Apoptosis DOI 10.1007/s10495-015-1111-7

ORIGINAL PAPER

Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells Haiyan Chen • Ji-ping Wang • Richard J. Santen Wei Yue

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Ó Springer Science+Business Media New York 2015

Abstract Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45a. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45a causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type

Electronic supplementary material The online version of this article (doi:10.1007/s10495-015-1111-7) contains supplementary material, which is available to authorized users. H. Chen  J. Wang  R. J. Santen  W. Yue (&) Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, P. O. Box 801416, Charlottesville, VA 22908, USA e-mail: [email protected] Present Address: H. Chen Department of Hematology, Dongfang Hospital Affiliated to Beijing University of Chinese Medicine, No. 6, Fangxinyuan, Fengtai District, Beijing 100078, China

MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45a. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy. Keywords Apoptosis  AMPK  FoxO3  Long-term estrogen deprivation  Estradiol

Introduction Patients with advanced estrogen receptor (ER) positive breast cancer frequently experience initial benefit from hormonal therapy with tamoxifen, aromatase inhibitors or surgical oophorectomy but secondary resistance invariably develops. Tumors begin to re-grow within a period of 12–18 months on average and become more aggressive with a higher metastatic potential upon recurrence. In a similar fashion, resistance also develops during adjuvant hormonal therapy. Signaling pathway up-regulation, a driver of proliferation, provides an adaptive mechanism for development of resistance [1]. A common strategy targets molecules that are specifically up-regulated during endocrine therapy to treat endocrine resistant, recurrent breast cancer. However, cancer cells rapidly adapt by using alternative signaling pathways so that they can evade each sequential therapy. During our studies on mechanisms of acquired endocrine resistance, we found that hormone dependent

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breast cancer cells can become hypersensitive to estradiol after long term estrogen deprivation (LTED) [2]. The hypersensitivity of LTED cells manifests itself in a proliferative response to E2 but importantly, also in apoptotic cell death in response to physiological concentrations of this sex steroid [3, 4]. The pro-apoptotic process appears to predominate in cells in vitro since reduction in cell number is the primary response to E2. In women, the pro-apoptotic process also occurs but only under certain conditions. Responses are limited to those developing breast cancer more than 5 years after the onset of menopause [5]. This clinical circumstance may represent the biologic equivalent of ‘‘long term estradiol deprivation’’ which serves as our in vitro model. We postulate that appropriate use of the pro-apoptotic effects of E2 in women would extend the therapeutic armamentarium useful for advanced, hormone dependent breast cancer. Our prior studies and those of others have shown that estradiol induced apoptosis in LTED cells is caspase-dependent and may involve both intrinsic and extrinsic death pathways [3, 4]. We have also demonstrated up-regulation of pro-apoptotic factors such as Bim, Bok, and Noxa and down regulation of anti-apoptotic factor, Mcl-1 in LTED cells when exposed to estradiol [6]. The causative mechanisms underlying these changes in molecular mediators remain unclear but are under intensive study by several laboratory groups. Our recent data suggest that E2 induces apoptosis through activation of AMPK and its downstream targets, FoxO3 and C-terminal binding protein 1 (CtBP-1), which is the focus of the current report. The FoxO family of forkhead transcription factors regulates target genes involved in glucose metabolism, cell cycle arrest, stress resistance and cell death. In particular, the expression of constitutively nuclear forms of FoxO proteins triggers cell death [7]. The proapoptotic factor Bim is an important target gene of FoxO and Fas ligand (FasL), that triggers extrinsic apoptosis, is also upregulated by FoxO. The expression of active forms of FoxO family members promotes cell cycle arrest at the G1/S and G2/M boundary. FoxO3 regulates the expression of DNA damage-inducible protein 45 (Gadd45), a factor that mediates G2/M arrest [8]. Based on these findings, we reasoned that the FoxO family of transcription factors might be particularly important in the pro-apoptotic effects of E2 in LTED cells because three FoxO target genes, Bim, FasL, and Gadd45a are also upregulated by E2 in these cells. FoxO proteins are mainly regulated by phosphorylationdependent nuclear/cytoplasmic shuttling. Both the PI3K/ Akt and AMP activated protein kinase (AMPK) pathways regulate FoxO3 but in opposite directions. Phosphorylation of FoxO proteins by Akt results in their cytoplasmic localization and promotion of cell survival [9]. In contrast, phosphorylation of FoxO3 by AMPK, a key energy sensor,

