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Endocrinology Vol. 148, No. 3 1171-1180
Copyright © 2007 by The Endocrine Society

The Effect of Estradiol on in Vivo Tumorigenesis Is Modulated by the Human Epidermal Growth Factor Receptor 2/Phosphatidylinositol 3-Kinase/Akt1 Pathway

Kevin Lehnes1, Abigail D. Winder1, Camille Alfonso, Natasha Kasid, Michael Simoneaux, Heather Summe, Elisha Morgan, Mary C. Iann, Jessica Duncan, Matthew Eagan, Raluca Tavaluc, Charles H. Evans, Jr., Robert Russell, Antai Wang, Fengming Hu and Adriana Stoica

Department of Human Science (K.L., A.D.W., C.A., N.K., M.S., H.S., E.M., M.C.I., J.D., M.E., R.T., C.H.E., A.S.), School of Nursing and Health Studies, Department of Oncology (R.R., A.S.), Lombardi Comprehensive Cancer Center, and Department of Bioinformatics, Biostatistics, and Biomathematics (A.W., F.H.), Georgetown University, Washington, D.C. 20007

Address all correspondence and requests for reprints to: Adriana Stoica, STM 260, 3700 Reservoir Road NW, Washington, D.C. 20057-1107. E-mail: stoicaa{at}georgetown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether the epidermal growth factor receptor 2 (ErbB2) and Akt1 can alter the in vivo growth of MCF-7 cells, parental cells or cells stably transfected with constitutively active Akt1 (myr-Akt1) or dominant-negative Akt1 mutants (K179M-Akt1 and R25C-Akt1) were implanted into athymic nude mice. Tumor growth was monitored in the presence or absence of the antiestrogen tamoxifen and the selective ErbB2 inhibitor, AG825. MCF-7 [parental or empty vector transfected, cytomegalovirus (CMV)] and myr-Akt1 cells formed tumors upon estradiol supplementation after 20–30 d (59-, 29-, and 17-fold increase in tumor volume, respectively). Tamoxifen and AG825 blocked the estradiol effect by 93 and 96% in MCF-7 xenografts, 88 and 81% in CMV xenografts, and 91% in myr-Akt1 xenografts. Furthermore, AG825 suppressed the growth of established tumors in CMV and myr-Akt1 inoculated animals by 68 and 75%, respectively, as compared with continued estrogen supplementation, suggesting a role for ErbB2. When K179M-Akt1 or R25C-Akt1 cells were injected into ovariectomized animals, tumor growth was reduced upon estradiol treatment by 95% and 98%, respectively, supporting a role for Akt1. In contrast to ovariectomized animals, in intact animals, myr-Akt1 cells could establish tumors without estradiol priming after 40–50 d (20-fold increase in tumor volume). Loss of Akt1 phosphorylation was associated with tumor growth inhibition. Immunohistochemical assays showed that in tumors from parental and CMV xenografts, estradiol decreased estrogen receptor-{alpha} expression and induced progesterone receptor expression and Akt phosphorylation, effects that were inhibited by tamoxifen, AG825, and R25C-Akt1 by 89, 82, and 77% for progesterone receptor expression and 48, 66, and 73% for pAkt expression, respectively. Cumulatively, our results suggest that Akt1 and ErbB2 are involved in in vivo tumorigenesis and modulation of estrogen receptor-{alpha} expression and activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH AND development of breast cancer is regulated by a complex set of factors, including hormones, growth factors, and their receptors. Estrogens are the most important etiological factors for many breast cancers, playing a critical role in tumorigenesis. Prolonged exposure of reproductive organs to estrogen leads to an increased risk for this disease (1). The effects of estrogens are mediated by direct binding to estrogen receptors (ERs) in the nucleus, after which the receptors dimerize and bind to specific estrogen response elements located in the promoters of target genes (classical genomic effect). Hormone binding causes a conformational change in the hormone binding domain that leads to recruitment of specific coactivators (2). Another genomic mechanism (nonclassical genomic effect) involves protein-protein interactions between ER and other DNA-binding transcription factors in the nucleus. In addition, membrane-associated nongenomic actions of estrogens can lead to altered functions of proteins in the cytoplasm and to regulation of gene expression (2). These actions are rapid and associated with activation of several protein kinase cascades that transduce the signal from the cytoplasm to the nucleus, activating nuclear transcription factors, and phosphorylating ER-{alpha}. Finally, growth factors can also activate protein kinase cascades, leading to phosphorylation and activation of nuclear ERs (ligand-independent activation) (2).

ER-{alpha} is the selection criterion for patients to endocrine therapy. Seventy percent of breast cancers express ER-{alpha} and progesterone receptors (PRs). However, most responsive tumors will eventually acquire resistance (3). The antiestrogen tamoxifen is a successful drug for ER-{alpha}-positive breast cancer treatment and is currently prescribed for the prevention of breast cancer in high-risk women (4). Many tumors that first respond to tamoxifen will eventually develop resistance to the drug (acquired tamoxifen resistance) (3). In contrast, the pure antiestrogen ICI 182,780 has no estrogenic properties because it causes destruction of the ER (5). Tamoxifen resistance is heterogeneous and multifactorial. A tamoxifen-stimulated phenotype may occur in some patients, other tumors may become resistant through changes in immunity, drug pharmacokinetics, or host endocrinology (6). Growth factor interactions may also occur, leading to breast tumor growth by growth factor-induced ER-{alpha} activity through activation of protein kinases resulting in ER-{alpha} phosphorylation (5).

