Estrogen Receptor Signaling Pathways Involved in Invasion and Colony Formation of Androgen-Independent Prostate Cancer Cells PC-3

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Article Estrogen Receptor Signaling Pathways Involved in Invasion and Colony Formation of Androgen-Independent Prostate Cancer Cells PC-3

Ana Paola G. Lombardi 1, Renan P. Cavalheiro 2, Catarina S. Porto 1,* and Carolina M. Vicente 1

1 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; [email protected] (A.P.G.L.); [email protected] (C.M.V.) 2 Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP 04039-032, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-11-55764976

Abstract: Castration-resistant prostate cancer (CRPC) is an advanced and androgen-independent form of prostate cancer. Recent studies of rapid actions mediated by estrogen in the prostate and its relationship with CRPC are emerging. We have previously shown that estrogen receptor (ER) promotes migration and invasion of the androgen-independent prostate cancer cells PC-3, but the signaling pathways involved in these events remain to be elucidated. Therefore, this study aimed to analyze the role of ERα and ERβ in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the PC-3 cells. Our results showed that the activation of ERα (using ERα-selective agonist PPT) and ERβ (using ERβ-selective agonist DPN) increased phosphorylation of SRC in PC-3 cells. In the presence of the selective inhibitor Citation: Lombardi, A.P.G.; for SRC-family kinases PP2, the effects of DPN and PPT on transmigration and soft agar colony Cavalheiro, R.P.; Porto, C.S.; Vicente, C.M. Estrogen Receptor Signaling formation assays were decreased. Furthermore, SRC is involved in the expression of the non- Pathways Involved in Invasion and phosphorylated β-catenin. Finally, using PI3K speciﬁc inhibitor Wortmannin and AKT inhibitor Colony Formation of MK2206, we showed that PI3K/AKT are also required for invasion and colony formation of PC-3 Androgen-Independent Prostate cells simulated by ER. This study provides novel insights into molecular mechanisms of ER in PC-3 Cancer Cells PC-3. Int. J. Mol. Sci. cells by demonstrating that ER, located outside the cell nucleus, activates rapid responses molecules, 2021, 22, 1153. https://doi.org/ including SRC and PI3K/AKT, which enhance the tumorigenic potential of prostate cancer cells, 10.3390/ijms22031153 increasing cell proliferation, migration, invasion, and tumor formation.

Academic Editors: Farzad Pakdel Keywords: estrogen receptor; SRC; PI3K/AKT; β-catenin; PC-3 cells Received: 27 November 2020 Accepted: 19 January 2021 Published: 25 January 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The androgen receptor (AR) is the classical target for prostate cancer treatment [1], and published maps and institutional afﬁl- estrogens and their receptors have recently been implicated in prostate cancer development iations. and tumor progression [2,3]. Initially, prostate cancer depends on androgen to evolve, but it can gradually progress to an androgen-independent form of the disease, also known as castration-resistant prostate cancer (CRPC) [2,4,5]. The molecular mechanisms involved in this stage of the disease are not fully understood and the current therapies are insufﬁcient to improve the survival of patients. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Previous studies from our laboratory have already shown that, in androgen-independent α This article is an open access article prostate cancer cells PC-3 [6] and DU-145 [7], estrogen receptors (ER) ER (ESR1) and distributed under the terms and ERβ (ESR2) are mostly located outside the nucleus of these cells, indicating the activa- conditions of the Creative Commons tion of rapid signaling pathways. In fact, the activation of these receptors increases the Attribution (CC BY) license (https:// phosphorylation of ERK1/2 (extracellular signal-regulated kinase1 and 2) in both cell creativecommons.org/licenses/by/ lines [6,7] and the phosphorylation of AKT (serine/threonine kinases) in PC-3 cells [8]. 4.0/). Furthermore, in PC-3 cells, the activation of ERβ decreases N-cadherin [9] and increases

Int. J. Mol. Sci. 2021, 22, 1153. https://doi.org/10.3390/ijms22031153 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1153 2 of 15

non-phosphorylated β-catenin levels [8,9]. The activation of ERβ promotes the increase of migration, invasion, and anchorage-independent growth of PC-3 cells through β-catenin pathway. The activation of ERα also plays a role on invasion and anchorage-independent growth of PC-3 cells [10]. However, the molecular mechanisms of the crosstalk between ER and β-catenin pathways in this cell remains to be elucidated. It is important to mention that 17β-estradiol impacts normal and malignant tissue development via ERα and ERβ, either through ligand-activated transcriptional regulation (genomic pathway) or by triggering cytoplasmic-signaling cascades (nongenomic pathway). The possible convergence of genomic and nongenomic pathways on target genes is an attractive mechanism by which ER can ﬁnely regulate gene expression in different cells [11,12]. Indeed, evidence indicates that a pool of ER located in the cytoplasm and/or at the plasma membrane forms multiprotein complexes leading to the activation of downstream signaling molecules. ER may interact with SRC (non-receptor tyrosine kinase) out of the nucleus to activate extranuclear signaling pathways, such as ERK1/2 and AKT, in breast cancer cells [11]. In addition, the extranuclear complex of ERα:SRC:PLCγ (phospholipase Cγ) plays a role in activation of the tumor-protective anticipatory UPR (unfolded protein response) (UPR), thereby increasing the resilience of breast cancer cells [13]. SRC, which is induced by various cellular signal molecules and has a great effect in regulating numerous processes, including cell growth, differentiation, adhesion and the migration signaling pathway [14–16], is highly expressed in several prostate cancer cell lines [17–19]; as well as in most tissues obtained from prostate cancer [17,19,20]. Studies have also demonstrated that the phosphorylation of tyrosine 654 from β-catenin by SRC reduces the association of β-catenin with E-cadherin and α-catenin [21,22]. When SRC phosphorylates E-cadherin at residue Y860 and β-catenin at residue Y654, no interaction between E-cadherin/β-catenin occurs. β-catenin is then degraded or remain stabilized in the cytoplasm, and E-cadherin will be directed via Haikai for degradation [23]. Whether ER activates SRC and plays a role in regulating the expression and/or activity of β-catenin remains to be investigated. SRC can combine with another non-receptor protein tyrosine kinase, FAK (focal adhesion kinase) to form a dual-kinase complex, which coordinate cell behavior through regulating downstream pathways and molecules, including AKT, p38 and ERK [24]. PI3K (phosphatidylinositol 3-kinase) and AKT are also involved in the development of prostate cancer and CRPC, but their functions are not yet fully elucidated [25]. The most common change in PI3K signaling in patients with advanced prostate cancer is the bi-allelic loss of tumor suppressor PTEN (phosphatase and tensin homolog) that occurs in 50% of patients. PTEN is the negative regulator of PI3K and its inactivation, by mutation or loss, results in the accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of AKT [26]. The phosphorylation of AKT activates mTOR (mammalian target of rapamycin), which leads to cell division [27]. In vitro studies with prostate cancer cells have shown that PI3K/AKT/mTOR signaling is not only involved with proliferation [28] and apoptosis [29], but also with migration and invasion [30]. Therefore, this study aimed to examine the role of estrogen receptor in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the androgen-independent prostate cancer cells PC-3.