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promotes nuclear translocation of FoxO3 and increases its transcriptional activity [10]. FoxO3A appears to be specifically important in certain neoplasms such as colorectal cancer in which the cell death induced by cisplatinum requires FoxO3 for this effect. Overexpression of FoxO3A has been shown to inhibit tumor growth in vitro and in vivo in breast cancer cells [11, 12]. Cytoplasmic localization of FoxO3A seems to correlate with poor survival in patients with breast cancer. An additional mechanism by which AMPK may enhance apoptosis involves deactivation of the transcription repressor, C-terminal binding protein (CtBP1) which results in up regulation of the pro-apoptotic factors Bax and Noxa [13]. Our current studies rest on the postulate that activation of the AMPK pathway mediates the pro-apoptotic effect of estradiol in hormone-dependent breast cancer after long term estrogen deprivation. We demonstrated that in LTED cells E2 stimulates AMPK activation and transcriptional activity of FoxO3 that results in up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45a. Treatment of wild type MCF-7 cells with the AMPK activator, AICAR, induced similar biological and molecular responses confirming the role of AMPK/FoxO3 axis in apoptosis.

Materials and methods Materials Estradiol (E2) was purchased from Steraloids (Newport, RI), ICI 182,780 (ICI) from Tocris Bioscience (Minneapolis, MN) and rapamycin from Sigma. Sources of antibodies for Western analysis and immunofluorescent microscopy are as follows: Thr172 phosphorylated and total AMPK antibody, FoxO3, Bim, FasL, and Bax were from Cell Signaling Technology (Beverly, MA). Antibodies against Gadd45a, Lamin A/C, and LDH-A were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against bactin was from Sigma Aldrich (St. Louis, MO). Near infrared dye conjugated secondary antibodies for western analysis were purchased from LI-COR INC (Lincoln, NE). Cell culture medium, Improved Minimum Essential Medium (IMEM) was from Cellgro through Fisher Scientific (Pittsburgh, PA). Fetal bovine serum, glutamine, and trypsin were from Invitrogen (Carlsbad, CA). MitoTracker Red CMXRos, Hoechst33324, and Alexa Fluor 488 goat antirabbit IgG were from Molecular Probes (Eugene, Oregon USA). All chemicals were obtained from Sigma. Assay for apoptosis Apoptosis was measured using the Cell Death Detection ELISA kit (Roche Diagnostics, Indianapolis, IN) following

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the manufacturer’s instructions. Briefly, cells were plated into 12-well plates at the density of 8 9 104 per well. Two days later, the cells were treated with testing compounds for desired periods of time. The cell lysates were prepared by incubation of the cell monolayer with 0.5 mL lysis buffer at room temperature for 30 min followed by centrifugation at 14,000 rpm for 10 min at 4 °C. A parallel set of plates with identical treatment was prepared for cell counting. The result was expressed as absorbance at 405 nm normalized by cell number. Determination of cell number Cells grown in dishes were rinsed twice with saline. Nuclei were prepared by sequential addition of 2 mL HEPESMgCl2 solution (0.01 mol/L HEPES and 1.5 mmol/L MgCl2) and 0.2 mL ZAP solution [0.13 mol/L ethylhexadecyldimethylammonium bromide in 3 % glacial acetic acid (v/v)], and were counted using a model Z1 Coulter counter. Assay for cellular proliferation Cell proliferation was determined by the rate of BrdU incorporation into DNA using the Cell Proliferation ELISA kit (colorimetric) from Roche (Indianapolis, IN). Cells (104 cells per well) were cultured in a 96-well plate with a volume of 100 lL/well in a humidified atmosphere at 37 °C for 1 day. Then the cells were treated with various test substances for 24–48 h. Two hours before the ending of the treatment, 10 lL/well BrdU labeling solution was added and incubated for 2 h at 37 °C. The labeling medium was removed and FixDenat (200 lL/well) was added to each well and incubated for 30 min at room temperature. The cells were then incubated for 90 min with 100 lL/well anti-BrdU-POD antibody followed by color development. Then we added 100 lL/well Substrate solution, incubated until color development was sufficient for photometric detection and measured the absorbance at 405 nm. Subcellular protein fractionation Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology Inc., Rockford, IL) following the manufacture’s instruction. Immunoblotting Cells grown in 60-mm dishes were washed with PBS, incubated on ice for 5 min with 0.5 mL lysis buffer [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate,