ER-positive breast cancer cells produce autocrine and/or paracrine growth factors that activate their intrinsic tyrosine kinases, initiating a complex cascade of signal transduction. The members of the epidermal growth factor receptor (EGFR) superfamily of tyrosine kinases are often activated in breast cancer (7, 8). Human epidermal growth factor receptor 2 (ErbB2) is overexpressed in approximately 30% of breast tumors (6, 9, 10). ErbB2 activation, via dimerization with other EGFR family members or itself, can lead to the activation of the phosphatidylinositol 3-kinase (PI 3-K) the serine/threonine protein kinase, Akt (11). Tumors with ErbB2 overexpression respond poorly to antiestrogen treatment (12). Preclinical data have demonstrated that inhibition of ErbB2 signaling can restore hormone responsiveness in ErbB2 overexpressing endocrine-resistant breast tumors, delay the development of endocrine resistance, and increase the antiproliferative effect of endocrine agents (13, 14, 15). These effects were, in part, mediated by the inhibition of the PI 3-K/Akt and MAPK pathways (16, 17, 18). Akt and MAPK activate ER-{alpha} by direct phosphorylation at S167 and S118, respectively (17, 18, 19).

The growth factor-coupled activation of PI 3-K and its central effector, Akt, is a critical pathway in oncogenesis. There are three isoforms of Akt in human cells: AKT1, AKT2, and AKT3, and all three have the same domain organization. Akt can be activated by growth factors that bind to their membrane receptors, phosphorylating their tyrosine residues and leading to PI 3-K activation. PI 3-K generates membrane-bound phoshoinositides, which act as second messengers and then recruit Akt and its upstream kinases, phosphoinositide-dependent kinase (PDK)1 and PDK2, to the membrane via its PH domain (20). This membrane localization facilitates Akt phosphorylation at T308 and S473 (20). Activated Akt then translocates to the cytosol and nucleus to phosphorylate several downstream protein targets (21). These phosphorylation events lead to cell survival, growth, cell cycle regulation, differentiation, angiogenesis, migration, invasion, and metabolism (8, 21). Constitutive activation of Akt, chromosomal amplification of Akt or PI 3-K, and the deletion of the phosphatase and tensin homolog deleted from chromosome 10 may play a role in tumor formation. ER-{alpha} but not ER-ß can directly bind to PI 3-K (22, 23) and estradiol stimulation increases ER-{alpha}-associated PI 3-K/Akt1 activity (22, 24, 25). The frequency of PI 3-K/Akt alterations found in breast tumors suggests the occurrence of hormone-dependent cancers with this pathway activated. Therefore, aberrant Akt signaling may block antiestrogen actions and those ER-positive cancers with Akt signaling might be candidates for antiestrogens in combination with Akt inhibitors (15).

We previously demonstrated a cross talk between growth factors and estrogen receptor signaling that converges in the ErbB2/PI 3-K/Akt pathway in hormone-dependent MCF-7 breast cancer cells (24, 26). The purpose of this study was to extend our in vitro observations (26) by examining whether the ErbB2/PI 3-K/Akt1/ER-{alpha} signal transduction pathway is similarly activated in vivo. In this study, we demonstrate that Akt1 and ErbB2 are involved in in vivo tumorigenesis and modulation of ER-{alpha} expression and activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture
MCF-7 cells were stably transfected with either dominant-negative Akt1 mutants (K179M-Akt1 or R25C-Akt1) or a constitutively active mutant of Akt1 (myr-Akt1) as described elsewhere (24). The expression vector alone, CMV, was used as a negative control. Monolayer cultures of MCF-7 cells (parental and stably transfected with CMV and the Akt1 mutants) were grown in improved MEM (IMEM) supplemented with 5% fetal calf serum in the presence or absence of 750 µg/ml geneticin (G418). At 80% confluence, the medium was replaced with phenol red-free IMEM containing 5% charcoal-treated calf serum (27) in the presence or absence of 750 µg/ml G418. The calf serum was pretreated with sulfatase and dextran-coated charcoal to remove endogenous steroids (28). After 2 d, the medium was changed into serum-free, phenol red-free IMEM supplemented with fibronectin, glutamine, HEPES, trace elements, and transferrin. Cells were harvested and inoculated into athymic nude mice.

Plasmids
The expression vector for wild-type Akt1 (29), the kinase defective Akt1 mutants, K179M-Akt1 (30) and R25C-Akt1 (30), and the constitutively active Akt1 mutant, myrAkt1, were generated as HindIII-BamHI inserts in pCMV-6 (30).

Western blot analysis
Snap-frozen tumors were crushed and minced under liquid nitrogen and homogenized by sonication (31) in Nonidet P-40 lysis buffer. The homogenate was incubated on ice for 30 min and then centrifuged at 100,000 x g for 30 min at 4 C. Protein loading buffer was added and lysates were heated to 95–100 C for 5 min. Protein concentration in the lysates was determined using the standard Bradford assay. Equal amounts of protein (100 µg) were loaded onto SDS-polyacrylamide gels. The samples were electrotransferred onto polyvinyl difluoride membranes and the membranes were washed in PBS four times at room temperature. Membranes were kept in blocking buffer overnight at 4 C and incubated with either an antiphospho-Akt antibody (S473) or an anti-Akt antibody (New England Biolabs, Beverly, MA) for 1 h at room temperature. After three additional washes in PBS, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:2000) in blocking buffer for 1 h at room temperature. Reactivity with a monoclonal anti-{alpha}-actin antibody (Chemicon, Temecula, CA) was used as a control for protein loading on each blot. Detection was performed by chemiluminescence, using the Super Signal chemiluminescent substrate (Pierce, Rockford, IL).

