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Research Article
Rho/ROCK/actin signaling regulates membrane androgenreceptor induced apoptosis in prostate cancer cells
Natalia Papadopouloua, Ioannis Charalampopoulosb, Konstantinos Alevizopoulosc,Achille Gravanisb, Christos Stournarasa,⁎aDepartment of Biochemistry, University of Crete Medical School, GR-71110 Heraklion, GreecebPharmacology, University of Crete Medical School, GR-71110 Heraklion, GreececMedexis-Biotech SA, Kryoneri, Athens, Greece
A R T I C L E I N F O R M A T I O N
⁎ Corresponding author. Fax: +302810394530.E-mail address: [email protected] (C. StouAbbreviations: mAR, membrane androgen re
Received 20 February 2008Revised version received 3 July 2008Accepted 15 July 2008Available online 25 July 2008
In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic
responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormoneinsensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selectiveagonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significantinduction of actin polymerization and apoptosis that can be reversed by actin disrupting agents inboth cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed bystimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actinorganization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated inmembrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Ourfindings suggest that Rho small GTPases are major membrane androgen receptor effectorscontrolling actin reorganization and apoptosis in prostate cancer cells.
mAR activationActin dynamicsRho-GTPase signalingApoptosisProstate cancer
Introduction
The traditional model of steroid hormone action involves bindingto specific intracellular steroid receptors, translocation to thenucleus, DNA binding, and subsequent modulation of transcriptionand protein synthesis [1]. This genomic response generallyrequires hours to be manifested. In recent years however, anumber of studies introduced the concept of non-genomic steroidhormone actions, explaining observations related to rapid steroideffects. According to the recently proposed classification for non-genomic steroid actions [2], a non-genomic effect occurs withinminutes, is present in cells devoid of functional classical intracel-
lular steroid receptors, and is insensitive to inhibitors of transcrip-tion and translation. Furthermore, activation of a non-genomiceffect may be triggered by non-permeable (e.g. bovine serumalbumin (BSA-covalently coupled) steroids and is, in most cases,insensitive to steroid antagonists. Non-genomic steroid actionshave been reported for most steroids including glucocorticoids [3–6], progesterone [7,8], estrogens [9,10], androgens [11–13] andneurosteroids [14–16]. For recent reviews, see [17–19].
We have recently reported the identification of functionalmembrane androgen receptors in the hormone-sensitive LNCaPhuman prostate cancer cell line, as well as in the hormoneinsensitiveDU145humanprostate cancer cell line [13,20]. Activation
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of these receptors in LNCaP cells by testosterone-BSA, a non-permeable testosterone conjugate was shown to induce rapid actincytoskeleton reorganization and to increase the secretion ofprostate-specific antigen (PSA) within minutes [13]. Furthermore,a specific non-genomic signaling cascade regulating the molecularmechanism of actin reorganization was identified, involvingphosphorylation of FAK, the association of FAK with PI-3K and thesubsequent activation of the latter, as well as the activation of thesmall guanosine triphosphatases Rac1/Cdc42 [21]. Finally, it wasshown thatmAR activation by BSA-coupled testosteronewas able toinduce: a) apoptosis of either androgen-dependent LNCaP orandrogen-independent DU145 cells and b) regression of prostatecancer cells both, in vitro and in vivo [20,22]. Similarly, stimulationof mAR by the impermeable androgen analog DHT-BSA wasassociated with apoptotic cell death in primary cortical astrocytes[23]. These findings collectively suggest that mAR activationmay bean important target for the apoptotic regression of a given cell ortissue.
To further analyze the signaling pathways downstream of mARand to correlate mAR-regulated events with intracellular androgenreceptor (iAR) function, we have used the DU145 and LNCaPprostate cancer cell lines. While LNCaP cells express functionalintracellular androgen receptors (iAR), the DU145 cell line has beenshown to express either non-functional iAR [24], or to be iAR-deficient [25,26] and which therefore fails to respond to androgentreatment. We compared cellular responses and pathways trig-gered by testosterone-BSA inmore detail. Specifically, we sought toelucidate the role of actin in mAR-dependent apoptotic cell deathand to identify key downstream effectors of mAR action regulatingboth actin reorganization and apoptosis. Our results provide newinsights into mAR function and point to a central role of actinremodeling and Rho small GTPases in mAR signaling. It isconcluded that the newly identified Rho/ROCK-dependent path-way and the observed actin reorganization operating in LNCaP andDU145 cells have a key functional role in regulating mAR-inducedapoptosis in prostate cancer cells independently of iAR functionalstatus.
Materials and methods
Preparation of steroid solution
Before each experiment testosterone-3-(O-carboxymethyl) oxime-BSA (Sigma), named testosterone-BSA, was dissolved in serum-freeculture medium to a final concentration of 10−5 M. This stocksolution was incubated for 30 min at room temperature with 0.3%charcoal and 0.03% dextran, centrifuged at 3000 ×g and passedthrough a 0.45 μm filter to remove any potential contaminationwith free steroid. The treated testosterone-BSA solution was usedin a final concentration of 10−7 M throughout all studies.
