Estrogen receptors localization and signaling pathways in DU-145 human prostate cancer cells

Molecular and Cellular Endocrinology 483 (2019) 11–23

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Estrogen receptors localization and signaling pathways in DU-145 human prostate cancer cells

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Deborah S. Souza, Ana Paola G. Lombardi, Carolina M. Vicente, Thaís Fabiana G. Lucas, Adolfo G. Erustes, Gustavo J.S. Pereira, Catarina S. Porto∗ Laboratory of Experimental Endocrinology, Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo 669, Vila Clementino, São Paulo, SP, 04039-032, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: ERα ERβ CRM1 ERK1/2 AKT Prostate cancer cell

The aim of the present study was to investigate the subcellular localization of estrogen receptors ERα and ERβ in androgen-independent prostate cancer cell line DU-145, and the possible role of exportin CRM1 on ERs distribution. In addition, we evaluated the ERs contribution to activation of ERK1/2 and AKT. Immunostaining of ERα and ERβ was predominantly found in the extranuclear regions of DU-145 cells. CRM1 inhibitor Leptomycin B reduced drastically the presence of ERα and ERβ in the extranuclear regions and increased in the nuclei, indicating the possible involvement of CRM1 on ERs nuclear-cytoplasmic shuttling. 17β-estradiol (E2), ERαselective agonist PPT and ERβ-selective agonist DPN induced a rapid increase on ERK1/2 phosphorylation. E2induced ERK1/2 activation was partially inhibited when cells were pretreated with ERα- or ERβ-selective antagonists, and blocked by simultaneous pretreatment with both antagonists, suggesting ERα/β heterodimers formation. Furthermore, E2 treatment did not activate AKT pathway. Therefore, we highlighted a possible crosstalk between extranuclear and nuclear ERs and their upstream and downstream signaling molecules as an important mechanism to control ER function as a potential therapeutic target in prostate cancer cells.

1. Introduction The treatment for advanced or metastatic prostate cancer includes androgen deprivation therapies to control key signaling pathways via androgen receptor (AR) and AR collaborative transcription factors (reviewed by Obinata et al., 2017; Gillessen et al., 2017); however, prostate cancer gradually evolves from an androgen-sensitive to an androgen-independent form of the disease, also known as castrationresistant prostate cancer (CRPC), during androgen deprivation therapies (Scher et al., 2004). The molecular mechanisms involved in CRPC are still not completely understood (reviewed by Scher and Sawyers, 2005; Parray et al., 2012) and new therapeutic strategies are required. The interest in estrogen receptors (ERs) biology in the prostate and its role in prostate cancer have increased after advances in the understanding of AR function in CRPC and the cross-talk that occurs between AR and the ERs subtypes, including ERα (ESR1, NR3A1) and ERβ (ESR2, NR3A2), in prostate cancer cells (reviewed by Grubisha and DeFranco, 2013). The traditional paradigm regarding the roles of the two ERs in the prostate is that ERα is oncogenic and promotes cell proliferation and

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survival, whereas ERβ is predominantly protective, being anti-carcinogenic and pro-apoptotic (reviewed by Prins and Korach, 2008; Nelson et al., 2014). However, increasing evidences have shown that ERβ may be potentially oncogenic in prostate cancer. In CRPC, it has been proposed the ERβ as a driver of AR-dependent gene transcription (Yang et al., 2015), along with a potential role in mediating the transition from hormone-sensitive to CRPC (Zellweger et al., 2013). The expression of ERβ is augmented in bone and lymph node metastases (Bouchal et al., 2011; Zhu et al., 2004) and high expression of ER correlates with poor clinical prognosis (Horvath et al., 2001; Zellweger et al., 2013). Dynamic changes in ERα and ERβ were detected in prostate cancer samples obtained from patients during the progression of prostate cancer and CRPC, but the relative levels of each receptor in different stages and functions remain controversial in the literature (reviewed by Nelson et al., 2014; Leach et al., 2016; Lau and To, 2016; Kowalska and Piastowska-Ciesielska, 2016; Fujimura et al., 2018). Thus, detailed studies are required to establish the cell-specific roles of ERα, ERβ and their isoforms in the prostate cancer progression and CRPC as well as the investigation of novel therapeutic agents that selectively target

Corresponding author. E-mail address: [email protected] (C.S. Porto).

https://doi.org/10.1016/j.mce.2018.12.015 Received 15 August 2018; Received in revised form 19 December 2018; Accepted 20 December 2018 Available online 17 January 2019 0303-7207/ © 2019 Elsevier B.V. All rights reserved.

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replaced by serum free medium for 24 h before the assays (Pisolato et al., 2016). At this stage, the cells were 85–90% confluent, and the amount of viable cells in each culture, as determined by trypan blue exclusion, was more than 90%. All experimental procedures were approved by the Research Ethical Committee at Escola Paulista de Medicina - Universidade Federal de São Paulo (#9164100615).