Athymic mouse model system
ER-positive MCF-7 human breast cancer cell lines [parental and stably transfected with the empty vector, CMV; constitutively active Akt1 (myr-Akt1); or dominant-negative Akt1 (K178M-Akt1 and R25C-Akt1) (24)] were cultured as described previously (31). The athymic nude mice were 4- to 6-wk-old female ovariectomized BALB/c-{nu}+/{nu}+ strain, approximately 15 g in weight and were purchased from Taconic (Germantown, NY). The animals were housed four to five per cage in a pathogen-free environment under controlled conditions of light (12-h light, 12-h dark cycle), temperature (22 ± 3 C), and humidity (60 ± 10%) and received sterilized food and water ad libitum. Mice were observed daily. Animal care was in accordance with a Georgetown University Institutional Animal Care and Use Committee-approved protocol.

Approximately 5 x 106 subconfluent MCF-7 cells (parental or stably transfected with the empty vector or with the Akt1 mutants), resuspended in 0.1 ml of medium (IMEM phenol-red free + 5% CCS ± 750 µg/ml G418) were injected sc into two sites of the flanks (on the left and right sides) of female nude mice to initiate tumor formation (32). Estradiol supplementation was provided in form of a 90-d release 0.25 mg estradiol pellet (Innovative Research of America, Sarasota, FL), placed sc in the interscapular region of the mice to stimulate tumor growth. The surgical incision was closed with a staple.

In the tumorigenesis experiments, animals were randomly divided into different treatment groups for the parental MCF-7 cells as well as for MCF-7 cells stably transfected with the empty vector, CMV, and myr-Akt1: 1) Vehicle treated [dimethylsulfoxide (DMSO)] control group, 2) estradiol-supplemented group, 3) tamoxifen-treated group (500 µg/d, Monday through Friday; sc injection), 4) AG825-treated group (50 µg/kg·d, ip injection), 5) estradiol supplemented in the presence of tamoxifen, and 6) estradiol supplemented in the presence of AG825. We chose ip dosing for AG825 to allow the component to reach maximum exposure regardless of its oral bioavailability. In addition, for the dominant-negative Akt1 transfected cell lines, K179M-Akt1 and R25C-Akt1, as well as for intact mice inoculated with either CMV or myr-Akt1 cells, animals were divided into two groups: 1) vehicle-treated animals (DMSO) and 2) estradiol supplemented animals. AG825 was dissolved in DMSO [1% (vol/vol)] and a corresponding volume of peanut oil was added to yield a final concentration of 50 µg/kg·d. Tamoxifen was dissolved in ethanol and peanut oil was added to yield its final concentration of 500 µg/d. Each group consisted of at least four to 10 animals inoculated bilaterally. Tumor volumes and body weight were measured twice a week with a Vernier caliper by a single observer. A trained observer closely monitored the clinical condition of the animals. Mean tumor volumes were determined by measuring tumors at all implantation sites. Volumes were calculated in cubic millimeters as the product of the width, length, and depth and reported to the percent of body weight of each animal.

The effect of AG825 on the growth of MCF-7 cells (parental, CMV, and myr-Akt1) established tumors was also studied after tumors had reached a size of approximately 200 mm3 (2–5 wk). At this time, tumor-bearing animals were randomly allocated to three treatment groups: 1) continued estradiol supplementation; 2) continued estradiol supplementation and treatment with AG825; and 3) removal of the estradiol pellet. The effect of AG825 was compared between groups 1 and 2 and between groups 2 and 3.

At the end of the experiment (52–104 d from the day of cell inoculation), mice were killed by cervical dislocation, tumors were excised, and all organs were examined macroscopically. Tumors, livers, and kidneys from each mouse were either immediately snap frozen in liquid nitrogen and stored at –70 C or were placed in 10% neutral buffered formalin until embedded in paraffin and processed for immunohistochemical staining or staining with hematoxylin and eosin.

Repeated ANOVA was used to test the significant difference between treatments or between experiments. ANOVA was used to test the significant difference between treatments at each day point. For animals inoculated bilaterally with cells treated with the same compound, mean value as one observation was considered.

Immunohistochemistry
For immunostaining of formalin fixed-paraffin embedded tumors, sections were cut at 5 µm thickness. The deparaffinized sections were pretreated for antigen retrieval in citrate buffer (pH 6.0) with incubation in a steamer for 20 min. After allowing to cool slowly to room temperature, slides were treated with 0.3% (wt/vol) hydrogen peroxide in methanol for 30 min to block endogenous peroxides activity, and then a blocking step was conducted by incubation of sections in 10% normal serum, the same as used in the secondary antibody. Then the sections were incubated for 1 h at room temperature in a humidified chamber with the appropriate dilution of the primary antibodies directed against either Akt (1:100; Cell Signaling Technology, Inc., Danvers, MA), pAkt (1:50; Cell Signaling Technology), PR (1:100, Dako, Carpinteria, CA), or ErbB2 (1:100; Dako). The secondary biotinylated antibody reagents were mouse and rabbit antibody, respectively (1:200 dilution). Sections were incubated for 30 min with the secondary antibody, followed by incubation in an ABC kit (Vector Laboratories, Ltd., Burlingame, CA). Then 3,3'-diaminobenzidine tetrahydrochloride substrate was used for detection. After washing, the slides were counterstained with hematoxylin for 20 sec, dehydrated, and mounted with a coverslip. Sections were evaluated for membrane, nuclear, or cytoplasmic staining. A semiquantitative score (negative, 0; weak, 1+; moderate, 2+; strong, 3+) was determined based on the staining intensity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dominant-negative Akt1 mutants and the selective ErbB2 inhibitor AG825 inhibit in vivo tumorigenesis in ovariectomized athymic nude mice
Because we have previously shown that Akt1 is a major downstream signaling molecule in MCF-7 cells (30, 32, 35), we further investigated in vivo regulation of Akt in a xenograft model of human breast cancer growth. Hormone-responsive breast tumors can be formed in ovariectomized athymic mice using MCF-7 cells and estrogen supplementation (36, 37). Female ovariectomized athymic mice implanted with MCF-7 cells are also an appropriate model of postmenopausal women with estrogen-dependent breast cancer because plasma levels of estradiol in these mice are similar with those observed in postmenopausal women (27–38 and 10–40 pg/ml, respectively) (38, 39).