Cell culture and transfections
The DU145 human prostate cancer cell line was obtained from theAmerican Type Culture Collection (Manassas, VA) and was studiedbetween passages 60 and 70. DU145 cells fail to respond toandrogen treatment owing to the expression of non-functional iAR[24], or to complete lack of iAR according to other studies [25,26].LNCaP cells were obtained from the German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany).LNCaP cells bear a functional iAR pathwayand respond to androgentreatment. Both cell lines were cultured in RPMI 1640 (Invitrogen)supplemented with 25 mM HEPES, 2 mM L-glutamine and 10% FBS(Biochrom KG). If not otherwise stated all treatments andincubations with testosterone-BSA including apoptosis assayswere performed in serum containing medium. Transient transfec-tions were performed according to the manufacturer's protocolusing Lipofectamine 2000 (Invitrogen) and the following mamma-lian expression vectors encoding for: (a) the N-terminally Myc-tagged dominant-negative RhoA, pEVX-MycRhoAN19 [27] (b) theN-terminally-Myc-tagged dominant-negative RhoB, pCDNA3-mycRhoBN19, a king gift from D. Kardasis (IMBB, Herakleion,Crete) (c) the N-terminally Myc-tagged dominant-negative Cdc42,pCMV-mycCdc42 (T17N) [28] or (d) GFP, pEGFP-C1 (BD BiosciencesClontech).
Immunofluorescence analysis and confocallaser-scanning microscopy
Cells were cultured on glass cover slips and treated withtestosterone-BSA for the timepoints indicated in the figure legends.For direct fluorescence microscopy of F-actin, cells were fixed with3% p-formaldehyde in PBS for 30 min, permeabilized with 0.5%Triton X-100 in PBS (10 min) and incubated with rhodamine–phalloidin (Molecular Probes, Eugene, OR) (1:100 dilution) for40min in the dark. For indirect immunofluorescence staining, cellswere incubated for 2 h at room temperature with mousemonoclonal anti-RhoA (26C4) (1: 100 dilution) or anti-c-myc(9E10) (1: 500 dilution) (Santa Cruz Biotechnology). SecondaryFITC-conjugated rabbit anti-mouse IgG (Chemicon)was used in a 1:50 dilution. Slides were mounted using the Slow Fade Antifade kit(Molecular Probes). All specimens were examined with a BH-2microscope (Olympus Corp., Lake Success, NY) equipped withepifluorescence illumination. Confocal microscopy was performedwith a confocal laser-scanning module (Leica Lasertechnik) andimages were analyzed with the instrument's software.
Measurement of G/total actin ratio by Triton X-100 fractionation
The Triton X-100 soluble G-actin containing and insoluble F-actincontaining fractions of cells exposed to testosterone-BSA in thepresence (1 h pre-treatment) or absence of 10 μM Y-27632(Calbiochem) were prepared as previously described [29]. Anincrease of the triton-insoluble (F) to triton-soluble (G) actin ratiois indicative of actin polymerization.
Immunoprecipitation and immunoblot analysis
Cells exposed or not to testosterone-BSA were washed twice withice-cold phosphate buffered saline and suspended in cold lysisbuffer containing 1% Nonidet P-40, 20 mM Tris (pH 7.4) and137 mM NaCl, supplemented with protease and phosphataseinhibitors. Cleared lysates were pre-adsorbed with protein A-Sepharose beads (Amersham) for 1 h at 4 °C. Equal amounts ofthe supernatants were subjected to immunoprecipitation usinganti-phosphotyrosine (PY20) antibody (Santa Cruz Biotechnol-ogy) and protein A-Sepharose beads. For immunoblot analysis,the immunoprecipitates and equal amounts of total proteinextracts were suspended in Laemmli's sample buffer and
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separated by SDS-PAGE. Proteins were transferred onto nitrocel-lulose membrane and blotted with rabbit polyclonal anti-FAK (A-17) (Santa Cruz Biotechnology) (1:100 dilution) and rabbitpolyclonal anti-pI3-kinase p85 (Upstate) (1:1000 dilution)respectively.
Affinity precipitation
To determine the activity of Rac1/Cdc42 or RhoA/B in testosterone-BSA treated versus untreated cells affinity precipitations with Rac/Cdc42 reagent (GST-PBD) and Rho Assay reagent (GST-RBD) fromUpstate were performed respectively, according to the manufac-turer's instructions. Briefly, cells were washed twice in ice-coldTBS and lysed in Mg 2+ lysis buffer (25 mM HEPES, pH 7.5, 150 mMNaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, 10% glycerol,25 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mMNa3VO4). Cleared cell lysates were incubated with 10 μg GST-PBDfor 1 h or with 30 μg GST-RBD for 45 min at 4 °C. Precipitates werewashed three times with Mg2+ lysis buffer and suspended inLaemmli's buffer. The precipitates and equal volumes of totalprotein extracts were separated by SDS-PAGE and immunoblottedwith either rabbit polyclonal anti-Rac 1 (C-11) (1:200 dilution)and mouse monoclonal anti-Cdc42 (B-8) (1:100 dilution), ormouse monoclonal anti-RhoA (26C4) and rabbit polyclonal anti-
Fig. 1 – Modulation of the dynamic equilibrium between G- and F-serum-starved cells were stimulatedwith 10−7 M androgen conjugatby immunoblot analysis after Triton X-100 subcellular fractionatiovalue±SE of four independent duplicate experiments (⁎P<0.05 and ⁎
analyzed by confocal laser-scanning microscopy. Each of the five mscanning sections. Bar, 20 μm.
RhoB (119) in a 1:100 dilution (all above antibodies were fromSanta Cruz Biotechnology).
