Molecular and Cellular Endocrinology 382 (2014) 314–324

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Molecular and Cellular Endocrinology

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Prolonged exposure to (R)-bicalutamide generates a LNCaP subclonewith alteration of mitochondrial genome

0303-7207/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mce.2013.10.022

⇑ Corresponding author. Address: Biosciences Laboratory, Istituto ScientificoRomagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via P. Maroncelli 40,Meldola 47014, Italy. Tel.: +39 0543 739263; fax: +39 0543 739221.

E-mail address: w.zoli@irst.emr.it (W. Zoli).

Sara Pignatta a, Chiara Arienti a, Wainer Zoli a,⇑, Marzia Di Donato b, Gabriella Castoria b, Elisa Gabucci c,Valentina Casadio a, Mirella Falconi d, Ugo De Giorgi a, Rosella Silvestrini a, Anna Tesei a

a Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italyb Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italyc Department of General Surgery and Organ Transplantation, University of Bologna, Italyd Department of Human Anatomy and Physiopathology of the Locomotor Apparatus, University of Bologna, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 July 2013Received in revised form 18 October 2013Accepted 18 October 2013Available online 25 October 2013

Keywords:Prostate cancer(R)-bicalutamideMtDNAHormone-resistanceMitochondrial fission

Advanced prostate cancers, initially sensitive to androgen deprivation therapy, frequently progress to thecastration-resistant prostate cancer phenotype (CRPC) through mechanisms not yet fully understood. Inthis study we investigated mitochondrial involvement in the establishment of refractoriness to hormonetherapy. Two human prostate cancer cell lines were used, the parental LNCaP and the resistant LNCaP-Rbic, the latter generated after continuous exposure to 20 lM of (R)-bicalutamide, the active enantiomerof Casodex�. We observed a significant decrease in mtDNA content and a lower expression of 8 mitochon-dria-encoded gene transcripts involved in respiratory chain complexes in both cell lines. We also foundthat (R)-bicalutamide differentially modulated dynamin-related protein (Drp-1) expression in LNCaP andLNCaP-Rbic cells. These data seem to indicate that the androgen-independent phenotype in our experi-mental model was due, at least in part, to alterations in mitochondrial dynamics and to a breakdownin the Drp-1-mediated mitochondrial network.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Prostate cancer is the most common cancer in men in NorthernEurope and the United States, and the second-leading cause ofmale cancer death in most western countries, with an estimated28,000 deaths occurring in 2012 (American Cancer Society,2012). Advances in screening and diagnosis have led to an 85%detection rate of early-stage disease for which therapeutic options,including surgery and radiotherapy, are potentially curative (At-tard et al., 2006; Harris et al., 2009; Marques et al., 2010). Con-versely, advanced disease, which is initially sensitive to androgendeprivation therapy, frequently progresses to a state of castrationresistance (castration-resistant prostate cancer, CRPC) (Osantoand Van Poppel, 2012), with a poor prognosis and a median sur-vival time of 16–18 months (Amaral et al., 2012).

The mechanisms underlying progression to CRPC are not fullyunderstood and represent a major research challenge. Mitochon-dria are essential and complex intracellular organelles that areequipped with their own genome. They are cellular compartmentswhere key physiological processes take place, e.g. energy produc-

tion, apoptosis and amino acid and lipid synthesis. For these rea-sons it has been hypothesized that mitochondrial metabolismdefects or alterations may play an important role in resistance totreatment and in the metastatic progression of different tumortypes, including breast, colon, pancreatic and prostate cancers(Abril et al., 2008).

Mitochondrial DNA (mtDNA) consists of a circular genome of16.6 kb encoding for 13 proteins, 22 tRNA and 2rRNA subunitsand containing the D-loop regulatory region necessary for tran-scription initiation (Shutt and Shadel, 2010). Furthermore, withincells mtDNA is composed of a mixture of relaxed circular, linearand supercoiled forms, the last needed to initiate mtDNA replica-tion and transcription.

There is increasing evidence that mtDNA mutations, includingpoint mutations, multiple deletions and depletion, play a pivotalrole in cancer progression and invasiveness (Simonnet et al.,2002) and, in particular, in prostate cancer tumorigenicity (Petroset al., 2005).

Notably, in their study of human prostate cancer (PCa), Chenet al. (2002) observed a high incidence of somatic mutations(90%) in the D-loop region which may have caused a reduction inmtDNA content. Different events can alter mtDNA content andthere is evidence that one of the possible causes of induction ofmtDNA deletion/depletion in prostate cancer cell lines is androgen

S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324 315

ablation (Higuchi et al., 2006). Furthermore, a growth advantage indifferent cancer cell histotypes harboring specific mtDNA altera-tions has been demonstrated in in vitro and in vivo models. In par-ticular, there is evidence to suggest that a reduction in mtDNAcontent may cause the shift from an androgen-dependent to anandrogen independent phenotype (Higuchi et al., 2006). Moreover,an in vitro study by Cook et al. (2012) reported that a reduction inmitochondrial genome content, normally present at 100–1000copies per cell caused the inhibition of oxygen consumption, a shiftfrom hypoxia to hyperoxia, the activation of the mevalonate path-way and proto-oncogene Ras, and the constitutive activation ofERK, AKT, NF-Kb and JNK, with progression to an advanced andaggressive phenotype (Cook et al., 2012).

Mitochondria homeostasis may be altered by two events, mito-chondrial fusion or mitochondrial fission. Mitochondrial fusion isrequired for the maintenance of the mitochondrial tubular net-work and function and is coordinated by transmembrane GTPasessuch as mitofusin (Mfn) 1 and 2 or OPA-1. Conversely, mitochon-drial fission requires the presence of dynamin-related protein 1(Drp-1), a large GTPase whose up-regulation/deregulation deter-mines a breakdown in the mitochondrial network, loss of mtDNAand respiratory defects.

The aim of the present study was to investigate, for the firsttime, the correlation between the presence of alterations inmtDNA, Drp-1 protein expression and refractoriness to hormonetherapy in an in vitro model of CRPC.

2. Materials and methods

2.1. Chemicals and reagents

(R)-bicalutamide and MDV3100 were kindly provided by Dr.Greta Varchi (CNR-ISOF Institute of Bologna, Italy). The drug wasdissolved in dimethylsulfoxide (DMSO) (Sigma Aldrich) to a con-centration of 10 mM, divided into aliquots and stored �20C�. Zyti-ga™ (Janssen-Cilag) was solubilized in DMSO to a finalconcentration of 15.96 mM, divided into aliquots and stored at�20C�. Drug stocks were freshly diluted in culture medium imme-diately before use. The final DMSO concentration never exceeded1% and this condition was used as control in each experiment.

2.2. Cell culture

The human prostate cancer cell line LNCaP (passage 10) waspurchased by the American Type Culture Collection (ATCC). Cellswere grown in RPMI medium supplemented with 10% fetal bovineserum and glutamine (2 mM) and checked periodically for myco-plasma contamination by MycoAlert™ Mycoplasma Detection Kit(Lonza). LNCaP-Rbic, the bicalutamide-resistant cell line derivedfrom LNCaP and isolated in our laboratory, was maintained inthe same way in continuous exposure to 20 lM of (R)-bicalutamide.

Cos 7 cells (ATCC) were grown in DMEM supplemented withphenol red, 5% fetal calf serum (FCS), insulin (6 ng/ml), l-glutamine(2 mM), penicillin (100 U/ml), streptomycin (100 U/ml) and hydro-cortisone (3.75 ng/ml). The cells were made quiescent by usingphenol red-free DMEM and charcoal-stripped calf serum, as previ-ously described (Castoria et al., 2003).

2.3. Transmission electron microscopy

LNCaP and LNCaP-Rbic cells were fixed in 2.5% glutaraldehydein 0.1 M of phosphate buffer for 2 h at 4 �C and post-fixed in 1%OsO4 in 0.1 M of phosphate buffer for 1 h at 4 �C. Subsequently,samples were dehydrated in a graded series of ethanol and embed-

ded in Epon resin (Sigma Aldrich). Ultrathin sections were counter-stained with uranyl acetate and lead citrate and observed under aPhilips CM10 electron microscope (FEI Company). Images weredigitally captured by SIS Megaview III CCD camera (FEI Company).