MCF-7 cells (parental and stably transfected with the empty vector, CMV, K179M-Akt1, R25C-Akt1, or myr-Akt1) were bilaterally inoculated into ovariectomized female nude mice. Tumor formation was monitored after estradiol treatment in the presence or absence of tamoxifen or AG825 (Fig. 1Go). Tumors were visible from 23 to 30 d after estradiol supplementation in mice inoculated with parental or CMV-transfected cells (Fig. 1Go, A and B). Tumor volume increased by 59- (P = 0.0013) and 29-fold (P < 0.0001) the next 68 d (Fig. 1AGo) and 52 d (Fig. 1BGo), respectively. Tamoxifen and AG825 caused a marked reduction in tumor development, with 93% (P = 0.0031) and 96% (P = 0.004) reduction in tumor size, respectively, in MCF-7 parental cells and with 88% (P < 0.0001) and 81% reduction (P < 0.0001), respectively, in CMV-transfected cells.


Figure 1
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  		FIG. 1. Effect of estrogen, tamoxifen, and AG825 on MCF-7 (parental or stably transfected with CMV and Akt1 mutants) tumorigenesis. Female ovariectomized (ovx) athymic nude mice were inoculated with MCF-7 cells [parental (A) and stably transfected with the empty vector CMV (B), the constitutively active Akt1 mutant, myr-Akt1 (C), and the dominant-negative Akt1 mutants K179M-Akt1 (D), and R25C-Akt1 (E)] on d 0 and randomly placed into six groups. One group received vehicle (DMSO) (designated control, C), another group treatment with a 17ß-estradiol (E2) pellet, a third group was tamoxifen treated (Tam) (500 µg/d, Monday through Friday, sc injection), a fourth group received AG825 treatment (AG825) (50 µg/d, ip injection), a fifth group was treated with estradiol in the presence of tamoxifen, and a sixth group received estradiol and AG825. Tumor volumes were determined at the times shown. Number of animals per group (n) are: A, D, and E, n = 8; B and C, n = 8–10; mean values ± SD. Statistical analysis was performed as described under Materials and Methods.

 
Additionally, growth of MCF-7 cells transfected with K179M-Akt1 or R25C-Akt1 cells after estradiol supplementation was markedly impaired [95% (P < 0.0001) and 98% (P < 0.0001) reduction, respectively] (Fig. 1Go, D and E). In two experiments, no tumors were formed until d 68 and 104, respectively, when ovariectomized mice were inoculated with K179M-Akt1 and supplemented with an estradiol pellet. In a third experiment, however, small tumors appeared, presenting a 3-fold increase in volume at d 101, compared with non-estradiol-treated animals. Tumor growth was substantially delayed because tumors were formed at 72 d upon estradiol treatment. When we used R25C-Akt1, in one experiment, no tumors were formed by d 68 in the presence of the estradiol pellet, whereas in another experiment tumors appeared in some animals at d 38 and represented a 82.9% inhibition when compared with CMV-inoculated and estradiol-treated animals.

In ovariectomized animals, myr-Akt1 cells did not show tumor development in the absence of estradiol. Only upon estradiol supplementation, tumors appeared after 20–34 d and showed a 17-fold increase in tumor volume (P < 0.0001) (Fig. 1CGo). AG825 inhibited the effect of estradiol, resulting in 94% (P < 0.0001) reduction in tumor volume.

These results implicate Akt1 and ErbB2 action as important mechanisms in tumorigenesis of MCF-7 cells in ovariectomized athymic nude mice.

AG825 inhibits the growth of established tumors in ovariectomized animals
It has been shown that estrogen withdrawal results in a partial regression of MCF-7 tumors due to an inhibition of cell proliferation and induction of apoptosis (40). To determine whether AG825 can also inhibit the growth of established tumors, another xenograft protocol was used. MCF-7 cells (stably transfected with empty vector and myr-Akt1) were injected into ovariectomized mice to initiate tumor formation by estrogen supplementation. Mice were divided into three treatment groups after tumors reached approximately 200 mm3 2–5 wk after inoculation: estrogen withdrawal (C), estrogen treatment in the absence (E2) or presence of AG825 (E2+AG825). A representative experiment is depicted in Fig. 2Go. AG825 suppressed the growth of established tumors in both CMV- and myr-Akt1-inoculated ovariectomized animals almost to the same extent as estrogen withdrawal. When compared with continued estrogen supplementation, the tumors in CMV- and myr-Akt1 xenografts were inhibited by 68% (P < 0.0001) (Fig. 2AGo) and 75% (P = 0.0004) (Fig. 2BGo), respectively, upon AG825 treatment. These results strongly suggest a role for ErbB2 in tumor growth.


Figure 2
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  		FIG. 2. Effects of estrogen withdrawal and AG825 on MCF-7 (CMV and myr-Akt1) tumor growth. Estrogen-supplemented ovariectomized (ovx) mice were inoculated with MCF-7 cells [stably transfected with CMV (A) and myr-Akt1 (B)]. On d 54 when tumor formation was observed, mice were randomly allocated to treatment by withdrawal of estrogen (C); continued estrogen supplementation [estradiol (E2)]; and estrogen supplementation and treatment with AG825 (E2+AG825) (50 µg/d, ip injection). Tumor volumes were determined at the times shown. A, n = 8; B, n = 4 mice/group with two sites of inoculated cells per animal; mean values ± SD. Statistical analysis was performed as described under Materials and Methods.