In vitro kinase assays
Cells were pre-treated or not with 10 μM Y-27632 for 1 h andstimulated with 10−7 M testosterone-BSA for the time pointsindicated in the figure legends. They were washed twice with ice-cold Tris-buffered saline, suspended in lysis buffer (50 mM Tris, pH7.5, 1% Triton X-100, 500 mMNaCl, 10% glycerol, 10 mMNaF, 25 mMβ-glycerophosphate, 1 mM phenyl-methyl-sulfonyl fluoride, 2 μg/mlaprotinin, 2 μg/ml leupeptin,1mMNa3VO4) and incubated for 20minon ice. After removing cell debris by centrifugation (10 min, 4 °C),cleared lysates were pre-adsorbed to G-protein agarose beads (SantaCruzBiotechnology) for 1hat4 °C. Thenequal protein amounts of thesupernatants were subjected to immunoprecipitation with poly-clonal rabbit anti-LIMK2 (H-78) (Santa Cruz Biotechnology) and G-protein agarose beads. Immunoprecipitated LIMK2 beads werewashed three times with kinase buffer (50 mM HEPES/NaOH, pH7.5, 5 mMMgCl2, 5 mMMnCl2, 25 mM β-glycerophosphate, 10 mMNaF, 1 mMNa3VO4) and incubated for 30min at 30° C in 20 μl kinasebuffer containing 15 μM ATP, 5 μCi [γ-32P]ATP (5000 Ci/mmolAmersham Biosciences). Proteins were subjected to 11% SDS-PAGEand transferred onto nitrocellulose. 32P-labeled proteins were
actin in testosterone-BSA stimulated DU145 cells. 24 he for the indicated time points. (A) F- and G-actinweremeasuredn. Each data value in the chart represents the F/G-actin mean⁎P<0.01). (B) Cells were stainedwith rhodamine–phalloidin andicrophotographs represents average projections of consecutive
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visualized by autoradiography. Identification of phosphorylatedproteins was carried out by immunoblotting with anti-LIMK2 (H-78) (1:100 dilution), goat polyclonal anti-LIMK2 (C-19) (Santa CruzBiotechnology) (1:100 dilution) and rabbit polyclonal anti-destrin/ADF (GV-13) (Sigma) (1:500 dilution).
Measurement of apoptosis
Cells (in RPMI 1640, supplemented with 25 mM HEPES, 2 mML-glutamine and 10% FBS) were cultured in 96-well plates for theAPOPercentage apoptosis assay (Biocolor Ltd., Belfast, Ireland) or in60 mm plates for FACS analysis. After pre-treatment with 10−7 Mcytochalasin B (Biomol Research Laboratories, PA), 100 nM wort-mannin (Sigma), or 10 μMY-27632, cells theywere stimulated or notwith 10−7 M testosterone-BSA in serum containing medium for the
Fig. 2 – FACS analysis shows inhibition of the apoptotic response inactin microfilaments. All experiments except the serum-free condicells were pre-treated with 10−7 M of cytochalasin B for 1 h, and theCells cultured in serum or 24 h serum-starved cells were used as a nwith Annexin V-FITC for apoptotic cells undergoing the flip–flop evfollowed by flow cytometric analysis. Viable cells are shown in theAnnexin positive/PI negative, while late apoptosis or already dead cpercentage of viable and apoptotic cells under each condition are ptestosterone-BSA stimulated DU145 and LNCaP cells in the presenc10−7 M cytochalasin B (cyt B) for 1 h, were exposed to 10−7 M testostchart legend on the right. Cells serum-starved for comparable periodthemean ODmeasured at 550 nm±SE of three independent experimsignificance of each condition versus the positive control (serum),testosterone-BSA alone condition versus the testosterone-BSA plus
time points indicated in the figure legends. Untreated cells culturedin serum-free mediumwere used as positive control for apoptotis.
FACS analysisAt the end of treatment cells were harvested in PBS and stainedwith the Annexin V-FITC Apoptosis Detection kit I (BD PharmingenTM, San Diego, CA) according to the manufacturer's instructions.Theywere analyzedwithin 1h by flowcytometry using a FACSArrayapparatus (BD Biosciences) and with CellQuest (BD Biosciences)and ModFit LT (Verify software, Topsham, MN) software.
APOPercentage apoptosis assayThis assay is based on the staining of the apoptotic cells undergoingthe membrane flip–flop event when phosphatidylserine is translo-cated to the outer leaflet. After staining according to the
duced by testosterone-BSA in the presence of agents disruptingtion were performed in serum containing medium. (A) DU145n stimulated with the androgen conjugate for 24 h as indicated.egative and positive control for apoptosis respectively. Stainingent and with the vitality marker propidium iodide (PI) wasAnnexin/PI negative quadrangle. Early apoptosis stage cells areells are both Annexin and PI positive. (B) The estimatedresented in the table. (C) APOPercentage apoptosis assay ofe of actin microfilament drugs. Cells pre-treated or not witherone-BSA for 2, 6, 12, 24 and 48 h respectively, according to thes of time served as a positive control for apoptosis. Bars presentents performed in triplicates (#P<0.01, represents the statistical⁎P<0.05 and ⁎⁎P<0.01 refer to the comparison between thecytochalasin B).
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manufacturer's instructions the amount of dye bound was releasedfrom the labeled cells into the solution and the concentration wasmeasured at a wavelength of 550 nm using a color filter microplatecolorimeter (MicroElisa reader, Dynatech Laboratories, Chantilly, VA).
To analyze actin responses in iAR negative DU145 cells,wemeasuredchanges in the triton-insoluble (F) to triton-soluble (G) actin ratio as
Fig. 3 – FAK/PI-3K signaling is not involved in mAR-induced actin rbetween G- and F-actin in testosterone-BSA stimulated DU145 cellspre-treated with 100 nM wortmannin for 1 h and then stimulated wmeasured by immunoblot analysis after Triton X-100 subcellular framean value±SE of three independent duplicate experiments (⁎⁎P<testosterone-BSA induced apoptosis in DU145 cells. Apoptosis waspre-treated with wortmannin (100 nM) for 30 min and then stimuserum or 24 h serum-starved cells were used as a negative and pos(C) Inhibition of the FAK/PI-3K pathway blocks testosterone-BSA indthe apoptosis assay–>APOPercentage apoptosis assay. Cells pre-treawith 10−7 M testosterone-BSA for 24 h. Cells cultured in serum or 2control for apoptosis respectively (n=4, ⁎⁎P<0.01). (D) Representatiand PI-3K. 24 h serum-starved DU145 cells were stimulated with 10cells lysis, equal amounts of proteins were immunoprecipitated (IPphosphorylated as well as equal amounts of total lysates was immuor the p85 subunit of PI-3K (right panels). The immunoblots wereand PI-3K bands was normalized to the intensity of the correspondindicates the fold phosphorylation of FAK or PI-3K respectively, wimean±SE of four independent experiments.