2.4. Population doubling time

To determine cell proliferation doubling time (PDT), 1 � 105

cells/well were plated into 6-well plates and counted after 24,48, 72, 96, 120, 144, 168 and 192 h. PDT was determined by theformula: log2 (Cv/Cs), where Cv is the number of viable cells atharvest and Cs is the number of cells seeded. The sum of all previ-ous population doublings determined the cumulative populationdoubling level at each passage. The Trypan blue exclusion testwas used to evaluate the percentage of viable cells, which alwaysexceeded 98% for the duration of the experiments.

2.5. In vitro chemosensitivity assay

The effects of different concentrations of (R)-bicalutamide (from0.02 to 20 lM) on cell proliferation of LNCaP-Rbic and its parentalcell line LNCaP were assessed by the Sulforhodamine B (SRB assay)according to the method by Skehan et al. (1990). Three indepen-dent experiments were performed in octuplicate. The optical den-sity (OD) of treated and untreated cells was determined at awavelength of 540 nm using a fluorescence plate reader.

The inhibitory effect of (R)-bicalutamide at each concentrationwas expressed as:

ðabsorbance of treated cells=absorbance of control cellsÞ � 100

The 50% inhibitory concentration (IC50) was calculated from adose–response curve obtained by plotting the percentage of sur-vival versus the concentrations using the GraphPad Prism program(version 4) (GraphPad Software).

2.6. Real-Time RT-PCR analysis

Total RNA was extracted from cells using TRIzol� Reagent,according to the manufacturer’s instructions (Invitrogen). RNApurity was evaluated by agarose gel electrophoresis and quantifiedusing the Nanodrop� ND-1000 spectrophotometer system. Reversetranscription (RT) reactions were performed in 20 ll of a solutioncontaining 160 ng of total RNA and using iScriptTM cDNA Synthe-sis kit (BIO-RAD). Gene expression was analyzed by Real-Time RT-PCR (MyiQ System, BIO-RAD). In particular, we analyzed 12S and16S ribosomal RNAs; MT-ND2, MT-ND4 and MT-ND6 mRNAs,which are polypeptides of the MRC-complex 1; cytochrome b(MT-CYB mRNA) of the MRC-complex III and ATP synthase subunit6, one of the peptides that forms the F0 subcomplex of MRC-com-plex V. Primers for mRNA amplification are described in Table S1(Abril et al., 2008).

The standard reaction volume was 25 ll containing 2 ll ofcDNA template, 1 � SYBR Green MIX and 5 lM of each primer.Gene marker expression was normalized to endogenous references(GAPDH and HPRT) using Gene Expression Macro Software (Ver-sion 1.1. BIO-RAD). Reference genes were chosen using the geNormVBA applet for Microsoft Excel to determine the most stable refer-ence genes. Gene-specific amplification efficiency was used to cal-culate the relative expression of target genes using GeneExpression Macro Software (Version 1.1) (BIO-RAD). Intra-experiment variability did not exceed 5%. The reproducibility ofReal-Time PCR results was verified in triplicate samples and thecoefficient of variation (CV), calculated on three Ct values, neverexceeded 1.5%.

316 S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324

2.7. Methylation-specific multiple ligation probe amplification (MS-MLPA)

MS MLPA was performed using at least 50 ng of genomic DNAdissolved in 1X TE buffer. We used the ME001C1 and ME003 A1kit (MRC-Holland) which simultaneously analyzes the methylationstatus of 40 tumor suppressor gene promoters. The manufacturer’sinstructions were adhered to; in brief, DNA was denatured (10 minat 98 �C) and cooled at 25 �C, after which the probe mix was addedto the samples and hybridization was performed by incubating at60 �C for 16–18 h. The reaction was then divided (equally) intotwo vials: one for ligase and the other for ligase-digestion reac-tions. A mix composed of Ligase-65 buffer, Ligase 65 enzyme andwater was added to the first vial, while the mix for the second vialconsisted of Ligase-65 Buffer, Ligase 65 enzyme, Hhal enzyme (Pro-mega) and water. Samples were then incubated at 49 �C for 30 min.At the end of ligase and ligase-digestion reactions, samples wereamplified by PCR by adding a mix of dNTPs, Taq polymerase andPCR buffer. The PCR reaction was performed under the followingconditions: 37 cycles at 95 �C for 30 s, 60 �C for 30 s and 72 �C for60 s. The final incubation was performed at 73 �C for 20 min.

Amplification products were analyzed by the ABI-3130 geneticAnalyzer (Applied Biosystems). Universally methylated and unme-thylated genomic DNA was used as positive and negative control,respectively. Electropherograms were analyzed using Gene Map-per software (Applied Biosystems) and the peak areas of eachprobe were exported to a home-made excel spreadsheet. As sug-gested in the manufacturer’s instructions, ‘‘intrasample data nor-malization’’ was performed by dividing the signal of each probeby the signal of every reference probe in the sample, thus creatingas many ratios per probe as there were reference probes. We thencalculated the median value of all these probe ratios per probe,obtaining the normalization constant (NC).

Finally, the methylation status of each probe was calculated bydividing the NC of a probe in the digested sample by that of thesame probe in the undigested sample, and multiplying this ratioby 100 to have a percentage value, as follows :

½NC ðdigested sampleÞ=NC ðundigested sampleÞ� � 100

2.8. mtDNA damage and copy number analysis

2.8.1. Supercoiling-sensitive qPCR methodQuantification of mtDNA structural damage/repair and copy

number change was performed using the new supercoiling-sensi-tive qPCR approach according to the protocol described by Chanand Chen (2009), and briefly described below.

Total DNA was extracted from at least 3 � 106 cells using theDNA extraction kit (Qiagen) according to the manufacturer’sinstructions and then measured by Nanodrop� ND-1000. ThemtDNA copy number quantification was performed by the MyiQ™Real-Time PCR detection system (BIO-RAD). The expression of eachgene marker was normalized to endogenous references (b-actin)and performed in triplicate. The primers used in the mtDNA anal-ysis are reported in Table S2. For each sample, an aliquot of 5 ng/llDNA was used to quantify the nuclear marker b-globin DNA copies.Furthermore, exploiting the different efficiencies in PCR amplifica-tion of supercoiled and relaxed mtDNA molecules (the supercoiledstructure inhibits PCR amplification, while relaxed DNA is readilyamplified), another DNA aliquot of about 2 ng/ll was split intotwo equal halves and used to quantify DNA copies of the mitochon-drial markers CO2 and D-Loop in two different experimental con-ditions. Half of the aliquot was used to maintain the mtDNA inits supercoiled original shape to highlight the presence of relaxedmtDNA, while the other half was pretreated at 95 �C for 6 min to

unfold any structure and to accurately quantify the total mtDNAcontent of specific mitochondrial genes.

2.9. Analysis of AR mutation status

The analysis of the androgen receptor mutation status was per-formed in LNCaP cell line and in the subclone LNCaP-Rbic asfollows:

2.9.1. Amplification of genomic DNAGenomic DNA was isolated from cell lines using QIAamp� DNA

Mini Kit (Qiagen). Aliquots of 50 ng of DNA were amplified by per-forming 30 cycles of PCR in 25 ll of reaction solution. Each reactionconsisted of 2.5 ll of Takara 10X Ex Taq Buffer, 2.5 ll of dNTP Mix(2.5 mM), 1 ll of Takara Ex Taq DNA polymerase (1 unit) (TakaraBiotechnology), 2 ll of template DNA, 1 ll and 5 lM of the appro-priate sense and antisense oligonucleotide primer pairs, respec-tively, and 15 ll of ddH2O. Primers used for genomic DNAamplification analysis are reported in Table S3 (Tilley et al.,1996). The current version of the androgen receptor (AR) genemutation database is available on the internet (http://androg-endb.mcgill.ca). After an initial denaturation step of 3 min at95 �C, cycle parameters were as follows:

1 min at 94 �C for denaturation, 1 min at 55–57 �C (temperatureoptimized for primer sequences) for primer annealing, and 1 minat 72 �C for primer extension.

The size and integrity of all PCR products were confirmed on 2%agarose gels.

2.9.2. DNA sequencingPCR products were purified using the Minielute PCR purification

kit (Qiagen) and then submitted to sequencing using the BigDyeTerminator 3.1 Reaction Cycle Sequencing kit (Applied Biosys-tems). Sequence reactions were purified using DyeEx 2.0 Spin kit(Qiagen) and separated by capillary electrophoresis with laser-induced fluorescence detection (ABI-3130 Genetic Analyzer,Applied Biosystems).

