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Endocrinology, doi:10.1210/en.2007-0756
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Endocrinology Vol. 149, No. 4 1942-1950
Copyright © 2008 by The Endocrine Society

The Regulation and Function of the Forkhead Transcription Factor, Forkhead Box O1, Is Dependent on the Progesterone Receptor in Endometrial Carcinoma

Erin C. Ward, Anna V. Hoekstra, Leen J. Blok, P. Hanifi-Moghaddam, John R. Lurain, Diljeet K. Singh, Barbara M. Buttin, Julian C. Schink and J. Julie Kim

Department of Obstetrics and Gynecology (E.C.W., J.J.K.), Division of Reproductive Biology Research, and Department of Obstetrics and Gynecology (A.V.H., J.R.L., D.K.S., B.M.B., J.C.S.), Division of Gynecologic Oncology, Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University, Chicago, Illinois 60611; and Department of Obstetrics and Gynecology (L.J.B., P.H.-M.), Erasmus University Medical Center, Rotterdam, The Netherlands 3000

Address all correspondence and requests for reprints to: J. Julie Kim Ph.D., Department of Obstetrics and Gynecology, Robert H. Lurie Comprehensive Cancer Center, 303 East Superior, Room 4-117, Northwestern University, Chicago, Illinois 60611. E-mail: j-kim4{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In many type I endometrial cancers, the PTEN gene is inactivated, which ultimately leads to constitutively active Akt and the inhibition of Forkhead box O1 (FOXO1), a member of the FOXO subfamily of Forkhead/winged helix family of transcription factors. The expression, regulation, and function of FOXO1 in endometrial cancer were investigated in this study. Immunohistochemical analysis of 49 endometrial tumor tissues revealed a decrease of FOXO1 expression in 95.9% of the cases compared with the expression in normal endometrium. In four different endometrial cancer cell lines (ECC1, Hec1B, Ishikawa, and RL95), FOXO1 mRNA was expressed at similar levels; however, protein levels were low or undetectable in Ecc1, Ishikawa, and RL95 cells. Using small interfering RNA technology, we demonstrated that the low levels of FOXO1 protein were due to the involvement of Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, given that silencing Skp2 increased FOXO1 protein expression in Ishikawa cells. Inhibition of Akt in Ishikawa cells also increased nuclear FOXO1 protein levels. Additionally, progestins increased FOXO1 protein levels, specifically through progesterone receptor B (PRB) as determined by using stably transfected PRA-specific and PRB-specific Ishikawa cell lines. Finally, overexpression of triple mutant (Tm) FOXO1 in the PR-specific Ishikawa cell lines caused cell cycle arrest and significantly decreased proliferation in the presence and absence of the progestin, R5020. Furthermore, TmFOXO1 overexpression induced apoptosis in PRB-specific cells in the presence and absence of ligand. Taken together, these data provide insight into the phosphoinositide-3-kinase/Akt/FOXO pathway for the determination of progestin responsiveness and the development of alternate therapies for endometrial cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL CARCINOMA IS the most common gynecological malignancy and the fourth most common malignancy in women in the developed world today (1). Although advances have been made in the field, it is estimated that approximately 7,400 women will die from endometrial carcinoma and 39,080 will be newly diagnosed within the next year, of which 70–80% of the cases will be type I endometrial carcinoma (2, 3). The transition from normal endometrium to carcinoma is thought to occur through a progression of alterations in genes involving cell proliferation, the inhibition of apoptosis, and angiogenesis (1). Although the chronological sequence of mutations and the final combination of defects differ significantly between type I endometrial carcinoma patients, the most common genetic changes include microsatellite instability or specific mutations in PTEN, K-ras, and β-catenin genes (3). Mutations in the PTEN gene are the most common genetic defects in endometrial carcinomas and seen in 83% of tumors (3).

PTEN, a tumor suppressor gene, is a lipid phosphatase that negatively regulates phosphoinositide-3-kinase (PI3K)/Akt-driven cell growth and survival (4). PTEN controls cell growth by dephosphorylating PI3K phosphorylation products, phosphatidylinositol-4,5 bisphosphate (PIP2) and phosphatidylinositol-3,4,5 triphosphate (PIP3), which in turn leaves Akt dephosphorylated and inactivated (4). When PTEN is mutated, Akt becomes constitutively active, inhibiting several downstream targets through phosphorylation, such as glycogen synthase kinase-3, BCLZ-antagonist of cell death, p27, and the Forkhead box O (FOXO) proteins (5).

FOXO1 is a transcription factor and a member of the FOXO subfamily of the Forkhead/winged helix family. The phosphorylation of FOXO1 by Akt leads to its inactivation through translocation from the nucleus to the cytoplasm (6, 7, 8, 9). It has also been shown in prostate cancer that FOXO1 is phosphorylated by Akt at Ser256, allowing Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, to associate and ubiquitinate the protein, targeting it to the proteasome for degradation (10). Under normal conditions, FOXO1 is constantly shuttled in and out of the nucleus, thereby contributing to the maintenance of homeostasis of the cell. Members of the FOXO family are involved in several different cellular functions such as differentiation, metabolism, proliferation, and survival (11). FOXO1 has been demonstrated to induce apoptosis through its localization to the nucleus and enhance subsequent transcription of several genes involved in the apoptotic pathway, such as BCL2-like 11, tumor necrosis factor (ligand) superfamily, member 10, Fas ligand, and TNFRSF1A-associated via death domain (12). In relation to the endometrium, it has been demonstrated that FOXO1 is an essential component in the decidualization process (13, 14, 15). It is a gene induced early during human decidualization and promotes expression of prolactin and IGF-binding protein 1 (IGFBP1) (13). In addition, it was recently demonstrated that cross talk between FOXO1 and progesterone receptor (PR) was important for decidualization (15) as well as the induction of apoptosis (16).