2.5 mmol/L sodium pyrophosphate, 1 % Triton X-100, 1 mmol/L b-glycerophosphate, 1 lg/mL leupeptin and aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)], pulse sonicated, and centrifuged at 14,000 rpm for 10 min at 4 °C. Cell lysates were stored at -80 °C until analysis. Total protein content of the lysate was determined by a standard Bradford assay using the reagent from BioRad Laboratories (Hercules, CA). Fifty micrograms of total protein were separated on 10 % SDS polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was probed with primary antibodies dissolved in TBS containing 5 % bovine serum albumin followed by incubation with IRDye conjugated secondary antibody (LiCor Inc., Lincoln, NE). Protein bands were visualized and quantified by Odyssey image scanner (Li-Cor Inc., Lincoln, NE). Immunofluorescent microscopy Cells grown on sterile glass cover slips in 6-well plate were rinsed with PBS and then incubated with MitoTracker (50 nM) in 1 % DCC-IMEM for 30 min at 37 °C. The cells were then fixed in 4 % paraformaldehyde in PBS at room temperature for 30 min. Cells were permeabilized in cold acetone for 5 min at -20 °C. After washing with PBS (5 min twice at room temperature), the cells were incubated with 5 % normal goat serum (NGS) in PBS for 1 h. Then, cells were incubated with primary antibodies overnight at 4 °C, washed three times in PBS, and then incubated with 488-Alexa-labeled (green) secondary antibodies (Molecular Probes, Eugene, OR). Nuclear staining was carried out in some experiments on coverslips incubated with Hoechst33324 (0.2 lg/mL in PBS) for 10 min before mounting. Images were taken using an Olympus IX81 microscope and SlideBook software. Real time PCR analysis Total RNA was extracted and purified using Qiagen RNeasy Mini Kit. cDNA was synthesized using Bio-Rad cDNA Synthesis Kit. Transcription of Bim, Gadd45a, and FasL genes was determined by qPCR using SYBR Green method. GAPDH was used as a house keeping gene for quantification. Relative mRNA copies were compared to vehicle control using DDCt method. The primer pairs used for qPCR were as follows. Bim: Forward 50 -CTGCAGTATGCGCCCA GAGAT-30 and Reverse 50 -CACCAGGCGGACAATGT AACG-30 ; Gadd45a: Forward 50 -CGCCTGTGAGTGA GTGC-30 and Reverse 50 -CTTATCCATCCTTTCGGTC TT-30 ; FasL: Forward 50 -CTTTCCTCCTTGATTTCTTCA TTCA-30 and Reverse 50 -GAAGGCCTAGCAAAGGCA GA-30 ; GAPDH: Forward 50 -ACCCACTCCTCCACCTT TG-30 and Reverse 50 -CTCTTGTGCTCTTGCTGGG-30 .

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Results AMPK activation To determine the effect of E2 on AMPK activation, LTED and wild type MCF-7 cells (noted hereafter as MCF-7 cells) were treated for 24 h in 5 % DCC-IMEM with various concentrations of estradiol. Phosphorylation at Thr172 of AMPK detected by western analysis served as an indicator of AMPK activation. As shown in Fig. 1, treatment with E2 increased AMPK phosphorylation in LTED cells in a dose-dependent fashion. In striking contrast, E2 inhibited AMPK phosphorylation dose-dependently in wild type MCF-7 cells. It should be noted that the basal levels of phosphorylated AMPK in MCF-7 cells were much higher than that of LTED cells. This is probably a response of MCF-7 cells to estrogen deprivation due to the shift of the culture medium from 5 % FBS to 5 % DCC. FoxO3: Phosphorylation of FoxO3 by AMPK leads to upregulation of its transcription activity which induces expression of several important factors involved in regulation of apoptosis and proliferation such as Bim, FasL and Gadd45a. As LTED cells respond to E2 with enhancement of AMPK and in MCF-7 cells a decline in this activity, we compared Bim, FasL and Gadd45a expression in response to E2 in LTED and MCF-7 cells. Consistent 0