 
Dominant-negative Akt1 mutants and AG825 inhibit tumor weight in xenograft ovariectomized animals
At the end of each of the two xenograft protocols (for tumorigenesis or inhibition of tumor growth in established tumors), animals were killed and tumors were weighed. Two representative experiments are shown in Fig. 3Go. In Fig. 3AGo the tumor weight was measured at the end of the tumorigenesis protocol. As in the case of tumor volume, the weight of tumors from ovariectomized animals inoculated with parental MCF-7 cells increased by 36-fold (P < 0.0005) upon estrogen supplementation. A similar growth upon continuous treatment with estrogen in median tumor weight was reported earlier in the same nude mouse MCF-7 xenograft model (41). The estradiol effect was inhibited by both tamoxifen as well as AG825 by 86% (P < 0.002) and 82% (P < 0.002), respectively (Fig. 3AGo). Tamoxifen and AG825 did not have any effect on tumor weight in the absence of estrogen supplementation. Inoculation of K179M-Akt1- and R25C-Akt1-expressing MCF-7 cells into nude mice also decreased tumor weight at the end of the experiment by 87% (P = 0.0056) and 74% (P = 0.002), respectively, in estradiol supplemented mice, confirming a role for Akt1.


Figure 3
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  		FIG. 3. Effect of estrogen, tamoxifen, and AG825 on MCF-7 (parental and stably transfected with CMV and Akt1 mutants) tumor weight. A, Effect on tumorigenesis. Ovariectomized mice were inoculated with MCF-7 cells (parental and stably transfected with dominant-negative Akt1 mutants K179M-Akt1 and R25C-Akt1) on d 0 and randomly divided into different treatment groups. One group received vehicle (DMSO) treatment (designated control, C), another group received treatment with a 17ß-estradiol (E2) pellet, a third group was tamoxifen treated (Tam) (500 µg/d, Monday through Friday, sc injection), a fourth group received AG825 treatment (AG825) (50 µg/d, ip injection), a fifth group received estradiol and tamoxifen, and a sixth group estradiol and AG825. B, Estrogen-supplemented ovariectomized mice were inoculated with MCF-7 cells (stably transfected with CMV and myt-Akt1). On d 54 when tumor formation was observed, mice were randomly allocated to treatment by withdrawal of estrogen (C); continued estrogen supplementation (E2); and estrogen treatment with AG825 (E2+AG825) (50 µg/d, ip injection). A and B, Animals were killed at the end of the experiment and tumor weight was measured for each animal in each treatment group. Tumor weight was normalized to each animal body weight. n = 4–5 mice/group with two sites of inoculated cells per animal; mean values ± SD. A two-tailed unpaired Student’s t test with unequal variances was used to determine the statistical significance or differences in tumor weight among treatment groups, compared with control. P < 0.05 was deemed significant. *, P < 0.05; **, P < 0.02; ***, P < 0.0005; a, C vs. E2; b, E2 vs. E2+Tam or E2 vs. E2+AG825.

 
Similarly, AG825 was able to inhibit established tumors by 52% (P = 0.0438) and 46% (P < 0.0001), respectively, in CMV- or myr-Akt1-inoculated animals when compared with animals that received continued estrogen supplementation (Fig. 3BGo).

These results corroborate with the data obtained for tumor volumes at the end of each experiment (Figs. 1Go, A, D, and E, and 2Go) and further suggest that AG825 is as effective as tamoxifen for inhibition of in vivo tumorigenesis in CMV xenografts. In addition, in contrast to tamoxifen, AG825 was also able to inhibit tumorigenesis in myr-Akt1 xenografts.

Cells expressing a constitutively active Akt1 form tumors in intact animals
We next determined the effect of Akt1 on in vivo tumorigenesis in intact animals. MCF-7 (empty vector or myr-Akt1 transfected) cells were injected into intact mice. Tumor formation was monitored for 101 d (Fig. 4Go). In contrast to ovariectomized mice, in intact animals, tumor formation was observed after 30 d, even in the absence of estrogen supplementation when animals were inoculated with myr-Akt1 cells (Fig. 4BGo), suggesting that Akt1 is tumorigenic in a physiological estrogen environment, when the steroid hormones were not removed by ovariectomy. The increase in tumor volume was 20-fold (P = 0.0049) in myr-Akt1 xenografts when compared with CMV-injected intact animals. As expected, in CMV-treated animals, estradiol supplementation also led to a similar increase in tumor volume with the induction observed in ovariectomized animals inoculated with the same cell transfectants [14-fold (P = 0.0275 increase in tumor volume] (Fig. 4AGo), and an even greater increase, although not statistically significant (P = 0.2125), was observed in myr-Akt1-injected animals (Fig. 4BGo).


Figure 4
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  		FIG. 4. Effect of estrogen, tamoxifen, and AG825 on MCF-7 (stably transfected with CMV and myr-Akt1) tumorigenesis. Intact mice were inoculated with MCF-7 cells [stably transfected with the empty vector CMV (A) and the constitutively active Akt1 mutant myr-Akt1 (B)] on d 0 and randomly divided into two groups. One group received vehicle (DMSO) treatment (designated control, C) and another group treatment with a 17ß-estradiol (E2) pellet. Tumor volumes were determined at the times shown. n = 4 mice/group with two sites of inoculated cells per animal; mean values ± SD. Statistical analysis was performed as described under Materials and Methods.

 
Regulation of Akt, and ErbB2 does not influence animal toxicity and body weight
Inoculation of dominant-negative Akt1 mutant transfected cells (K179M-Akt1 and R25C-Akt1) and AG825 administration were well tolerated by animals for the entire time of the experiments and did not result in gross toxicity, such as altered growth of the host or any drug- or cell line inoculation-related death. Furthermore, no significant changes in body weight gain between treatment groups or between different cell transfectants were observed at the end of each experiment (data not shown).