In this study, we demonstrate that FOXO1 expression is decreased in endometrial carcinoma. One mechanism for this decrease is by posttranslational Skp2 ubiquitination of the FOXO1 protein. Furthermore, the effect of FOXO1 on cell cycle progression and apoptosis of endometrial cancer cells is differentially influenced by PRA and PRB, supporting the importance of FOXO1 and PR cross talk in the endometrium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Endometrial cancer tissues and cell lines
Normal human endometrial tissue was obtained from hysterectomies or biopsies from premenopausal women with no clinically documented abnormalities of the endometrium. Human stromal cells (HSCs) were isolated as previously described (17). Endometrial tumors were obtained from women undergoing hysterectomies at Northwestern Memorial Hospital. Patients gave consent before surgery, and these studies were approved by the Human Subject Committee of our institution in accordance with U.S. Department of Health regulations. Ishikawa and ECC1 cells were obtained from B. Lessey (Greenville Hospital System, Greenville, SC), and Hec1B and RL95 cells were obtained from American Type Culture Collection (Rockville, MD). Ishikawa and Hec1B cells were maintained in MEM (Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate, penicillin/streptomycin, and 10% fetal bovine serum (FBS), ECC1 cells were maintained in MEM/F12 (Invitrogen) supplemented with sodium pyruvate, penicillin/streptomycin, and 10% FBS, and RL95 cells were maintained in DMEM/F12 supplemented with supplemented with sodium pyruvate, penicillin/streptomycin, 0.005 mg/ml insulin, and 10% FBS. Ishikawa cell lines that were stably transfected with PRA (PRA14), PRB (PRB23), or both PRA and PRB (PRAB36; L. Blok, The Netherlands) were maintained in DMEM/F12 supplemented with sodium pyruvate, penicillin/streptomycin, and 10% FBS. The parental Ishikawa cell line from which these stable PR cell lines were created did not express endogenous PRA or PRB (18).

Immunohistochemistry
Tissues were fixed in formalin and paraffin embedded, and 4-µm tissue sections were placed on glass slides. Tissue sections were incubated in 3% hydrogen peroxide at room temperature and rinsed with Tris-buffered NaCl solution with 0.1% Tween 20 [TBS-T; 0.2 M Tris (pH 7.6), 1.37 M NaCl]. Protein Block (Dako, Fort Collins, CO) was applied, and slides were incubated in primary FOXO1 antibody (1:1000; Bethyl Laboratories Inc., Montgomery, TX) overnight at 4 C in a humidified chamber. Slides were rinsed in TBS-T, and antirabbit secondary antibody conjugated to a dextran-labeled polymer and horseradish peroxidase (Dako) was applied. TBS-T was then used to rinse the slides twice, and diaminobenzidine (DAB) solution (Dako) was applied. Slides were rinsed in distilled water and counterstained in Mayer’s hematoxylin (Sigma Chemical Co., St. Louis, MO) followed by a rinse in running tap water. After another rinse in running distilled water, slides were incubated in ammonia water (4 ml 28% ammonium hydroxide in 1 liter distilled water) and then rinsed in tap and distilled running water. Dehydration and clearing of the slides was accomplished through two changes of 95% ethanol, two changes of 100% ethanol, and two changes of xylene (10 dips per solution). Slides were mounted using the xylene-based Cytoseal-XYL (Richard-Allan Scientific, Kalamazoo, MI). Using a multispectral imaging camera (Nuance, Burlington, MA) mounted to an Axioskop 50 microscope (Zeiss, Jena, Germany), images were acquired that simultaneously detected the overlapping spectra of the hematoxylin stain and the FOXO1 DAB stain. A component spectra library was then created based on each stain, which allowed for the separation of the hematoxylin and DAB spectra into individual channels. These unmixed, FOXO1 DAB stain images were then analyzed with Metamorph 6.0 by creating regions around the glandular epithelial cells and measuring the intensity of the FOXO1 stain per region. These regional intensities were then averaged to acquire the overall average intensity of FOXO1 stain per sample.

RT-PCR
Cells were lysed using TriReagent (Sigma), and total RNA was extracted using the manufacturer’s protocol. After RNA reverse transcriptase using 1 µg RNA per sample, PCR was performed with 2 µl cDNA and primers for the housekeeping gene 36B4 and FOXO1 as previously reported (13). The PCR consisted of a preliminary denaturing step for 3 min at 94 C, followed by 35 cycles consisting of 30 sec at 94 C, 30 sec at 55 C, and 30 sec at 72 C. A final extension for 7 min at 72 C concluded the reaction. Samples were then electrophoresed on a 1.5% agarose gel and visualized using ethidium bromide staining.