2. Materials and Methods 2.1. Cell Culture The human androgen-independent prostate cancer cell lines PC-3 (derived from bone metastasis) and DU-15 (derived from brain metastasis) were obtained from the American Type Culture Collection (Manassas, VA, United States). PC-3 and DU-145 cells were used in passages under 46 and 69, respectively, and cultures were carried out as previously described [6,8]. PC-3 and DU-145 cells were grown in RPMI 1640 medium without phenol red (GIBCO®, Rockville, MD, USA), supplemented with 10% of fetal bovine serum, HEPES (5.95 mg/mL) and gentamicin (0.02 mg/mL), in a humidiﬁed atmosphere with 5% CO2:95% air, at 37 ◦C, for 72 h. Then, the culture medium was replaced by serum free medium 24 h Int. J. Mol. Sci. 2021, 22, 1153 3 of 15

before the experiments. At this stage, the cells were 85–90% conﬂuent, and the number 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 (#4330100615, 11 December 2015).

2.2. Western Blot Analysis for Detection of Total and Phosphorylated SRC and Non-Phosphorylated β-Catenin PC-3 cells in culture medium without serum were incubated in the absence (control) and presence of 17β-estradiol (E2, 10 nM; Sigma Chemical Co., St.Louis, MO, USA) for 5, 15, 30 min and 1 and 2 h; ERβ-selective agonist DPN (10nM; 2,3-bis(4-hydroxyphenyl)- propionitrile, Tocris Bioscience, Bristol, UK) for 30 min and 1, 2, 4, and 24 h; ERα- selective agonist PPT (10 nM; 4,40,4”-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, Tocris Bioscience) for 30 min and 1, 2, and 24 h. The cells were also untreated or pretreated with the selective inhibitor for SRC-family kinases PP2 (5 nM; 4-amino-3-(4-chlorophenyl)-1-(t- butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4 chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d]pyrimidine, Calbiochem, Darmstadt, Germany) for 30 min. Incubation was continued in the absence and presence of DPN (10 nM) for 30 min or PPT (10 nM) for 1 h. 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[1,5- a]pyrimidin-3-yl]phenol, Tocris Bioscience) for 30 min. Incubation was continued in the absence and presence of DPN (10 nM) for 30 min or PPT (10 nM) for 1 h. At these concentrations, the agonists and antagonists are highly selective, as previously reported [6,31–33]. Western blot analyses were performed as previously described [6,8], using rabbit monoclonal antibody raised against a synthetic phosphopep- tide corresponding to Tyr419 of human SRC (Phosphorylated SRC #6943, Cell Signaling Technology, Boston, MA, USA, 1:1000 dilution) or antibody polyclonal raised against a synthetic peptide corresponding to human SRC (Total SRC, #2108, Cell Signaling Tech- nology, 1:1000 dilution) or rabbit polyclonal antibody raised against a synthetic peptide corresponding to Ser 37 (Sr33/37/Thr41) of human non-phosphorylated β-catenin (#4270, Cell Signaling Technology, 1:600 dilution) or monoclonal rabbit antibody raised against a synthetic peptide corresponding to the amino-terminal of the human β-tubulin (#2128, Cell Signaling Technology, 1:2000 dilution) overnight at 4 ◦C. Apparent molecular masses were determined from molecular mass standards. Band intensities of phosphorylated SRC, total SRC, non-phosphorylated β-catenin, 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 based on expression of total SRC or β-tubulin in each sample and plotted (mean ± SEM) in relation to control (C = 1).

2.3. Protein Assays Protein concentration was determined with the Bio-Rad protein assay, using bovine serum albumin as standard (Bio Rad Laboratories Inc., Hercules, CA, USA).

2.4. Cell Invasion Analysis PC-3 or DU-145 cells (2 × 105 cells) in serum free culture medium were seeded in Thin- certR chambers (Greiner Bio-one, Kremsmünster, Austria) with polyethylene terephthalate membranes (8 mm pore size) pre-coated with 50 mL of phenol red-free Matrigel (1:10, BD, Corning). These chambers were placed in 24-well plates containing culture medium with 10% FBS in the lower chamber [10,34]. PC-3 or DU-145 cells in upper chambers were incubated in the absence (control) and presence of 17β-estradiol (E2, 10 nM) or DPN (10 nM) or PPT (10 nM) for 48 h at 37 ◦C. The cells were also untreated or pretreated with PP2 (5 nM) or PI3K speciﬁc inhibitor Wortmannin (1 µM; Sigma-Aldrich Co., St. Louis, MO, USA) or AKT inhibitor MK2206, [200 nM, 8-(4-(1-aminocyclobutyl)phenyl)]-9-phenyl- [1,2,4]triazolo[3,4f][1,6]naphthyridin-3(2H)-one, Selleck Chemicals, Kirby Drive, Houston, Int. J. Mol. Sci. 2021, 22, 1153 4 of 15

TX, USA) for 30 min. Incubation was continued in the absence and presence of E2 (10 nM) or DPN (10 nM) or PPT (10 nM) for 48 h at 37 ◦C. Cell invasion analyses were performed as previously described [10]. The chambers were washed thoroughly with 10 mM PBS, ﬁxed in 4% paraformaldehyde for 30 min, and stained with 0.2% crystal violet for 10 min. Non-invading cells, from the membrane upper surface, were removed using a cotton swab. The membranes containing the invaded cells (under the surface of membrane), were photographed. Images of three random microscope ﬁelds were captured in duplicate, using an inverted optical microscope (Floid Cell Imaging Station, Life Technologies, Carlsbad, CA, USA). The areas of cell invasion were determined by Image J software. Results were plotted (mean ± SEM) in relation to control (C = 100) or agonists subtracted from the control (agonists = 100).