ERα- and ERβ-dependent pathways (reviewed by Nelson et al., 2014). Recent studies from our laboratory have shown that androgen-independent prostate cancer cells PC-3, used in vitro and in xenograft implants as CRPC models, expressed both ERα and ERβ (Pisolato et al., 2016). The ERβ activation by 17β-estradiol (E2) or ERβ-selective agonist DPN (diarylpropionitrile) decreased N-cadherin (Silva et al., 2018) and increased non-phosphorylated β-catenin levels, which plays an important role on PC-3 cell proliferation (Lombardi et al., 2016). Interestingly, ERα and ERβ were mostly found in the extranuclear region of PC-3 cells, and the treatment with E2 (0.1 nM) for 24 h was not able to modulate the localization of these receptors (Pisolato et al., 2016). Localization of ERs, extranuclear versus nuclear has been reported to provide differential prognostic information at least in breast cancer in vivo (Shaaban et al., 2008; reviewed by Boonyaratanakornkit et al., 2018), but it remains to be explored in prostate cancer. The nucleocytoplasmic transport of several proteins is highly regulated and coordinated by exportin CRM1 (Chromosome region maintenance 1), also known as XPO1 (reviewed by Turner et al., 2014; Dickmanns et al., 2015). Proteins exported from the nucleus must possess a hydrophobic nuclear export signal (NES) peptide that binds to a hydrophobic groove containing an active site Cys528 in the CRM1 protein (Fung and Chook, 2014). NES was mapped within the 444–456 sequence of ERα and it is responsible for E2-dependent interaction of ERα with CRM1 (Lombardi et al., 2008). The involvement of CRM1 in the nucleocytoplasmic transport of ERs in androgen-independent prostate cancer cells remains unexplored. Estrogen receptors ERα and ERβ, both nuclear and membrane/cytoplasmic pools, have been associated to the regulation of some key genes (reviewed by Acconcia and Marino, 2011; Arnal et al., 2017; Boonyaratanakornkit et al., 2018), and these receptors showed a similar affinity for the binding of steroid ligand in different cell types (reviewed by Levin, 2014, 2018). In prostate cancer, there are few studies reporting the importance of membrane/cytoplasmic localization of ER and rapid signaling activation (nongenomic action). In androgen-dependent prostate cancer cells LNCaP, androgen or 17β-estradiol treatment promotes ERK-2 (extracellular signal-regulated kinase 2) activation and proliferative effects by inducing the rapid assembly of a signaling complex containing classical AR, ERβ and Src (Migliaccio et al., 2000). In androgen-independent prostate cancer cells PC-3, the activation of ERα and ERβ by selective agonists increased the ERK1/2 phosphorylation (Pisolato et al., 2016). Thus, this study was performed to investigate the cellular localization of ERα and ERβ in the androgenindependent prostate cancer cell line DU-145 and the role of exportin CRM1 in this regulation. In addition, we evaluated the contribution of these receptors to the activation of ERK1/2 (MAPK3/1) and AKT (serine/threonine kinases).

2.2. Immunofluorescence analysis for the detection of estrogen receptors DU-145, PC-3, PNT1A and MCF-7 cells were grown as described above on coverslips coated with gelatin (0.1%, w/v) and placed into six-well plates. DU-145 and MCF-7 cells in culture medium without serum were incubated in the absence (control) and presence of CRM1 inhibitor, Leptomycin B (Sigma-Aldrich Chemical Co) (LMB 4.5 nM), for 24 h at 37 °C. After that, the cells were washed with ice-cold PBS, fixed in 2% formalin (formaldehyde EM grade, Electron Microscopy Sciences, Hatfield, PA, USA) for 20 min at room temperature and washed with PBS containing 0.1 M glycine (Sigma Chemical Co.). The immunofluorescence analysis were performed as previously described (Lucas et al., 2008; Pisolato et al., 2016), using rabbit polyclonal antibody raised against a peptide mapping at the carboxy-terminal of ERα of mouse origin, similar to human ERα (MC-20, sc-542, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and goat polyclonal antibody against epitope mapping near the carboxy-terminal of ERβ of human origin (L20, Santa Cruz Biotecnology Inc.), or mouse monoclonal antibody that recognizes all forms of ERβ (MC10, Thermo Fisher Scientific, Waltham, MA, USA) at 1:50 dilution in PBS, containing 0.01% saponin (w/v) and 1% BSA (w/v), for 1 h at room temperature. Cells were also incubated with Alexa Fluor 594-labeled secondary antibody (anti-rabbit and antigoat antibodies) or Alexa Fluor 488-labeled secondary antibody (antimouse) (1:300; Molecular Probes®, Invitrogen, NY, EUA). Nuclear staining was performed with DAPI (4’,6-diamidino- 2-phenylindole, Sigma Chemical Co). Negative controls were performed in the absence of primary antibody or with the primary antibodies pre-adsorbed with blocking peptide. Immunostaining of estrogen receptors was visualized under a confocal microscope Leica Microsystems TCSSP8 (Leica Inc., Wetzlar, Germany). Images of five random microscope fields were captured, in duplicate, in each assay (three independent experiments) and analyzed using the software LAS-AF and ImageJ. 2.3. Western blot analysis for detection of estrogen receptors DU-145 cells in culture medium without serum were incubated in the absence (control) and presence of CRM1 inhibitor, Leptomycin B (Sigma- Aldrich Chemical Co) (LMB 18, 9, 4.5 and 2.25 nM), for 24 h at 37 °C. Nuclear and extranuclear (containing membrane, cytoplasm and organelles) extracts were obtained as previously described by Royer et al. (2012). Briefly, the cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P40, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, 10 mM Na2P2O7, 1 μg/ml leupeptin, and 1 μg/ml aprotinin, Sigma Chemical Co.). Nuclear and extranuclear fractions were separated using a buffer containing 1.0 M sucrose. Afterward, the nuclear content was extracted with buffer containing 10 mM HEPES, 200 mM NaCl, pH 7.9, 1.5 mM MgCl2, 0.1 mM EDTA, 5% glycerol, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, 10 mM Na2P2O7, 1 μg/ml leupeptin, and 1 μg/ml aprotinin (Sigma Chemical Co.). The nuclear and extranuclear extracts of the DU-145 cells (30 μg protein/lane) were incubated with sample buffer containing β-mercaptoethanol (Sigma Chemical Co.), and subjected to 10% SDS/PAGE (BioRad Laboratories, Richmond, CA, USA). Western blot analysis were performed as previously described (Pisolato et al., 2016), using rabbit polyclonal antibody raised against a peptide mapping at the carboxyterminal of ERα of mouse origin, similar to human ERα (MC-20, sc-542, Santa Cruz Biotechnology Inc.), and goat polyclonal antibody against

