Oncogene (2014) 33, 4213–4225 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE b upregulates FOXO3a and causes induction of apoptosis through PUMA in cancer

P Dey1, A Stro¨m1 and J-Å Gustafsson1,2

Estrogen receptor b (ERb) is emerging as a critical factor in understanding biology. Although reduced in prostate cancer above Gleason grade 3, ERb is a potential drug target at the initial stage of the disease. In human prostate cancer cells, we found that ERb causes apoptosis by increasing the expression of pro-apoptotic factor p53-upregulated modulator of apoptosis (PUMA), independent of p53, but dependent on the forkhead transcription factor class-O family member, FOXO3a. FOXO3a has previously been shown to induce PUMA after growth factor withdrawal and inhibition of the Akt pathway. Surprisingly, the phosphorylation of FOXO3a remained unchanged, while the mRNA and total protein levels of FOXO3a were increased in response to ERb expression or treatment of PC3, 22Rv1 and LNCaP cells with the ERb-specific ligands 3b-Adiol (5a-androstane-3b,17b-diol), DPN (diarylpropionitrile) or 8b-VE2 (8-vinylestra-1,3,5 (10)-triene-3,17b-diol). Knockdown of FOXO3a or ERb expression abolished the increase of PUMA in response to 3b-Adiol in LNCaP and PC3 cells, suggesting that FOXO3a mediates the apoptotic effect of 3b-Adiol-activated ERb. Moreover, the ventral prostate of ERb À / À mice had decreased expression of FOXO3a and PUMA compared with the ERb þ / þ mice, indicating a relationship between ERb and FOXO3a expression. The regulation of FOXO3a by ERb in normal basal epithelial cells indicates a function of ERb in cell differentiation and maintenance of cells in a quiescent state. In addition, the expression of ERb, FOXO3a and PUMA is comparable and higher in benign prostatic hyperplasia than in prostate cancer Gleason grade 4 or higher, where there is substantial loss of ERb, FOXO3a and PUMA. We conclude that ERb induces apoptosis of prostate cancer cells by increasing transcription of FOXO3a, leading to an increase of PUMA and subsequent triggering of apoptosis via the intrinsic pathway involving caspase-9. Furthermore, we conclude that ligands specifically activating ERb could be useful pharmaceuticals in the treatment of prostate cancer.

Oncogene (2014) 33, 4213–4225; doi:10.1038/onc.2013.384; published online 30 September 2013 Keywords: ERb; apoptosis; p53; FOXO3a; PUMA

INTRODUCTION (phosphatase and tensin homolog), leading to cell survival in 2,5 Prostate cancer is the leading cause of cancer-related death in cancers. In the absence of phosphoinositide 3-kinase/Akt men in the United States.1 Although the frontline treatment signaling, FOXO3a localizes to the nucleus and initiates the remains deprivation, the tumor eventually becomes transcription of its target genes PUMA and GADD45A.4,6 Further- resistant to anti-androgen therapy. In the healthy prostate, andro- more, miRNA-155 has been shown to repress FOXO3a protein gen withdrawal causes apoptosis by a process called androgen- expression by directly interacting with its 30-untranslated region.7 withdrawal-induced apoptosis, which helps to maintain tissue In a study of breast cancer MCF-7 cells, FOXO3a was found to homeostasis.2 In advanced prostate cancer androgen-withdrawal- inhibit -dependent and a-mediated induced apoptosis is abrogated, leading to androgen-independent cell proliferation.8 prostate cancer. Estrogen receptor b (ERb) acts as a tumor suppressor in many The p53-upregulated modulator of apoptosis (PUMA) is a cancers and this is supported by knockout studies of the receptor potent pro-apoptotic factor, which belongs to the Bcl-2 homology in mice.9–12 Studies of ERb function in cell lines have revealed: 3-only subgroup. PUMA was initially identified as a p53 target (1) an anti-proliferative effect,13–16 (2) promotion of apoptosis gene and is induced upon genotoxic stress through induction of via extrinsic pathway in castration-resistant basal epithelial cells p53.3 PUMA can also be upregulated in a p53-independent in the of aromatase knockout mice17,18 and (3) increased manner by the forkhead transcription factor class-O family -induced cell death by upregulation of the pro-apoptotic member, FOXO3a, upon withdrawal of cytokine/growth factor gene Bik.19 All the above findings suggest an apoptotic effect of signaling.4 The apoptotic action of FOXO3a is inhibited by Akt- ERb, although its molecular mechanism still remains unclear. mediated phosphorylation, upon which the FOXO3a protein In the present study, we have examined the role of ERb in interacts with a 14-3-3 member and is translocated to the cyto- apoptosis. We found that the apoptotic function of ERb is plasm, where it undergoes proteasomal degradation. The phos- mediated by upregulation of FOXO3a (a member of FOXO family). phoinositide 3-kinase/Akt pathway is upregulated in cancer, Upon transcriptional upregulation of FOXO3a, its downstream either due to a gain-of-function mutation in the phospho- target PUMA is increased, leading to activation of the apoptosis inositide 3-kinase enzyme or by deletion mutation of PTEN machinery.

1Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, USA and 2Department of BioSciences and Nutrition, Karolinska Institutet, Huddinge, Sweden. Correspondence: Dr A Stro¨ m, Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, 3605 Cullen Boulevard, Science and Engineering Research Center Building 545, Houston, TX 77204-5056, USA. E-mail: [email protected] Received 25 January 2013; revised 5 August 2013; accepted 9 August 2013; published online 30 September 2013 ERb induces apoptosis via FOXO3a P Dey et al 4214 RESULTS PC3 cells (Figures 1f and g), respectively. Immunocytochemistry of Expression of estrogen receptors in prostate cancer cell lines ERb-expressing cells showed nuclear localization of the protein in ERb expression is reduced during progression of prostate cancer20 22Rv1 (Figure 1e) and PC3 cells (Figure 1h). and cells isolated from prostate cancer express low levels of ERb.21 In the present study, we used the prostate cancer cell lines PC3, LNCaP and 22Rv1, the first two of which express low levels of ERb, ERb induces apoptosis in prostate cancer cells whereas 22Rv1 cells are ERb negative (Figure 1a). In LNCaP cells, Although several reports have shown an apoptotic effect of ERb in ERb was detected in the nucleus by immunocytochemistry cancer cells, no detailed mechanism has been described. In order (Figure 1b). Further, to understand how ERb causes apoptosis in to study the apoptotic effect of ERb, we used the ERb-over- prostate cancer cells, we developed a doxycycline-regulated expressing PC3 and 22Rv1 cells. By using a flow-cytometry-based expression system for ERb in PC3 and 22Rv1 cells. Following the terminal transferase uridyl nick-end labeling (TUNEL) assay, we removal of doxycycline, we observed a robust induction of ERb at showed a representative plot where 12.5% of the cells expressing both the mRNA and protein levels in 22Rv1 (Figures 1c and d) and ERb were labeled with 5-bromo-2’-deoxyuridine compared with

Figure 1. Expression of ERb in PC3, LNCaP and 22Rv1 cells. (a) The endogenous expression of ERb in 22Rv1, LNCaP and PC3 cells. Lane 1 is the recombinant ERb as positive control; lane 2, 4 and 6 are vehicle-treated cells, and lane 3, 5 and 7 are the 3b-Adiol-treated cells. (b) Immunocytochemistry of ERb in LNCaP cells showing nuclear staining in the left panel, DAPI (4’,6-diamidino-2-phenylindole) in the center panel and merged in the right panel. (c)ERb mRNA expression in the 22Rv1-ERb cells compared with the 22Rv1-control cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.), calculated using Student’s t-test, *Pp0.03). (d)ERb protein expression in 22Rv1-ERb cells compared with 22Rv1-control cells as detected by western blotting. (e) Expression of ERb in 22Rv1-ERb cells compared with 22Rv1-control cells verified by immunocytochemistry using the ERb antibody (503). Scale bars, 50 mm. (f)ERb mRNA expression in PC3-ERb cells compared with PC3-control cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.), calculated using Student’s t-test, ***Pp0.0007). (g)ERb protein expression in PC3-ERb cells compared with PC3-control cells as detected by western blotting. (h) Expression of ERb in PC3-ERb cells compared with PC3-control cells verified by immunocytochemistry using the ERb antibody (503). Scale bars, 100 mm. All the experiments were performed n ¼ 3.

Oncogene (2014) 4213 – 4225 & 2014 Macmillan Publishers Limited ERb induces apoptosis via FOXO3a P Dey et al 4215 1.3% in control cells (Figure 2a). The quantification of TUNEL- was observed in control tumors. Furthermore, there was a higher positive cells (n ¼ 3) showed a 9.6-fold increase of apoptosis in frequency of TUNEL staining in tumors expressing ERb compared ERb-expressing cells compared with control cells (Figure 2b). with control tumors (Figure 3c). The apoptotic index (number of Furthermore, we showed that the sub-G1 peak, a feature of apoptotic cells/total number of cells  100%) showed an approxi- apoptotic cells, was increased in ERb-expressing PC3 (Figures 2c mately threefold increase in TUNEL-positive cells expressing ERb and d) and 22Rv1 cells (Figures 2e and f) compared with control compared with control cells (Figure 3d). (n ¼ 3). Moreover, analysis of annexin V using fluorescence- activated cell sorting of PC3 cells showed an increase in annexin-V-positive cell population upon treatment with 3b-Adiol Endogenously expressed ERb in LNCaP cells induces apoptosis (5a-androstane-3b,17b-diol) compared with the vehicle-treated after treatment with ERb-specific ligands group (See Supplementary Figures S1A and B). The use of an ERb antibody made in our laboratory (ERb503 IgY To estimate tumor-based apoptosis, we analyzed the xenografts chicken) allowed detection of nuclear staining in LNCaP cells by from a previous study (Dey et al.14). Briefly, we used athymic nude immunocytochemistry (Figure 1b), and transfection with ERb- mice to grow subcutaneous xenografts of PC3-control and PC3- specific small interfering RNA (siRNA) resulted in a significant ERb cells. The cells (2  105 per 100 ml in 1:1 cells:matrigel) were reduction in nuclear immunostaining of ERb, indicating the injected into the flank of the mice and visible tumors developed presence of ERb in these cells (Figure 4a). We then sought to after 40 days (Figure 3a). There was a reduction in ki67 staining in determine whether ERb-specific ligands could induce apoptosis ERb-expressing tumors (Figure 3b). The inset in Figure 3b shows by treating LNCaP cells with the 5a- apoptotic bodies (arrows) in ERb-expressing tumor, whereas none metabolite 3b-Adiol, a known endogenous ligand of ERb22,23