2.5. Colony Formation Analysis (Soft Agar) PC-3 cells (6 × 103 cells) in culture medium containing 10% FBS and 0.35% agarose (low melting, Sigma Chemical Co.) were seeded in 24-well plates pre-coated with 300 mL of 0.7% agarose at 4 ◦C for 30 min [10,34]. Cells were incubated at 37 ◦C for 2 h. Afterward, this culture medium was replaced by culture medium containing 10% SFB, pretreated with activated charcoal (0.25%) and dextran T-70 (0.0025%), for 24 h at 37 ◦C. PC-3 cells were incubated in the absence (control) and presence of E2 (10 nM), DPN (10 nM) or PPT (10 nM) for 3 weeks, with regular change in medium on every alternate day, at 37 ◦C. Cells were also untreated or pretreated with PP2 (5 nM), Wortmannin (1 µM) or MK2206 (200 nM), for 30 min. Incubation was continued in the absence and presence of E2 (10 nM) DPN (10 nM) or PPT (10 nM) for 3 weeks at 37 ◦C[10]. Colony formation analyses were performed as previously described [10]. Images of three random microscope ﬁelds, in duplicate, were captured using an inverted optical microscope (Floid Cell Imaging Station, Life Technologies). Image J software was used to determine the area of each colony. Only the spheroid-shaped colonies were considered for area calculation. Star- and spheroid- shaped colonies above 50 µm were counted using software Zen. Images are representative of three different experiments.

2.6. Immunofluorescence Analysis for the Detection of Non-Phosphorylated β-Catenin PC-3 cells were grown as described above on coverslips coated with gelatin (0.1%, w/v) and placed into six-well plates. PC-3 cells in serum free culture medium were incubated in the absence (control) and presence of DPN (10 nM) or PPT (10 nM), for 2 h at 37 ◦C. The cells were also untreated or pretreated with PP2 (5 nM) for 30 min. Incubation was continued in the absence and presence of PPT (10 nM) or DPN (10 nM), for 2 h at 37 ◦C. The medium was removed. The cells were washed with PBS, fixed in 2% paraformaldehyde for 20 min at room temperature and washed with PBS containing 0.1 M glycine. Cells were then permeabilized with 0.01% saponin and blocked with PBS containing 1% bovine serum albumin (BSA) for 10 min at room temperature. The immunofluorescence analyses were performed as previously described [6,8] using a rabbit polyclonal antibody raised against a synthetic peptide corresponding to Ser 37 (Sr33/37/Thr41) of human non-phosphorylated β-catenin (#4270, Cell Signaling Technology) at 1:200 dilution and Alexa Fluor 488-labeled secondary antibody (anti rabbit antibody; Molecular Probes, Invitrogen, Carlsbad, CA, USA) at 1:300 dilution. Nuclear staining was performed with DAPI (40,6-diamidino-2- phenylindole, Sigma Chemical Co). Negative control was performed in the absence of the primary antibody. Images of five random microscope fields were captured, in duplicate, in each assay and analyzed using the software LAS-AF and Image J. Images are representative of at least three different experiments performed in duplicate.

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. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 16

488-labeled secondary antibody (anti rabbit antibody; Molecular Probes, Invitrogen, Carlsbad, CA, USA) at 1:300 dilution. Nuclear staining was performed with DAPI (40,6- diamidino-2-phenylindole, Sigma Chemical Co). Negative control was performed in the absence of the primary antibody. Images of five random microscope fields were captured, in duplicate, in each assay and analyzed using the software LAS-AF and Image J. Images are representative of at least three different experiments performed in duplicate.

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 com- Int. J. Mol. Sci. 2021, 22, 1153 pare the differences between two data. p values < 0.05 were accepted as significant. 5 of 15

3. Results 3.1. Activation of ERα and ERβ Increases the Phosphorylation of SRC in PC-3 Cells 3. Results 17β-estradiol (E2) induced an increase in the phosphorylation of SRC (Tyr419) in PC- 3.1. Activation of ERα and ERβ Increases the Phosphorylation of SRC in PC-3 Cells 3 cells (Figure 1). The maximum increase (about 2-fold increase compared with control) β was observed17 -estradiol at 30 min (E2) after induced treatment. an increase ERα-selective in the phosphorylation agonist PPT and of SRCERβ (Tyr419)-selective in ag- PC-3 onistcells DPN (Figure also1 increased). The maximum the phosphorylation increase (about of 2-foldSRC at increase 30 min and compared 1 h, respectively with control) (Fig- was observed at 30 min after treatment. ERα-selective agonist PPT and ERβ-selective agonist ure 2A). DPN also increased the phosphorylation of SRC at 30 min and 1 h, respectively (Figure2A ).

β FigureFigure 1. Effects 1. Effects of 17 ofβ 17-estradiol-estradiol (E2) (E2) on SRC on SRC (Tyr419) (Tyr419) phos phosphorylationphorylation in androgen-independent in androgen-independent prostateprostate cancer cancer cells cells PC-3. PC-3. The Thecells cells were were incubate incubatedd in the in absence the absence (C, control) (C, control) and andpresence presence of E2 of E2 ◦ (10 (10nM) nM) for for5, 15, 5, 15, 30 30min, min, 1 and 1 and 2 h 2 hat at 37 37 °C.C. Phosphorylated Phosphorylated SRC SRC (p-SRC) (p-SRC) and and total total SRC were detectedde- tectedby Westernby Western Blot. Blot. The The immunoassay immunoassay was was performed performed with with anti-phosphorylated anti-phosphorylated SRC SRC (upper (upper panel) panel)and and anti-total anti-total SRC SRC antibodies antibodies (lower (lower panel). pane Thel). The relative relative position position of SRC of SRC was was determined determined from the frommolecular the molecular weight weight standard. standard. The data The showndata sh areown representative are representative of three of three independent independent experiments. experiments.Densitometric Densitometric analysis analysis was performed was performed of the results of the results obtained obtained from each from band, each normalizedband, normal- by the ized by the expression of the total SRC and expressed in relation to the control (C = 1). Results expression of the total SRC and expressed in relation to the control (C = 1). Results were plotted were plotted (mean ± SEM) of three independent experiments. * Significantly different from the (mean ± SEM) of three independent experiments. * Significantly different from the values obtained values obtained in relation to the control (C) (p < 0.05, ANOVA and Newman-Keuls). † Signifi- cantlyin relationdifferent to from the E2 control 5 min (C) (p < ( p0.05,< 0.05, ANOVA ANOVA and Newman-Keuls and Newman-Keuls).). ‡ Significantly† Significantly different different from E2 5 min (p < 0.05, ANOVA and Newman-Keuls). ‡ Significantly different from E2 15 min (p < 0.05, ANOVA and Newman-Keuls). • Significantly different from E2 2 h (p < 0.05, ANOVA and Newman-Keuls). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 16

Int. J. Mol. Sci. 2021, 22, 1153 6 of 15 from E2 15 min (p < 0.05, ANOVA and Newman-Keuls). • Significantly different from E2 2 h (p < 0.05, ANOVA and Newman-Keuls).