2.10. siRNA transfection

A validated double strand Drp-1-targeting siRNA oligonucleo-tides (siRNA ID #s19559, Ambion) was used to silence the Drp-1gene. A validated Negative Universal Control™ (Invitrogen) wasused as a control for transfection. The transfection condition show-ing the highest knockdown efficiency of Drp-1 mRNA was used forthe experiments. To perform transfection, cells were seeded in25-cm2 flasks at a density of 2.5 � 103 cells (60% confluence). Thetransfection was carried out using Lipofectamine™ RNAi MAX(Invitrogen) and Opti-MEM GlutaMax medium (Invitrogen) with-out antibiotics. The incubation time for the oligonucleotide/lipo-fectamine 2000 complexes was 20 min.

2.11. Western blotting

Cell proteins were extracted with M-PER Mammalian ProteinExtraction Reagent (Thermo Fisher Scientific) supplemented withHalt Protease Phosphatase Inhibitor Cocktail (Thermo Fisher Scien-tific). Mini-PROTEAN� TGX™ precast gels (4–20%) (BIO-RAD) wererun using Mini-PROTEAN Tetra electrophoresis cells and then elec-troblotted by Trans-Blot� Turbo™ Mini PVDF Transfer Packs (BIO-RAD). The unoccupied membrane sites were blocked with T-TBS 1X(Tween 0.1%) and 5% non-fat dry milk to prevent nonspecific bind-ing of antibodies and probed with specific primary antibodies over-night at 4 �C. This was followed by incubation with the respectivesecondary antibodies. The antibody-antigen complexes were de-tected with Immun-Star™ WesternC™ kit (BIO-RAD).

S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324 317

The following antibodies were used: anti-42 kDa actin (dilution1:5000, Sigma–Aldrich, #A2066), anti-27 kDa Drp-1 (dilution1:500, BD Biosciences, #611738), anti-110 kDa AR (dilution 1:500,Cell Signaling, #3202), anti-29 kDa PSA (dilution 1:500, Cell Signal-ing, #5365), goat anti-rabbit IgG-HRP (dilution 1:5000, Santa Cruz,#sc-2004), goat anti-mouse IgG-HRP (dilution 1:5000, Santa Cruz,#sc-2005), and precision Protein™ StrepTactin-HRP Conjugate(dilu-tion 1:10,000, BIO-RAD). Precision Plus Protein™ WestrernC™Standards were used as molecular weight standards (BIO-RAD#161-0376). Where indicated, cell lysates (2 mg/ml protein concen-tration) were prepared, as previously described (Castoria et al.,2012), and AR was detected using a rabbit polyclonal anti-AR anti-body (C-19; Santa Cruz), as already reported (Castoria et al., 2011).Immunoreactive proteins were revealed using the ECL detectionsystem (GE Healthcare).

2.12. PSA analysis of culture media

A PSA ELISA kit #KA0208 supplied by Abnova (Taiwan Corpora-tion) was used according to the manufacturer’s instructions. PSAconcentrations were measured spectrophotometrically at 450 nmin culture medium.

2.13. Constructs, transfection and transactivation assay

cDNA encoding the wild-type hAR was in pSG5 (Chang et al.,1988). The 3416 construct, containing four copies of the wild-typeslp-HRE2 (59-TGGTCAgccAGTTCT-39), was cloned in the NheI sitein pTK-TATA-Luc (Verrijdt et al., 2000). This construct was agenerous gift from Dr. F. Claessens (Molecular Endocrinology Lab-oratory, Department of Cellular and Molecular Medicine, KU Leu-ven, Campus Gasthuisberg, BE-3000 Leuven, Belgium). In thetransactivation assay, LNCaP and LNCaP-Rbic were plated in100 mm plates (Falcon) at 60% confluence in RPMI containing10% fetal calf serum. After 4 h, cells were extensively washed andthen placed in phenol-red free RPMI containing 10% charcoal-stripped fetal calf serum. Cos-7 cells were plated at 60% confluencein DMEM containing 5% fetal calf serum. After 4 h, the cells wereextensively washed and then placed in phenol red-free DMEM con-taining 5% charcoal-stripped fetal calf serum (Castoria et al., 2011).LNCaP, LNCaP-Rbic and Cos-7 cells were then transfected by Super-fect (Qiagen) with 4 lg of 3416-pTK-TATA-Luc. When indicated,1 lg of pSG5 empty plasmid or pSG5-hAR-expressing plasmidwere included. After 72 h, transfected cells were left unstimulatedor were stimulated with 10 nM of the synthetic androgen R1881(Perkin–Elmer) for 18 h. R1881 was dissolved in 0.001% ethanol (fi-nal concentration) and control cells were treated with the vehiclealone. Cell lysates were prepared and luciferase activity was mea-sured, as previously reported (Castoria et al., 2011), using a lucifer-ase assay system (Promega). The results were corrected usingCH110-expressed-galactosidase activity. Values were obtainedfrom several independent experiments, each performed intriplicate.

2.14. Data mining

Human cDNA microarray data sets available at the onlineOncomine database (http://www.oncomine.com/) were used tocompare the mRNA expression profiles of the Drp-1 gene in pros-tate cancers and normal counterparts (Rhodes et al., 2007). Datawere summarized as fold changes with statistic p value.

2.15. Statistical analysis

Data are presented as the mean ± standard deviation (SD). Dataobtained from the analysis of cytotoxic activity, mtDNA content

and PSA dosage were analyzed by the Student’s t-test (unpaired,two-tailed). Data obtained from transactivation assays were ana-lyzed using the Student’s test for paired observations. A p value<0.05 was considered significant. Data obtained from quantitativeReal-Time PCR experiments were analyzed by comparing groups ofmean values using one-way ANOVA with Tukey’s multiple compar-ison test. Data were processed using the GrafPad Prism program(version 4) (GraphPad Software).

Significant differences, were defined at ⁄p 6 0.05, ⁄⁄p 6 0.01.

3. Results

3.1. Establishment of LNCaP-Rbic subclone

LNCaP cells were continuously exposed to 20 lM of (R)-bicalu-tamide for 8 months and after only a few days LNCaP cell prolifer-ation was strongly hampered. Such conditions persisted for theentire 8-month period, after which cells started to proliferate againand in only a few weeks a subclone of actively proliferating cellswas isolated, established and called LNCaP-Rbic. This clone exhib-ited a faster growth than that of the parental cell line and a sub-stantially reduced doubling time from 55 to 37 h (Fig. S1).

The SRB assay was used to assess the effects of increased con-centrations of (R)-bicalutamide on cell proliferation of LNCaP-Rbicand its parental line LNCaP. After exposure to (R)-bicalutamide,naïve LNCaP cells showed a dose-dependent reduction in survivalstarting from a concentration of 0.02 lM, reaching an IC50 valueof about 7 lM (Fig. 1A). Conversely, (R)-bicalutamide exerted apoor antiproliferative effect on LNCaP-Rbic and only at the highestconcentration of 20 lM (Fig. 1A).

We also investigated the antiproliferative effect of Zytiga™ andMDV3100 in LNCaP and LNCaP-Rbic. Cells were exposed for 144 hto scalar drug concentrations ranging from 0.07 nM to 700 nM(Zytiga™) and from 0.02 lM to 20 lM (MDV3100), respectively(Fig. S2). Zytiga showed a poor cytotoxic effect, never reachingIC50 values in either cell line.

Conversely, MDV3100 induced a dose-related, strong cytotoxiceffect in both the parental and derived cell line, reaching an IC50

value of 0.44 lM (LNCaP) and 1.13 lM (LNCaP-Rbic), respectively.

3.2. Biomolecular characterization of LNCaP-Rbic

We investigated expression levels of the androgen receptor (AR)and its target gene (PSA) in both LNCaP and LNCaP-Rbic cell lines.AR expression was higher in Rbic-resistant cells than in the paren-tal cell line, whereas PSA levels were significantly reduced in theRbic-resistant cell line (Fig. 1B). To confirm these data we analyzedthe protein expression of AR and PSA by western blot technique. Asexpected, AR protein expression was increased in LNCaP-Rbic cells,while PSA protein was only weakly expressed (Fig. 1C). A signifi-cant reduction in PSA secretion from 93.2 ng/ml to 10.8 ng/mlwas also observed in the culture medium of LNCaP-Rbic cell linewith respect to that found in LNCaP culture medium (Fig. 1D). Suchdata suggest that the AR transactivation function is not fully main-tained in these cells.