2. Materials and methods 2.1. Cell culture The human post pubertal prostate epithelial cell line (PNT1A) was obtained from the Culture Collections, Public Health England (Porton Down, Salisbury, UK). The human androgen-independent prostate cancer cell lines DU-145 (derived from brain metastasis) and PC-3 (derived from bone metastasis) and breast cancer cells MCF-7 (used as a control of some experiments) were obtained from the American Type Culture Collection (Manassas, VA, USA). PNT1A, DU-145 and PC-3 were obtained in passages 57, 60 and 30, respectively, and only used below passages 62, 69 and 45. PNT1A, DU-145, PC-3 and MCF-7 cells (2 × 105 cells/ml) were grown in RPMI 1640 medium without phenol red (GIBCO®, Life Technologies Co., Grand Island, NY, USA), supplemented with 10% of fetal bovine serum (GIBCO®, Life Technologies Co.), HEPES (5.95 mg/ml) and gentamicin (0.02 mg/ml) (Sigma Chemical Co. St. Louis, MO, USA) in a humidified atmosphere with 5% CO2:95% air, at 37 °C, for 72 h. Afterwards, the culture medium was 12

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agonist DPN (10 nM; 2,3-bis(4-hydroxyphenyl)-propionitrile, Tocris Bioscience), for 5 min at 37 °C. The cells were also untreated or pretreated with the ERα-selective antagonist MPP (10 nM; 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride, Tocris Bioscience) or with the ERβ-selective antagonist PHTPP (10 nM; 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5a]pyrimidin-3-yl]phenol, Tocris Bioscience) or with both antagonists, MPP (10 nM) and PHTPP (10 nM), for 30 min. Incubation was continued in the absence and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM), for 5 min at 37 °C. At these concentrations, the agonists and antagonists are highly selective, as previously reported (Stauffer et al., 2000; Meyers et al., 2001; Lucas et al., 2008; Pisolato et al., 2016). Western blot analysis were performed as previously described (Lucas et al., 2008; Royer et al., 2012; Pisolato et al., 2016; Lombardi et al., 2016), using rabbit polyclonal antibody raised against a synthetic peptide (KLH-coupled) derived from the sequence in the carboxy-terminus of rat 44 MAP kinase, that recognize 42 and 44 MAP kinases, which cross-reacts with human kinases (#9102; Cell Signaling Technology Inc.; 1:2500 dilution), or polyclonal antibody raised against a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase, (#9101; Cell Signaling Technology Inc.; 1:1000 dilution), or monoclonal antibody produced in rabbit by immunizing animals with a synthetic peptide corresponding to residues in the carboxy-terminal sequence of mouse AKT (total AKT #4691, Cell Signaling Technology Inc., 1:2000 dilution) or polyclonal antibody produced in rabbit by immunizing animals with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser473 of mouse AKT (p-AKT #9271, Cell Signaling Technology Inc., 1:1000 dilution), which cross-reacts with human kinases. The antibodies were incubated overnight at 37 °C. Apparent molecular masses were determined from molecular mass standards (#26634 Thermo Fisher Scientific Inc.) Band intensities of ERK1/2, phospho-ERK1/2, AKT and phospho-AKT from individual experiments were detected by a chemiluminescence detection system (UVITEC, Cambridge, UK) and quantified by UVI-1D software or by densitometric analysis of linear-range autoradiograms, using an Epson Expression 1680 scanner and the quick Scan 2000 WIN software (Helena Laboratories Co.). Results were normalized based on expression of total ERK1/2 in each sample and plotted (mean ± SEM) in relation to control (C = 1).