Figure 2. Effects of ERβ- (DPN) or ERα-selective agonists (PPT) on SRC (Tyr419) phosphorylation in Figureandrogen-independent 2. Effects of ERβ- (DPN) prostate or cancerERα-selective cells PC-3. agonists The cells (PPT) were on incubatedSRC (Tyr419) in the phosphorylation absence (C, control) in androgen-independentand presence of DPN or prostate PPT (10 cancer nM) for cells 30 min PC-3. and The 1h cells at 37 we◦C(re Aincubated). The cells in werethe absence incubated (C, in the control)absence and (C, presence control) of and DPN presence or PPT of (10 DPN nM) (10 for nM) 30 formin 30 and min 1 (hB at) or 37 PPT °C ( (10A). nM)The (cellsC) for were 1 h. incu- The cells batedwere in alsothe absence pre-treated (C, withcontrol) the selectiveand presence inhibitor of DPN for SRC-family(10 nM) for kinases30 min (PP2(B) or 5 PPT nM, (10 30 min)nM) and(C) then for 1 h. The cells were also pre-treated with the selective inhibitor for SRC-family kinases (PP2 5 incubated or not with DPN for 30 min (B) or PPT for 1 h (C). Phosphorylated SRC (p-SRC) and total nM, 30 min) and then incubated or not with DPN for 30 min (B) or PPT for 1 h (C). Phosphory- SRC were detected by Western Blot. The immunoassay was performed with anti-phosphorylated lated SRC (p-SRC) and total SRC were detected by Western Blot. The immunoassay was per- formedSRC (upperwith anti-phosphorylated panel) and total anti-SRC SRC (upper antibodies panel) (lowerand total panel). anti-SRC The antibodies relative position (lower ofpanel). SRC was Thedetermined relative position from theof SRC molecular was determined weight standard. from the The molecular data shown weight are standard. representative The data of fourshown to six areindependent representative experiments. of four to six Densitometric independent analysisexperiments. was performed Densitometric of the analysis results was obtained performed from each of theband, results normalized obtained by from the expressioneach band, ofnormalized the total SRC by the and expr expressedession of in the relation total toSRC the and control ex- (C = 1). pressedResults in wererelation plotted to the (mean control± SEM)(C = 1). of Results four to were six independent plotted (mean experiments. ± SEM) of *four Significantly to six inde- different pendentfrom controlexperiments. (C) (p <* Significantly 0.05, ANOVA different and Newman-Keuls). from control (C) ** Significantly(p < 0.05, ANOVA different and from Newman- PPT 30 min Keuls).(p < 0.05,** Significantly ANOVA and different Newman-Keuls). from PPT 30 # Significantlymin (p < 0.05, different ANOVA from and DPN Newman-Keuls). for 30 min or # PPT Signif- for 1 h icantly different from DPN for 30 min or PPT for 1 h (p < 0.05, ANOVA and Newman-Keuls). (p < 0.05, ANOVA and Newman-Keuls).

In Inthe the presence presence of of the the selective selective inhibitor inhibitor for for SRC-family SRC-family kinases (PP2,(PP2, 55 nM),nM), the the effects effectsof of DPN DPN and and PPT PPT on on phosphorylation phosphorylation of SRCof SRC were were blocked blocked (Figure (Figures2B,C). 2B,C). TheThe involvement involvement of each of each ER ER (ER (ERα andα and ER ERβ) onβ) onphosphorylation phosphorylation of SRC of SRC was was detected, detected, usingusing the the respective respective selective selective antagonists antagonists MPP MPP (10 (10 nM) nM) and and PHTPP PHTPP (10 (10 nM). nM). The effects of of PPT and and DPN DPN on on phosphorylation phosphorylation of of SRC SRC we werere blocked blocked by by their their respective respective antagonists antagonists (Figure(Figure 3).3 It). is It isimportant important to tomention mention that that PP2, PP2, MPP MPP and and PHTPP PHTPP were were also also incubated incubated with with thethe cells cells in the in the absence absence of the of the agonists, agonists, and and the the observed observed effects effects were were similar similar to the to the control control (Figures(Figures 2 and2 and 3).3 ).