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Figure 2. Expression of ERb increases apoptosis as analyzed by flow cytometry. (a) The induction of apoptosis was evaluated using TUNEL assay by flow cytometry. The x-axis indicates DNA content (propidium iodide staining) and the y-axis indicates 5-bromo-2’-deoxyuridine (BrdU) labeling. The DNA that has incorporated BrdU is detected by anti-BrdU-APC antibody. Representative (n ¼ 3) flow cytometry profile of PC3-ERb cells compared with PC3-control cells is shown. The top right quadrant represents BrdU-labeled cells. There are 12.5% of BrdU-labeled cells in the PC3-ERb panel (right) compared with 1.3% of the PC3-control panel (left). (b) Quantification of flow cytometry data shows that PC3-ERb cells have 9.6-fold TUNEL-positive cells compared with PC3-control cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.), calculated using Student’s t-test, ***Pp0.0004). (c) PC3 stable cells were analyzed for apoptosis by propidium iodide staining and flow cytometry. Apoptotic cells exhibited sub-G1 DNA and viable cells displayed G1 and G2 DNA content. PC3-ERb cells showed an increase in sub-G1 population compared with control cells. (d) Quantification of flow cytometry data shows that PC3- ERb cells have a twofold increase in the sub-G1 population. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.), calculated using Student’s t-test, *Pp0.018). (e) 22Rv1-ERb cells showed an increase in sub-G1 population compared with control cells. (f) Quantification of flow cytometry data shows that 22Rv1-ERb cells have a five-fold increase in sub- G1 population. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **Pp0.0017).

& 2014 Macmillan Publishers Limited Oncogene (2014) 4213 – 4225 ERb induces apoptosis via FOXO3a P Dey et al 4216 PC3 stable cells xenograft

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PC3-ER PC3-Control Figure 3. ERb increases apoptosis in xenograft tumor of PC3 stable cells in nude mice. (a) Nude mice injected with PC3 cells shows visible tumors in the flank region (arrow) after 40 days of growth (n ¼ 3). (b) ki67 staining of PC3-control and PC3-ERb tumors. The inset is a higher magnification. The inset in the right panel shows apoptotic bodies (arrow) in PC3-ERb tumor, whereas none is observed in PC3-control tumor (left panel). (c) The induction of apoptosis in vivo was evaluated by TUNEL staining using histological samples from 40 days of cell inoculation in nude mice.14 More TUNEL-positive cells were observed in the PC3-ERb tumor compared with the PC3-control tumor. The upper right panel is the negative control where the cells were treated without terminal transferase enzyme. The lower right panel is the positive control where the cells were treated with DNase I. (d) Quantification of the apoptotic index of the xenograft tumor samples. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, ***Po0.0001).

(see Supplementary Figure S2 for 3b-Adiol dose response). ERb increases expression of PUMA independently of p53 Treatment of LNCaP cells transfected with control siRNA or ERb We initially observed that prostate cancer cell lines stably siRNA for 72 h with 3b-Adiol (48 h treatment) showed extensive expressing ERb are sensitive to serum starvation and show signs apoptosis in the control (siControl)-transfected cells whereas the ERb of apoptosis. This prompted us to look for apoptotic factors siRNA (siERb)-transfected cells showed increased protection against involved. After assaying expression of the most common factors apoptosis (Figure 4b). Furthermore, a survival assay with LNCaP cells involved in apoptosis, we found PUMA to be changed by ERb treated with 3b-Adiol for 24 h showed a reduction in colony expression. Treatment of LNCaP cells with 3b-Adiol resulted in formation after 20 days. A similar result was obtained with PC3 and increased PUMA mRNA and protein levels (Figures 6a and b). 22Rv1 cells stably expressing ERb compared with control (Figure 4c). Treatment of PC3 cells with 3b-Adiol resulted in increased PUMA In order to understand the mechanism underlying the observed protein levels (Figure 6c). In addition, 22Rv1 cell lines stably apoptosis, we investigated regulation of factors involved in expressing ERb showed increased levels of PUMA protein the apoptotic pathway. We observed that the expression of the (Figure 6d). Knockdown of ERb using siRNA caused a decrease anti-apoptotic Bcl-2 was reduced in response to treatment of in PUMA expression, abolishing the 3b-Adiol-specific effect LNCaP cells with 3b-Adiol (Figure 5a), in 22Rv1 cells stably (Figures 6e-g). As PUMA is an established p53 controlled gene, we expressing ERb and treated with 3b-Adiol (Figure 5b) and in PC3 investigated whether ERb regulates p53. To our surprise, we found cells stably expressing ERb (Figure 5c). On the other hand, the pro- that p53 expression was downregulated by 3b-Adiol in LNCaP cells apoptotic factor Bax remained unchanged, except in 22Rv1 cells, (Figures 6h and i) and in 22Rv1-ERb stable cells (Figures 6j and k). which showed an increase in Bax expression (Figure 5b). As p53 is not upregulated by ERb, it became apparent that the Furthermore, the ERb-specific ligands 3b-Adiol, DPN or 8b-VE2 regulation of PUMA must occur via an alternate pathway. PC3 cells (8-vinylestra-1,3,5 (10)-triene-3,17b-diol) decreased Bcl-2 expres- are null for p53 expression, which further strengthens the idea sion in PC3 cells at a concentration of 10 nM (Figures 5d–f). that the regulation of PUMA by ERb is independent of p53. In a Caspase-9 is the first caspase to respond to mitochondrial- report by You et al.,4 FOXO3a was shown to upregulate PUMA induced release of cytochrome c, following changes in the activity independently of p53, upon withdrawal of cytokines or growth of pro-apoptotic factors. We showed that cleaved caspase-9 factors. FOXO3a’s activity is regulated via phosphorylation by Akt (Asp330) was increased in PC3 cells treated with 3b-Adiol, DPN or (pT32, pS253 and pS315), whereupon it is exported from the 8b-VE2, strongly implying a role for ERb in apoptosis (Figures 5g–i). nucleus and inactivated.24 Reduction of threonine 32 phospho- The antibody used to detect cleaved caspase-9 (Cell Signaling rylation leads to retention of active FOXO3a in the nucleus and Technology, Danvers, MA, USA; number 7237) recognizes the p37 upregulation of PUMA and GADD45A, which are direct targets of subunit only, upon cleavage at the Asp330 site. Moreover, caspase FOXO3a.25 We found that total FOXO3a protein increased after 3/7 showed an increase in activity in the presence of 3b-Adiol or treatment with ERb-specific ligands in LNCaP (Figures 7b–d) and DPN in LNCaP cells (Figure 5j). PC3 cells (Figures 7g–i) and in ERb-overexpressing 22Rv1 cells