Western blot analysis
Total cell lysates were obtained by lysing cells with RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL (Sigma), 0.1% SDS] plus protease inhibitors (Sigma) on ice. Nuclear and cytoplasmic proteins were isolated using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL). Protein concentration was measured using the Micro BCA protein assay kit (Pierce). Isolated protein samples were run on 7.5 or 10% Tris-HCl acrylamide precast gels (Bio-Rad, Hercules, CA) and transferred onto polyvinylidene difluoride membranes (Whatman). Membranes were blocked in 5% nonfat milk made in TBS-T at room temperature and incubated in FOXO1 primary antibody (1:5000 from Bethyl Laboratories or 1:1000 from Cell Signaling, Beverly, MA) or p27 primary antibody (1:2500; BD Transduction Laboratories, San Jose, CA) in 1% nonfat milk (with TBS-T) overnight at 4 C. After the incubation, membranes were washed three times with TBS-T and incubated in either secondary peroxidase-conjugated goat antirabbit IgG or goat antimouse IgG (1:10,000; Bio-Rad) in 1% nonfat milk made with TBS-T. Membranes were then washed and developed with the ECL Plus Western Blot Detection System kit (Amersham, Piscataway, NJ). Membranes were stripped with Restore Western Blot Stripping Buffer (Pierce) and reprobed with a monoclonal antibody to β-actin (1:10,000; Sigma).

Immunofluorescent staining
Cells were grown on glass coverslips and treated with 12 or 24 µM Akt inhibitor IX, API-59CJ-OMe (AI-IX; Calbiochem, Gibbstown, NJ) for 48 h. Cells were fixed with 4% paraformaldehyde (Sigma), and coverslips were then washed with phosphate-buffered NaCl solution (0.138 M NaCl, 2.7 mM KCl) and permeablized with 0.1% Triton-0.1% deoxycholate (Sigma). Cells were blocked with 5% BSA (Sigma) made in PBS. Subsequently, the FOXO1 primary antibody (1:50; Cell Signaling) made in filtered 5% BSA was added to each sample and incubated overnight at 4 C in a humidified chamber. A fluorescein secondary peroxidase-conjugated goat antirabbit IgG (1:50; Vector Laboratories Inc., Burlingame, CA) was used. Cells were then mounted with Vectashield Hard Set mounting medium for fluorescence (Vector) and visualized using a fluorescent inverted microscope, Axiovert 200 (Zeiss).

Small interfering RNA (siRNA)
Cells were grown in 60-mm dishes to 50% confluence. Dharmacon (Lafayette, CO) SMARTpool siRNA specific to Skp2 was transiently transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol for siRNA. siRNA specific to the firefly luciferase protein (Dharmacon) was used as a control. Mock transfections, where no oligonucleotides were transfected, were used as a negative control. Cells were transfected for 4 h, the transfection media was removed and replaced with MEM (Invitrogen) supplemented with 2% charcoal-stripped FBS, and the cells were cultured for an additional 48 h. After incubation, the cells were harvested with RIPA buffer for Western blot analysis. Silencing of Skp2 was verified by immunoblot analysis using Skp2 antibody (1:1000; Cell Signaling).

Expression vectors and reporter gene constructs
The pPRE/GRE.E1b.Luc vector, containing the proximal promoter regions of progesterone and glucocorticoid receptors along with a luciferase gene expression region, was given to us by M. J. Tsai and B. O’Malley (Houston, TX) and constructed as previously described (19). The human triple mutant (Tm) FOXO1 expression vector was a gift from T. G. Unterman (Chicago, IL). This vector is mutated at three Akt phosphorylation sites, Thr-24, Ser-256, and Ser-319, to alanines creating a constitutively active form of FOXO1 as previously described (20). PRA and PRB cDNAs were gifts from P. Chambon (Strasbourg, France).

Cell transfection and reporter gene studies
Ishikawa cells were transiently transfected with the pPRE/GRE.E1b.Luc vector with an empty pcDNA 3.1+ vector (Promega, Madison, WI) as a control, or with TmFOXO1 and/or PRA or PRB expression vectors, along with a β-galactosidase reporter plasmid (pCMV SPORT; Promega), which was used as an internal control for normalization. After 4 h, the medium was replaced with MEM (Invitrogen) and 1% charcoal-stripped FBS with 1 µM medroxyprogesterone acetate (MPA). Cells were incubated for an additional 24 h, harvested, and then analyzed for luciferase activity using the Luciferase Assay kit (Promega). β-Galactosidase activity was measured using the β-Galactosidase Enzyme Assay kit (Promega). Normalized relative luciferase units (RLU) were calculated as luciferase units/β-galactosidase units.

Cell cycle analysis
Cells were grown in six-well plates until 50% confluency. Cells were infected with adenoviral constructs of either AD-CMV (an empty vector) or AD-TmFOXO1 (containing the cDNA encoding the constitutively active FOXO1 and created as previously described (12) at 50 multiplicity of infection (MOI) per well and incubated for 24 h. Cells were then treated with 1 µM R5020 in serum-free medium and cultured for 48 h. Cells were trypsinized and fixed with 75% ethanol for 2 h. Cells were resuspended in 1 ml propidium iodide (PI) staining solution containing 50 µg/ml PI (Sigma), 2 mg RNase A (Invitrogen), and 0.1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) made in PBS. Samples were incubated for 20 min at 37 C and analyzed for G0/G1, S, and G2/M fractions on a Coulter EPICS-XL flow cytometer (Beckman Coulter, Fullerton, CA).