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Figure 3. Effects of ERβ- (PHTTP) or ERα-selective antagonists (MPP) on SRC (Tyr419) expression Figureand phosphorylation3. Effects of ERβ- in (PHTTP) androgen-independent or ERα-selective prostate antagonists cancer (MPP) cells on PC-3 SRC induced (Tyr419) by expression DPN or PPT. andThe phosphorylation cells were incubated in androgen-i in the absencendependent (C, control) prostate and cancer presence cells of PC-3 DPN induced (10 nM) by for DPN 30 min or PPT. (A) or ThePPT cells (10 were nM) incubated for 1 h (B )in at the 37 absence◦C. The (C, cells cont wererol) also and pre-treated presence of with DPN PHTPP (10 nM) (10 for nM) 30 min (A) ( orA) MPP or(10 PPT nM) (10 (nM)B) for for 30 1 min,h (B) and at 37 then °C. incubatedThe cells were or not, also respectively, pre-treated with with DPN PHTPP for 30(10 min nM) ( A(A)) or or PPT for MPP (10 nM) (B) for 30 min, and then incubated or not, respectively, with DPN for 30 min (A) or 1 h (B). Phosphorylated SRC (p-SRC) and total SRC were detected by Western Blot. The immunoassay PPT for 1 h (B). Phosphorylated SRC (p-SRC) and total SRC were detected by Western Blot. The was performed with anti-phosphorylated SRC (upper panel) and anti-total SRC antibodies (lower immunoassay was performed with anti-phosphorylated SRC (upper panel) and anti-total SRC antibodiespanel). The (lower relative panel). position The rela of SRCtive position was determined of SRC was from determ the molecularined from weight the molecular standard. weight The data standard.shown are The representative data shown are of three representative independent of experiments.three independent Densitometric experiments. analysis Densitometric was performed of analysisthe results was obtainedperformed from of the each results band, obtained normalized from by each the expressionband, normalized of the total by the SRC expression and expressed of in therelation total SRC to the and control expressed (C = in 1). relation Results wereto the plotted control (mean (C = 1).± ResultsSEM) of were three plotted independent (mean experiments.± SEM) of three* Signiﬁcantly independent different experiments. from control* Significantly (C) (p < different 0.05, ANOVA from control and Newman-Keuls). (C) (p < 0.05, ANOVA # Signiﬁcantly and Newman-Keuls).different from DPN # Significantly for 30 min different or PPT for from 1 h DPN (p < 0.05, for 30 ANOVA min or andPPT Newman-Keuls).for 1 h (p < 0.05, ANOVA and Newman-Keuls). 3.2. SRC is Involved on Invasion and Colony Formation of PC-3 Cells Induced by ER Activation 3.2. SRCTo is analyze Involved the on participationInvasion and Colony of SRC Formation in the tumorigenicity of PC-3 Cells of Induced PC-3 cells, by ER we Activation performed invasionTo analyze and colonythe participation formation of assays. SRC in Thethe tumorigenicity treatment with of ER PC-3β-selective cells, we agonist performed DPN invasion(10 nM) and and colony ERα-selective formation agonist assays. PPT The (10 treatment nM) for 48 with h increased ERβ-selective the invasion agonist of DPN PC-3 (10 cells nM)(Figure and 4ER). Inα-selective the presence agonist of the PPT selective (10 nM) inhibitor for 48 h for increased SRC-family the kinasesinvasion PP2 of PC-3 (5 nM), cells the (Figureeffects 4). of In DPN the presence were blocked of the (100%) selective and inhibitor PPT decreased for SRC-family 79%, suggesting kinases PP2 the participation(5 nM), the effectsof SRC of DPN on the were invasion blocked of (100%) PC-3 cells and (FigurePPT decreased4). Cells 79%, were suggesting also incubated the participation with PP2 in ofthe SRC absence on the invasion of ER agonists, of PC-3 andcells the(Figure observed 4). Cells effects were were also similarincubated to thewith control PP2 in (datathe absencenot shown). of ER agonists, and the observed effects were similar to the control (data not shown).The participation of SRC was also analyzed on invasion of DU-145 cells induced by activationThe participation of ER. The treatmentof SRC was with also 17 βanalyzed-estradiol on (E2 invasion 10 nM) forof DU-145 48 h increased cells induced the invasion by activationof DU-145 of cellsER. The (Supplementary treatment with Figure 17β-estradiol S1). In the (E2 presence 10 nM) for of the48 h selective increased inhibitor the inva- for sionSRC-family of DU-145 kinases cells (Sup PP2plementary (5 nM), the Figure effects S1). of In E2 the were presence blocked of the (100%), selective suggesting inhibitor the forinvolvement SRC-family ofkinases SRC also PP2 on (5 thenM), invasion the effects of DU-145 of E2 were cells blocked (Supplementary (100%), suggesting Figure S1). the In the colony formation assay, treatment with E2 (10 nM) increased the size of the involvement of SRC also on the invasion of DU-145 cells (Supplementary Figure S1). colonies when compared to the control, in the third week of treatment. The pretreatment with PP2 blocked the effect of E2 (Figure5). The treatment with E2 also increased the number of colonies when compared to control, in the third week of treatment. The pretreatment with PP2 blocked this effect (Figure5). Cells were also incubated with PP2 in the absence of ER agonists, and the observed effects were similar to the control (data not shown). These results suggest the involvement of SRC in the formation of colonies of PC-3 cells stimulated by E2.

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3.3. The Increase in the Expression of the Non-Phosphorylated β-Catenin Induced by Activation of ERβ is Mediated by SRC We previously demonstrated that ERs promote migration, invasion, and colony formation of PC-3 cells through β-catenin [10]. To analyze the participation of SRC on the expression of the non-phosphorylated β-catenin, we performed immunoﬂuorescence analysis. The treatment with the ERβ-selective agonist DPN (10 nM) for 2 h increased the immunostaining of the non-phosphorylated β-catenin in the cytoplasm, near to the plasma membrane and cell nucleus, conﬁrming our previous results [8] (Figure6). This effect of DPN was blocked by the selective inhibitor for SRC-family kinases (PP2, 5 nM). In the presence of only PP2, the immunostaining of the β-catenin was similar to that detected in basal conditions (control, C). No immunostaining was detected in the negative control, performed in the absence of the primary antibody (insert). To confirm the effect of the ERβ-selective agonist DPN on the expression of the non- phosphorylated β-catenin, Western blot analysis was also performed (Supplementary Figure S2). The activation of ERβ by DPN increased the expression of the non-phosphorylated β- catenin (Supplementary Figure S2). It is important to mention that the activation of ERα Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWby PPT for 2 and 24 h did not alter the expression of the non-phosphorylated β-catenin.8 of 16 However, an increase in the expression of this protein was observed only after 48 h of incubation with PPT (data not shown).

Figure 4. Effects of the selective inhibitor for SRC-family kinases (PP2) on the invasion of androgen-independent prostate Figurecancer 4. Effects cells PC-3 of the induced selective by DPNinhibitor and PPT.for SRC-family Cells were incubatedkinases (PP2) in the on absence the invasion (C, control) of androgen-independent and in the presence of prostate DPN ◦ cancer(10 nM)cells (PC-3A) or induced PPT (10 nM) by DPN (B) for and 48 hPPT. at 37 CellsC. Thewere cells incubated were pre-treated in the absence with PP2 (C, (5 control) nM) for and 30 min in the and presence then incubated of DPN (10 nM)or not (A with) or thePPT DPN (10 nM) (A) or (B PPT) for ( B48) forh at 48 37 h. °C. The The membranes cells were containing pre-treated the with invaded PP2 cells(5 nM) (under for 30 the min surface and ofthen membrane), incubated or notwere with photographed. the DPN (A) or Images PPT ( ofB) threefor 48random h. The membranes microscope containing ﬁelds, in duplicate, the invaded were cells captured (under using the surface an inverted of membrane), optical weremicroscope. photographed. The areas Images of invaded of three cells rand wereom determinedmicroscope by fields, Image in J software.duplicate, Results were werecaptured plotted using (mean an ±invertedSEM of threeoptical microscope.independent The experiments)areas of invaded in relation cells were to the determined DPN or PPT by subtracted Image J software. from the controlResults (DPNwere =plotted 100) (A (mean) or (PPT ± SEM = 100) of (threeB). independent# Signiﬁcantly experiments) different in from relation DPN (top

In the colony formation assay, treatment with E2 (10 nM) increased the size of the colonies when compared to the control, in the third week of treatment. The pretreatment with PP2 blocked the effect of E2 (Figure 5). The treatment with E2 also increased the number of colonies when compared to control, in the third week of treatment. The pretreatment with PP2 blocked this effect (Figure 5). Cells were also incubated with PP2 in the absence of ER agonists, and the observed effects were similar to the control (data not shown). These results suggest the involvement of SRC in the formation of colonies of PC- 3 cells stimulated by E2.