Oncogene (2014) 4213 – 4225 & 2014 Macmillan Publishers Limited ERb induces apoptosis via FOXO3a P Dey et al 4217

Figure 4. siRNA knockdown of ERb reverses the apoptotic effect of 3b-Adiol. (a) Knockdown of ERb in LNCaP cells using siRNA. Left panel: ERb staining using specific antibody, the central panel is the DAPI (4’,6-diamidino-2-phenylindole) staining and the right panel is the merged one. The top panel cells are transfected with siControl and the bottom panel cells are transfected with siERb.(b) Microscopy of the LNCaP cells transfected with siControl (top panel) and siERb (bottom panel). Cells transfected with siControl and treated with 3b-Adiol show extensive cell death compared with cells transfected with siERb and treated with 3b-Adiol. (c) Survival assay of LNCaP cells shows a decrease in the number of viable colonies after treatment with 3b-Adiol for 20 days compared with vehicle-treated cells. Similarly, PC3 and 22Rv1 stable cells expressing ERb shows a decrease in the number of viable colonies compared with the control after 12 days of propagation (n ¼ 3).

(Figure 7j). Upregulation of FOXO3a also occurred at the mRNA ERb’s regulation of FOXO3a is required for the upregulation level, indicating a pretranslational regulation of FOXO3a by ERb of PUMA (Figures 7a and f). Time course of FOXO3a expression in 3b-Adiol- Finally, we wanted to examine whether ERb’s regulation of treated LNCaP cells showed a peak at 12 h, matching PUMA’s FOXO3a could explain ERb’s control of PUMA. We used siRNA to expression (Figure 7e). Furthermore, phosphorylated FOXO3a knock down expression of FOXO3a in LNCaP cells and PC3 cells remained unchanged in ERb ligand-treated compared with - (Figures 8a and b). Here we showed that FOXO3a knockdown untreated cells (Figures 7b–d and g) or in cells not expressing ERb abolished the effect of 3b-Adiol on PUMA expression in LNCaP and compared with cells expressing ERb (data not shown). PC3 cells. These data strongly indicate that FOXO3a expression is We also investigated the effect of 3b-Adiol on phosphorylation required for the regulation of PUMA by ERb. Moreover, combined of Akt at Thr 308 and Ser 473 in LNCaP cells, and observed knockdown of ERb and FOXO3a abolished the effect of 3b-Adiol on reduced phosphorylation at Thr 308 and reduced phospho c-raf, PUMA expression in PC3 cells (see Supplementary Figure S3). indicating less active Akt. However, phosphorylation of Akt at Ser To investigate whether ERb regulates FOXO3a in prostate epi- 473 was unchanged from control. Moreover, total Akt (pan) thelial cells in vivo, we used the ERb À / À mouse and compared remained unchanged (Figure 7k). it with the ERb þ / þ mouse. Deleting ERb (Figures 9b, d and f)

& 2014 Macmillan Publishers Limited Oncogene (2014) 4213 – 4225 ERb induces apoptosis via FOXO3a P Dey et al 4218

Figure 5. ERb decreases the Bcl-2/Bax ratio. (a) LNCaP cells treated with 3b-Adiol show a reduction in Bcl-2 protein expression, although no change in Bax protein expression is observed. (b) 22Rv1 stable cells expressing ERb show a reduction in Bcl-2 protein expression compared with the control cells. Both cell lines were treated with 3b-Adiol. Moreover, the expression of pro-apoptotic Bax protein increases in the 22Rv1 cells expressing ERb compared with the control cells. (c) PC3 stable cells expressing ERb show a reduction in Bcl-2 protein expression compared with the control cells; no change in Bax protein expression is observed. (d–f) PC3 cells treated with 3b-Adiol, DPN and 8b-VE2 show a reduction in Bcl-2 expression, although no change in Bax expression is observed. (g–i) The expression of cleaved caspase-9 in PC3 cells is increased upon treatment with 3b-Adiol, DPN and 8b-VE2. (j) Caspase 3/7 assay of LNCaP cells treated with 3b-Adiol and DPN show an increase in cleaved caspase 3/7 activity. The graph shows the data as an increase in luminescence (caspase activity) of ligand-treated cells compared with the vehicle treatment (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *Pp0.046 (3b-Adiol) and *Pp0.01 (DPN)).

from epithelial basal cells drastically reduced expression of FOXO3a FOXO3a and ERb expression is reduced in higher Gleason grade and PUMA, indicating a direct relationship between ERb and prostate cancer. FOXO3a expression (Figures 9a, c and e) (additional immuno- To assess expression of FOXO3a and ERb in human prostate, we histochemistry staining figures can be found in Supplementary stained for both factors in samples from benign prostatic Figure S7A–H). Moreover, co-localization of ERb and FOXO3a by hyperplasia (BPH) and prostate cancer Gleason grade 4 and 5. double immunostaining showed that both ERb and FOXO3a are Immunohistochemical staining of BPH samples revealed the located in the nuclei of the epithelial cells of mouse ventral presence of ERb, FOXO3a and PUMA in the prostate epithelium, prostate (see Supplementary Figure S4A–H). whereas the samples from prostate cancer Gleason grade 4 and 5