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with the effects expected in MCF-7 cells, E2 did not increase Bim and Fasl expression in these cells whereas these factors were stimulated in LTED cells (Fig. 2). As expected with the increased expression of Bim, FasL, and Gadd45a, FoxO3 levels were increased in LTED cells but not in MCF-7 cells (Fig. 2 and Fig. S1). A major component of regulation of FoxO3 activity is the shuttling between nucleus and cytoplasm. Nuclear translocation of FoxO3 is associated with an increase of its transcriptional activity. To assess its intracellular distribution, we examined localization of FoxO3 in LTED cells using immunofluorescent microscopy. MitoTracker was chosen as a counterstain to outline the nucleus without confounding FoxO3 staining. FoxO3 in vehicle treated cells was evenly distributed in the cytoplasm and nuclei (Fig. 3a). Ten-hour treatment with a physiological concentration of E2 promoted nuclear translocation of FoxO3 (Fig. 3a and Fig. S2). The percentage of cells with FoxO3 mainly located in the nucleus was increased by E2 from 52.2 to 86.9 % (p \ 0.005). Concomitant treatment with E2 plus the selective estrogen receptor down regulator (SERD), ICI 182780, antagonized the E2-induced nuclear translocation of FoxO3 (Figs. 3a, b) (p \ 0.005). E2 promoted nuclear translocation of FoxO3 was confirmed by western blot analysis of nuclear and cytoplasmic fractionation shown in Fig. 3c. Correspondingly, the levels of messenger RNA of Bim, Gadd45a and FasL were upregulated by E2 (Fig. 3d). We showed earlier that E2 increased the phosphorylation of AMPK, nuclear localization of FoxO3 and the expression of FoxO3-target genes. Accordingly, we would expect the levels of FoxO3 in E2-treated LTED cells to increase. This occurred starting at 2 h, peaking at 8–16 h and remaining high at 48 h (Fig. 3e). As expected, E2 did not increase the level of FoxO3 in MCF-7 cells (Figs. 2 and S1).

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Fig. 3 Intracellular distribution and function of FoxO3 in LTED cells. a Fluorescent microscopy images of LTED cells stained with FoxO3 antibody and MitoTracker after 10 h treatment with E2 (10-10 M) plus or minus ICI 182, 780 (10-8 M) (960, water medium). b Quantification of the cells with FoxO3 distributed in the nuclear and cytoplasmic areas. For each slide, about 200 cells in 15–25 fields were counted. Differences in FoxO3 distribution in LTED cells with different treatments were compared using v2 analysis. The experiment was repeated twice with similar results. c Western blots of FoxO3 of nuclear and cytoplasmic fractions of LTED cells treated with or without E2 for 10 h. d Effects of E2 (24 h treatment) on mRNA expressions of Bim, Gadd45a, and FasL (average fold increase of three experiments each). e Time course of changes in protein levels of FoxO3 and its downstream targets, Bim, FasL and Gadd45a, in LTED cells exposed to E2 (10-8 M)

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Our prior studies [6] have shown that in LTED cells E2 stimulates expression of Bim, a pro-apoptotic factor that is regulated by FoxO3. In this study, we further examined the effect of E2 on expression of two other targets of FoxO3, FasL and Gadd45a. We found that FasL and Gadd45a were increased by E2 in a time dependent fashion in LTED cells (Fig. 3e). Expression of all three targets of FoxO3 increased at 4 h of E2 treatment and remained at high levels at 48 h. Our studies had shown that E2 did not stimulate apoptosis in MCF-7 cells, presumably due to the lack of

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stimulation of AMPK. However, we reasoned that this lack of response could be due to factors other than AMPK. To refute this possibility, we used the known AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), in MCF-7 cells to see if similar apoptotic effects could be induced as seen in E2 treated LTED cells. As shown in Fig. 4a, there was a dose-dependent increase in apoptosis of MCF-7 cells in response to AICAR. Consistent to what was observed in E2-treated LTED cells AICAR treated MCF-7 cells displayed increased phosphorylation of AMPK and increased levels of FoxO3. As a