Int.Int. J. Mol. J. Mol. Sci. Sci. 20212021, 22, 22, x, 1153FOR PEER REVIEW 9 of 159 of 16

FigureFigure 5. Effects of the selectiveselective inhibitorinhibitor for for SRC-family SRC-family kinases kinases (PP2) (PP2) on on size size and and number number of of the the colonycolony formed by androgen-independentandrogen-independent prostate prostate cancer cancer cells cells PC-3 PC-3 induced induced by by 17β 17-estradiolβ-estradiol (E2). (E2). CellsCells were were incubated incubated in the absence (C,(C, control) andand inin thethe presencepresenceof of17 17ββ-estradiol-estradiol (10 (10 nM) nM) for for 3 weeks3 weeks at at37 37 °C.◦C. The The cells cells were were also also pre-treated pre-treated wi withth PP2 (5(5 nM)nM) forfor 30 30 min min and and then then incubated incubated or or not with E2. Representative image of PC-3 cell colony formation assays. The images were ac- not with E2. Representative image of PC-3 cell colony formation assays. The images were acquired, quired, and the area of each colony was measured by the Image J software, and then the values and the area of each colony was measured by the Image J software, and then the values obtained were obtained were expressed in relation to E2 and subtracted from the control (E2 = 1). The colonies expressed in relation to E2 and subtracted from the control (E2 = 1). The colonies were also counted were also counted with Zen software and the values of the number of colonies were expression in with Zen software and the values of the number of colonies were expression in relation to E2 and relation to E2 and subtracted from the control (E2 = 1). Results are expressed as (mean ± SEM) of ± threesubtracted independent from the experiments. control (E2 = # 1). Significantly Results are expresseddifferent from as (mean E2 (p

3.3.3.4. The PI3K/AKT Increase are in also the RequiredExpression for of Invasion the Non-Phosphorylated and Colony Formation β-Catenin of PC-3 Induced Cells Simulated by Activation ofby ER ERβ is Mediated by SRC WeThe previously treatment withdemonstrated ERβ-selective that agonist ERs prom DPNote (10 migration, nM) and invasion, ERα-selective and agonistcolony for- mationPPT (10 of nM) PC-3 for 48cells h increasedthrough theβ-catenin invasion [10]. of PC-3To analyze cells. In the the participation presence of PI3K of SRC speciﬁc on the expressioninhibitor (Wortmannin, of the non-phosphorylated 1 µM) and AKT inhibitor β-catenin, (MK2206, we performed 200 nM), theimmunofluorescence effects of DPN analysis.were blocked The (100%)treatment by both with inhibitors the ERβ-selective and the effects agonist of PPT DPN decreased (10 nM) by for 80%, 2 h suggesting increased the immunostainingthe participation ofof PI3K/AKT the non-phosphorylated on the invasion ofβ-catenin PC-3 cells in stimulated the cytoplasm, by both agonistsnear to the µ plasma(Figure7 membrane). Moreover, and the pretreatmentcell nucleus, ofconfirming PC-3 cells withour previous Wortmannin results (1 M)[8] and(Figure MK2206 6). This (200 nM) blocked the effects of both agonists on size and number of the colonies (Figure8). effect of DPN was blocked by the selective inhibitor for SRC-family kinases (PP2, 5 nM). These results suggest the involvement of PI3K/AKT in the colony formation of PC-3 cells In the presence of only PP2, the immunostaining of the β-catenin was similar to that de- stimulated by DPN and PPT. tected in basal conditions (control, C). No immunostaining was detected in the negative control, performed in the absence of the primary antibody (insert).

Int. J. Mol. Sci. 2021, 22, 1153 10 of 15 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 16

FigureFigure 6. Effects 6. Effects of the ofselective the selective inhibitor inhibitorfor SRC-family for ki SRC-familynases (PP2) on kinases the expression (PP2) onof non- the expression of non- phosphorylated β-catenin in androgen-independent prostate cancer cells PC-3 induced by DPN. Cellsphosphorylated were incubated inβ -cateninthe absence in (C, androgen-independent control) and in the presence prostate of DPN (10 cancer nM) for cells 2 h PC-3at 37 induced by DPN. °C.Cells The cells were were incubated also pre-treated in the absencewith PP2 (5 (C, nM control)) for 30 min and and in thethen presence incubated ofor DPNnot with (10 the nM) for 2 h at 37 ◦C. DPNThe for cells 2 h. werePositive also immunost pre-treatedaining withfor non-phosphorylated PP2 (5 nM) for 30 β-catenin min and (green) then was incubated detected or not with the DPN using the polyclonal antibody produced in rabbit by immunization with the synthetic peptide correspondingfor 2 h. Positive to the immunostainingregion around Serine for 37 non-phosphorylated(Ser33/37/Thr41) of humanβ-catenin β-catenin. (green) Nuclei were was detected using the stainedpolyclonal with DAPI antibody (blue). producedNegative control, in rabbit in the by absence immunization of the primary with antibody the synthetic (detail). Re- peptide corresponding sultsto theare representative region around of Serinefour independent 37 (Ser33/37/Thr41) experiments. of human β-catenin. Nuclei were stained with DAPI Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW(blue). Negative control, in the absence of the primary antibody (detail). Results11 areof 16 representative of To confirm the effect of the ERβ-selective agonist DPN on the expression of the non- phosphorylatedfour independent β-catenin, experiments. Western blot analysis was also performed (Supplementary Fig- ure S2). The activation of ERβ by DPN increased the expression of the non-phosphorylated β-catenin (Supplementary Figure S2). It is important to mention that the activation of ERα by PPT for 2 and 24 h did not alter the expression of the non-phosphorylated β- catenin. However, an increase in the expression of this protein was observed only after 48 h of incubation with PPT (data not shown).