previously described [29]. Incubation of cells with testosterone-BSAresulted in a significant increase of the F/G-actin ratio, indicatingstrong actin polymerization. This effect was evident 5 min upontestosterone-BSA treatment; it persisted for up to 24 h and returnedto nearly control levels after 48 h (Fig.1A). Ourquantitative datawerefully supported by confocal laser-scanning microscopic analysis. Acomplete submembranuous redistribution of microfilamentousstructures and the formation of stress fibers and filopodia becameevident in testosterone-BSA treated cells, as shown in representativemicrographs of Fig. 1B, II–IV. From these results we conclude thatmAR activation leads to rapid and potent actin cytoskeletonreorganization in prostate cancer cells, independently of theexpression of intracellular androgen receptors.
eorganization in DU145 cells. (A) The dynamic equilibriumpre-treated with wortmannin. 24 h serum-starved cells wereith 10−7 M androgen conjugate for 30 min. F- and G-actin werectionation. Each data value in the chart represents the F/G-actin0.01). (B) Inhibition of the FAK/PI-3K pathway does not blocksmeasured by using the APOPercentage apoptosis assay. Cellslated with 10−7 M testosterone-BSA for 24 h. Cells cultured initive control for apoptosis respectively (n=4, ⁎⁎P<0.01).uced apoptosis in LNCaP cells. Apoptosis wasmeasured by usingted with wortmannin (100 nM) for 30 min and then stimulated4 h serum-starved cells were used as a negative and positiveve experiments showing constant levels of phosphorylated FAK−7 M testosterone-BSA for the indicated time points. Following) with an anti-phosphotyrosine (pTyr) antibody. The tyrosine-noblotted (IB) with specific antibodies against FAK (left panels)analyzed by densitometry. The intensity of phosphorylated FAKing total FAK and PI-3K bands. The number below each laneth that of untreated cells taken as 1. Each value represents the
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Actin reorganization is required for the testosterone-BSAinduced apoptotic response of DU145 cells
We have recently shown that activation of mAR by testosterone-BSA resulted in a significant induction of apoptosis in both LNCaPand DU145 prostate cancer cell lines [20]. In addition, actinreorganization was reported to control apoptotic responses invarious cell systems [28,30–32]. Since actin reorganization is amain downstream event inmAR stimulated LNCaP and DU145 cells[13,20], we studied the apoptotic responses following mARactivation in the presence of agents blocking actin polymerizationsuch as cytochalasin B. As shown by FACS analysis, the pre-incubation of DU145 cells with the anti actin agent resulted in aclear inhibition of the apoptotic response induced by testosterone-BSA (Figs. 2A, B). This apoptotic action remained unchanged upon
Fig. 4 – (A–D) Rapid GTP-loading of Cdc42, RhoA and RhoB but noRepresentative experiments showing the amount of GTP-bound Cddown assays in lysates derived from 24 h serum-starved cells (0)indicated time points. Affinity precipitated Rac1/Cdc42 (AP: GST-PBlysates were immunoblotted (IB) with antibodies specific for theincluded in each assay. The intensity of GTP bands was normalizednumber below each lane indicates the fold GTP-loading for each Grepresents the mean±SE of three or four independent experimentestosterone-BSA stimulated LNCaP cells. Representative experimedetermined by GST pull down assays in lysates derived from 24 h se(10−7 M) for the indicated time points. Affinity precipitated RhoA/immunoblotted (IB) with antibodies specific for the respective GTPnormalized to the intensity of the corresponding total protein banfor each GTPase with that of untreated cells taken as 1. Each valu(⁎P<0.05).
testosterone-BSA treatment, strongly indicating that the steroidaction is totally abolished in the presence of cytochalasin B. Itshould be noticed here that necrotic cell death was also increased;however this is most probably due to the experimental manipula-tions, since it was observed under serum-control conditions too,that normally do not induce any type of cell death. The FACSanalysis findings were corroborated by the quantitative APOPer-centage apoptosis assay, which showed a clear inhibition oftestosterone-BSA stimulated apoptosis in the presence of theanti-cytoskeletal agent (Fig. 2C, upper panel). Similar results wereobtained in LNCaP cells (Fig. 2C, lower panel) indicating that actinredistribution is a mandatory step for the apoptotic response ofmAR stimulated prostate cancer cells independently of their iARstatus. Cytochalasin B alone showed also a pro-apoptotic effect,being clearly less extensive than testosterone-BSA.