Oncogene (2014) 4213 – 4225 & 2014 Macmillan Publishers Limited ERb induces apoptosis via FOXO3a P Dey et al 4219

Figure 6. ERb increases the expression of pro-apoptotic factor PUMA. (a) mRNA expression of PUMA increased by 3b-Adiol in LNCaP cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **Pp0.0073). (b) Protein expression of PUMA increased by 3b-Adiol in LNCaP cells. (c) Protein expression of PUMA increased by 3b-Adiol in PC3 cells. (d) Protein expression of PUMA increased in 22Rv1 cells stably expressing ERb compared with control cells. (e) Knockdown of ERb using siRNA in LNCaP cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *Pp0.013) (3b-Adiol-treated siControl cells vs vehicle-treated siERb cells). ERb mRNA expression of LNCaP cells showing a decrease in ERb expression after knockdown using siRNA. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **Pp0.0094). (f) The knockdown of ERb also decreased the PUMA mRNA level. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *Pp0.014 (vehicle- vs 3b-Adiol-treated siControl cells)). (g)ERb protein expression of LNCaP cells showing a decrease in ERb expression after knockdown using siRNA. Treatment of LNCaP cells with ERb siRNA attenuated 3b-Adiol regulation of PUMA protein level. (h and j) mRNA expression of p53 in LNCaP±3b-Adiol and 22Rv1±ERb cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, PpNS (LNCaP cells) and *Pp0.02 (22Rv1 cells)). (i and k) Protein expression of p53 in LNCaP±3b-Adiol and 22Rv1±ERb cells. showed absence of ERb, FOXO3a and PUMA (Figures 10a–i) cancer cells upon ERb expression. ERb was detected in the nucleus (additional immunohistochemistry figures are found in Supple- of LNCaP, PC3 and 22Rv1 cell lines in agreement with a nuclear mentary Figure S8A–F) localization of ERb in PC3 cells, as reported by Leung et al.21 The very low levels of ERb found in PC3 and LNCaP cells are consistent with the decrease in expression of ERb observed during DISCUSSION progression of prostate cancer. The second estrogen receptor, ERb, was originally discovered in Furthermore, we observed that PC3 and 22Rv1 cells expressing rat prostate in 1996.26 Multiple studies during the last 16 years exogenous ERb showed a larger sub-G1 peak in flow cytometry have described ERb as being anti-proliferative and pro- analysis and a higher number of apoptotic cells determined by apoptotic.16–18,27–29 While the mechanism underlying ERb’s anti- TUNEL assay. proliferative effect has been described in several studies14–16 the The three-dimensional xenograft model offers an in vivo-like pro-apoptotic effect is less clear. We observed that high environment compared with the two-dimensional exogenous ERb expression levels obtained in breast and colon dishes, and we demonstrated that, as expected, tumors expressing cancer cell lines are difficult to achieve in prostate cancer cell lines, ERb showed an increase in apoptotic cells. When analyzing various which require an inducible system or expression at very low levels pathways involved in apoptosis, we found the apoptotic factor by using a weak promoter to avoid apoptosis. This prompted us to PUMA to be induced by exogenous ERb expression in PC3 and study the cause for increased apoptosis observed in prostate 22Rv1 stable cells, and, also upon addition of 3b-Adiol, DPN or 8b-

& 2014 Macmillan Publishers Limited Oncogene (2014) 4213 – 4225 ERb induces apoptosis via FOXO3a P Dey et al 4220

Figure 7. ERb increases the expression of FOXO3a. (a) FOXO3a mRNA increased by 3b-Adiol in LNCaP cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *Pp0.03). (b–d) Protein expression of FOXO3a increased by 3b-Adiol, DPN and 8b-VE2 in LNCaP cells. (e) Time course with 3b-Adiol treatment of LNCaP cells shows an increase in FOXO3a expression with maximal level at 12 h. Moreover, PUMA expression increases with a little delay after FOXO3a expression. (f) mRNA expression of FOXO3a increased by 3b-Adiol in PC3 cells. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **Pp0.0018). (g–i) FOXO3a protein increased by 3b-Adiol, DPN and 8b-VE2 in PC3 cells. (j) FOXO3a protein increased in 22Rv1 cells stably expressing ERb compared with control. (k) PanAkt expression remained unchanged upon treatment with 3b-Adiol in LNCaP cells. Phosphorylation of Akt (T308) decreased by 3b-Adiol in LNCaP cells, but pAktS473 remained unchanged. The Akt target gene cRaf showed a decrease upon treatment with 3b-Adiol, but no change was observed for another Akt target gene, pGSK3b (requires phosphorylation of Akt at S473).

Figure 8. Knockdown of FOXO3a abolishes the PUMA regulation by 3b-Adiol. (a) Upon knockdown of FOXO3a using siRNA, the regulation of PUMA by 3b-Adiol is lost in LNCaP cells. The left panel is the siControl-transfected cells±3b-Adiol, and the right panel is the siFOXO3a- transfected cells±3b-Adiol. (b) Following knockdown of FOXO3a in PC3 cells, 3b-Adiol is unable to induce PUMA expression. Lane 1 and 2 is transfected with siControl±3b-Adiol, and lane 3 and 4 is transfected with siFOXO3a±3b-Adiol.