3.4. PI3K/AKT are also Required for Invasion and Colony Formation of PC-3 Cells Simulated by ER The treatment with ERβ-selective agonist DPN (10 nM) and ERα-selective agonist PPT (10 nM) for 48 h increased the invasion of PC-3 cells. In the presence of PI3K specific inhibitor (Wortmannin, 1 µM) and AKT inhibitor (MK2206, 200 nM), the effects of DPN were blocked (100%) by both inhibitors and the effects of PPT decreased by 80%, suggesting the participation of PI3K/AKT on the invasion of PC-3 cells stimulated by both agonists (Figure 7). Moreover, the pretreatment of PC-3 cells with Wortmannin (1 µM) and MK2206 (200 nM) blocked the effects of both agonists on size and number of the colonies (Figure 8). These results suggest the involvement of PI3K/AKT in the colony formation of FigurePC-3Figure cells 7. Effects 7. stimulatedEffects of the ofPI3K by the DPN specific PI3K and specificinhibitor PPT. (Wor inhibitortmannin) (Wortmannin) and AKT inhibitor and (MK2206) AKT inhibitor on the (MK2206) on the invasioninvasion of androgen-independent of androgen-independent prostate prostatecancer cells cancer PC-3 induced cells PC-3 by DPN induced and PPT. by DPNCells and PPT. Cells were were incubated in the absence (C, control) and presence of DPN (10 nM) (A) or PPT (10 nM) (B) forincubated 48 h at 37 °C. in The the cells absence were pre-treated (C, control) with and Wortmannin presence (1 ofµM) DPN or MK2206 (10 nM) (200 (nM)A) orfor PPT30 (10 nM) (B) for ◦ min,48 and h at then 37 incubatedC. The cells or not were with pre-treated DPN (A) or PPT with (B) Wortmannin for 48 h. Representative (1 µM) or image MK2206 of PC-3 (200 nM) for 30 min, celland invasion. then incubatedThe membranes or notcontaining withDPN the invaded (A) or cells PPT (under (B) forthe surface 48 h. Representativeof membrane), were image of PC-3 cell photographed. Images of three random microscope fields, in duplicate, were captured using an invertedinvasion. optical The microscope. membranes The areas containing of invaded the cells invaded were determined cells (under by Image the J surfacesoftware. of Re- membrane), were sultsphotographed. were plotted (mean Images ± SEM) of in three relation random to DPN microscope or PPT and subtracted fields, in from duplicate, the control were (DPN captured using an = 100)inverted (A) or optical PPT = 100) microscope. (B). # Significantly The areas different of invaded from DPN cells (p were< 0.05, determined ANOVA and by Newman- Image J software. Results Keuls). + Significantly different from PPT (p < 0.05, ANOVA and Newman-Keuls). Images are rep- were plotted (mean ± SEM) in relation to DPN or PPT and subtracted from the control (DPN = 100) resentative of three different experiments. (A) or PPT = 100)(B). # Significantly different from DPN (p < 0.05, ANOVA and Newman-Keuls). + Significantly different from PPT (p < 0.05, ANOVA and Newman-Keuls). Images are representative of three different experiments.

Int. J. Mol. Sci. 2021, 22, 1153 11 of 15 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 12 of 16

FigureFigure 8.8.Effects Effects ofof thethe PI3KPI3K specificspecific inhibitorsinhibitors (Wortmannin)(Wortmannin) andand AKTAKT inhibitorinhibitor (MK2206)(MK2206) onon sizesize andand numbernumber ofof thethe colonycolony formedformed byby androgen-independentandrogen-independent prostateprostate cancer cells PC3 induced by by DPNDPN oror PPT.PPT. Cells Cells were were incubated incubated in in the the absence absence (C, (C, control) control) and and presence presence of of DPN DPN (10 (10 nM) nM) (A (A) and) PPTand (10PPT nM) (10 nM) (B) for (B) 3 for weeks 3 weeks at 37 at◦ C.37 The°C. The cells cells were were also also pre-treated pre-treated with with Wortmannin Wortmannin (1 µ (1M) µM) or MK2206or MK2206 (200 (200 nM) nM) for for 30 min,30 min, and and then then incubated incubated or or not not with with DPN DPN (A ()A or) or PPT PPT (B ).(B Representative). Representa- tive image of PC-3 cell colony formation assays. The images were acquired, and the area of each image of PC-3 cell colony formation assays. The images were acquired, and the area of each colony colony was measured by the Image J software and the values obtained were expressed in relation was measured by the Image J software and the values obtained were expressed in relation to the to the DPN or PPT and subtracted from the control (DPN = 1) (A) or (PPT = 1) (B). The colonies DPNwere oralso PPT counted and subtracted with Zen fromsoftware the control and the (DPN values = 1)of (theA) ornumber (PPT =of 1) colonies (B). The were colonies expression were also in countedrelation withto DPN Zen (A software) or PPT ( andB) and the subtracted values of thefrom number the control of colonies (DPN=1) were (A)expression or (PPT=1) in(B). relation Re- tosults DPN are (A expressed) or PPT (asB) (mean and subtracted ± SEM) of from three the independent control (DPN experiments. = 1) (A) or # (PPTSignificantly = 1) (B). different Results are expressedfrom DPN as (p (mean < 0.05,± ANOVASEM) of and three Newman-Keuls). independent experiments. + Significantly # Significantly different from different PPT (p from < 0.05, DPN (ANOVAp < 0.05, ANOVAand Newman-Keuls). and Newman-Keuls). + Significantly different from PPT (p < 0.05, ANOVA and Newman-Keuls). 4. Discussion 4. Discussion Recent studies of rapid actions mediated by estrogen in the prostate and its relation- shipRecent with the studies development of rapid actionsof prostate mediated cancer by or estrogen with CRPC in the are prostate emerging. and itsOur relationship laboratory withshowed the that development in androgen-independent of prostate cancer prostate or with cancer CRPC PC-3 are and emerging. DU-145 Our cells, laboratory the estro- showedgen receptors that in ER androgen-independentα and ERβ are mostly prostate located outside cancer PC-3 the ce andll nucleus DU-145 [6,7]. cells, The the activation estrogen