t Rac1 in testosterone-BSA stimulated DU145 cells.c42 (A), Rac1 (B), RhoA (C) or RhoB (D) determined by GST pullor cells stimulated with testosterone-BSA (10−7 M) for theD) or RhoA/B (AP: GST-RBD) as well as equal volumes of total
respective GTPases. A positive control loaded with GTPγS wasto the intensity of the corresponding total protein band. TheTPase with that of untreated cells taken as 1. Each value
ts (⁎P<0.05). (E, F) GTP-loading of RhoA and RhoB innts showing the amount of GTP-bound RhoA (E) or RhoB (F)rum-starved cells (0) or cells stimulated with testosterone-BSAB (AP: GST-RBD) as well as equal volumes of total lysates wereases. The intensity of GTP bands analyzed by densitometry wasd. The number below each lane indicates the fold GTP-loadinge represents the mean±SE of three independent experiments
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mAR induces actin reorganization and apoptosis withouttriggering active FAK/PI-3K signaling in DU145 cells
In iAR-positive LNCaP cells, mAR stimulation was recently shownto induce rapid non-genomic activation of the FAK/PI-3K/Cdc42/Rac1 signaling pathway controlling actin redistribution [21]. Toassess the functional role of this pathway in DU145 cells, we pre-treated cells with 100 nM of the PI-3K inhibitor wortmannin andsubsequently stimulated them with testosterone-BSA. Surpris-ingly, pharmacologically active doses of wortmannin [21,33]affected neither mAR-induced actin reorganization (Fig. 3A), norapoptotic response of DU145 cells upon testosterone-BSA treat-ment (Fig. 3B). In contrast, wortmannin clearly blocked PI-3Ksignaling in LNCaP cells [21, and data not shown] and mAR-dependent apoptosis of LNCaP cells (Fig. 3C). Further to the aboveobservations, FAK was constitutively phosphorylated in DU145cells and following incubation with the BSA-conjugated androgen
Fig. 5 – Active RhoGTPases are necessary for testosterone-BSA inducthe actin cytoskeleton in DU145 cells exposed (+) or not (−) to 10−7
plasmids expressing control GFP (row A), the dominant-negative mRhoA-N19 (row C), or the dominant-negative mutant RhoB-N19 (roGFP-transfected control (1) or testosterone-BSA treated cells (3). Coexpressing Cdc42(T17N), RhoA-N19, RhoB-N19mutants or GFP, treate2.1, 4.1, 4.2, represent magnifications of the corresponding areas inrhodamine–phalloidin specific for actin microfilaments are showntestosterone-BSA treated cells. Arrows point towards the transfecte
we could not detect any apparent additional phosphorylation ofFAK (Fig. 3D, left panels), or of the p-85 subunit of PI-3K (Fig. 3D,right panels). Taken together, these findings suggest that, FAK/PI-3K signaling may be dispensable for induction of actin reorganiza-tion and initiation of the apoptotic response in DU145 cells.
Activation of mAR in DU145 cells induces rapid activation ofRho family small GTPases,which regulate actin reorganization
The Rho family of small GTPases holds a prominent role inregulating rapid actin reorganization and apoptosis, induced byvarious effectors [34–36]. To evaluate the potential role of Rho inmediating testosterone-BSA-induced actin reorganization andapoptosis in DU145 cells, we performed affinity precipitationassays with: (I) a GST-fusion protein corresponding to the p21-binding domain of PAK1 (GST-PBD) that specifically binds to andprecipitates Rac1/Cdc42-GTP, as an indicator of Rac1/Cdc42
ed actin reorganization. Confocal laser-scanning microscopy ofM testosterone-BSA for 1 h, after transient transfection withutant cdc42 (T17N) (row B), the dominant-negative mutantw D). Columns 1 and 3 show GFP autofluorescence inlumns 2 and 4 show actin microfilament staining in cellsd (column 4) or not (column 2) with testosterone-BSA. Columnscolumns 2 and 4 respectively. Actin organization stained within detail in column 2.1 for untreated and columns 4.1 and 4.2 ford cells. Bar, 20 μm.
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activation [37] or (II) the rhotekin Rho-binding domain fused to GST(GST-RBD) to assess RhoA/B GTP-loading [38]. GTP-bound proteinsas well as total protein extracts were revealed with specificantibodies. The data in Figs. 4A–D show representative resultsfrom the corresponding experiments, whereas the fold-increasevalues indicate mean measurements from three (Fig. 4C) or four(Figs. 4A, B, D) independent experiments. Treatment of DU145 cellswith 10−7 M testosterone-BSA induced a very fast (within 3 min)and robust (2.35-fold) activation of Cdc42 (Fig. 4A). The amount ofGTP-bound Cdc42 returned to baseline 30 min post-stimulation.Even more prominent activation of RhoA (3.2 fold) was evidentafter 3 min treatment of DU145 cells with testosterone-BSA (Fig.4C). RhoB activation was moderate in comparison (1.95 fold), butsustained for longer period (60 min post-stimulation Fig. 4D). Incontrast, we could not detect GTP-loading of Rac1 (Fig. 4B).
Having established an effect of testosterone-BSA treatment onRhoA/B and Cdc42 activation, we addressed their functional role inmAR-induced actin reorganization. For this purpose, we transi-ently overexpressed dominant-negative mutants of Cdc42, RhoA,RhoB or GFP-control in DU145 cells and we analyzed actinreorganization following mAR stimulation. Results are depictedin Fig. 5. Cells expressing dominant-negative Cdc42, RhoA or RhoBmutants (Figs. 5B3, C3, D3 indicated by arrows) were unable toreorganize the actin network and to form stress fibers in responseto testosterone-BSA treatment (Fig. 5B4 and 4.1, C4 and 4.1, D4 and4.1, indicated by arrows). Otherwise, actin reorganization occurrednormally in androgen stimulated non-transfected surroundingcells (Figs. 5B4 and 4.2, C4 and 4.2, D4 and 4.2), or in control cellstransfected with GFP (Fig. 5A4, 4.1 and 4.2). These findings suggestthat Rho family GTPase's activation is necessary for actinpolymerization induced by mAR activation in DU145 cells.
mAR signaling activates Rho family small GTPases in LNCaP cells
To further investigate the role of Rho GTPase in iAR-positive LNCaPcells, we measured mAR-dependent RhoA and RhoB stimulation in
Fig. 6 – ROCK is involved in actin polymerization induced by testostDU145 (A) and LNCaP cells (B) was determined by immunoblot anaserum-starved cells (control), cells stimulated with 10−7 M testostepre-treated with Y-27632 for 1 h and then stimulated with the andvalues±SE of five independent experiments (⁎P<0.05 ⁎⁎P<0.01).
those cells. The data in Figs. 4E, F show representative results froman indicative experiment, whereas the fold-increase valuesindicate mean measurements from three independent experi-ments. Our results clearly showed prominent RhoA (Fig. 4E) andRhoB (Fig. 4F) activation. Interestingly, in LNCaP cells both GTPaseswere stimulated later (after 15 min) and the stimulation wassustained much longer (at least 120 min) when compared toDU145 cells (Figs. 4C, D) indicating differential activation kinetics.From these results we conclude that Rho GTPases are importanteffectors activated in response to mAR stimulation in LNCaP cells.