Oncogene (2014) 4213 – 4225 & 2014 Macmillan Publishers Limited ERb induces apoptosis via FOXO3a P Dey et al 4221

Figure 9. Knockout of ERb (ERb À / À ) in mice decreases the expression of FOXO3a in ventral prostate. (a) Staining of ventral prostate from wild-type (ERb þ / þ ) mice with ERb antibody. Positive nuclear staining of ERb is observed. (b) Staining of ERb À / À mice with ERb antibody. (c) Staining of wild-type (ERb þ / þ ) mice with FOXO3a antibody. Positive nuclear staining of FOXO3a is observed. (d) Staining of ERb À / À mice with FOXO3a antibody. (e) Staining of wild-type (ERb þ / þ ) mice with PUMA antibody. Positive extranuclear staining of PUMA is observed. (f) Staining of ERb À / À mice with PUMA antibody. Magnification  200. Scale bars, 200 mm. All the experiments were performed n ¼ 6.

VE2 to PC3 and LNCaP cells, which are known to express low levels active in cell lines in the absence of ligand, whereas the low of endogenous ERb.30,31 Neither PC3 nor LNCaP cells responded to endogenous levels of ERb found in PC3 and LNCaP cells need 17b-estradiol (E2) or 5a-dihydrotestosterone treatment as evident ligand to be activated. We are currently unable to explain this by unchanged levels of FOXO3a and PUMA protein (see difference in ligand activation between exogenously expressed Supplementary Figures S5A and B). The non-responsiveness to ERb and endogenous ERb. Nonetheless, there is growing evidence E2 was an unexpected finding, indicating that the ERb/FOXO3A/ that prostate tumors may maintain low level of ERb, which is not PUMA signaling pathway shows ligand specificity towards active in the absence of the natural 3b-Adiol. Moreover, 3b-Adiol and the other tested ERb-specific ligands; interestingly, 3b-Adiol is reduced in cancer because of lower conversion from a similar ligand specificity has been described for the anti- 5a-dihydrotestosterone, because of decreased 3b-hydroxysteroid inflammatory action of ERb in the microglia.32 Moreover, dehydrogenase activity,33 or a higher metabolism of 3b-Adiol by 5a-dihydrotestosterone was unable to regulate the expressions 7a-hydroxylase (CYP7B1), a member of the cytochrome P450 of FOXO3a and PUMA, indicating that this pathway is independent superfamily of enzymes.34 of . To investigate the involvement of ERa,we The mechanism of ERb-induced apoptosis in prostatic cancer treated PC3 cells simultaneously with the ERa-specific ligand apparently involves the pro-apoptotic PUMA, a well-characterized propyl pyrazole triol and with 3b-Adiol. We found that under these target gene of p53.35 However, surprisingly, the p53 levels were conditions, 3b-Adiol no longer increased the expression of PUMA, not changed or rather decreased following ERb activation and, suggesting an antagonistic action of ERa, perhaps explaining why thus, p53 regulation could not explain the induction of PUMA. This E2 was inactive (because of the activation of both receptors; see was further supported by experiments with PC3 cells, which, upon Supplementary Figure S5c). In addition, GPR30 agonist G-1 alone activation of ERb, respond by upregulating PUMA, although these demonstrated some degree of positive effect on PUMA regulation, cells lack p53. This prompted us to look for an alternate pathway. but the effect was lost in the presence of 3b-Adiol (see It is known that PUMA can be induced by the withdrawal of Supplementary Figure S5c). The exogenously expressed ERb is growth factors and this mechanism is dependent on activation of

& 2014 Macmillan Publishers Limited Oncogene (2014) 4213 – 4225 ERb induces apoptosis via FOXO3a P Dey et al 4222

Figure 10. Higher Gleason grade tumors show a decrease in FOXO3a expression in parallel to that of ERb.(a) Staining of benign prostatic hyperplasia (BPH) with ERb antibody shows a strong nuclear staining in the prostatic epithelium. (b) Staining of BPH with FOXO3a antibody shows a strong nuclear staining in the prostatic epithelium. (c) Staining of BPH with PUMA antibody shows a strong extranuclear staining in the prostatic epithelium. (d) The staining of ERb is reduced in Gleason grade 4. (e) The staining of FOXO3a is also reduced in Gleason grade 4. (f) Consistent with the loss of FOXO3a staining, the extranuclear staining of PUMA is also lost in Gleason grade 4. (g) The staining of ERb is undetected in Gleason grade 5. (h) The staining of FOXO3a is also undetected in Gleason grade 5. (i) Similarly, the extranuclear staining of PUMA is also lost in Gleason grade 5. Magnification  200. Scale bars, 200 mm. The experiments were performed with n ¼ 6 BPH samples, n ¼ 3 Gleason grade 4 samples and n ¼ 2 Gleason grade 5 samples, respectively.