To further address this issue, we transfected an ARE-responsivegene into Bic-resistant LNCaP cells. The transfected cells were leftunstimulated or were stimulated with 10 nM of R1881 and theluciferase activity of ARE-reporter gene was evaluated (Fig. 2).Challenging the quiescent LNCaP-Rbic cells with 10 nM of R1881increased the transcriptional activity of AR 2.5-fold, while a 3.4-fold increase in the androgen-triggered transcriptional activity ofAR was observed in the parental LNCaP cells (Fig. 2A). Thesefindings are consistent with the results obtained by measuringPSA levels in the same cells (Fig. 1). As expected, we observed astrong increase in the luciferase activity of the ARE reporter gene

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Fig. 1. Biomolecular characterization of LNCaP-Rbic subclone. (A) Dose–response curves in LNCaP-Rbic (solid line) and LNCaP (dashed line) cell lines after a 144-h exposure to(R)-bicalutamide. Each point indicates the mean of at least three experiments. Standard deviation never exceeded 5%, ⁄p < 0.05. (B) Expression of the AR and PSA genes. Real-Time PCR analysis in LNCaP-Rbic (normalized to GAPDH and HPRT) relative to control (LNCaP). Bars represent the mean of three independent experiments. The statisticalsignificance of results was evaluated by the Student’s t-test and p values 60.05 were considered significant. (C) AR and PSA protein expression. Cells were grown to 80%confluence and whole-cell lysates were prepared. Protein (40 lg) was subjected to SDS–PAGE followed by Western Blot analysis and chemiluminescence detection, asdescribed in Section 2. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for b-actin. (D) PSA levels were measured by Elisa assay. Valuesare the mean ± SD of three independent experiments. The statistical significance of results was evaluated by the Student’s t-test and p values 60.05 were consideredsignificant. (E) AR mutation status. The AR sequencing analysis was performed on LNCaP cells and the LNCaP-Rbic subclone obtained after 8 months of continuous exposure to20 lM of (R)-bicalutamide. The sequencing chromatograms show the T877A point mutation harbored by naive LNCaP cells and, as expected, by the derived subclone. Arrowsindicate sites of mutation.

318 S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324

in a control reporter assay which consisted of ectopically express-ing hAR in Cos-7 cells (Fig. 2B). The Western blot of AR from thecorresponding cell lysates is shown in Fig. 2C. In brief, AR tran-scriptional function was retained in Bic-resistant cells, albeit to alesser degree than in parental LNCaP cells.

In an attempt to identify the molecular mechanisms underlyingthe onset of resistance to the antiandrogen, we sequenced the ARgene but found no differences in the DNA sequences of either cellline. The T877A mutation was the only one detected in both lines(Fig. 1E).

The promoter methylation profiles of genes involved in cell pro-liferation, DNA repair, apoptosis and tumor-suppressor pathwaysare shown in Supplementary Table S4. Methylation was only ob-served in APC, CASP8, RARB, RASSF1, CD44, GSTP1, CCND2,SGB3A1, ID4 and RUNX3 promoter genes, with no differences be-tween the parental cell line and LNCaP-Rbic.

3.3. Ultrastructural analysis

Both cell lines showed typical cellular morphology with wellcharacterized nuclei, a clearly visible nuclear envelope and a finechromatin structure with multiple nucleoli (Fig. 3A and B). Highermagnification highlighted alterations in mitochondrial morphol-ogy in the LNCaP-Rbic cells with respect to naïve LNCaP cells

(Fig. 3C and D). In particular, all mitochondrions in LNCaP-Rbiccells were tubular-shaped and showed mitochondrial cristaeenlargement (Wheater et al., 2007; Prince, 1999).

3.4. Correlation between resistance to (R)-bicalutamide andalterations in mitochondrial dynamics and gene expression profile

We also used the supercoiling-sensitive qPCR approach toinvestigate whether (R)-bicalutamide exposure influenced mtDNAcontent or the expression profile of specific genes belonging toLNCaP-Rbic mtDNA.

mtDNA content of D-Loop and CO2 was low in the originalmtDNA template and did not vary significantly in the two cell lines,indicating that the continuous exposure of cells to (R)-bicaluta-mide did not cause structural damage to mtDNA. Conversely, thecopy number of D-Loop and CO2 genes in pre-heated templates,which represented the total mtDNA content, was significantly low-er in LNCaP-Rbic than in the parental line LNCaP (Fig. 4A). Further-more, analysis of the expression of a number of mitochondrialgenes involved in the respiratory chain complexes, performed byReal-Time RT-PCR at different times during the continuous expo-sure of LNCaP to (R)-bicalutamide, highlighted a gradual decreasein their mRNA levels over time. In particular, all the genes investi-gated (Fig. 4B) showed a reduction in expression starting 2 months

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C

B

Fig. 2. Analysis of androgen-triggered transcriptional activity of AR in LNCaP, LNCaP-Rbic and Cos-7 cells. (A) LNCaP and LNCaP-Rbic cells were transfected with 3416 ARE-Luc construct as described in the Materials and Methods. (B) Cos-7 cells were transfected with 3416 ARE-luc construct in the absence (pSG5) or presence of hAR (hAR)-expressing plasmid. (A and B) Cells were left unstimulated or were stimulated for 18 h with 10 nM of R1881. Luciferase activity was assayed, normalized using beta-gal as aninternal control, and expressed as fold induction. Three independent experiments were performed in triplicate. Means and standard error of mean (SEM) are shown; nrepresents the number of experiments. (⁄) p value <0.05. (C) Lysates from transfected LNCaP, LNCaP-Rbic or Cos-7 cells were prepared as previously reported in (Castoria et al.,2012). Protein lysates (at 2 mg/ml) were analyzed by Western blot for AR expression using the rabbit polyclonal C-19 anti-AR antibody. Filters were then reprobed with theanti- tubulin antibody, as a loading control. AR, androgen receptor; tub, tubulin.

S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324 319

after the beginning of drug exposure, with the exception of 16s and12s genes in which a decrease was registered only after 8 months.

We also investigated whether Drp-1 protein, highly expressedin both cell lines at baseline, might be implicated in the processthat led to a reduction in mtDNA in LNCaP-Rbic cell line. Expres-sion levels of Drp-1 gene in LNCaP and LNCaP-Rbic cells main-tained in normal medium culture without R1881 were evaluatedafter exposure to 20 lM of (R)-bicalutamide. A significant increasein Drp-1 expression was observed in LNCaP cells but not in LNCaP-Rbic (Fig. 5A). Similar results were obtained by western blot anal-ysis in which we also observed an important increase in AR in bothcell lines after the same drug exposure (Fig. 5B). We also silencedDrp-1 by the gene-silencing technique (Fig. 6A and B). SilencedLNCaP cells showed poor sensitivity to (R)-bicalutamide (IC50 valuenot reached), with a chemosensitivity profile similar to that ob-served in LNCaP-Rbic cells (Fig. 6C).

We queried the Oncomine database to compare mRNA expres-sion profiles of Drp-1 genes in prostate cancer and normal tissue.The database filtered 16 studies among which only 4 showed a sig-nificantly different gene expression value (p 6 0.05) in canceroustissues compared to normal counterparts. In particular, Drp-1 genewas significantly overexpressed in 3 different studies Table 1(Tomlins et al., 2007; Welsh et al., 2001; Singh et al., 2002).

4. Discussion

Androgen depletion therapy is the most widely used treatmentfor advanced prostate cancer but is rarely curative because at this

stage of the disease the tumor almost invariably progresses to themore aggressive castration-resistant prostate cancer phenotype(CRPC). The aim of the present work was to investigate the mech-anisms of resistance to endocrine treatment to identify potentialnew targets for innovative therapeutic strategies. We used a newsubclone of LNCaP prostate cancer cell line, LNCaP-Rbic, isolatedin our laboratory after prolonged exposure of parental cells to(R)-bicalutamide, the active enantiomer of the racemic mixtureof Casodex�. This subclone was refractory to the antiproliferativeeffect of (R)-bicalutamide, as previously demonstrated in in vitroand in vivo experimental models (Tesei et al., 2013). We also testedtwo of the most promising drugs for the treatment of CRPC, abira-terone acetate (Zytiga™) and the second-generation antiandrogenMDV3100 (End et al., 2013; Richards et al., 2012). The subcloneLNCaP-Rbic was still sensitive to MDV3100, albeit to a lesser de-gree than the parental line LNCaP. The data obtained confirmedthe usefulness of LNCaP-Rbic to study the mechanisms of actionof second-generation antiandrogens.