an epitope mapping near the carboxy-terminal of ERβ of human origin (L-20, Santa Cruz Biotechnology Inc.), at 1:200 dilution, overnight at 4 °C. Proteins were visualized by enhanced chemiluminescence reagent (ECL, GE Healthcare) after incubation, for 1 h at room temperature, with the appropriate HRP-conjugated antibodies (GE Healthcare, Chalfont, St. Giles, United Kingdom or Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA) (1:3000 dilution). Lamin A, used as a nuclear protein loading control, was detected with a rabbit antibody raised against a synthetic peptide derived from residues 563–664 Lamin A (sc-20680, Santa Cruz Biotechnology Inc.). β-tubulin, used as cytoplasmic protein loading control, was detected with a monoclonal rabbit antibody raised against a synthetic peptide corresponding to the amino-terminal of the human β-tubulin (#2128, Cell Signaling TechnologyInc., Danvers, MA, USA). To detect any cross contamination between nuclei and cytoplasm, Lamin A and β-tubulin were also assayed in extranuclear and nuclear fractions, respectively. No cross contamination was detected. Apparent molecular masses were determined from molecular mass standards (#26634 Thermo Fisher Scientific Inc.). Band intensities of ERα, ERβ, Lamin A and β-tubulin from individual experiments were quantified by densitometric analysis of linear-range autoradiograms, using an Epson Expression 1680 scanner and the quick Scan 2000 WIN software (Helena Laboratories Co., Beaumont, TX, USA). Results were normalized to the respective expression of Lamin A or β-tubulin and plotted (mean ± SEM) in relation to control of each extract (C = 100%). Western blot analysis for detection of estrogen receptors in DU-145, PC-3 and PNT1A cells was also performed using total cell lysates (Pisolato et al., 2016) and mouse monoclonal antibody that recognizes all forms of ERβ (MC10, Thermo Fisher Scientific) (1:300). 2.4. Immunoprecipitation analysis of estrogen receptors Rabbit polyclonal antibody raised against a peptide mapping at the carboxy-terminal of ERα of mouse origin, similar to human ERα (MC20, sc-542, Santa Cruz Biotechnology Inc.) (8 μg) was first bound to Protein A Agarose beads (Cell Signaling Technology, Inc., Beverly, MA, USA) (80 μL) by incubation for 4 h at 4 °C, under gentle agitation. DU145 cells in culture medium without serum were incubated in the absence (control) and presence of 17β-estradiol (E2, 0.1 nM; Sigma Chemical Co.) for 5 min. After that, cells were lysed in ice-cold lysis buffer as previously described (Pisolato et al., 2016). DU-145 lysates (2 mg of total cell protein) from control or treated cells were added to the complex anti-ERα antibody-Protein A Agarose beads, and immunoprecipitation was performed overnight at 4 °C, under gentle agitation. The samples were centrifuged at 400 g for 5 min, and the pellets were washed five times with 1 ml of ice-cold lysis buffer. The immunocomplexes were eluted from Protein A Agarose beads with 60 μL of glycine (100 mM, pH 2.4, 15 min), and submitted to 12% SDS-PAGE. Immunoblot for the detection of ERα and ERβ were performed as previously described (Pisolato et al., 2016), using rabbit polyclonal antibody raised against a peptide mapping at the carboxy-terminal of ERα of mouse origin, similar to human ERα (MC-20, sc-542, Santa Cruz Biotechnology Inc.) (1:200) or mouse monoclonal antibody that recognizes all ERβ isoforms (MC10, Thermo Fisher Scientific) (1:300). Negative control of immunoprecipitation (IP) was performed as described above without total cell lysates.

2.6. Protein assays Protein concentration was determined with the Bio-Rad protein assay, using bovine serum albumin as standard (Bio Rad Laboratories Inc.). 2.7. Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was carried out by ANOVA followed by the Newman-Keuls test for multiple comparisons or by Student t-test to compare the differences between two data. P values < 0.05 were accepted as significant. 3. Results 3.1. Estrogen receptors ERα and ERβ are mostly located in the extranuclear region of DU-145 cells

2.5. Western blot analysis for detection of total and phosphorylated ERK1/ 2 and AKT

In androgen-independent prostate cancer cells DU-145, immunostaining of ERα was predominantly found in the plasma membrane and cytoplasm (extranuclear region), and weakly in the nuclei (Fig. 1A, top panel). No immunostaining was observed in the negative control, performed with the primary antibody pre-adsorbed with blocking peptide (Fig. 1A, bottom panel). On the other hand, immunostaining of ERα was predominantly nuclear in human post

DU-145 and PC-3 cells in culture medium without serum were incubated in the absence (control) and presence of 17β-estradiol (E2, 0.1 nM; Sigma Chemical Co.) for 5, 10, 15, 20, 30 min and 1 and 2 h; ERα-selective agonist PPT (10 nM; 4,4′,4”-(4-propyl-(1H)-pyrazole1,3,5-triyl)trisphenol, Tocris Bioscience, Bristol, UK) or ERβ-selective 13

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Fig. 1. Expression of ERα in DU-145 (A) and PNT1A (B) cells. Immunostaining for ERα (red) was detected using antibody against the carboxy-terminal region of ERα (MC-20, Santa Cruz) (A and B top panels). Nuclei were stained with DAPI (blue). Negative controls were performed with the primary antibody against the carboxy-terminal region of ERα pre-adsorbed with blocking peptide (A and B, bottom panels). Scale bar as indicated. The data shown are representative of three independent experiments. 14

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Fig. 2. Expression of ERβ in DU-145 (A) and PNT1A (B) cells. Immunostaining for ERβ (red) was detected using antibody against the carboxy-terminal region of ERβ (L-20, Santa Cruz) (A and B top panels). Nuclei were stained with DAPI (blue). Negative controls were performed with the primary antibody against the carboxyterminal region of ERβ pre-adsorbed with blocking peptide (A and B, bottom panels). Scale bar as indicated. The data shown are representative of three independent experiments. 15