Cell proliferation and viability assay
Cells were infected with 100 MOI of either AD-CMV or AD-TmFOXO1 and incubated for 24 h after which time they were treated with 1 µM R5020 for 24 and 48 h. PRB23 cells were also treated with 10 nM R5020 after infection with AD-CMV or AD-TmFOXO1 for 24 and 48 h. Using the Quick Cell Proliferation Assay Kit (BioVision, Mountain View, CA), 10 µl WST-1/ECS (electrocoupling solution) was added per well and incubated at 37 C. Samples were read on the Synergy HT from Bio-Tek (Winooski, VT) with the KC4 3.4 software at 420nm to determine cell proliferation.

Annexin V analysis
Cells were infected with 50 MOI of either AD-CMV or AD-TmFOXO1 and incubated for 24 h after which time they were treated with 1 µM R5020 for 48 h. Cells were trypsinized and resuspended in annexin-binding buffer [10 mM HEPES (Invitrogen), 140 mM NaCl, 2.5 mM CaCl2 (pH 7.4)] to a concentration of approximately 1 x 106 cells/ml. Annexin V, Alexa Fluor 647 conjugate (Invitrogen, Carlsbad, CA) and 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA) were added to each cell solution, and samples were analyzed using the CyAn flow cytometer (Dako, Fort Collins, CO) for early and late apoptosis.

Statistical analysis
Statistical analysis was performed using the paired t test, unpaired t test assuming unequal variances, and one-way ANOVA followed by the Dunnett’s multiple comparison test or Tukey’s multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FOXO1 expression in normal and malignant endometrium
The levels of FOXO1 protein were detected using immunohistochemical analysis of four normal endometrial tissues and 49 grade I and II endometrial adenocarcinomas. In normal endometrial tissue, FOXO1 staining was intense in the glandular epithelial cells with punctate staining in the stromal cells (Fig. 1AGo). In 47 of the 49 grade I and II endometrial adenocarcinoma samples (95.9%), FOXO1 protein levels were decreased (Fig. 1AGo). The intensity of the FOXO1 stain was measured in both normal endometrial tissue and grade I and II endometrial cancer tissue. The average intensity of the FOXO1 stain in grade I and II endometrial cancer tissue was significantly lower than the average intensity seen in normal endometrial tissue (Fig. 1AGo).


Figure 1
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  		FIG. 1. FOXO1 expression in endometrial cancer tissues and cell lines. A, Immunohistochemical staining for FOXO1 was done in normal endometrial tissue (n = 4) and grade I and II endometrial adenocarcinoma (n = 49) (magnification, x400). Intensity of the FOXO1 stain was measured in each sample, and the unpaired t test assuming unequal variance was used to demonstrate statistical significance. *, P < 0.05. B, FOXO1 mRNA was measured in ECC1, Hec1B, Ishikawa, and RL95 cell lines using RT-PCR. The housekeeping gene, 36B4, was used as a control. The figure is representative of four experiments. C, Whole-cell lysates were subjected to Western blot analysis for FOXO1. HSCs were used as a positive control for FOXO1, and 20 µg protein were loaded per sample. The figure is representative of three experiments.

 
The expression of FOXO1 was investigated in four endometrial cancer cell lines by RT-PCR and Western blot analysis. All cells tested, ECC1, Hec1B, Ishikawa, and RL95, expressed FOXO1 mRNA, although a slight decrease was observed in Ishikawa and RL95 cells (Fig. 1BGo). FOXO1 protein expression, however, was not detectable in any of the cells with the exception of Hec1B, compared with that of HSCs, the positive control (Fig. 1CGo). These data suggest that posttranslational degradation of the FOXO1 protein is occurring in the endometrial cancer cells.

Skp2 proteasomal degradation of FOXO1
It has previously been shown that Skp2 was responsible for the ubiquitination of FOXO1 in prostate cancer (10). Also, high levels of Skp2 correlate with poor prognosis in endometrial endometrioid adenocarcinoma (21). Skp2 protein levels were measured in the four endometrial carcinoma cell lines and in HeLa cells, used as a positive control. Skp2 expression was the most abundant in ECC1 and Ishikawa cells, with RL95 cells demonstrating weaker expression (Fig. 2AGo). Hec1B cells expressed very little Skp2 protein (Fig. 2AGo). Interestingly, Skp2 protein levels in the four endometrial cancer cell lines were the inverse of FOXO1 protein levels (Figs. 2AGo and 1CGo).