We characterized the two cell lines for AR and PSA mRNAexpression to investigate the extent to which alterations in andro-gen/AR signaling affect the ability of tumor cells to respond to hor-mone therapy. As expected, LNCaP cells showed high levels of bothAR and PSA, whereas an inverse correlation in marker expression,i.e. an increase in the expression of AR and a reduction of PSA,was observed in the LNCaP-Rbic cell line. In our model, increasedAR expression, a typical feature of the CRPC phenotype, was notaccompanied by a concomitant increase in PSA levels, which, incontrast, actually decreased. This would seem to indicate that,

LNCaP-RbicLNCaP

2600x

13500x

A

C

B

D

Fig. 3. Ultrastructural analysis of LNCaP and LNCaP-Rbic by transmission electron microscopy. Nuclei, nuclear envelope and chromatin structure in (A) LNCaP and in (B)LNCaP-Rbic cell lines. Details of mitochondria morphology in (C) LNCaP and in (D) LNCaP-Rbic. White arrows indicate normal mitochondria morphology in LNCaP (C) andmitochondria with tubular-shape and typical cristae enlargement in LNCaP-Rbic (D). Images were digitally captured by SIS Megaview III CCD camera (FEI Company).

320 S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324

during the shift from antiandrogen sensitivity to resistance, ARtransactivation function was not fully maintained. In our reporterassay, we observed that androgen-stimulated AR transcriptionalactivity was significantly reduced in Bic-resistant LNCaP cells com-pared to that observed in parental LNCaP cells. However, AR wasstill active as a transcription factor, albeit to a lesser degree, andmay nevertheless be required for Bic-resistant cell functions. Thus,the decrease we observed in PSA levels cannot be exclusively dueto the loss of AR transcriptional activity in LNCaP- Rbic cells. Otherdifferent mechanisms (i.e. post-transcriptional effects, mRNA sta-bility, proteolytic degradation) may be responsible for the differ-ences in PSA levels observed in LNCaP-Rbic cells compared toparental LNCaP cells. The fact that LNCaP-Rbic cells were sensitiveto the cytotoxic effect of MDV3100 (a drug that specifically targetsAR) further confirms that the transcriptional activity of AR in thisline is still active and needed for cell survival. Such findings, inagreement with literature data, indicate that AR signalling axis isstill a driving force in CRPC (Li et al., 2013).

It is known that changes in the AR sequence can occur in pros-tate cancer and that specific point mutations of AR exons are capa-ble of altering its canonical regulation and functionality(Gnanapragasam et al., 2000; Lamont and Tindall, 2011). We thusexplored the possibility that the cell line generated in our labora-tory harbored AR mutations which would make it insensitive tothe effect of the antiandrogen. Although AR mutations in Caucasianpatients are rarely found in untreated localized prostate cancer(<2%), they are frequently detected in hormone-refractory, andro-gen-ablated and metastatic tumors (Gottlieb et al., 2004; Linjaand Visakorpi, 2004). In addition, it has been seen that severalantiandrogens, including bicalutamide may behave as agonistsand activate the AR when specific mutations are present in itsligand binding domain (Hara et al., 2003; Shi et al., 2002;

Veldscholte et al., 1990). For this reason, we sequenced the AR genein both cell lines in an attempt to detect the well known mutations(i.e. W741C or WW741L) or to identify novel genetic lesions thatmay be responsible for the antiandrogen’s switch from antagonistto agonist. The only alteration in the mutational status of the ARgene in our experimental models was found in the well knownpoint mutation at codon 877 (thr ? ala) (Tilley et al., 1996), har-bored by both cell lines. We also evaluated the methylation profilesof a panel of gene promoters known to be involved in tumor sup-pression, cell proliferation, DNA repair, apoptosis, cell cycle pro-gression and protein synthesis regulation. In particular, ouranalysis focused on gene promoters at specific sites of CpG islandswith high mutation rates in cancer tissue (Albany et al., 2011). Nosignificant differences were observed between the parental lineand the antiandrogen-resistant subclone, even in RASSF1, GSTP1and RARB gene promoters whose methylation status is frequentlyaltered in prostate cancer (Meiers et al., 2007; Hesson et al., 2007;Wang et al., 2005).

Ultrastructural alterations in mitochondrial morphology thatsupport the hypothesis of metabolic hyperactivity and evidencefrom the literature highlighting the role of mtDNA alterations incancer progression led us to investigate the potential involvementof mtDNA alterations behind the switch to antiandrogen resistanceof the subline LNCaP-Rbic (Wheater et al., 2007; Prince, 1999). Anumber of degenerative diseases, aging and several tumor histo-types (including prostate cancer) (Bossy-Wetzel et al., 2003;Higuchi, 2007) are characterized by a decline in mitochondrialfunction with a decrease in oxidative phosphorylation and ATPsynthesis, an increase in ROS production and the onset of mtDNAmutations. Furthermore, the presence of transcription hormoneresponse elements (HREs) capable of specifically binding to theAR hormone receptor complex within some mitochondrial gene

100

90

80

70

60

50 LNCaP

LNCaP-Rbic40

30R

elat

ive

ampl

ific

atio

n of

rt-

PC

R

20

10**

0 **D-loop D-loop heated CO2 CO2 heatedβ-globin

4.5 16s

4

3.5

3

2 months2.5

2

4 months

26 months

8 months

12s

* 8MT-ATP6 MT-CYB MT-ND2

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Rel

ativ

e m

RN

A e

xpre

ssio

n

** ** **

******1

0.5

0

A

B

MT-ND6MT-ND4

Fig. 4. Analysis of mitochondrial DNA. (A) mtDNA damage and copy number analysis was performed in the two different prostate cancer cell lines using the supercoiling-sensitive qPCR approach. The D-loop and CO2 markers were normalized with the nuclear marker b-actin and a second nuclear marker b-Globin was used to indicate the lackof structural change in the target nuclear gene. Bars represent the mean of three independent experiments. The statistical significance of results was evaluated by Student’s t-test, ⁄⁄p 6 0.01. (B) Real time RT-PCR analysis of mitochondrial gene expression in LNCaP cell line after different exposure times to (R)-bicalutamide. Values are the mean ± SDof three independent experiments. Gene expression was normalized to GAPDH and HPRT. Gene expression was relative to LNCaP naïve (control). The statistical significance ofresults was evaluated by one-way ANOVA with Tukey’s multiple comparison test, ⁄p<0.05; ⁄⁄a p value <0.01 was considered significant.

S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324 321

promoters and in the D-loop regulatory region indicates a possiblerole of the androgens in the mtDNA initiation and transcriptionprocess (Scheller and Sekeris, 2003). The process that regulatesthe interaction between androgens and mtDNA is not yet fullyunderstood. Mitochondria perform multiple functions that areessential for the maintenance of cellular homoeostasis whose dys-function leads to disease. Elaborate control mechanisms haveevolved for protecting, repairing or eliminating damaged mito-chondria. The right balance of these quality control mechanismsis needed for optimal functioning of these cellular organelles(Taylor and Rutter, 2011). The morphology, number and functionof mitochondria are regulated by numerous cellular events suchas autophagy, fusion and fission, and mitochondrial proteolysis(Bossy-Wetzel et al., 2003).

Recently, several authors suggested that the GTPase dynamin-related protein 1 (Drp-1) may play a pivotal role in the regulationof mitochondrial fission. The over-stimulation of this process, alsodue to high Drp-1 protein expression, determines a breakdown inthe mitochondrial network, loss of mtDNA and respiratory defects

(Han et al., 2011). A recent study also highlighted a critical role ofAR in the transcriptional and post-translational regulation of Drp-1, suggesting a modulating effect of androgens on mitochondrialmorphology (Choudhary et al., 2011).