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Fig. 3. Effects of Leptomycin B (LMB) on expression and cellular localization of ERα and ERβ in DU-145 cells. Cells were incubated in the absence (control, C) and presence of CRM1 inhibitor, Leptomycin B (LMB 18, 9, 4.5 and 2.5 nM) for 24 h at 37 °C. Extranuclear (A and C) and nuclear (B and D) extracts (30 μg of protein/ lane) were resolved in 10% SDS/PAGE, transferred to PVDF membrane, and probed with antibody against the carboxy-terminal region of ERα (MC-20, Santa Cruz) (top panels) (A and B), with antibody against the carboxy-terminal region of ERβ (L-20, Santa Cruz) (top panels) (C and D), with antibodies that recognize cytoplasmic protein β-tubulin or nuclear protein Lamin A (bottom panels) (A, B, C and D). The relative positions of ERα, ERβ, β-tubulin and Lamin A are shown at the right. The data shown are representative of three independent experiments. Bars represent the densitometric analysis of the Western blot assays. Results were normalized to β-tubulin or Lamin A expression in each sample and plotted (mean ± SEM) in relation to control (C = 100%). *Significantly different from control, C (P < 0.05, Student t-test).

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3.3. Activation of ERα and ERβ increases the phosphorylation of ERK1/2, but not the phosphorylation of AKT in DU-145 cells

pubertal prostate epithelial cell line PNT1A, used as a control (Fig. 1B, top panel). No immunostaining was observed in the negative control (Fig. 1B, bottom panel). Immunostaining of ERβ was mainly detected in the cytoplasm (extranuclear region) and less intense in the nuclei of DU-145 cells (Fig. 2A, top panel, Supplemental Fig. 1) and also in PNT1A cells (Fig. 2B, top panel). No immunostaining was observed in the negative controls, performed with the primary antibody pre-adsorbed with blocking peptide (Fig. 2A and B, bottom panels). The expression and localization of ERα and ERβ were confirmed using extranuclear (plasma membrane, cytoplasm and organelles) and nuclear extracts of DU-145 cells in Western blot analysis (Fig. 3). ERα was detected as a single protein band of about 66 kDa (Fig. 3A and B). In control cells (C, untreated DU-145 cells), the expression of ERα was higher (5-fold) in the extranuclear (Fig. 3A) than in the nuclear (Fig. 3B) extracts. ERβ was detected as a single protein band of about 56 kDa (Fig. 3C and D). The expression of ERβ was also higher (4-fold) in the extranuclear (Fig. 3C) than in the nuclear (Fig. 3D) extracts of DU-145 cells. It is recognized that there is wide variability in the sensitivity and specificity of ERβ antibodies (Nelson et al., 2017; Andersson et al., 2017), which may contribute to the controversial expression of this receptor in cells and tissues. To confirm our results, it was used the validated MC10 ERβ antibody (Wu et al., 2012; Nelson et al., 2017) and the expression of ERβ was detected in three prostate cell lines, PNT1A, DU-145 and PC-3 by Western blot and immunofluorescence analyses (Supplemental Fig. 1). Furthermore, the expression of this receptor is higher in DU-145 than in PC-3 cells (Supplemental Fig. 1), suggesting distinct androgen-independent mechanisms involved in the regulation of the ERβ expression.

The presence of ERα and ERβ in the extranuclear region of DU145 cells suggests that both ERs may play a role in membrane-initiated rapid signaling pathways. Several proteins are involved in this process, such as ERK1/2 and AKT. Activity and expression of ERK1 and ERK2 in DU-145 cells lysates were assessed by immunoblotting using phosphorylation state-dependent (top panels) and -independent (total ERK1/2, bottom panels) antibodies (Fig. 5). The treatment with 17β-estradiol (E2, 0.1 nM, for 5 and 10 min) increased ERK1/2 phosphorylation in DU-145 cells. The activity of ERK1/2 returned to control levels at 20 min (Fig. 5A). The activation of ERK1/2 induced by a 5-min treatment with E2 (0.1 nM) was partially blocked by ERα- (MPP, 10 nM) or ERβ- (PHTPP, 10 nM) selective antagonists (Fig. 5B). A complete blockade of ERK1/2 activation was only achieved by simultaneous pretreatment of DU-145 cells with both, MPP and PHTPP (Fig. 5C), suggesting that ERα/β heterodimers may play a role in the activation of ERK1/2. Treatment of cells with the ERα-selective agonist PPT (10 nM) (Fig. 5D) or the ERβ-selective agonist DPN (10 nM) (Fig. 5E), for 5 min, also increased the phosphorylation state of ERK1/2 in the DU-145 cells. These effects were blocked by pretreatment with the respective ERα or ERβ antagonist (Fig. 5D and E). Activity and expression of AKT in DU-145 cell lysates were measured by immunoblotting using phosphorylation state-dependent (top panels) and -independent (total AKT, bottom panels) antibodies (Fig. 6). The expression of phosphorylated AKT at Ser473 was neither detected in control cells nor in those treated with 17β-estradiol (E2) (0.1 nM) for 10, 15, 30 min and 1 and 2 h (Fig. 6A and B, top panels) or for 2 and 5 min (data not shown). However, the expression of total AKT was detected in DU-145 cells (Fig. 6A and B, bottom panels). Using the same antibodies, phosphorylated (top panel) and total (bottom panel) AKT were detected in PC-3 cells (Fig. 6B), confirming our previous study (Lombardi et al., 2016).