ROCK mediates actin reorganization in mAR activatedDU145 and LNCaP cells
To analyze the downstream events of Rho GTPase's activation, weinvestigated whether ROCK1 [39], was involved in the testoster-one-BSA induced actin reorganization. We therefore performedquantitative analysis of actin cytoskeleton dynamics in cell extractsderived from DU145 or LNCaP cells pre-treated with the specificROCK inhibitor Y-27632 and subsequently stimulated withtestosterone-BSA. Cells were pre-treated with Y-27632 for30 min before assessing actin dynamics in order to facilitate directcomparison with the short term signaling effects on Rho smallGTPases presented so far. Interestingly, testosterone-BSA wasunable to induce actin polymerization in DU145 and LNCaP cellspre-treated with Y-27632, as documented by quantitative analysisof the F/G-actin ratio respectively (Figs. 6A, B). Taken togetherthese results suggest that ROCK1 is activated upon mAR stimula-tion in prostate cancer cells and have a functional role in mediatingtestosterone-BSA-induced actin reorganization.
Testosterone-BSA induces activation of Lim Kinase 2 (LIMK2)and phosphorylation of destrin in DU145 cells
It is known that LIMK2, a Ser/Thr kinase, can be activated by Rho-and/or Cdc42-GTPases via phosphorylation of Thr 505 and induces
erone-BSA in DU145 and LNCaP cells. (A, B) The F/G-actin ratio inlysis after Triton X-100 subcellular fractionation in 24 hrone-BSA for 30 min or treated with 10 μM Y-27632 for 1 h orrogen conjugate. The results are presented in bars as the mean
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actin reorganization by inactivating–via phosphorylation–actindepolymerizing factors such as ADF/cofilin [40]. We thereforeperformed in vitro kinase assays for LIMK2 in extracts of DU145cells treated with or without testosterone-BSA. Incubation ofLIMK2 immune complexes with [γ-32P] ATP and visualizationof 32P-labeled proteins by autoradiography revealed increasedlevels of phosphorylated LIMK2 that occurred 5 min post-mARstimulation only in testosterone-BSA treated cells (Fig. 7A). Thiswas followed by an increase in phosphorylation of destrin, anisoform of cofilin (Fig. 7B). The phosphorylation kinetics of bothproteins was similar and reached a maximum 15 min post-treatment. Figs. 7A and B depict representative experiments, whilethe fold-increase values indicate mean values from three inde-pendent experiments. We further investigated whether ROCK1was involved in the testosterone-BSA induced phosphorylation ofLIMK2 and destrin. We performed in vitro kinase assays usingextracts derived from DU145 cells pre-treated with the specificROCK inhibitor Y-27632 and then stimulated with testosterone-BSA. In the pre-treated cells, no significant changes in thephosphorylation levels of LIMK2 (Fig. 7C) or destrin (Fig. 7D)could be detected. These data strongly support the hypothesis thatmAR activation in DU145 cells rapidly activates LIMK2, which in
Fig. 7 – LIMK2 and destrin are rapidly phosphorylated in response tstimulated with 10−7 androgen conjugate for the indicated time polysates were assayed for kinase activity in vitro. The phosphorylatevisualized by autoradiography as shown in representative experimThe membrane was then immunoblotted (IB) with a goat polyclonarabbit polyclonal anti-destrin/ADF antibody (B, lower panel). The redensitometry analysis. The number below each lane indicates the funtreated cells taken as 1. Each value represents the mean±SE of timmune complexes derived from24 h serum-starved cells treated (+1 h, and then stimulated with 10−7 M androgen conjugate for the iThe phosphorylated proteins that co-immunoprecipitated with LIMpanels) for LIMK2 and destrin/ADF respectively, and analyzed by dengoat polyclonal LimK2 antibody (C, lower panel), stripped and repropanel). The numbers below each lane indicate the protein fold pho
turn inhibits destrin via phosphorylation. Most probably this earlyand transient activation of LIMK is sufficient to mediate thedownstream effects and suggest that destrin inhibition may beresponsible for the observed rapid induction of actin polymeriza-tion and microfilament reorganization.
To explore the involvement of the Rho/ROCK1 signaling in mAR-induced apoptosis, we tested the apoptotic responses of DU145and LNCaP cells after blocking ROCK1. We initially assessedapoptosis in DU145 cells by FACS analysis (Figs. 8A–G). As shownin Fig. 8E, Y-27632 blocked the apoptotic effect of testosterone-BSA. Interestingly, wortmannin failed to inhibit testosterone-BSAinduced apoptosis (Fig. 8G). This observation is in line with thedata shown in Fig. 3B and supports the hypothesis that PI-3Ksignaling does not participate in mAR-mediated actin reorganiza-tion and apoptosis in iAR-deficient DU145 cells. The APOPercen-tage apoptosis assay in DU145 cells (Fig. 8I) confirmed the FACSanalysis data, indicating that actin reorganization produced bystimulation of the Rho/ROCK pathway is a critical step inpropagating the pro-apoptotic response of DU145 cells to
o testosterone-BSA in DU145 cells. 24 h serum-starved cells wereints. Anti-LIMK2 immune complexes precipitated from the celld proteins that co-immunoprecipitated with LIMK2 wereents for LIMK2 and destrin respectively (A, B upper panels).l LIMK2 antibody (A, lower panel), stripped and reprobed with alative phosphorylation of both proteins was estimated byold phosphorylation of LIMK2 (A) and destrin (B) with that ofhree independent experiments (⁎P<0.05). (C, D) Anti-LIMK2) or not (−) with 10 μMof the specific ROCK inhibitor Y-27632 forndicated time points were assayed for kinase activity in vitro.K2 were visualized by autoradiography, as shown in (C, D, uppersitometry. The membrane was then immunoblotted (IB) with abed with a rabbit polyclonal anti-destrin/ADF antibody (D, lowersphorylation with that of untreated cells taken as 1.