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consequence of FoxO3 activation, Bim, FasL and Gadd45a were upregulated (Fig. 4b). AICAR treatment also increased the levels of Bax (both 20 kd and 18 kd isoforms, Fig. 4b) and mitochondrial localization of Bax (Fig. 4b). These data provided strong support for the role of AMPK and its downstream targets in apoptosis in LTED cells. To demonstrate that the effects observed were consistent across different cell lines, we examined the effect of AICAR in LTED cell line developed in our laboratory (LTED) and an LTED clone from Dr. Jordan’s (MCF-7-5C) [14]. Exposure of these two additional cell lines to AICAR resulted in apoptosis as seen in wild type MCF-7 cells (Fig. 5a). Interestingly, the apoptotic effect was more potent in LTED and MCF-7-5C cells at lower concentrations of AICAR than in MCF-7 cells. In contrast, cell counts of all three cell lines were consistently reduced independently of AICAR concentrations (Fig. 5b). These data suggest that in addition to apoptosis, effects on proliferation might also be taking place. Indeed, AICAR inhibited cell proliferation as determined by BrdU incorporation in LTED cells and confirmed in MCF-7-5C cells (Fig. 5c). Inhibition of cell proliferation might be a result of upregulation of Gadd45a expression. Expression of Gadd45a leads to cell cycle arrest at the G2/M phase. Our data suggest that activation of AMPK/FoxO3 pathway in hormone-dependent breast cancer cells reduces cell number by promoting apoptosis and inhibiting proliferation.

Discussion Long term deprivation of estrogen (LTED) induces serial, adaptive changes involving specific signaling pathways and epigenetic changes [1, 15] in hormone dependent breast

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cancer cells. Systematic studies in MCF-7 model systems have demonstrated several biological consequences of these adaptive changes. Some but not all of the cell lines develop hypersensitivity to the proliferative effects of very low levels of estradiol [2, 16]. More consistently, physiologic concentrations of estradiol induce apoptosis, not only in model LTED systems but also in patients treated with estrogen many years past the menopause [5]. Interestingly, estradiol inhibits apoptosis in wild type MCF-7 cells grown long term in the presence of this steroid. A series of studies have demonstrated pro-apoptotic effects of estradiol on LTED cells through extrinsic, death receptor pathways as well as intrinsic mitochondrial mediated events. The death receptor ligand, FasL, has an upstream estrogen responsive element which may partially explain its estrogen mediated increase. However, the precise mechanisms triggering both the death receptor and mitochondrial events have remained incompletely understood. Adenosine monophosphate activated protein kinase (AMPK) is the primary regulator of the cellular response to lowered ATP levels in eukaryotic cells [17, 18]. Through phosphorylation of its downstream targets, AMPK activation results in the up-regulation of ATP-producing catabolic pathways and the down-regulation of ATP-consuming processes, such as protein synthesis, to restore energy homeostasis [19]. Sustained activation of AMPK leads to cell cycle arrest and apoptosis in a variety of cells including cancer cells [20–22]. In hormone sensitive breast cancer cells, such as MCF-7, E2 stimulates proliferation and inhibits apoptosis. Accordingly, AMPK is inhibited by E2 (Fig. 1) which is consistent with what was reported by Brown and colleagues [23]. In striking contrast, E2 stimulated AMPK in LTED cells. The differential response

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Fig. 4 Activation of AMPK by AICAR in MCF-7 cells recapitulates pro-apoptotic effect of E2 in LTED cells. a AICAR induced apoptosis in MCF-7 cells (48 h treatment). *p \ 0.05, **p \ 0.005, ***p \ 0.0005 compared with vehicle control; b Western analysis of phosphorylated AMPK, FoxO3, and related pro-apoptotic factors

in MCF-7 cells exposed to various concentrations of AICAR for 24 h; c Fluorescent microscopy images of MCF-7 cells stained with Bax antibody and MitoTracker after 24 h treatment with AICAR (0.4 mM) (960, water medium). The experiments were repeated for at least three times with consistent results

to E2 on AMPK is the only effect identified to the date that distinguishes LTED cells from wild type MCF-7 cells. Our current studies suggest that the activation of AMPK in LTED cells is a key upstream mechanism for E2-induced apoptosis. The activation of AMPK leads to enhanced transcriptional activity of FoxO3, with resulting up-regulation of Bim, FasL, and Gadd45a. These results provide a novel mechanism for the pro-apoptotic effect of estrogen under the circumstances of long term estrogen deprivation. Specifically, downstream targets of FoxO3 have been shown previously to mediate pro-apoptotic effects. A major mediator of mitochondrial disruption in LTED cells is Bim. E2 increases Bim in LTED but not in wild type MCF-7 cells. As evidence of mechanistic causality, knock down of Bim using siRNA completely abolished apoptosis [6]. The other downstream target of FoxO3, FasL, also contributes

to apoptosis as shown by our previous knock down studies [6]. Other data suggest that FoxO3 is important for a number of processes regulating neoplastic tissues. FoxO3 is a member of the forkhead-box family of transcription factors that play important roles in tumor suppression, regulation of energy metabolism and stress response. Transcriptional regulation of the pro-apoptotic Bcl-2 family member Bim by FoxO3 has been reported to control apoptosis in a variety of experimental systems such as trophic factor withdrawal in neurons [24] and cytokine deprivation in lymphocytes [25]. In breast cancer cell lines, paclitaxel induced apoptosis is associated with upregulation of Bim. Cell lines that express low levels of FoxO3 are resistant to paclitaxel [26]. FoxO3 activity is regulated by both the PI3K/Akt and AMPK pathways. Akt directly phosphorylates FoxO3 and