Figure 2
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  		FIG. 2. Regulation of FOXO1 by Skp2 in endometrial cancer. A, Skp2 protein levels were measured by Western blot in four endometrial cancer cell lines using HeLa cell lysates as a positive control. Twenty micrograms of protein were loaded per lane, and Skp2 antibody was used at a 1:500 dilution. The figure is representative of two experiments. B, Skp2 protein was measured after a mock transfection and transient transfection of siCONT or siSkp2 oligonucleotides in Ishikawa cells. Fifty micrograms of protein were loaded per lane, and Skp2 antibody was used at a 1:1000 dilution. The figure is representative of seven experiments. C, Immunoblotting for FOXO1 and p27 in Ishikawa whole-cell lysates mock transfected and transiently transfected with siCONT or siSkp2 was performed. Twenty micrograms of HSC cell lysate were used as a positive control for FOXO1 protein. Fifty micrograms of protein were loaded per lane. The figure is representative of seven experiments.

 
To determine whether FOXO1 was a Skp2 target molecule in endometrial cancer cells, Skp2 was knocked down using a specific siRNA oligonucleotide in Ishikawa cells. This siRNA was effective in decreasing Skp2 protein levels (Fig. 2BGo). As a consequence of the Skp2 knockdown, levels of FOXO1 protein increased similarly to that of p27, which has been demonstrated to be a target of Skp2 (Fig. 2CGo) (22). These data establish that Skp2 is involved in the decreased expression of FOXO1 protein in Ishikawa cells.

Akt inhibition on FOXO1 localization and expression in Ishikawa cells
It has previously been shown that phosphorylation of FOXO1 at Ser-256 by Akt is not only responsible for its translocation from the nucleus to the cytoplasm but also required for Skp2 ubiquitination and degradation of FOXO1 (10). In hopes to inhibit degradation and restore nuclear FOXO1 expression in Ishikawa cells, we used an Akt inhibitor to prevent phosphorylation of FOXO1. Ishikawa cells were treated with increasing amounts of an Akt inhibitor, API-59CJ-OMe (AI-IX), and FOXO1 protein was visualized with immunofluorescence. In untreated cells, weak, dispersed staining for FOXO1 protein was seen throughout the cytoplasm, with no obvious nuclear staining (Fig. 3AGo). After treatment with 12 µM AI-IX, FOXO1 protein was observed within the nucleus with weak staining in the cytoplasm (Fig. 3BGo). After treatment with 24 µM AI-IX, there was a further increase in FOXO1 staining in the nucleus (Fig. 3CGo). Additionally, Western blot analysis demonstrated an increase in FOXO1 protein expression in the nucleus after treatment with AI-IX (Fig. 3DGo). These results demonstrate an increase in nuclear FOXO1 protein expression after AI-IX treatment.


Figure 3
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  		FIG. 3. FOXO1 expression in response to an Akt inhibitor, AI-IX, in Ishikawa cells. A–C, Immunofluorescent staining for FOXO1 in Ishikawa cells was done after treatment with vehicle (A), 12 µM AI-IX (B), and 24 µM AI-IX (C) (magnification, x63). Figures are representative of three experiments. D, Nuclear and cytoplasmic protein fractions were isolated from Ishikawa cells treated with 1 µM AI-IX (+). Fifteen micrograms of HSC whole-cell lysate were used as a positive control for FOXO1. Forty micrograms of protein were loaded per lane for nuclear and cytoplasmic fractions. The figure is representative of five experiments.

 
FOXO1 and PR in endometrial carcinoma
Progesterone is considered to be inhibitory to endometrial growth and preventative for endometrial cancer. We previously demonstrated that cross talk between FOXO1 and PR was associated with decidualization of human endometrial stromal cells. To determine whether a similar interaction occurred in endometrial cancer, Ishikawa cells, which express endogenous levels of both PRA and PRB, were transiently transfected with an empty pcDNA 3.1+ vector, as the control, or with TmFOXO1 and PRB or PRA expression vectors along with a PR-responsive promoter, pPRE/GRE.E1b.Luc reporter. Although TmFOXO1 did not affect promoter activity of the pPRE/GRE.E1b.Luc compared with the control, both liganded PRA and PRB increased activity of pPRE/GRE.E1b.Luc (Fig. 4AGo). Most noteworthy was the synergistic up-regulation of pPRE/GRE.E1b.Luc by PRA or PRB with TmFOXO1, supporting the existence of an interaction between these two transcription factors and suggesting that FOXO1 potentiates the function of PR on progesterone response element (PRE) responsive genes.


Figure 4
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  		FIG. 4. Transactivation of pPRE/GRE.E1b.Luc by PR and FOXO1. A, Expression vectors for PRA or PRB were cotransfected with TmFOXO1 and the luciferase reporter pPRE/GRE.E1b.Luc in Ishikawa cells. Cells were treated with 1 µM MPA, and luciferase activity was measured after 24 h. The control sample was transfected with an empty pcDNA 3.1+ vector and treated with ethanol (vehicle control) for 24 h. B, PRAB36, PRB23, and PRA14 cells were treated with 1 µM AI-IX, 1 µM R5020, or AI-IX and R5020 in combination. Nuclear and cytoplasmic fractions were subjected to Western blot analysis for FOXO1. Fifteen micrograms of HSC whole-cell lysate were used as a positive control for FOXO1. Forty micrograms of protein were loaded per lane for nuclear and cytoplasmic lysates. The figure is representative of two experiments.