3.2. CRM1 is involved in the export of ERs from the nucleus to the cytoplasm in DU-145 cells To explore a possible involvement of CRM1 in the nuclear export of ERα and ERβ in DU-145 cells, we used Leptomycin B (LMB), which has been shown to interact with CRM1 inhibiting protein export from the nucleus to the cytoplasm (Kudo et al., 1999). In the presence of CRM1 inhibitor LMB (18, 9, 4.5 and 2.25 nM), the expression of ERα (Fig. 3A) and ERβ (Fig. 3C) decreased in the extranuclear lysates of DU-145 cells when compared to control cells. On the other hand, the expression of these receptors increased in the nuclear extract of DU-145 cells treated with LMB (Fig. 3B and D) compared with control cells. Immunofluorescence analysis confirmed the results of the Western blot. In control cells (untreated DU-145 cells), immunostaining of ERα was predominantly found in plasma membrane and cytoplasm (extranuclear region) and weak immunostaining of ERα was also detected in the nuclei (Fig. 4A). On the other hand, immunostaining of ERα was drastically reduced in the plasma membrane and cytoplasm in the presence of LMB. Immunostaining of ERα was predominantly detected in the nuclei after treatment of DU-145 cells with LMB (Fig. 4A, bottom panel). Breast cancer MCF-7 cells were used as a control, since ERα is localized in the nuclei and membrane/cytoplasm of these cells (Nonclercq et al., 2007; Lombardi et al., 2008). In the presence of LMB, ERα immunostaining was drastically reduced in the plasma membrane and cytoplasm and it was predominantly found in the nuclei of these cells (Supplemental Fig. 2). In DU145 cells, immunostaining of ERβ was mainly detected in the cytoplasm and nuclei of control cells (untreated DU-145 cells) (Fig. 4B). In the presence of CRM1 inhibitor (LMB 4.5 nM), immunostaining of ERβ was detected predominantly in the nuclei of DU-145 cells (Fig. 4B, bottom panel). These results indicated that CRM1 protein is involved with the localization of ERs in DU-145 cells.

3.4. ERα/β heterodimers are present in DU-145 cells It has become important to evaluate the status of estrogen receptor dimers in DU-145 cells in order to obtain a better understanding of effects of 17β-estradiol (E2) upon ERK1/2 phosphorylation. We first assessed the co-localization of ERα and ERβ, using a confocal microscope (Fig. 7A). Co-localization of ERα and ERβ was detected mostly in the extranuclear region of DU-145 cells (Fig. 7A). In addition, total cell lysates were immunoprecipitated with anti-ERα antibody and blotted with anti-ERα (Fig. 7B, top panel) or anti-ERβ (Fig. 7B, bottom panel). ERα co-immunoprecipitated with ERβ in control (untreated DU145 cells) and E2-treated cells, indicating the presence of functionally ERα/β heterodimers. 4. Discussion The variability of ERα and ERβ expression in different grades and stages of prostate cancer (reviewed by Nelson et al., 2014; Leach et al., 2016) and their re-emergence in prostate cancer metastasis present some difficulty in deciphering the underlying mechanisms and functions of these receptors in prostate carcinogenesis. To improve outcomes for patients, there is an urgent need for detailed understanding of these mechanisms. In the present study, we showed the presence of ERα and ERβ in the extranuclear regions, the involvement of CRM1 on ERs nuclear-cytoplasmic shutting and modulation of ERK1/2 activation in androgen-independent prostate cancer cells DU-145. The mRNAs for ERα and ERβ were detected in DU-145 cells (Linja et al., 2003) and in the present study both receptors (protein) were detected. Furthermore, the expression of ERβ is higher in DU-145 than in PC-3 cells, suggesting distinct androgen-independent mechanisms 17

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Fig. 4. Effects of Leptomycin B (LMB) on cellular localization of ERα (A) and ERβ (B) in DU-145 cells. Cells were incubated in the absence (control) and presence of CRM1 inhibitor, Leptomycin B (LMB 4.5 nM) for 24 h at 37 °C. Immunostaining for ERα (red) and ERβ (red) was detected using antibody against the carboxy-terminal region of ERα (MC-20, Santa Cruz) (A) and ERβ (L-20, Santa Cruz) (B). Nuclei were stained with DAPI (blue). Scale bar as indicated. The data shown are representative of three independent experiments. 18

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Fig. 5. Effects of 17β-estradiol (E2), PPT and DPN on ERK1/2 phosphorylation in DU-145 cells. Cells were incubated in the absence (control, C) and presence of 17β-estradiol (E2) (0.1 nM) for different times (A, B and C), ERα-selective agonist PPT (10 nM) (D) or ERβ-selective agonist DPN (10 nM) (E) for 5 min at 37 °C. Cells were also untreated or pretreated with the ERα-selective antagonist MPP or with the ERβ-selective antagonist PHTPP (B, D and E) or with both antagonists, MPP (10 nM) and PHTPP (10 nM), (C) for 30 min at 37 °C. Incubation was continued in the absence and presence of E2 (0.1 nM) (B and C), PPT (D) or DPN (E) for 5 min at 37 °C. Total cell lysates (30 μg per lane) were resolved in 10% SDS/PAGE, transferred to PVDF membrane, and probed with antibody specific for phosphorylated ERK1/2 (Thr202/Tyr204, #9101; Cell Signaling) (top panels) or with antibody that recognizes total (phosphorylation state-independent) ERK1/2 (p44/p42 MAP kinase, Erk1/Erk2, #9102; Cell Signaling) (bottom panels). The relative positions of phosphorylated ERK1/2 and total ERK1/2 proteins are shown at the right. The data shown are representative of four independent experiments. Bars represent the densitometric analysis of the Western blot assays. Results were normalized to total ERK1/2 expression in each sample and plotted (mean ± SEM) in relation to control (C = 1). Open bars ERK1 and closed bars ERK2. *Significantly different from control (P < 0.05, Student t-test). **Significantly different from E2 (P < 0.05, Student t-test).