ER-{alpha} expression in tumors is modulated by ErbB2 and Akt1
To determine the effect of AG825 on ER-{alpha} expression in tumors from animals inoculated with parental and CMV-transfected MCF-7 cells, immunohistochemical analyses were performed in paraffin sections using an anti-ER-{alpha} antibody (Fig. 5Go). All tumors were carcinomas and expressed ER-{alpha}, Akt and ErbB2. In both parental and empty vector transfected xenograft tumors, estrogen treatment led to a 49% (P < 0.0005) and 51% (P = 0.0004) decrease in ER-{alpha} expression. This effect was inhibited by 95% (P = 0.0055), 90% (P = 0.0299), and 80% (P = 0.00853), respectively, upon tamoxifen, AG825, or R25C-Akt1 treatment. In myr-Akt1-transfected xenograft tumors, ER-{alpha} expression was inhibited by 62% (P < 0.05). Estradiol treatment did not further decrease ER-{alpha} expression (63.3%, P < 0.05). Whereas AG825 did not have any effect on ER-{alpha} expression, it was able to block the effect of estradiol, suggesting that myr-Akt1 mimics the effect of estradiol in this xenograft model. These results are similar to those obtained in our previous study in vitro (26).


Figure 5
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  		FIG. 5. Effect of estrogen, tamoxifen, and AG825 on ER-{alpha} expression in MCF-7 xenograft tumors. MCF-7 cells (parental or stably transfected with CMV or myr-Akt1) were injected sc into the flanks of ovariectomized nude mice. Animals were randomly placed into groups, receiving vehicle (DMSO, C), an estradiol pellet (E2) in the presence or absence of tamoxifen (Tam) and AG825. Mice were killed after 52–104 d. Tumors were kept in 10% formalin, and paraffin sections from each tumor were prepared. Immunohistochemical staining was performed using an anti-ER-{alpha} antibody. The immunohistochemical staining intensity was categorized into: – = negative; 1+ = weak; 2+ = moderate; and 3+ = strongly positive. The final score resulted from the product of staining intensity and percentage of stained cells, mean values ± SD. Statistical analysis was performed as described in Fig. 3Go.

 
ER-{alpha} activity in tumors is modulated by ErbB2 and Akt
To determine the effect of AG825 on ER-{alpha} activity in tumors from animals inoculated with MCF-7 cells (stably transfected with CMV or AKT1 mutants), immunohistochemical analyses were performed in paraffin sections using an anti-PR antibody (Fig. 6Go). Staining intensity was categorized into negative (–), weakly positive (+1), moderately positive (2+), and strongly positive (≥3+). The final score resulted from the product of staining intensity and percentage of stained cells and was compared with percent vehicle-treated cells (percent control, C). ErbB2 staining showed that tumors had low ErbB2 levels, similar to parental MCF-7 cells. In tumors from CMV xenografts, PR expression was 55-fold (P < 0.0005) elevated upon estrogen supplementation when compared with no estrogen treatment. Tamoxifen and AG825 inhibited this effect by 89% (P = 0.0003) and 82% (P < 0.0001), respectively. R25C also inhibited PR expression by 77% (P = 0.0038). These results suggest that ER-{alpha} activity can be modulated by Akt1 and ErbB2.


Figure 6
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  		FIG. 6. Effect of estrogen, tamoxifen, and AG825 on ER-{alpha} activity, and Akt phosphorylation in MCF-7 xenograft tumors. MCF-7 cells (stably transfected with CMV or R25C-Akt1) were injected sc into the flanks of ovariectomized nude mice. Animals were randomly placed into groups, receiving vehicle (DMSO, C), an estradiol pellet (E2) in the presence or absence of tamoxifen (Tam) and AG825. Mice were killed after 52–104 d. Tumors were kept in 10% formalin, and paraffin sections from each tumor were prepared. Immunohistochemical staining (IHC) was performed using an anti-PR, Akt, pAkt, or ErbB2 antibody. Cellular localization of PR, ErbB2, Akt, and pAkt was also determined (A). The immunohistochemical staining intensity in each cellular compartment was categorized into: – = negative; 1+ = weak; 2+ = moderate; and 3+ = strongly positive. The final score resulted from the product of staining intensity and percentage of stained cells (B). Statistical analysis was performed as described in Fig. 3Go. cyt, Cytoplasmic; nucl, nuclear.

 
Tumor formation is accompanied by elevated Akt activity and can be inhibited by AG825
To determine Akt activity in the xenograft tumors, immunohistochemical analyses were performed using an antiphospho-Akt antibody as well as an antitotal Akt antibody (Fig. 6Go). Akt was both localized in the cytoplasm and in the nucleus. A statistically significant increase (5-fold) (P = 0.0135) was observed for cytoplasmic pAkt level in tumors from estrogen supplemented CMV-inoculated ovariectomized nude mice when compared with nonsupplemented animals. Tamoxifen, AG825, and R25C-Akt1 inhibited cytoplasmic pAkt level by 48% (P = 0.0459), 66% (P = 0.0046), and 73% (P = 0.0089), respectively. Although nuclear pAkt levels presented the same trend with cytoplasmic pAkt, the results were not statistically significant.