 
We continued to explore the cross talk of FOXO1 and PR by determining whether progestins influenced FOXO1 protein expression in Ishikawa cell lines stably expressing PRA (PRA14), PRB (PRB23), or both PRA and PRB (PRAB36). These cell lines were generated from a parental cell line that did not express endogenous PRA or PRB (18). In response to 1 µM R5020, the PRAB36 cells expressed higher levels of both nuclear and cytoplasmic FOXO1 protein (Fig. 4BGo). The PRB23 or PRA14 was used to decipher which PR isoform was responsible for the increase in FOXO1 protein expression after R5020 treatment. Although R5020 increased protein expression in both nuclear and cytoplasmic fractions in the PRB23 cell line, compared with the controls, R5020 treatment did not increase FOXO1 protein expression in the PRA14 cell line (Fig. 4BGo). Additionally, AI-IX treatment did not increase FOXO1 protein expression in any of the three cell lines (Fig. 4BGo).

The relationship between FOXO1 and progestins was further explored in a physiological context. The PR stably transfected Ishikawa cell lines were used to overexpress TmFOXO1 using an adenoviral construct of TmFOXO1 (AD-TmFOXO1). These cells were then treated with or without R5020. Cell cycle analysis demonstrated a significant increase in the percentage of cells in the G0/G1 phase in PRB23 and PRA14 cells after infection with AD-TmFOXO1 with or without R5020 treatment compared with those infected with AD-CMV (Fig. 5AGo). The PRAB36 cell line showed no significant difference in the amount of cells in the G0/G1 phase after infection with AD-TmFOXO1 (Fig. 5AGo). Strikingly, there were significant decreases seen in the S phase in all cell lines after infection with AD-TmFOXO1 with or without R5020 treatment (Fig. 5BGo). No significant differences were seen in G2/M phase among any of the cell lines (Fig. 5CGo). To confirm cell cycle arrest, a cell proliferation and viability assay was performed in the PRAB36, PRB23, and PRA14 cell lines after infection with AD-TmFOXO1 and treatment with or without R5020. In the PRAB36 and PRB23 cell lines, there was a significant fold decrease in the number of viable cells remaining after infection with AD-TmFOXO1 with or without R5020 treatment (Fig. 5DGo). A similar decrease was also observed in the PRA14 cell line, but only after infection with AD-TmFOXO1 and R5020 treatment (Fig. 5DGo). A cell proliferation and viability assay was also conducted on PRB23 cells after infection with AD-TmFOXO1 and treatment with a lower dose (10 nM) of R5020. Under these treatment conditions, there was a significant fold decrease in the number of viable cells remaining after infection with AD-TmFOXO1 and treatment with or without R5020 (Fig. 5DGo). These results are consistent with those observed in PRB23 cells treated with a higher dose of R5020 (Fig. 5DGo).


Figure 5
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  		FIG. 5. Cell cycle analysis in response to AD-TmFOXO1 overexpression and progestin treatment. PRAB36, PRB23, and PRA14 were infected with either AD-CMV (used as a control) or AD-TmFOXO1 for 24 h and then treated with 1 µM R5020. After 48 h of treatment, cells were analyzed for cell cycle using PI. A–C, Graphs depict the percentage of cells in the G0/G1 phase (A), S phase (B), or G2/M phase (C) of the cell cycle. Figures represent the mean ± SEM of three experiments. D, A cell proliferation and viability assay was conducted on PRAB36, PRB23, and PRA14 cells lines after infection with either AD-CMV or AD-TmFOXO1 for 24 h and treatment with 10 nM R5020 (PRB23 cells only) or 1 µM R5020 for 24 and 48 h. Graphs depict the fold change of viable cells remaining after AD-TmFOXO1 infection and R5020 treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figures represent the mean ± SEM of five experiments for the PRAB36 and PRB23 cell lines (treated at 1 µM R5020), three experiments for the PRA14 cell line, and six experiments for the PRB23 cell line treated at 10 nM R5020.

 
Additionally, the effect of FOXO1 and PR on apoptosis was investigated using annexin V/DAPI analysis. In the PRB23 cell line, there were significant fold increases in the number of cells in both early and late stages of apoptosis after infection with AD-TmFOXO1 with or without R5020 treatment (Fig. 6Go, A and B). No significant differences in living cells were found among treatments in the PRB23 cell line (Fig. 6CGo). There were no significant differences found in the other cell lines in early or late apoptosis or in living cells (Fig. 6Go, A–C). These data clearly demonstrate differential roles of PRA vs. PRB in terms of FOXO1 function for cell cycle progression and apoptosis. The importance of PR and FOXO1 cross talk in endometrial cancer is supported by these data.