protein, events that are involved in the transport of the ERα/FKHR complex from the nucleus to the cytoplasm in breast cancer cells (Lombardi et al., 2008). In PC-3 cells, the expression of total and phosphorylated AKT was detected, and E2 increased the phosphorylation of AKT (Lombardi et al., 2016). AKT may play a role in the extranuclear localization of ERs in PC-3 cells, this mechanism remain to be explored. On the other hand, phosphorylated AKT was not detected in DU-145 cells. Although PI3K and its lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3) have been shown to activate multiple downstream signaling proteins, the vast majority of studies have focused on the AKT as the dominant effector of PI3K signaling. However, recent studies have shown three PI3K-dependents, but AKT-independent, signaling branches with important roles in promoting phenotypes associated with malignancy (reviewed by Lien et al., 2017). The nuclear export of ERs could represent an important switch between tumor suppression and oncogenic functions of these receptors in prostate cancer cells. In breast cancer cells MCF-7, the accumulation of ER in the nuclear compartment induced by Leptomycin B resulted in a decrease rather than in an increase of transactivation activity (Nonclercq et al., 2007). ERs interact with Src out of the nucleus to activate extranuclear signaling pathways, such as ERK1/2 and AKT. These studies are emerging in androgen-independent prostate cancer cells. In the present study, 17β-estradiol (E2), ERα-selective agonist PPT (Stauffer et al., 2000) and ERβ-selective agonist DPN (Meyers et al., 2001) induced a rapid increase in the phosphorylation of ERK1/2 in DU-145 cells. The phosphorylation of ERK1/2 induced by E2 was partially blocked by the ERα- or ERβ-selective antagonists, MPP or PHTPP, respectively. On the other hand, this effect was fully blocked by simultaneous pretreatment with both, MPP and PHTPP. These results indicated the presence of functionally ERα/β heterodimers, activating a rapid MAPK pathway in DU-145 cells. In fact, ERα/β heterodimers were detected in control (untreated cells) and in DU-145 cells treated with E2. Furthermore, colocalization of ERα and ERβ was shown mostly in the extranuclear region of DU-145 cells. It has been reported that E2 induced similar levels of ERα/α and ERβ/β homodimers and ERα/β heterodimers

involved in the regulation of ERβ expression. It is important to emphasize that PC-3 cells are derived from bone metastasis and DU145 cells from brain metastasis, suggesting that the microenvironmental stimuli may play different role in the regulation of ERβ expression. In human post pubertal prostate epithelial cell line PNT1A, immunostaining of ERα was predominantly nuclear while immunostaining of ERβ was detected in the nuclei and also in the cytoplasm. On the other hand, in androgen-independent prostate cancer cells DU-145 (present study) and PC-3 (Pisolato et al., 2016) predominant presence of ERα and ERβ were shown in the extranuclear region. Nuclear-cytoplasmic trafficking of proteins is a significant factor in the development of cancer and resistance to endocrine therapy (reviewed by Turner et al., 2014; Dickmanns et al., 2015). In addition to nuclear signaling, ERs mediate hormone-induced rapid extranuclear signaling at the cell membrane or in the cytoplasm which initiates downstream signaling to regulate either rapid or extended cellular responses in breast cancer cells (reviewed by Boonyaratanakornkit et al., 2018). We showed that the preferential extranuclear localization of ERs in DU-145 cells involves a CRM1-mediated nuclear export of ERα and ERβ. CRM1 is functionally inactivated by Leptomycin B without a loss of protein expression in DU-145 and LNCaP cells (Mendonca et al., 2014). CRM1 is overexpressed in prostate cancer cells (LNCaP, PC-3 and DU-145 cells) compared with normal prostate epithelial cells or prostate fibroblasts (Mendonca et al., 2014). The mechanisms through which ERs is exported from the nucleus to the cytoplasm are not completely understood to date. In breast cancer cells, E2 induces Src (a non-receptor tyrosine kinase)-mediated phosphorylation of ERα at tyrosine 537, which in turn enhances ERα-CRM1 interaction to facilitate the nuclear-cytoplasmic shuttling of ERα (Lombardi et al., 2008; Castoria et al., 2012). Src is expressed in PC-3 and DU-145 cells (Rice et al., 2012) and may be involved in the phosphorylation of ERα and ERβ, interaction of these receptors with CRM1 and nuclear-cytoplasmic shuttling of ERs. It has also been described that E2 activates the PI3K/AKT pathway, followed by the AKT-mediated phosphorylation of the FKHR (Forkhead in rhabdomyosarcoma)