To validate our immunohistochemical results for pAkt, Western blot analysis performed from tumor extracts from ovariectomized animals inoculated with MCF-7 cells (stably transfected with CMV or myr-Akt1) without or with an estradiol pellet in the presence or absence of AG825 were also analyzed for Akt phosphorylation and compared with the parental cell line, MCF-7, nontreated or treated with estradiol or heregulin-ß1 (HRG-ß1) (Fig. 7AGo). Similar to cells grown in vitro, tumors growing in the presence of estradiol in CMV-inoculated animals as well as myr-Akt1 inoculated animals without estradiol supplementation had high Akt phosphorylation. This phosphorylation was significantly reduced when animals received estradiol plus AG825, suggesting that loss of Akt phosphorylation was paralleled by inhibition of tumor growth. In tumors from xenografts of MCF-7 cells stably transfected with CMV, phosphorylation of Akt was accompanied by phosphorylation of ErbB2 and ErbB3 (Fig. 7BGo), and AG825 treatment also inhibited ErbB2 and ErbB3 phosphorylation. These results suggest that, similar to MCF-7 cells in vitro (26), estradiol treatment results in dimerization and activation of the ErbB2*ErbB3 complex.


Figure 7
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  		FIG. 7. Effect of estrogen and AG825 on Akt phosphorylation in MCF-7 xenograft tumors. Western blot analysis of parental MCF-7 cell extract from vehicle-treated (C) or treated with estradiol (E2) or HRG-ß1 (A and C) or tumor extracts from ovariectomized (ovx) nude mice inoculated with MCF-7 cells stably transfected with CMV and receiving an estradiol pellet (E2) in the presence or absence of AG825 or with myr-Akt1 and not receiving an estradiol pellet (A–C). Numbers represent different animals receiving the treatments shown. Representative immunoblots: A and B, lanes 1–3, MCF-7 cells, nontreated (1 ) or treated with E2 (2 ) and HRG-ß1 (3 ); lanes 4–17, tumor cell extracts from ovariectomized nude mice; lanes 4, 5, and 8–17, tumor cell extracts from ovariectomized nude mice inoculated with MCF-7 cells stably transfected with CMV and treated with vehicle (C) (4 and 5 ), an estradiol pellet in the absence (8 , 9 , and 12 13 14 ) or presence of ip injection with AG825 (10 , 11 , and 15 16 17 ); lanes 6 and 7, tumor cell extracts from ovariectomized nude mice inoculated with MCF-7 cells stably transfected with myr-Akt1 and treated with vehicle (C). C, The ratio between pAkt and actin was determined by densitometry and results are presented as percent of control cells ± SD. A two-tailed unpaired Student’s t test was used to determine the statistical significance in pAkt among treatment groups. P <0.05 was considered significant. *, P < 0.05; ***, P < 0.0005; a, CMV vs. E2 or myr-Akt1; b, E2 vs. E2+AG825.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Athymic female nude mice were used to investigate the effects of the selective ErbB2 inhibitor, AG825, and Akt1 mutant-transfected cells (MCF-7-parental and stably transfected cells with empty vector, CMV; constitutively active Akt1, myr-Akt1; and dominant-negative-Akt1, K179M-Akt1 and R25C-Akt1) on in vivo growth. Overall, AG825 treatment and use of both dominant-negative Akt1 mutant-transfected cells inoculated into animals resulted in inhibition of tumor growth (Figs. 1–4GoGoGoGo). The growth-inhibitory effects of AG825 and the dominant-negative Akt1 mutant-transfected cells correlated with decreased Akt phosphorylation in tumors (Figs. 5Go and 6Go), a decrease in PR expression (Fig. 6Go), and an increase in ER-{alpha} expression (Fig. 5Go). In intact animals without estradiol supplementation but not in ovariectomized ones, myr-Akt1 promoted tumorigenesis (Fig. 4Go). Collectively, these results reinforce the concept that cross talk among estradiol, ER-{alpha}, and Akt1 signaling occurs in vivo.

Akt is a central effector of many signaling pathways in breast cancer, involving ER-{alpha}, ErbB2, and EGFR (35, 36, 37). AKTs are amplified in breast cancer (38) and have elevated activity and/or expression [in ER-negative breast cancers, AKT3 (42); MCF-7, AKT1 (41), and 40–94% of human tumors, AKT1 and AKT2 (17, 43)]. High activity of AKT in breast cancer was associated with poor prognosis (43) and pathological phenotype (44), and hormone and chemotherapeutic resistance (45). Although expression of constitutively active Akt (T308D and S473D, Akt-DD) interfered with normal mammary gland development by attenuating apoptosis, tumors were not observed in transgenic mice carrying the Akt-DD transgene (46). However, coexpression of Akt-DD with a middle T antigen, decoupled from PI 3-K resulted in a dramatic acceleration of mammary tumorigenesis (44). This suggests that tumorigenesis requires the constitutive activation of other signaling pathways that are recruited by the mutant polyoma virus middle T antigen, PyVmT, oncogene. Similarly, in our study, constitutively active Akt (myr-Akt1) was not able to induce tumor formation in ovariectomized animals without estrogen priming (Fig. 1Go). These results are also corroborated by other recent studies that suggested that overexpression of AKT1 was not enough to induce transformation or tumorigenesis (47). Isografts of PDK1- or protein kinase C{alpha}-expressing COMMA-1D mouse mammary epithelial cells but not AKT1-expressing cells in syngeneic mice led to formation of poorly differentiated breast carcinomas. Moreover, constitutively active AKT3- but not AKT1-expressing MCF-7 cells produced tumors in ovariectomized nude mice in the absence of estradiol with a reversal of estrogen and tamoxifen response (48). However, in intact animals, we were able to observe tumor growth when animals were inoculated with myr-Akt1 cells in the absence of estradiol treatment (Fig. 4Go). These results are in agreement with the findings reported by deGraffenried et al. (49). They have also shown that MCF-7 cells expressing myr-Akt1 could proliferate under reduced estrogen conditions, being resistant to the growth inhibitory effects of tamoxifen (49).