Fig. 8 – Inhibition of the Rho/ROCK pathway blocks testosterone-BSA induced apoptosis in DU145 and LNCaP cells. All experimentsexcept the serum-free condition were performed in serum containing medium. FACS analysis of cells pre-treated with the specificROCK inhibitor Y-27632 (10 μM) for 1 h (D, E) or with wortmannin (100 nM) for 30 min (F, G) and then stimulated with 10−7 Mtestosterone-BSA for 24 h (C, E and G). Cells cultured in serum (B) or 24 h serum-starved cells (A) were used as a negative and positivecontrol for apoptosis respectively. Cells were co-stained with Annexin V-FITC for apoptotic cells undergoing the flip–flop event andthe vitalitymarker propidium iodide (PI). In the charts, viable cells are shown in the Annexin/PI negative quadrangle. Early apoptosisstage cells are Annexin positive/PI negative, while late apoptosis or already dead cells are both Annexin and PI positive. (H) Theestimated percentage of viable and apoptotic cells under each condition are presented in the table. (I, J) DU145 (I) and LNCaP (J)prostate cancer cells were pre-treated with the specific ROCK inhibitor Y-27632 (10 μM) for 1 h and then stimulated with 10−7 Mtestosterone-BSA for 24 h. Cells cultured in serum or 24 h serum-starved cells were used as a negative and positive control forapoptosis respectively. Apoptosis was measured by the APOPercentage apoptosis assay at a wavelength of 550 nm as described inMaterials and methods. Bars represent the mean OD±SE of three independent experiments performed in triplicates (⁎⁎P<0.01).
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testosterone-BSA. Similar results were obtained in LNCaP cells pre-treated with the ROCK inhibitor and then stimulated withtestosterone-BSA (Fig. 8J).
Discussion
Two human prostate cancer cell lines that express functional mAR(20), namely iAR insensitive DU145 and iAR sensitive LNCaP cells,served in the present study as cellular models to show that a Rho/ROCK-dependent signaling mechanism controls mAR-inducedactin reorganization and apoptotic cell regression. Several linesof evidence support our hypothesis. First, we showed thatapoptosis of LNCaP and DU145 cells induced by mAR stimulationwas abolished in the presence of cytochalasin B, an actin disruptingagent. Second, pharmacological inhibition of ROCK1 through thespecific inhibitor Y-27632 blocked both the testosterone-BSA
induced actin reorganization and the apoptotic response. Finally,dominant-negative Rho alleles block actin reorganization inresponse to testosterone-BSA in DU145 treated cells. Therefore,actin remodeling regulated by specific signaling events seems to bethe key link between mAR activation and apoptosis of prostatecancer cells treated with mAR agonists. It should be noted thatactin redistribution and the pro-apoptotic effects were alsodocumented in DU145 cells upon mAR activation through addi-tional stimuli including non-BSA-conjugated androgens such astestosterone or dihydrotestosterone (DHT) as previously reported[20 and data not shown].
Our results further emphasize the hypothesis postulatedrecently that actin cytoskeleton dynamics regulate apoptoticresponses in various cell systems [41,42]. Indeed, the polarizationof the CD95 receptor in human T lymphocytes mediated by ezrinand the actin cytoskeleton was reported to regulate apoptosis inthese cells [32]. Furthermore, actin and tubulin cytoskeleton
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control cooperatively Akt phosphorylation, Bcl-2 expression andapoptosis in capillary endothelial cells [30]. Actin reorganizationstimulated by TNFα and regulated by PI-3K/Cdc42/PLCγ1 signalingwas reported to control NF-κB nuclear translocation and transcrip-tional regulation of TNFα induced anti-apoptotic response inopossum kidney cells [28]. Finally, changes in actin cytoskeletondynamics have proved to be crucial for apoptosis in yeast [31, 43].
