic phenotype of mammary tumors (17). In that study, MMTV-HER-2/neu tumors that developed in Id-deficient mice were larger, exhibited central necrosis, and became cystic with a viable rim of tumor cells surrounding a nonviable core of cellular debris (17). We checked Id-1 expression in tumors from three different studies to see if 2ME2 could down-regulate Id-1 expression. Although expression of Id-1 in late-stage tumors was quite heterogeneous in the in vivo studies using 2ME2, we showed that 2ME2 does significantly inhibit Id-1 transcription in mouse and human breast cancer cell lines using Id-1 promoter-reporter constructs.

Elevated Id-1 expression is strongly associated with breast cancer and poor prognosis (28, 29). A recent report showed that Id-1 may contribute to both primary breast tumorigenicity and metastatic potential (35). Whether 2ME2 can reduce Id-1 expression in a subset of human breast tumors will require further study.

It is not clear why cystic tumor formation was not observed in mice treated with 2ME2 at later stages of tumor development in the intervention studies. Because 2ME2 was administered at a point when these tumors already reached a stage with some degree of vascularization and were treated for a shorter period of time, large cystic tumors may not have been able to develop. 2ME2 was able to reduce tumor growth and multiplicity when administered at later stages, primarily through an induction of apoptosis in the tumor cells as discussed above. It is possible that 2ME2 may, therefore, be useful as a therapeutic agent in combination with other drugs.

In vitro data from other studies indicate that unlike estradiol, 2ME2 does not promote proliferation of ER-positive breast cancer cells (8, 9), and that the affinity of 2ME2 to ER is extremely low (8). Expression of ER and progesterone receptor in mammary tumors did not change by 2ME2 treatment (data not shown). Unlike a recent study in which increased uterine and liver weights were observed (36), we did not observe increased uterine weight in treated animals compared with controls (data not shown). These results are consistent with other studies that have shown that 2ME2 does not have an estrogenic effect (8).

In summary, we show that 2ME2 may inhibit tumorigenesis through different mechanisms, depending upon the stage of cancer development, which has important implications for its use in clinical settings. Although tumor growth at late stages seemed to be reduced by 2ME2 through an increase in apoptosis, administration of 2ME2 at very early stages may have a more profound effect on the establishment of angiogenesis, resulting in cystic tumor formation. This is relevant because 2ME2 is being evaluated in clinical trials. It is possible that 2ME2 used in combination with agents that affect other pathways important in cancer growth may lead to additional strategies to prevent tumor progression or the treatment of established tumors.


   Acknowledgments
 
Grant support: Fulbright-Ministerio de Educacion y Ciencia fellowship at NIH (A. Calvo).

The costs of publication of this article were defrayed in part by the 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.

We thank Lisa Birely for animal care, Andrea Waghelstein for excellent technical assistance, Dr. Pierre-Yves Desprez for Id-1 promoter constructs, Dr. Junya Fukuoka and Lalage Wakefield for useful discussions, and Theresa M. LaVallee, Glenn Swartz, Victor S. Pribluda, and Patricia A. Burke (EntreMed, Inc., Rockville, MD) for providing 2-methoxyestradiol and their helpful discussions related to this study.


   Footnotes
 
Note: J-I. Huh and A. Calvo contributed equally to this work. A. Calvo is currently at the Division of Oncology, Center for Applied Medical Research and Department of Histology and Pathology, University of Navarra, Pamplona, Spain.

Received 7/12/04. Revised 12/20/05. Accepted 1/16/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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