Mammary gland-directed expression of either constitutively active or wild-type AKT1 in transgenic animals has failed to induce a malignant phenotype (38, 50, 51), suggesting that additional factors or signaling pathways are required for tumorigenesis. Nevertheless, introduction of antisense AKT2 or dominant-negative forms of AKT1 can inhibit MCF-7 (52) or ZR75–1 (53) tumor formation in nude mice. In our study, we demonstrate herein that both dominant-negative Akt1 mutant (K179M-Akt1 and R25C-Akt1)-containing MCF-7 cells, implanted into ovariectomized animals, blocked tumor formation. These results were also confirmed by Beliakoff et al. (54) using other Akt inactive mutants, T308A and S473A. They showed that the Akt inhibitor geldamycin and its clinically relevant derivative, 17-allyl amino-17-demethoxygeldamicin, led to inhibition of MCF-7 and tamoxifen-resistant MCF-7 tumor xenografts in severe combined immunodeficient mice. Introduction of an Akt-kinase dead mutant directly into tumor cells can also inhibit tumor formation in nude mice (52). However, these effects were not observed in normal cells (53) or the MCF10 normal counterpart (52). Thus, Akt inhibition may not be toxic to normal cells. In our study, we also did not observe toxic effects or any significant changes in body weight gain upon inoculation of MCF-7 cells stably transfected with dominant-negative Akt1 mutants into nude mice. These results suggest that Akt is an integral component of the phenotypic signaling during tumorigenesis and an important signaling node that regulates many downstream effectors. Therefore, targeting the PI 3-K/Akt1 pathway simultaneously with ER-{alpha} signaling may lead to inhibition of in vivo tumorigenesis in not only hormone-dependent breast cancers but also hormone-resistant forms of this disease.

Deregulation of ErbB2 is also frequently associated with aggressive clinical course of breast cancer, decreased disease-free survival, poor prognosis, and development of hormone independence as well as increased metastasis (48). Treatment with a combination of tamoxifen and an ErbB2 kinase inhibitor, AG 1478, reduced tumor MAPK activity in ErbB2-overexpressing MCF-7 xenografts in athymic mice (19). These results suggest that inhibition of ErbB2 and MAPK signaling may enhance tamoxifen action and abrogate antiestrogen resistance. Similarly, we demonstrate in this study that even in xenograft tumors from MCF-7 cells, which contain low concentrations of ErbB2, a selective ErbB2 inhibitor was able to block in vivo tumorigenesis (Figs. 1Go and 4Go). In addition, AG825 was also capable of inhibiting tumorigenesis when a constitutively active Akt1 construct-transfected MCF-7 cells were inoculated into ovariectomized nude mice. Therefore, AG825 is more effective than the antiestrogen tamoxifen in these cells. Moreover, AG825 significantly decreased Akt phosphorylation in these tumors (Figs. 6Go and 7Go), suggesting that ErbB2 is coupled with Akt1. Our results indicate that, in cells overexpressing myr-Akt1, the membrane localization of constitutively active Akt1 may permit a direct interaction with ErbB2 and membrane ER-{alpha}, as opposed to the known signaling of ErbB2 via PI 3-K/Akt under conditions when myr-Akt1 is not overexpressed (MCF-7 and CMV transfected cells). These two different pathways may either be alternative or concurrent, their balance depending largely on cell conditions. In agreement with our findings, MDA-MB435 cells stably transfected to express a constitutively active ErbB2 also led to increased Akt phosphorylation (55). Whereas it is very clear that ErbB2 can activate Akt and that Akt activity is important for tumor growth, the ability of AG825 to block growth of tumors containing myr-Akt1 strongly suggests that other signaling pathways, independent of Akt, could also be activated by ErbB2.

We have previously shown that in MCF-7 cells, the blockade of ErbB2, but not the EGFR signaling, inhibits the effect of estradiol on ER-{alpha} expression and activity. Our in vitro results suggested that the nongenomic effects of estradiol are coupled and/or synergize with its genomic effects (26). In this study, we demonstrate that similar to our in vitro results, the effect of estradiol on ER-{alpha} expression and activity in xenograft tumors, can be abrogated by the selective ErbB2 inhibitor (Figs. 5Go and 6Go). AG825 inhibited ER-{alpha} decrease and PR induction. Additionally, R25C-Akt1 cells inoculated into nude mice also inhibited PR expression (Fig. 6Go), suggesting that both ErbB2 and Akt1 can modulate ER-{alpha} expression and activity. Our results show for the first time that the ErbB2/PI 3-K/Akt signaling pathway plays a major role in estrogen-mediated signaling in vivo and that activation of this pathway may therefore set the stage for later genomic actions of estradiol. In our model, the nongenomic estradiol effect may complement and/or synergize with its genomic actions. Therefore, simultaneous interruption of the ErbB2 and Akt1 signaling pathways may enhance the inhibitory effects of antiestrogens on both ER-{alpha}-mediated transcription and tumor cell proliferation.


   Acknowledgments
 
We thank A. Foxworth, C. Benitez, and S. Abdulah for technical assistance with the animal studies; Vernon Daily with immunohistochemistry; and Drs. S. Byers and R. Clarke for critical reading of the manuscript.


   Footnotes
 
Support for tissue culture, animal, and histopathology facilities was provided by PA50-CA-58185 and P30-CA-51008.

Disclosure Summary: The authors have nothing to disclose.

First Published Online November 30, 2006

1 K.L. and A.D.W contributed equally to the manuscript. Back

Abbreviations: DMSO, Dimethylsulfoxide; EGFR, epidermal growth factor receptor; ER, estrogen receptor; ErbB2, epidermal growth factor 2; HRG-ß1, heregulin-ß1; IMEM, improved MEM; PDK, phosphoinositide-dependent kinase; PI 3-K, phosphatidylinositol 3-kinase; PR, progesterone receptor.

Received August 28, 2006.

Accepted for publication November 22, 2006.


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