FOXO3a through reduced phosphorylation by Akt.4 Although we and showed that this sequence upregulates FOXO3a transcription found that phospho-Akt (pT308) was reduced by the activation (see Supplementary Figure S6). Furthermore, ERb À / À mice of ERb, phosphorylation of FOXO3a was unchanged by ERb showed a strong decrease in expression of FOXO3a. In addition, expression, whereas both mRNA and protein levels of FOXO3a we found ERb and FOXO3a to be co-expressed in BPH, but a were increased. To establish the importance of the observed strong reduction of both factors in higher grade prostate cancer. regulation of FOXO3a mRNA and protein levels for subsequent Because of the low number of cancer samples used in this study, induction of PUMA, we used siRNA to knock down FOXO3a we cannot conclude that high Gleason grade prostate cancer expression, resulting in a loss of PUMA regulation by ERb. Our generally loses ERb expression. Some studies suggest that ERb present study indicates that ERb-stimulated upregulation of might be involved in driving an aggressive prostate cancer FOXO3a produces enough unphosphorylated, active FOXO3a phenotype.39,40 It is possible that the contradictory findings may protein to induce the expression of PUMA. It is interesting to depend on differences in function of ERb splice variants that are note that PUMA has been shown to be acutely downregulated by also knocked down by ERb siRNA and react with an N-terminal estrogen in breast cancer cell lines, while, on the other hand, antibody. Further studies with a larger number of clinical samples being transcriptionally upregulated by tamoxifen independent of will help to increase our understanding of ERb’s function in p53 status,36 indicating that estrogen receptors regulate FOXO3a prostate. An overview of the proposed novel ERb/FOXO3a/PUMA and PUMA also in other tissues than prostate. model is outlined in Figure 11. Exploration of this newly The pathway connecting ERb to FOXO3a is an unexpected and discovered pathway connecting ERb to FOXO3a and PUMA might novel finding helping to explain some of our previous studies with help to develop new targeted drugs in the clinical management of breast, colon and prostate cancer cells where we have shown that prostate cancer. ERb regulates p45Skp2, p21 and p27.14,16,37,38 These are all known target genes of FOXO3a, and ERb might indirectly regulate these genes via FOXO3a. MATERIALS AND METHODS In summary, this is the first study demonstrating that induction Reagents, cell culture and generation of stable ERb-expressing of FOXO3a expression has a role in its effect on apoptosis besides cells the well-known post-translational modification of FOXO3a by The PC3, LNCaP and 22Rv1 cell lines were obtained from the American phosphorylation. We identified two estrogen response element Type Culture Collection and maintained in RPMI-1640 (Invitrogen Inc., (ERE) half sites and an AP1 site downstream of the FOXO3a gene, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum

Oncogene (2014) 4213 – 4225 & 2014 Macmillan Publishers Limited ERb induces apoptosis via FOXO3a P Dey et al 4223 transferred to a nitrocellulose membrane after electrophoretic separation. The membranes were blocked with 5% non-fat powdered milk in 0.1% Tris-buffered saline and Tween 20 buffer. The blots were then probed with various primary antibodies. The primary antibodies were used at dilutions of 1:200–1000, and the secondary antibody was used at a dilution of 1:10,000. More details and lists about the antibodies can be found in Supplementary Table 1.

RNA extraction and real-time PCR RNA extraction was performed with Qiagen mRNA extraction kit (Qiagen Sciences, Fredrick, MD, USA) according to standard protocol. cDNA was synthesized from 1 mg of total RNA with First Strand System according to standard protocol (Invitrogen Inc.). Real-time PCR was performed with SYBR Green I dye master mix (Applied Biosystems, Foster City, CA, USA). The list of primers used can be found in the Supplementary Table 2.