Fig. 6. Effects of 17β-estradiol (E2) on AKT phosphorylation in DU-145 and PC-3 cells. A. DU-145 cells were incubated in the absence (control, C) and presence of 17β-estradiol (E2) (0.1 nM) for different times at 37 °C. B. PC-3 and DU-145 cells were incubated in the absence and presence of E2 (0.1 nM) for 5 min at 37 °C. Total cellular proteins (30 μg per lane) were resolved in 10% SDS/PAGE, transferred to PVDF membrane, and probed with antibody specific for phosphorylated (p) AKT (Ser473) (p-AKT #9271, Cell Signaling) (top panels) or with antibody that recognizes total (phosphorylation state-independent) AKT (total AKT #4691, Cell Signaling) (bottom panels). The data shown are representative of two to four independent experiments. 20

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(caption on next page)

et al., 1999). It has become pivotal to evaluate the status of ER dimers in prostate cancer patients in order to obtain a better understanding of potential effects of E2 upon cell proliferation, migration and invasion. Endocrine therapy by blocking pathways linked to nuclear export of estrogen receptors (as Src inhibitors) is a promise for treatment of

(Powell and Xu, 2008). ERα/β heterodimers have been detected in vivo using molecular imaging techniques (Paulmurugan et al., 2011) and in breast cancer tissues using proximity ligation assay (Iwabuchi et al., 2017). ERα/β heterodimer is transcriptionally active and may regulate a distinct set of genes from their respective homodimers (Tremblay

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Fig. 7. Co-localization of ERα and ERβ and immunoprecipitation of ERα/β heterodimers in DU-145 cells. A. Representative photomicrographs illustrating double immunofluorescence localization of ERα and ERβ in DU-145 cells. Immunostaining for ERα (red) was detected using antibody against the carboxy-terminal region of ERα (MC-20, Santa Cruz). The same cells were incubated with monoclonal antibody that recognizes all forms of ERβ (MC10, Thermo Fisher Scientific) (immunostaining for ERβ, green). Co-localization of the red and green labels is shown in merged images (at right). Nuclei were stained with DAPI (blue). Negative controls were performed in the absence of primary antibody (inserts). The data shown are representative of two independent experiments. B. DU-145 cells were incubated in the absence (untreated cells, control, C) and presence of 17β-estradiol (E2) (0.1 nM) for 5 min at 37 °C. Total cell lysates (50 μg of protein/lane) (input) and the sample obtained from the immunoprecipitation of total cell lysates-anti-ERα antibody-Protein A Agarose (IP:ERα) were submitted to 12% SDS-PAGE, transferred to PVDF membrane, and probed with antibody directed against ERα (MC-20, Santa Cruz) (top panel) and ERβ (MC10, Thermo Fisher) (bottom panel). The relative positions of ERα and ERβ are shown at the right. Negative control of immunoprecipitation was performed as described in methods without total cell lysates (IP:Negative Control). The data shown are representative of two independent experiments.

Grant support

breast cancer (reviewed by Guest et al., 2016). However, in patients with prostate cancer, Src inhibition by dasatinib, saracatinib, and KX2391 has been tested in phase II clinical trials. Nevertheless, disappointing results have been obtained by these clinical trials, which suggest that Src inhibition cannot be used as monotherapy, but as part of a combinatorial therapy strategy in treating patients with CRPC (reviewed by Varkaris et al., 2014). Important advances in identification of AR binding partners (as Src and FlnA, Filamin A) have prompted the development of new approaches. Small peptides that mimic receptor sequences of AR interacting with Src or FlnA have been designed and successfully used in preclinical models of human prostate cancer (reviewed by Castoria et al., 2017). CRM1 inhibitors have also been proposed as therapeutic agents for several cancer types in which CRM1 is overexpressed (reviewed by Gravina et al., 2014; Yang et al., 2014). It is also clear that more studies are required to elucidate the molecular basis of ERs subcellular transport in prostate cancer cells and its role in, or its interconnection with other mechanisms that influence its expression, abundance, and activity in the prostate cancer development and CRPC. Further studies are needed to fully develop a valid marker and therapeutic target in prostate cancer. In conclusion, ERα and ERβ are located in the extranuclear region of the androgen-independent prostate cancer cells DU-145, differing from their usual nuclear localization in the majority of estrogen target cells. CRM1 is involved in the nuclear-cytoplasmic shuttling of ERs in these cells. The presence of ERs in the extranuclear region is important for activation of rapid signaling pathways, as ERK1/2 phosphorylation, but not AKT. We highlighted a possible crosstalk between extranuclear and nuclear ERs and their upstream and downstream partners as an important mechanism to control ER function in prostate cancer cells.

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Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Acknowledgements We thank Priscila Veronica Sartorio, Elizabeth Kanashiro and Caroline Zito Romera for technical assistance and Dr. Maria de Fatima M. Lazari for helpful discussions and critical reading of the manuscript. Confocal microscope Leica Microsystems TCSSP8, facility from the Instituto Nacional de Farmacologia e Biologia Molecular (INFAR) was supported by Financiadora de Estudos e Projetos (FINEP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Research (C.S.P.) and Doctoral (A.P.G.L.) fellowships were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Master fellowship (D.S.S.) was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Postdoctoral fellowships (T.F.G.L.) were supported by CAPES/PNPD and (A.G.E) by CNPq. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mce.2018.12.015. 22

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