In our study, exposure to 20 lM of the antiandrogen (R)-bicaluta-mide caused an increase in Drp-1 protein expression in LNCaP cellsbut not in LNCaP-Rbic. To explain this, we hypothesized the absenceof the regulatory function of Drp-1 on mitochondrial physiology dueto prolonged exposure to (R)-bicalutamide. Our data, in fact, re-vealed that, when Drp-1 gene was silenced in LNCaP cells, the cytoc-idal effect of (R)-bicalutamide was reduced and the cells showed asimilar chemosensitivity profile to that of LNCaP-Rbic cells. The in-crease in Drp-1 protein expression caused by prolonged exposureto (R)-bicalutamide may have led to an excessive fission processand an alteration in mitochondrial biogenesis . Our results wouldseem to confirm literature data on the close correlation betweenDrp-1 overexpression and mitochondrial fission (Frank et al.,2001; Barsoum et al., 2006). In effect, we highlighted a depletionof mtDNA content and, in particular, a dramatic reduction in

Drp-11.20

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LNCaP LNCaP-Rbic Drp-1-HCT116PC3

- -+ + Ctrl- Ctrl-Ctrl+(R)-bic

Drp-1 27 kDa

AR 110 kDa

42 kDaβ-actin

A

B

Fig. 5. Drp-1 and AR expression in LNCaP and LNCaP-Rbic. (A) Real time RT-PCRanalysis of Drp-1 gene expression in LNCaP and LNCaP-Rbic cell line after exposureto (R)-bicalutamide 20 lM. Values are the mean ± SD of three independentexperiments. Gene expression was normalized to GAPDH and HPRT. ⁄p <0.05. (B)Total cell lysates were prepared and 30 lg of protein was subjected to SDS–PAGEfollowed by Western Blot analysis. Equal loading of protein was confirmed bystripping the immunoblot and reprobing it for b-actin. Cells were treated with20 lM of (R)-bicalutamide. HCT-116 was used as positive control for Drp1, Drp-1-silenced LNCaP was used as negative control for Drp-1, and PC3 was used asnegative control for AR. Digital images were captured with Chemidoc XRS systemunder Quantity-One software control (BIO-RAD).

Table 1Drp-1 gene expression in the Oncomine database.

References Cases Foldchange

p

Normalprostate

Prostatecancer

Tomlins et al. (2007) 28 49 1.365 0.049Welsh et al. (2001) 9 25 1.245 0.018Singh et al. (2002) 50 52 1.382 0.026Holzbeierlein et al.

(2004)3 29 �1.034 0.634

Magee et al. (2001) 4 11 1.433 0.418Luo (2002) 15 15 1.725 0.084Yu et al. (2004) 23 89 1.019 0.314Vanaja et al. (2003) 8 32 1.140 0.045Wallace et al. (2008) 20 69 �1.002 0.512LaTulippe et al. (2002) 3 32 �1.418 0.958Grasso et al. (2012) 28 93 1.031 0.258Liu et al. (2006) 13 44 �1.016 0.569Lapointe et al. (2004) 41 70 �1.155 1.000Arredouani et al.

(2009)8 13 1.079 0.198

Varambally et al.(2005)

6 13 1.031 0.471

Taylor et al. (2010) 29 155 �1.081 0.895

Drp-1 expression profiles in 16 cDNA microarray datasets. The fold induction ofgene expression data in prostate cancer was compared with that of gene expressiondata in normal prostate tissue by extracting the information from the Oncomineonline database along with the publication citations, the case numbers, and thestatistical p values.

322 S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324

mitochondrial gene D-Loop and CO2 expression. These data are alsoin agreement with recent evidence that mitochondrial homeostasisis altered by environmental stimuli or pathological conditions(Taylor and Rutter, 2011). Furthermore, analysis of data from theOncomine database revealed an overexpression of Drp-1 in prostatecancer with respect to normal counterparts.

We also analyzed expression levels of genes involved in mito-chondrial respiratory chain complexes. A decrease in OXPHOScomplexes was observed 2 months after the start of treatmentwhen cell replication was strongly hampered by exposure to the

1.4

121.2 Ctrl

110

0.8

k-

Drp-1-

70.6

0.4

50.2

Rel

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A e

xpre

ssio

n(t

o co

ntro

l)

Surv

ival

(%

)

02Ctrl k- Drp-1-

Drp-1 27 kDa

ββ-actin 42 kDa

A

B

C

Fig. 6. Survival of LNCaP cells after Drp-1 gene silencing. (A) Real Time RT-PCR analysisand Drp-1-silenced LNCaP (Drp-1-). Gene expression was normalized to GAPDH and HPanalysis of Drp-1 protein expression after gene-silencing (normalized to b-actin). (C) CytsiRNA (k-), and Drp-1-silenced LNCaP (Drp-1-). SRB assay was used to assess the effectsexperimental conditions. Each point indicates the mean of at least three experiments. S

antiandrogen. This reduced expression was stably maintained inthe actively proliferating (R)-bicalutamide resistant subclone,which has a much lower doubling time that of the parental line.

Our results provide new evidence of the fact that, under specificconditions, mtDNA alterations in prostate carcinoma may be asso-ciated with the acquisition of hormone-resistant phenotype. Therole of these alterations in other treatments such those based ontaxanes or novel antiandrogens requires further investigation tobetter elucidate the mechanisms of tumor resistance.

5. Conclusions

In conclusion, in our experimental model the mitochondriaseem to have played a pivotal role in overcoming the pharmacolog-ically-induced androgen blockade. In particular, we observed that

(R)-bicalutamide Ctrl5

k-Drp-1-

0

* *

5

0

5

00 0.02 0.2 2 20 µM

of Drp-1 mRNA levels in LNCaP (Ctrl), LNCaP transfected with scrambled siRNA (k-),RT. Values are the mean ± SD of three independent experiments. (B) Western blototoxic effect of (R)-bicalutamide in LNCaP (Ctrl), LNCaP transfected with scrambledof increased concentrations of (R)-bicalutamide on cell proliferation under differenttandard deviation never exceeded 5%, ⁄p < 0.05.

S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324 323

the resistance or sensitivity to antiandrogen exposure was mir-rored in alterations in mtDNA content and mitochondrial morphol-ogy and dynamics, this last regulated by Drp-1 protein. Aprognostic/predictive role of changes in mtDNA content is proba-bly unrealistic in view of the substantial molecular and clinicalheterogeneity of CRPC and the varied mechanisms of action ofanti-CRPC therapeutics (Grasso et al., 2012; Lee et al., 2012).However, despite limitations in our understanding of the patholog-ical and clinical significance of mitochondrial alterations, theidentification of novel mechanisms correlating mtDNA contentwith AR signaling deregulated in prostate cancer could undoubt-edly facilitate their application in clinical practice.

Funding

Marzia Di Donato and Gabriella Castoria were funded by TheItalian Association for Cancer Research (A.I.R.C.; Grant No. IG11520) and the Italian Ministry for University and Scientific Re-search (P.R.I.N 2010–2011; Grant No. 2010NFEB9L_002). The fund-ers had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Acknowledgements

We thank Greta Varchi (Istituto CNR per la Sintesi Organica eFotoreattività I.S.O.F., Bologna, Italy) for providing (R)-bicalutamideand Ursula Elbing for editing the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.mce.2013.10.022.

References

Abril, J., de Heredia, M.L., González, L., Clèries, R., Nadal, M., Condom, E., Aguiló, F.,Gómez-Zaera, M., Nunes, V., 2008. Altered expression of 12S/MT-RNR1, MT-CO2/COX2, and MT-ATP6 mitochondrial genes in prostate cancer. Prostate 68,1086–1096.

Albany, C., Alva, A.S., Aparicio, A.M., Singal, R., Yellapragada, S., Sonpavde, G., Hahn,N.M., 2011. Epigenetics in prostate cancer. Prostate Cancer 2011, 580318.

Amaral, T.M., Macedo, D., Fernandes, I., Costa, L., 2012. Castration-resistant prostatecancer: mechanisms, targets, and treatment. Prostate Cancer 2012, 327253.

American Cancer Society, 2012. Cancer Facts & Figures 2012. American CancerSociety, Atlanta.

Arredouani, M.S., Lu, B., Bhasin, M., Eljanne, M., Yue, W., Mosquera, J.M., Bubley, G.J.,Li, V., Rubin, M.A., Libermann, T.A., Sanda, M.G., 2009. Identification of thetranscription factor single-minded homologue 2 as a potential biomarker andimmunotherapy target in prostate cancer. Clin. Cancer Res. 15, 5794–5802.

Attard, G., Sarker, D., Reid, A., Molife, R., Parker, C., de Bono, J.S., 2006. Improving theoutcome of patients with castration-resistant prostate cancer through rationaldrug development. Br. J. Cancer 95, 767–774.

Barsoum, M.J., Yuan, H., Gerencser, A.A., Liot, G., Kushnareva, Y., Gräber, S., Kovacs, I.,Lee, W.D., Waggoner, J., Cui, J., White, A.D., Bossy, B., Martinou, J.C., Youle, R.J.,Lipton, S.A., Ellisman, M.H., Perkins, G.A., Bossy-Wetzel, E., 2006. Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases inneurons. EMBO J. 25, 3900–3911.