We have recently identified a non-genomic signaling cascade iniAR-positive LNCaP prostate cancer cells, which regulates rapidtestosterone-BSA induced responses that include actin reorganiza-tion and PSA secretion [21]. This signaling pathway involved rapidphosphorylation of FAK and concomitant downstream activationof PI-3K, Cdc42 and Rac1. In the present study, we have tested thefunctional implication of this pathway in the response of mARstimulated DU145 cells. Using wortmannin, a PI-3K inhibitorpreviously shown to block mAR signaling in LNCaP cells [21], wesuggest that PI-3K is not involved in mAR-dependent actinredistribution and apoptosis in DU145 cells (Figs. 3A, B). Moreover,unlike LNCaP cells, DU145 cells express constitutively active FAK(Fig. 3C) and treatment with testosterone-BSA has no effect on FAKphosphorylation or on the activation of the downstream effectorsPI-3K and Rac1 (Figs. 3C, D and4B). These results collectivelyindicate that FAK/PI-3K signaling is not involved in DU145 cellresponses targeting actin remodeling and apoptosis induced bymAR activation. The presented results may reflect the highlytumorigenic and metastatic potential reported for DU145 cells[24]. FAK, is an oncogenic factor frequently expressed in prostatetumor cells [44] and implicated in cancer invasion and metastasis[44,45]. Increased FAK phosphorylation was also reported invarious tumor cells [46,47], as well as in tissues from metastaticprostate cancer patients [46] and has been linked to theirmetastatic potential, probably due to the activation of the anti-apoptotic PI-3K/Akt pathway. Our findings showing highly phos-phorylated FAK and PI-3K levels in metastatic, iAR-deficient DU145cells are in line with these observations
To identify the signaling pathways mediating the mAR-induced actin rearrangement and apoptosis in DU145 cells, wefocused on the Rho family small GTPases and their downstreameffector ROCK1 [48,49]. Activated ROCK1 phosphorylate andactivate LIM-kinases 1 and 2, which in turn inactivate–viaphosphorylation–actin depolymerizing proteins of the ADF/cofilinfamily thus allowing actin polymerization [39,40]. We report herethat testosterone-BSA treatment of DU145 cells resulted in rapidactivation of Cdc42, RhoA and RhoB GTPases, followed by LIMK2and ADF/destrin phosphorylation via ROCK1. Inhibition of thispathway by dominant-negative RhoA/B and Cdc42 mutants, or bya specific ROCK1 inhibitor abrogated the observed actin poly-merization and blocked the apoptotic response of DU145 cells inresponse to mAR activation. Based on these findings we suggestthat Rho/ROCK1/LIMK2/destrin is the predominant signalingcascade, triggered by mAR activation in iAR-deficient tumorigenicDU145 prostate cancer cells.
Pointing to a central role of Rho/ROCK in the mAR-inducedevents resulting to actin reorganization and apoptosis in prostatecancer cells, we show here that both, RhoA and RhoB are activatedupon mAR stimulation in LNCaP cells. Furthermore, pharmacolo-gical inhibition of ROCK impacts on mAR-induced events in LNCaPcells similarly to what was shown for DU145 cells. These dataindicate a potential involvement of the Rho/ROCK signaling in theapoptotic response of prostate cancer cells, although they should
be interpreted with caution since the ROCK inhibitor may alsoinhibit certain isoforms of PKC that may influence cell viability.
There are significant differences in terms of pathway activationbetween LNCaP and DU145 cells, which suggest that the expres-sion of iAR may influence the functional significance of mARactivation in prostate cancer. The hypothesis that the iARcooperates with the mAR to elicit testosterone-BSA effects wasaddressed in our previous work in LNCap cells [20]. It was shownthat the anti-proliferative effect of this steroid conjugate was notinhibited either after pre-treatment of the cells with the anti-androgen flutamide, or following down regulation of iAR withspecific antisense oligonucleotides. These data indicated that thePI-3K signaling reported in LNCaP cells [21] is mainly due to mARactivation and does not require interactions with iAR. Although wecannot exclude an interaction between iAR and mAR in DU145cells, we believe that this is unlikely, since iAR in DU145 cells arenot functional [24]. In addition, since PI3K is constitutivelyphosphorylated and no differences could be detected upon mARstimulation (Fig. 3D), we assume rather an indirect role of PI-3Kpathway in mAR signaling in DU145 cells. Our findings indicatethat the rapid Rho/ROCK activation is the main regulatory event,leading frommAR activation to actin reorganization and apoptoticresponses. However, the molecular link between mAR activationand Rho signaling in highly metastatic iAR-deficient DU145 cellsremains still unclear and is under investigation in our laboratory.On the other hand, in iAR-positive and hormone-sensitive LNCaPcells that have a lower tumorigenic ability, FAK expression wasmuch lower, while activation via phosphorylation, followed bydownstream stimulation of PI-3K, Rac and Cdc42 was observedupon mAR stimulation [21]. Since Rac and Rho are known toregulate each other in various systems [50], it is formally possiblethat in LNCaP cells, Rho activation occurs downstream of FAK/PI-3K/Rac. This hypothesismay also account for the late stimulation ofRhoA/B documented in LNCaP cells. Although it remains stillunclear and is under investigation whether Rho GTPases areregulated by Cdc42/Rac or directly by mAR stimulation, theinhibition of actin remodeling (21, and data not shown) in thepresence of the PI-3K inhibitor wortmannin, suggest that Rho/ROCK activation is manifested downstream of the FAK/PI-3K/Rac/Cdc42 signaling. It should be also noted that our present results donot specify, whether the observed inhibition of mAR-inducedapoptosis in LNCaP cells pre-treated with wortmannin (Fig 3C), ismanifested following possible down regulation of the pro survivalPI-3K/Akt signaling, or following inhibition of actin remodeling, orvia coordinated involvement of both signaling events triggered bymAR. Experiments are now in progress in our laboratory to addressthis issue.
In conclusion, in the present work we provide novel mechan-istic insights addressing the role of mAR activation in humanprostate cancer cells. Our data indicate that Rho/ROCK signaling,acting either downstream of PI-3K/Rac (as this is most probably thecase in LNCaP cells) or directly uponmAR activation (as this may bethe case in DU145 cells), is the prominent regulatory mechanismlinking mAR activation to actin reorganization and apoptosis inhuman prostate cancer cells. These findings may contribute to amore concise understanding of the differences in the behaviorbetween androgen sensitive and androgen insensitive humanprostate cancer cells in vivo. Understanding the functional role ofmAR activation is an important frontier for the development ofnovel and targeted therapies addressing prostate tumors.
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Acknowledgments
We are grateful to Dr. D. Kardasis (University of Crete) for providingthe expression plasmid pCDNA3-mycRhoBN19. We thank Dr. R.Buchanan and Dr. E.A. Papakonstanti for critical reading of themanuscript. The European Social Fund and National recourses(Herakleitos Program) supported this work.
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