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compared with vehicle control. The experiments were repeated for three times with consistent results

causes its relocalization to the cytoplasm and degradation. Phosphorylation of FoxO3 by AMPK, in contrast, enhances its transcription activity [10]. Enhanced FoxO3 activity was demonstrated by upregulation of three proteins encoded by FoxO3 targeted genes, Bim, FasL and Gadd45a in LTED cells exposed to E2. Activation of AMPK-FoxO3 pathway by E2 in LTED cells was recapitulated in MCF-7

by AICAR, a known AMPK activator. Because of the dual pathway regulation, the activity of FoxO3 in a given cell line is determined by the sum effect of each regulatory pathway. For example, when LTED cells are exposed to E2, AMPK and FoxO3 activation predominate and override the inhibitory effect of the PI3K/Akt pathway. In contrast, in wild type MCF-7 cells, E2 inhibits AMPK activation and

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stimulates PI3K/Akt both of which lead to down regulation of FoxO3 activity. Bim plays a critical role in E2-induced apoptosis in LTED cells. We postulated that activation of FoxO3 via AMPK serves as an important mechanism because there is no known ERE at the promoter region of the Bim gene. We have shown that ICI treatment significantly promoted distribution of FoxO3 out of the nucleus and complete blockade of E2 stimulated Bim mRNA transcription. While two effects of ICI were associated, these experiments do not exclude other potential mechanisms and further studies are required. It is necessary to be cautious in interpreting data from the experiments using ICI as a pure antiestrogen. Our prior studies and those of others have shown that ICI may exert non-genomic effect on signal transduction pathways when interacting with membrane associated ER [27]. It is not clear how AMPK is activated by E2 and more in depth investigation is warranted to dissect out precise mechanisms. It has been reported that carboxyl-terminal binding protein (CtBP) is a corepressor that selectively represses epithelial cell adhesion and proapoptotic genes including Noxa and Bax [28]. Kim et al. reported that activation of AMPK led to phosphorylation of CtBP1 on Ser158 and subsequent ubiquitination of CtBP1, which attenuates the repressive effect of CtBP on Bax expression [13]. We have shown that Noxa is up-regulated by E2 in LTED cells [6]. In the current studies, we found that Bax is increased by E2. Correlation between up-regulation of Bax and Noxa and activation of AMPK implicates the important role of AMPK in E2-induced apoptosis of LTED cells. Inhibition of mTOR activity is another result of AMPK activation which leads to shutting down of the energy consuming processes and halt of cell growth. However, inhibition of mTOR also leads to autophagy that compromise the proapoptotic effect of AMPK. This could be the explanation why the proapoptotic effect of AICAR diminished at higher concentrations (Fig. 5a). In anti-estrogen resistant breast cancer cells, switching off autophagy could resume sensitivity of these cells to the proapoptotic effect of pure antiestrogen, ICI182780 [29, 30]. Our LTED cells have shown up-regulatoion of multiple signaling pathways including the mTOR pathway during adaption to estrogen deprivation [1, 31]. We have not observed inhibition of mTOR activity in LTED cells when treated with E2 (data not shown). Enhanced basal mTOR activity might partially explain why physiological concentrations of estradiol are proapoptotic to LTED cells. Taken together, our studies have shown that long term estrogen deprived breast cancer cells become sensitive to proapoptotic effect of E2. E2 induced apoptosis in LTED cells are characterized by up-regulation of proapoptotic factors, Bim, FasL, Bax and Noxa. Activation of AMPK is

a central molecule integrating diverse mechanisms involved in regulation of these factors. Acknowledgments The current studies were supported by Department of Defense Grant (W81XWH-10-1-0030). The authors acknowledge Dr. Craig Jordan for providing MCF-7-5C cells. Conflict of interest of interest.

The authors declare that they have no conflict

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