Bossy-Wetzel, E., Barsoum, M.J., Godzik, A., Schwarzenbacher, R., Lipton, S.A., 2003.Mitochondrial fission in apoptosis, neurodegeneration and aging. Curr. Opin.Cell Biol. 15, 706–716.

Castoria, G., Lombardi, M., Barone, M.V., Bilancio, A., Di Domenico, M., Bottero, D.,Vitale, F., Migliaccio, A., Auricchio, F., 2003. Androgen-stimulated DNA synthesisand cytoskeletal changes in fibroblasts by a nontranscriptional receptor action.J. Cell Biol. 161, 547–556.

Castoria, G., D’Amato, L., Ciociola, A., Giovannelli, P., Giraldi, T., Sepe, L., Paolella, G.,Barone, M.V., Migliaccio, A., Auricchio, F., 2011. Androgen-induced cellmigration: role of androgen receptor/filamin A association. PLoS One 6, e17218.

Castoria, G., Giovannelli, P., Lombardi, M., De Rosa, C., Giraldi, T., de Falco, A., Barone,M.V., Abbondanza, C., Migliaccio, A., Auricchio, F., 2012. Tyrosinephosphorylation of estradiol receptor by Src regulates its hormone-dependentnuclear export and cell cycle progression in breast cancer cells. Oncogene 31,4868–4877.

Chan, S.W., Chen, J.Z., 2009. Measuring mtDNA damage using a supercoiling-sensitive qPCR approach. Methods Mol. Biol. 554, 183–197.

Chang, C.S., Kokontis, J., Liao, S.T., 1988. Structural analysis of complementary DNAand amino acid sequences of human and rat androgen receptors. Proc. Natl.Acad. Sci. USA 85, 7211–7215.

Chen, J.Z., Gokden, N., Greene, G.F., Mukunyadzi, P., Kadlubar, F.F., 2002. Extensivesomatic mitochondrial mutations in primary prostate cancer using lasercapture microdissection. Cancer Res. 62, 6470–6474.

Choudhary, V., Kaddour-Djebbar, I., Lakshmikanthan, V., Ghazaly, T., Thangjam, G.S.,Sreekumar, A., Lewis, R.W., Mills, I.G., Bollag, W.B., Kumar, M.V., 2011. Novelrole of androgens in mitochondrial fission and apoptosis. Mol. Cancer Res. 9,1067–1077.

Cook, C.C., Kim, A., Terao, S., Gotoh, A., Higuchi, M., 2012. Consumption of oxygen: amitochondrial-generated progression signal of advanced cancer. Cell Death Dis.3, e258.

End, D., Molina, A., Todd, M., Meyers, M.L., 2013. Interactions of abiraterone,eplerenone, and prednisolone with wild-type and mutant androgen receptor: arationale for increasing abiraterone exposure or combining with MDV3100.Cancer Res. 73, 2926.

Frank, S., Gaume, B., Bergmann-Leitner, E.S., Leitner, W.W., Robert, E.G., Catez, F.,Smith, C.L., Youle, R.J., 2001. The role of dynamin-related protein 1, a mediatorof mitochondrial fission, in apoptosis. Dev. Cell 1, 515–525.

Gnanapragasam, V.J., Robson, C.N., Leung, H.Y., Neal, D.E., 2000. Androgen receptorsignalling in the prostate. BJU Int. 86, 1001–1013.

Gottlieb, B., Beitel, L.K., Wu, J.H., Trifiro, M., 2004. The androgen receptor genemutations database (ARDB): 2004 update. Hum. Mutat. 23, 527–533.

Grasso, C.S., Wu, Y.M., Robinson, D.R., Cao, X., Dhanasekaran, S.M., Khan, A.P., Quist,M.J., Jing, X., Lonigro, R.J., Brenner, J.C., Asangani, I.A., Ateeq, B., Chun, S.Y.,Siddiqui, J., Sam, L., Anstett, M., Mehra, R., Prensner, J.R., Palanisamy, N., Ryslik,G.A., Vandin, F., Raphael, B.J., Kunju, L.P., Rhodes, D.R., Pienta, K.J., Chinnaiyan,A.M., Tomlins, S.A., 2012. The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243.

Han, X.J., Tomizawa, K., Fujimura, A., Ohmori, I., Nishiki, T., Matsushita, M., Matsui,H., 2011. Regulation of mitochondrial dynamics and neurodegenerativediseases. Acta Med. Okayama 65, 1–10.

Hara, T., Miyazaki, J., Araki, H., Yamaoka, M., Kanzaki, N., Kusaka, M., Miyamoto, M.,2003. Novel mutations of androgen receptor: a possible mechanism ofbicalutamide withdrawal syndrome. Cancer Res. 63, 149–153.

Harris, W.P., Mostaghel, E.A., Nelson, P.S., Montgomery, B., 2009. Androgendeprivation therapy: progress in understanding mechanisms of resistance andoptimizing androgen depletion. Nat. Clin. Pract. Urol. 6, 76–85.

Hesson, L.B., Cooper, W.N., Latif, F., 2007. The role of RASSF1A methylation in cancer.Dis. Markers 23, 73–87.

Higuchi, M., 2007. Regulation of mitochondrial DNA content and cancer.Mitochondrion 7, 53–57.

Higuchi, M., Kudo, T., Suzuki, S., Evans, T.T., Sasaki, R., Wada, Y., Shirakawa, T.,Sawyer, J.R., Gotoh, A., 2006. Mitochondrial DNA determines androgendependence in prostate cancer cell lines. Oncogene 25, 1437–1445.

Holzbeierlein, J., Lal, P., LaTulippe, E., Smith, A., Satagopan, J., Zhang, L., Ryan, C.,Smith, S., Scher, H., Scardino, P., Reuter, V., Gerald, W.L., 2004. Gene expressionanalysis of human prostate carcinoma during hormonal therapy identifiesandrogen-responsive genes and mechanisms of therapy resistance. Am. J.Pathol. 164, 217–227.

Lamont, K.R., Tindall, D.J., 2011. Minireview: alternative activation pathways for theandrogen receptor in prostate cancer. Mol. Endocrinol. 25, 897–907.

Lapointe, J., Li, C., Higgins, J.P., van de Rijn, M., Bair, E., Montgomery, K., Ferrari, M.,Egevad, L., Rayford, W., Bergerheim, U., Ekman, P., DeMarzo, A.M., Tibshirani, R.,Botstein, D., Brown, P.O., Brooks, J.D., Pollack, J.R., 2004. Gene expressionprofiling identifies clinically relevant subtypes of prostate cancer. Proc. Natl.Acad. Sci. USA 101, 811–816.

LaTulippe, E., Satagopan, J., Smith, A., Scher, H., Scardino, P., Reuter, V., Gerald, W.L.,2002. Comprehensive gene expression analysis of prostate cancer revealsdistinct transcriptional programs associated with metastatic disease. CancerRes. 62, 4499–4506.

Lee, D.J., Cha, E.K., Dubin, J.M., Beltran, H., Chromecki, T.F., Fajkovic, H., Scherr, D.S.,Tagawa, S.T., Shariat, S.F., 2012. Novel therapeutics for the management ofcastration-resistant prostate cancer (CRPC). BJU Int. 109, 968–985.

Li, Y., Chan, S.C., Brand, L.J., Hwang, T.H., Silverstein, K.A., Dehm, S.M., 2013.Androgen receptor splice variants mediate enzalutamide resistance incastration-resistant prostate cancer cell lines. Cancer Res. 73, 483–489.

Linja, M.J., Visakorpi, T., 2004. Alterations of androgen receptor in prostate cancer. J.Steroid Biochem. Mol. Biol. 92, 255–264.

Liu, P., Ramachandran, S., Ali Seyed, M., Scharer, C.D., Laycock, N., Dalton, W.B.,Williams, H., Karanam, S., Datta, M.W., Jaye, D.L., Moreno, C.S., 2006. Sex-determining region Y box 4 is a transforming oncogene in human prostatecancer cells. Cancer Res. 66, 4011–4019.

Luo, J.H., 2002. Gene expression alterations in human prostate cancer. Drugs Today(Barc) 38, 713–719.

Magee, J.A., Araki, T., Patil, S., Ehrig, T., True, L., Humphrey, P.A., Catalona, W.J.,Watson, M.A., Milbrandt, J., 2001. Expression profiling reveals hepsinoverexpression in prostate cancer. Cancer Res. 61, 5692–5696.