Apoptosis detection by TUNEL assay The apoptotic cells were quantified for DNA fragmentation, by using an In-situ Cell Death Detection kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. In brief, excised tumor tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded tissues were cut into 5-mm thick sections. The sections were deparaffinized and rehydrated according to standard protocol. The tissue sections were then antigen retrieved at 0.1 M citrate buffer for B5 min in a pretreatment module (pH 6.0) and then rinsed twice in PBS. The slides were then treated with 50 ml of ‘TUNEL reaction mixture’ and incubated at 37 1C for 60 min The slides were then Figure 11. Proposed model of how ERb causes apoptosis by rinsed 3  with PBS. Negative and positive slides were included. For regulating FOXO3a. ERb interacts with its ligand 3b-Adiol and negative slides, only 50 ml of ‘Label solution’ (without terminal transferase upregulates FOXO3a expression pretranslationally. FOXO3a then enzyme) was used and for positive slides, the slides were treated with transcriptionally upregulates PUMA, which further activates the DNase I before proceeding with treating with ‘TUNEL reaction mixture’. For apoptosis pathway. FHRE: forkhead responsive element. microscopic image capture, four high-power fields were randomly selected and the apoptotic index was calculated as equal to number of positive cells/total number of cells  100%. (Sigma, St Louis, MO, USA), 2 mML-glutamine and 25 mM HEPES buffer (Invitrogen Inc.). For ligand treatment, the medium was changed to phenol Annexin V assay red-free RPMI-1640 supplemented with 1% dextran-coated charcoal- treated fetal bovine serum (Sigma). All experiments used the cells below Cells were plated on a six-well plate and treated for 12 h with ERb-specific passage 30. For LNCaP cells, the experiments were conducted using cells ligand (3b-Adiol). For flow-cytometry-based analysis, FlowCollect annexin below passage 15. The ERb-specific ligands 3b-Adiol, DPN and 8b-VE2, and red kit from Millipore (Billerica, MA, USA) was used. Sample collection and ERa ligand propyl pyrazole triol were obtained from Tocris Bioscience annexin staining of the cells were done according to manufacturer’s (Ellisville, MO, USA). GPR30 agonist G-1 was obtained from Azano instruction. Briefly, cells were washed with 1  PBS, then with 1  assay Pharmaceuticals (Albuquerque, NM, USA). buffer. The cells were then incubated for 15 min with annexin VCF674 and the DNA was stained with propidium iodide for another 15 min. The stained samples were then analyzed using FACS Aria II (BD Biosciences, San Construction of an inducible system for ERb Jose, CA, USA). Prostate cancer cell lines LNCaP and PC3 express minute amounts of endogenous ERb, and 22Rv1 expresses none. To elucidate the molecular Survival assay mechanism behind the apoptotic function of ERb, a transposon-based For survival assays, 5000 cells were seeded on a 60-mm dish. The cells were overexpression system was used to stably transfect PC3 and 22Rv1 treated with ligands or vehicle for 24 h in 1% serum stripped media. After prostate cancer cells with the ERb constructs or with empty vector 24 h treatment, the cells were grown for another 20 days in a complete as a control. All plasmids were transfected using Lipofectamine 2000 medium. The cells were then washed twice with cold PBS and fixed in cold (Invitrogen Inc.) in Opti-Mem medium (Invitrogen Inc.) according to the methanol for 10 min. The cells were then stained with 0.25% crystal violet manufacturer’s protocol. Complete RPMI-1640 medium with 10% fetal in 50% methanol. The plates were then washed with water, air-dried and bovine serum medium was added after 6 h of incubation with the photographed. transfection reagents. Further details about the transposon-based over- expression system can found in Dey et al.14 Human samples Prostate biopsies were obtained from the Department of Urology at Protein extract preparation Danderyd Hospital, Stockholm, Sweden. Samples were fixed in buffered Nuclear extracts from PC3, 22Rv1 and LNCaP cells were prepared as paraformaldehyde, dehydrated and imbedded in paraffin. Samples from 41 described previously. To prepare whole-cell extracts, the cells were six different patients with BPH, three patients with prostate cancer Gleason washed twice with phosphate-buffered saline (PBS), lysed in 10 times grade 4 and two patients with prostate cancer Gleason grade 5 were used packed cell volume of lysis buffer (0.1% Nonidet P-40, 250 mM KCl, 5 mM for this study. Hepes, pH 7.9, 10% (vol/vol) glycerol, 4 mM NaF, 4 mM sodium orthovana- date, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethyl- sulfonyl fluoride, protease inhibitor cocktail and PhosStop (Roche, Immunohistochemistry and immunofluorescence of knockout Indianapolis, IN, USA)) for 30 min on ice and then centrifuged at 14 000 g mice and tumor xenograft for 10 min. For immunohistochemistry, 5 mm sections from 12-month-old ventral prostate of wild-type and ERb knockout (ER À / À ) mice were used. ERb was detected using an antibody raised in chickens (ERb 503). Slides were Western blotting and antibodies deparaffinized with 3  xylene and rehydrated through ethanol gradient. Twenty micrograms of protein were loaded on an SDS–polyacrylamide Antigen retrieval was performed in pretreatment module in 0.01 mol/l gel electrophoresis 10% Bis-Tris gel with Tris running buffer42 and citric acid (pH 6.0) for 15 min followed by 0.5% Triton-X treatment for

& 2014 Macmillan Publishers Limited Oncogene (2014) 4213 – 4225 ERb induces apoptosis via FOXO3a P Dey et al 4224 15 min. Endogenous peroxidase activity was blocked by incubating between two groups. The significance is presented as *Po0.05, **Po0.005 sections with shaking in 1% hydrogen peroxide in 50% methanol for and ***Po0.001, and non-significant differences are presented as NS. 30 min and then blocked in 3% bovine serum albumin (BSA) in 0.1% NP40 for 10 min at room temperature. For ERb, primary antibody was diluted 1:100 in 3% BSA in 0.1% NP40 and incubated overnight at 4 1C. Sections CONFLICT OF INTEREST were then washed consecutively in 0.1% NP40 for 30 min followed by PBS The authors declare no conflict of interest. for 10 min. The slides were then incubated for 1 h with biotinylated goat anti-chicken antibody (Abcam, Cambridge, MA, USA) diluted to 1:200 in 3% BSA in 0.1% NP40, washed in 0.1% Nonidet P-40 in PBS for 30 min and incubated with Vectastain ABC (Vector Labs, Burlingame, CA, USA) for 1 h. ACKNOWLEDGEMENTS The slides were then stained in 3,30-diaminobenzidine and counterstained This work was supported by The Cancer Prevention and Research Institute of Texas with Mayer hematoxylin (Sigma) before dehydration through ethanol, and (CPRIT) grants HIRP100680 and RP110444, the Texas Emerging Technology Fund mounted in Pertex (Histolab, Goteborg, Sweden). For FOXO3a, antigen under Agreement number 300-9-1958, the Robert A. Welch Foundation (E-0004) and retrieval was performed in pretreatment module for 7 min. Endogenous the Swedish Cancer Fund. We are grateful to Linda Waage, Department of Urology at peroxidase activity was blocked as mentioned before and then blocked in Danderyd Hospital, Stockholm, Sweden, for providing us the human prostate cancer 3% BSA and 0.1% NP40 for 10 min at room temperature. For FOXO3a, samples, and Ulf Bergerheim for Gleason grading the tissue samples. primary antibody was diluted 1:800 (Santa Cruz, Dallas, TX, USA) in 3% BSA and 0.1% NP40, and incubated overnight at 4 1C. Sections were then washed consecutively in 0.1% NP40 for 30 min and with PBS, followed by REFERENCES incubation with a Rabbit-on-Rodent HRP polymer (Biocare Medical, 1 Siegel R, Naishadham D, Jemal A. Cancer statistics 2012CA Cancer J Clin 2012; 62: Concord, CA, USA) for 30 min at room temperature. Sections were then 10–29. washed consecutively in 0.1% NP40 for 30 min and with PBS for 10 min. 2 Cornforth AN, Davis JS, Khanifar E, Nastiuk KL, Krolewski JJ. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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