Figure 6
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  		FIG. 6. Effect of AD-TmFOXO1 and R5020 on apoptosis. PRAB36, PRB23, and PRA14 cells were infected with either AD-CMV (used as a control) or AD-TmFOXO1 for 24 h and then treated with 1 µM R5020 for 48 h. Cells were analyzed by flow cytometry using annexin V/DAPI for the determination of early (A) and late (B) apoptosis and viable cells (C). Graphs depict the fold increase of cells from those infected with AD-CMV and treated with the vehicle in early and late apoptosis. *, P < 0.05; **, P < 0.01. Figures represent the mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Due to the prevalence of PTEN mutations in type I carcinoma, we focused our studies on one downstream target of the PTEN/Akt pathway, FOXO1. Our results show that FOXO1 expression is decreased in endometrial tumors compared with normal endometrium and that this decrease involves the Skp2 ubiquitination system. Inhibition of Akt, as well as progestin treatment, were individually able to increase nuclear FOXO1 protein expression. FOXO1 is of particular importance in the endometrium in that it is a major player in decidualization and menstruation, thus maintaining normal reproductive function (13, 15, 16). However, the role of FOXO1 in endometrial cancer has not been investigated. Giatromanolaki et al. (23) demonstrated that 24 of 82 cases of endometrial cancer exhibited a significant loss of FOXP1. More recently, it has been shown that FOXO1 expression in endometrial cancer is decreased compared with normal endometrium, confirming our observations (24). Given that FOXOs have been associated with cell proliferation, oxidative stress, and apoptosis (25, 26, 27), its decreased expression in endometrial carcinoma has numerous implications and may contribute to tumorigenesis.

FOXO1 is a protein that has been shown to be modified at the posttranslational level by phosphorylation, acetylation, and ubiquitination (28) with the ubiquitin-proteasome system being responsible for its regulation when overabundance is detected (29, 30). Recently, Huang et al. (10) illustrated that in prostate carcinoma cells, Skp2, a member of the Skp1/Cul1/F-box protein ubiquitin complex, binds to FOXO1 and induces its ubiquitination after phosphorylation at Ser-256 by Akt (10). We have demonstrated in four endometrial cancer cell lines that the expressions of Skp2 and FOXO1 were inversely correlated and that silencing Skp2 restored FOXO1 protein expression to some degree, implicating its association with FOXO1 protein degradation. A recent study by Goto et al. (24), showing an inverse correlation between Skp2 and FOXO1 in Hec1B and Ishikawa cells, supports our findings. It has also been reported that high expression of Skp2 is correlated with poor prognosis in endometrial cancer (21). Additionally, Akt inhibition resulted in an increase in FOXO1 protein levels in the nuclear fraction of Ishikawa cells, demonstrating that inhibition of Akt prevents the phosphorylation of FOXO1, resulting in nuclear retention as well as rescuing the protein from Skp2 ubiquitination. Although silencing Skp2 and inhibiting Akt increased FOXO1 protein levels slightly, the Skp2 ubiquitin complex may not be solely responsible for the loss of FOXO1 protein expression in endometrial carcinoma. As previously mentioned, FOXO1 is posttranslationally modified by several pathways (28), suggesting that other mechanisms are involved in its regulation. Also, it was curious that there was a differential effect of AI-IX treatment on FOXO1 localization in parental Ishikawa cells compared with the PR stably transfected Ishikawa cell lines. It is possible that the heightened expression of PRA and PRB in the stable Ishikawa cell lines interrupts the signaling events associated with retention of FOXO1 in the nucleus.

It is well known that progesterone plays an influential role in the endometrium by stimulating glandular and stromal differentiation and by inhibiting estrogen-stimulated proliferation of the epithelium (31, 32). In a clinical setting, progesterone has been effectively administered to reverse endometrial hyperplasia and to decrease the growth of endometrial tumors (33). Progesterone acts through its two receptors, PRA and PRB, which are transcribed from the same gene, but due to alternative transcriptional start sites, PRA lacks 164 amino acids from the N terminus (34). The mechanism by which PR inhibits growth of endometrial tumors remains unclear. In our study, we demonstrate that PR and FOXO1 can cooperatively up-regulate a PRE-responsive promoter, and that FOXO1 and PR act coordinately to inhibit cell cycle progression and increase apoptosis. In all of our studies involving R5020, we have chosen to use 1 µM concentration for the following reasons. In cycling women, the circulating progesterone levels range from 6–70 nmol/liter during the secretory phase and up to 1 µmol/liter in pregnancy (35). In addition to the high circulating levels of progesterone, the localized concentration in the uterine cavity could be even higher given the proximity of the uterus to the corpus luteum or the placenta and the connecting vasculature and remains to be elucidated. Progestin therapy for women with endometrial hyperplasia or endometrial cancer involves milligram doses of progestin per day, which results in circulating levels of approximately 100 ng/ml (36). Thus, the use of 1 µM R5020 in our cell system is biologically supported. Interestingly, 10 nM R5020 had similar effects as 1 µM R5020 in reducing the number of viable cells that were infected with AD-TmFOXO1 (Fig. 5DGo). Further analysis will be required to determine whether there are differential effects of high vs. low concentrations of progestins in promoting PR/FOXO1 cooperativity.

We have recently reported that during the process of decidualization of human endometrial stromal cells, the interplay of FOXO1 and PR is important for the regulation of many decidua-specific genes (15). Also, Labied et al. (16) demonstrated that MPA increases FOXO1 protein levels in endometrial stromal cells only when treated in combination with cAMP. Here we have shown that R5020 does in fact increase FOXO1 alone. Other than cell type specificity, this difference could be due to biochemical and molecular alterations as a result of tumorigenesis.