Marques, R.B., Dits, N.F., Erkens-Schulze, S., van Weerden, W.M., Jenster, G., 2010.Bypass mechanisms of the androgen receptor pathway in therapy-resistantprostate cancer cell models. PLoS One 5, e13500.

Meiers, I., Shanks, J.H., Bostwick, D.G., 2007. Glutathione S-transferase pi (GSTP1)hypermethylation in prostate cancer: review 2007. Pathology 39, 299–304.

Osanto, S., Van Poppel, H., 2012. Emerging novel therapies for advanced prostatecancer. Ther. Adv. Urol. 4, 3–12.

324 S. Pignatta et al. / Molecular and Cellular Endocrinology 382 (2014) 314–324

Petros, J.A., Baumann, A.K., Ruiz-Pesini, E., Amin, M.B., Sun, C.Q., Hall, J., Lim, S., Issa,M.M., Flanders, W.D., Hosseini, S.H., Marshall, F.F., Wallace, D.C., 2005. MtDNAmutations increase tumorigenicity in prostate cancer. Proc. Natl. Acad. Sci. USA102, 719–724.

Prince, F.P., 1999. Mitochondrial cristae diversity in human Leydig cells: a revisedlook at cristae morphology in these steroid-producing cells. Anat. Rec. 254,534–541.

Rhodes, D.R., Kalyana-Sundaram, S., Mahavisno, V., Varambally, R., Yu, J., Briggs, B.B.,Barrette, T.R., Anstet, M.J., Kincead-Beal, C., Kulkarni, P., Varambally, S., Ghosh,D., Chinnaiyan, A.M., 2007. Oncomine 3.0: genes, pathways, and networks in acollection of 18,000 cancer gene expression profiles. Neoplasia 9, 166–180.

Richards, J., Lim, A.C., Hay, C.W., Taylor, A.E., Wingate, A., Nowakowska, K., Pezaro,C., Carreira, S., Goodall, J., Arlt, W., McEwan, I.J., de Bono, J.S., Attard, G., 2012.Interactions of abiraterone, eplerenone, and prednisolone with wild-type andmutant androgen receptor: a rationale for increasing abiraterone exposure orcombining with MDV3100. Cancer Res. 72, 2176–2182.

Scheller, K., Sekeris, C.E., 2003. The effects of steroid hormones on the transcriptionof genes encoding enzymes of oxidative phosphorylation. Exp. Physiol. 88, 129–140.

Shi, X.B., Ma, A.H., Xia, L., Kung, H.J., de Vere White, R.W., 2002. Functional analysisof 44 mutant androgen receptors from human prostate cancer. Cancer Res. 62,1496–1502.

Shutt, T.E., Shadel, G.S., 2010. A compendium of human mitochondrial geneexpression machinery with links to disease. Environ. Mol. Mutagen. 51, 360–379.

Simonnet, H., Alazard, N., Pfeiffer, K., Gallou, C., Béroud, C., Demont, J., Bouvier, R.,Schägger, H., Godinot, C., 2002. Low mitochondrial respiratory chain contentcorrelates with tumor aggressiveness in renal cell carcinoma. Carcinogenesis23, 759–768.

Singh, D., Febbo, P.G., Ross, K., Jackson, D.G., Manola, J., Ladd, C., Tamayo, P.,Renshaw, A.A., D’Amico, A.V., Richie, J.P., Lander, E.S., Loda, M., Kantoff, P.W.,Golub, T.R., Sellers, W.R., 2002. Gene expression correlates of clinical prostatecancer behavior. Cancer Cell 1, 203–209.

Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T.,Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assayfor anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112.

Taylor, E.B., Rutter, J., 2011. Mitochondrial quality control by the ubiquitin–proteasome system. Biochem. Soc. Trans. 39, 1509–1513.

Taylor, B.S., Schultz, N., Hieronymus, H., Gopalan, A., Xiao, Y., Carver, B.S., Arora, V.K.,Kaushik, P., Cerami, E., Reva, B., Antipin, Y., Mitsiades, N., Landers, T., Dolgalev, I.,Major, J.E., Wilson, M., Socci, N.D., Lash, A.E., Heguy, A., Eastham, J.A., Scher, H.I.,Reuter, V.E., Scardino, P.T., Sander, C., Sawyers, C.L., Gerald, W.L., 2010.Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22.

Tesei, A., Leonetti, C., Di Donato, M., Gabucci, E., Porru, M., Varchi, G., Guerrini, A.,Amadori, D., Arienti, C., Pignatta, S., Paganelli, G., Caraglia, M., Castoria, G., Zoli,

W., 2013. Effect of small molecules modulating androgen receptor (SARMs) inhuman prostate cancer models. PLoS One 8, e62657.

Tilley, W.D., Buchanan, G., Hickey, T.E., Bentel, J.M., 1996. Mutations in the androgenreceptor gene are associated with progression of human prostate cancer toandrogen independence. Clin. Cancer Res. 2, 277–285.

Tomlins, S.A., Mehra, R., Rhodes, D.R., Cao, X., Wang, L., Dhanasekaran, S.M.,Kalyana-Sundaram, S., Wei, J.T., Rubin, M.A., Pienta, K.J., Shah, R.B., Chinnaiyan,A.M., 2007. Integrative molecular concept modeling of prostate cancerprogression. Nat. Genet. 39, 41–51.

Vanaja, D.K., Cheville, J.C., Iturria, S.J., Young, C.Y., 2003. Transcriptional silencing ofzinc finger protein 185 identified by expression profiling is associated withprostate cancer progression. Cancer Res. 63, 3877–3882.

Varambally, S., Yu, J., Laxman, B., Rhodes, D.R., Mehra, R., Tomlins, S.A., Shah, R.B.,Chandran, U., Monzon, F.A., Becich, M.J., Wei, J.T., Pienta, K.J., Ghosh, D., Rubin,M.A., Chinnaiyan, A.M., 2005. Integrative genomic and proteomic analysis ofprostate cancer reveals signatures of metastatic progression. Cancer Cell 8, 393–406.

Veldscholte, J., Ris-Stalpers, C., Kuiper, G.G., Jenster, G., Berrevoets, C., Claassen, E.,van Rooij, H.C., Trapman, J., Brinkmann, A.O., Mulder, E., 1990. A mutation in theligand binding domain of the androgen receptor of human LNCaP cells affectssteroid binding characteristics and response to anti-androgens. Biochem.Biophys. Res. Commun. 173, 534–540.

Verrijdt, G., Schoenmakers, E., Haelens, A., Peeters, B., Verhoeven, G., Rombauts, W.,Claessens, F., 2000. Change of specificity mutations in androgen-selectiveenhancers. Evidence for a role of differential DNA binding by the androgenreceptor. J. Biol. Chem. 275, 12298–12305.

Wallace, T.A., Prueitt, R.L., Yi, M., Howe, T.M., Gillespie, J.W., Yfantis, H.G., Stephens,R.M., Caporaso, N.E., Loffredo, C.A., Ambs, S., 2008. Tumor immunobiologicaldifferences in prostate cancer between African–American and European–American men. Cancer Res. 68, 927–936.

Wang, Y., Yu, Q., Cho, A.H., Rondeau, G., Welsh, J., Adamson, E., Mercola, D.,McClelland, M., 2005. Survey of differentially methylated promoters in prostatecancer cell lines. Neoplasia 7, 748–760.

Welsh, J.B., Sapinoso, L.M., Su, A.I., Kern, S.G., Wang-Rodriguez, J., Moskaluk, C.A.,Frierson, H.F., Hampton, G.M., 2001. Analysis of gene expression identifiescandidate markers and pharmacological targets in prostate cancer. Cancer Res.61, 5974–5978.

Wheater, P.R., Joung, B., Lowe, J.S., Stevens, A., Heath, J.W., 2007. Le ghiandoleendocrine, Istologia e anatomia microscopica. Elsevier, Milan, Edizione V, pp.328–346.

Yu, Y.P., Landsittel, D., Jing, L., Nelson, J., Ren, B., Liu, L., McDonald, C., Thomas, R.,Dhir, R., Finkelstein, S., Michalopoulos, G., Becich, M., Luo, J.H., 2004. Geneexpression alterations in prostate cancer predicting tumor aggression andpreceding development of malignancy. J. Clin. Oncol. 22, 2790–2799.