Int. J. Mol. Sci. 2021, 22, 1153 12 of 15

receptors ERα and ERβ are mostly located outside the cell nucleus [6,7]. The activation of ERα and ERβ can activate rapid cell signaling pathways in these cells, including an increase in the phosphorylation of ERK1/2 in PC-3 and DU-145 cells [6,7] and AKT in PC-3 cells [7,8], but not in DU-145 cells [7]. We now report that ER induces activation of SRC and PI3K/AKT, increases the expression of the non-phosphorylated β-catenin and enhances the invasion and colony formation of the PC-3 cells. SRC and the non-receptor protein tyrosine kinases are downstream targets for cell surface receptors, and function as a link between the membrane receptors and the cytoplasmic signaling machinery, thereby regulating many fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals [15]. All these processes, if deregulated, lead to tumor progression [35–37]. E2-ERα complex can enhances kinase activity by inducing binding of phosphotyrosine 537 of ERα to SH2 domain of SRC, changing the inactive conformation of SRC to active conformation [38]. In fact, in the present study, using the PC-3 cells, the activation of ER by E2, ERα- (PPT) or ERβ-selective agonists (DPN) leads to the phosphorylation of SRC (Tyr419). Furthermore, the selective inhibitor for SRC-family kinases (PP2) blocked the invasion of the PC-3 cells stimulated by DPN or PPT and also the invasion of the DU-145 cells stimulated by E2, indicating the involvement of ERβ-SRC and ERα-SRC on the invasion of both cells. In addition, activation of both ER by E2 increases the size and number of the colony formed by PC-3 cells [10] and present study. The pretreatment with the selective inhibitor for SRC-family kinases PP2 blunted these effects induced by E2, indicating the involvement of ER/SRC on tumor formation in vitro. The possible convergence of nongenomic and genomic pathways on target genes involved with invasion and colony formed by androgen-independent prostate cancer cells may be occurring. It is important to mention that the activation of ERβ increases the expression of the non-phosphorylated β-catenin [8], and present data and ERβ-β-catenin-TCF/LEF complex is involved in proliferation of the PC-3 cells [8], migration, invasion, size and number of the colony formed by PC-3 cells [10]. In the present study, the pretreatment with the selective inhibitor for SRC-family kinases PP2 blocked the effect induced by DPN on expression of the non-phosphorylated β-catenin, indicating the involvement of SRC in the regulation of this protein. It is important to mention that the antibody used in the detection of the non-phosphorylated β-catenin is against the Ser33/37/Thr41 region, this region may be phosphorylated by GSK3β [39] and not by SRC. SRC can phosphorylate β-catenin in Tyr654 [23]. Phosphorylation of β-catenin by members of the SRC family reduces the association of β-catenin with E-cadherin and α-catenin [21,22] increasing the levels of cytoplasmic β-catenin and translocation to the nucleus, where it interacts with the TCF/LEF transcription factor, resulting on the activation of target genes [40]. Whether ERβ-SRC complex also plays a role on the activation of β- catenin in Tyr654 in PC-3 cells remains to be determined. In the present study, we show that ERβ induces activation of SRC (30 min) (rapid action, nongenomic) and increases the levels of the expression of the non-phosphorylated β-catenin in the cytoplasm of PC-3 cells (2 h, genomic action). Consistent with our results, study has shown that selective inhibitor for SRC-family kinases PP2 suppressed migration, invasion, and angiogenesis of PC3 and LNCAP cells via FAK [41]. Whether 17β-estradiol-ER-SRC plays a direct role or together with β-catenin and/or FAK on migration, invasion, and angiogenesis of PC3 cells remains to be explored. Recently, new emerging roles for SRC have been described in the nuclear compartment. In the nucleus of normal and cancer cells, SRC is involved in several activities involving both its enzymatic activity as tyrosine kinase and its capability to interact with other protein thereby forming protein complexes. SRC participates in the regulation of chromatin reorganization and transcriptional activity of transcription factors, and it is surely involved in the oncogenic transformation of tumoral cells, by repressing some oncosuppressors [42]. The roles of SRC in the nuclear compartment of the prostate cancer cells remains to be explored. Int. J. Mol. Sci. 2021, 22, 1153 13 of 15

Although several partners of extranuclear ER have been described in different cell types, the most conserved partners are SRC and PI3K [43]. The pathway, characterized by the formation of ER/SRC/PI3K and the subsequent activation of AKT, is present in normal breast tissue and is hyperactivated in aggressive breast tumors [44]. In PC-3 cells, our laboratory showed that E2 increases the phosphorylation of AKT [8]. In the present study was shown that the inhibitors of PI3K or AKT blocked the increase in cell invasion stimulated by DPN (100%) or PPT (80%), suggesting the involvement of ERβ-PI3K/AKT and ERα-PI3K/AKT on the invasion potential of PC-3 cells. In addition, the activation of ERβ by DPN or ERα by PPT increases the size and number of the colony formed by PC-3 cells [10 and present study). These effects were also blocked by inhibitors of PI3K or AKT. It is also important to mention that the phosphorylation of the β-catenin in the Ser552 by AKT may increase β-catenin/TCF-LEF activation possibly by association with histone acetylase [23]. In addition, coactivators recruited by β-catenin can determine which target genes are activated, and this differential recruitment can be regulated by phosphorylation [45]. Whether the complex ER/PI3K and the subsequent activation of AKT plays a role in expression and/or activation of β-catenin remains to be explored. In conclusion, this study provides novel insights into molecular mechanisms of ER in androgen-independent prostate cancer cells. In PC-3 cells, ER activates rapid responses molecules, including SRC and PI3K/AKT. SRC is involved on the expression of the non- phosphorylated β-catenin. These events enhance the tumorigenic potential of prostate cancer cells PC-3, increasing cell proliferation, migration, invasion, and tumor formation. The complete mechanism by which ER are involved in CRPC is not fully understood, but it represents a promising new therapeutic avenue for advanced prostate cancer.

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-0 067/22/3/1153/s1, Figure S1: Effects of the selective inhibitor for SRC-family kinases (PP2) on the invasion of androgen-independent prostate cancer cells DU-145 induced by 17β-estradiol. Figure S2. Effects of DPN on non-phosphorylated β-catenin expression in androgen-independent prostate cancer cells PC-3. Author Contributions: A.P.G.L. and C.M.V. performed the experiments, organized the database, performed the statistical analysis, and wrote the first draft of the manuscript; R.P.C. contributed to immunofluorescence images acquisition; C.S.P. revised the data and literature about the topic, performed funding acquisition, and was the head investigator of the study. All authors contributed to the concept and design of the study, manuscript revision, and read and approved the submitted version. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2014/05292-2 and 2017/16060-3 to CSP). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and ap-proved by the Research Ethics Committee of Escola Paulista de Medicina-Universidade Federal de São Paulo (protocol code 4330100615, date: 11/12/2015). Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We thank Priscila Veronica Sartorio, Elizabeth Kanashiro and Caroline Zito Romera for technical assistance. Confocal microscope Leica Microsystems TCSSP8, facility from the Instituto Nacional de Farmacologia e Biologia Molecular (INFAR) was supported by Finan- ciadora de Estudos e Projetos (FINEP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Research (C.S.P.) and (A.P.G.L.) fellowships were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. Sci. 2021, 22, 1153 14 of 15

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