With the use of stably transfected PR cell lines, we were able to decipher the different roles of PRA vs. PRB for FOXO1 expression and function on cell cycle progression and apoptosis. Previous studies have shown that PRA and PRB exhibit different activating properties and mediate the transcription of different sets of genes in endometrial cancer cells (37, 38). Smid-Koopman et al. (38) demonstrated that in the presence of progesterone, PRB-expressing Ishikawa cells displayed almost complete inhibition of cell growth, whereas PRA-expressing Ishikawa cells displayed only 50% inhibition of cell growth. In an additional study by Hanekamp et al. (18), it was demonstrated that whereas PRB-expressing Ishikawa cells caused more tumor growth in vivo than PRA-expressing Ishikawa cells, tumor growth was inhibited after administration of MPA only in the tumors expressing PRB. Interestingly, a recent study in breast cancer demonstrated that liganded PRB was responsible for regulating the transcription of FOXO1, as opposed to liganded PRA (39), supporting our observations.

It has been demonstrated that unliganded PRB and PRA are also functionally distinct. In the breast cancer study previously mentioned, Jacobsen et al. (39) showed that genes are differentially regulated by unliganded and liganded PR. Of the genes regulated by unliganded PR, 58% are regulated only by PRA, 7% are regulated only by PRB, and 36% are regulated by both, with the majority regulated more strongly by PRA (39). The genes regulated only by PRA encode proteins involved in extracellular matrix binding, cell-cell communication, and membrane signaling and are markers of tumor aggressiveness (39). We recently showed that in human endometrial stromal cells, cAMP-mediated regulation of genes involved unliganded PR (15). The differential response to TmFOXO1 in the PRB vs. PRA cell lines for cell proliferation and apoptosis, particularly in the absence of progestin, was especially intriguing. The overexpression of TmFOXO1 was able to decrease cell proliferation in PRB cells whether or not R5020 was present. Additionally, TmFOXO1 overexpression induced apoptosis only in the PRB cells in the absence and presence of ligand. These data suggest that unliganded PRB may influence AD-TmFOXO1 function. Here, we have demonstrated that FOXO1 and PR act coordinately to regulate cell cycle progression and apoptosis providing insight on PR responsiveness in endometrial cancer and the implication this has in regard to patient treatment methods.

As previously mentioned, progesterone is used to effectively inhibit tumor growth during the early, noninvasive stages of endometrial cancer (40, 41); however, it has been demonstrated that tumors expressing only PRB are responsive to this treatment in mice. Our study demonstrates that when nuclear FOXO1 is expressed, endometrial cancer cells expressing PRA, PRB, or both respond to progestin treatment differently in terms of cell proliferation or apoptosis. There is an ongoing debate as to the PR status in endometrial tumors with one study suggesting that PRB is predominant in advanced endometrial tumors (42), another study pointing to the loss of both isoforms in advanced endometrial cancer (43), and a third study that indicates only PRA is expressed in poorly differentiated endometrial carcinoma cell lines (44). Our study suggests that progesterone treatment would benefit patients expressing either or both isoforms upon restoration of FOXO1 in endometrial cancer.

The data presented here give promise to targeting the PI3K/Akt/FOXO pathway for the development of alternate therapies for endometrial carcinoma. It is well known that activated Akt is an essential survival factor in vitro (5); therefore, Akt inhibitors, targeting kinase activity and Akt mRNA, have been shown to effectively induce apoptosis in several tumor types. Recent data (45) have demonstrated a novel synergistic relationship between AI-IX and carboplatin in promoting apoptosis in endometrial carcinoma and that FOXO1 was involved. On the other hand, Akt is a molecule that is involved in several cellular functions necessary for normal activity, thus targeting Akt has the possibility for disrupting processes necessary for homeostasis in other systems. It has been documented that throughout the development of PI3K pathway inhibitors, toxicity decreased as targets further downstream and more selective outputs are inhibited (46). Compounds that specifically target FOXO1 by inhibiting its phosphorylation and restoring its activity in carcinomas could be effective in preventing endometrial cancer progression.


   Acknowledgments
 
We thank Dr. Terry Unterman for the AD-TmFOXO1 construct, as well as Jennifer Hardt for her technical support. Additionally, we are grateful to Dr. Tian Chuang for her expertise and assistance in the immunohistochemical analysis of normal endometrium and endometrial adenocarcinoma. We also acknowledge the Robert H. Lurie Comprehensive Cancer Center Pathology and Flow Cytometry Core Facilities at Northwestern University.


   Footnotes
 
This work was supported by Northwestern Memorial Foundation, Friends of Prentice, and HD044715 from the National Institutes of Health.

Disclosure Statement: E.W., A.V.H., L.J.B., P.H.-M., J.R.L., B.M.B., J.C.S., and J.J.K. have nothing to disclose. D.K.S. received lecture fees from Merck.

First Published Online December 20, 2007

Abbreviations: DAB, Diaminobenzidine; FBS, fetal bovine serum; FOXO, Forkhead box O; HSC, human stromal cell; MOI, multiplicity of infection; MPA, medroxyprogesterone acetate; PI, propidium iodide; PI3K, phosphoinositide-3-kinase; PR, progesterone receptor; PRE, progesterone response element; siRNA, small interfering RNA; TBS-T, Tris-buffered NaCl solution with 0.1% Tween 20; TmFOXOl, triple mutant FOXOl.

Received June 8, 2007.

Accepted for publication December 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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