The Prostate 70:457^ 466 (2010)

Establishment andCharacterizationof anAndrogenReceptor-Dependent, Androgen-IndependentHuman

ProstateCancerCell Line,LNCaP-CS10

Nobuyuki Ishikura,* Hiromitsu Kawata, Ayako Nishimoto,Ryo Nakamura, Nobuya Ishii, and Yuko Aoki

ResearchDivision,Kamakura Research Laboratories, Pharmaceutical ResearchDepartment 2,Chugai Pharmaceutical Co.Ltd.,Kamakura,Kanagawa, Japan

BACKGROUND. Hormone refractoriness is a lethal event for advanced prostate cancerpatients, but the mechanisms of the disease are not well elucidated, especially for the so-called‘‘outlaw’’ pathways of androgen receptor (AR)-dependent, androgen-independent hormone-refractory prostate cancer.METHODS. Androgen-dependent prostate cancer LNCaP cells were treated with bicaluta-mide under an androgen-depleted condition to obtain refractory cells. In the obtained cell line,LNCaP-CS10, we analyzed the effects of androgen and bicalutamide on cell growth andprostate-specific antigen (PSA) production. In addition, AR gene mutation, AR expressionlevels, and AR subcellular localizations were analyzed.RESULTS. In LNCaP-CS10, cell growth and PSA production were found under an androgen-depleted condition and were induced by both R1881 and bicalutamide. Knocking down AR bysiRNAs did suppress the growth and PSA production of LNCaP-CS10 cells in the androgen-depleted condition. In comparison to LNCaP, amplification or additional new mutations werenot found in the AR genes, but AR nuclear translocation induced by bicalutamide was identifiedin the LNCaP-CS10 cells. The growth and PSA production of xenografted LNCaP-CS10 tumors,which secrete PSA not only in non-castrated SCID mice but also in castrated SCID mice, were notinhibited by bicalutamide.CONCLUSIONS. We have generated a bicalutamide-resistant and androgen-independentprostate cancer cell line, LNCaP-CS10, with outlaw activation both in vitro and in vivo. TheLNCaP-CS10 cell line is beneficial for elucidating outlaw pathway mechanisms and evaluatingthe efficacy of new therapeutics for hormone-refractory prostate cancer. Prostate 70: 457–466,2010. # 2009 Wiley-Liss, Inc.

KEY WORDS: prostate cancer; androgen receptor; outlaw pathway; bicalutamideresistant; hormone refractory

INTRODUCTION

Prostate cancer is the most common epithelialcancer and the second leading cause of cancer deathsin males in the United States [1]. Androgen depletiontherapies such as surgical castration or luteinizinghormone-releasing hormone analogs are initially effec-tive against this disease. But, because adrenal androgensare still produced by adrenal glands under suchconditions, the patients additionally require an andro-gen receptor (AR) antagonist in order to achieve totalandrogen blockade [2,3]. However, the response to totalandrogen blockade therapy is often less than several

years [4,5]. To date, there is no efficacious hormonaltherapy for hormone-refractory prostate cancer.

Disclosure Statement: All authors are employed by ChugaiPharmaceutical Co. Ltd.

*Correspondence to: Nobuyuki Ishikura, Kamakura ResearchLaboratories, Chugai Pharmaceutical Co. Ltd., 200 Kajiwara,Kamakura, Kanagawa 247-8530, Japan.E-mail: ishikuranby@chugai-pharm.co.jpReceived 24 April 2009; Accepted 24 September 2009DOI 10.1002/pros.21079Published online 9 November 2009 in Wiley InterScience(www.interscience.wiley.com).

/ 2009 Wiley-Liss, Inc.

Recent analysis revealed that expression of ARremained in most hormone-refractory prostate cancerspecimens and these patients were still prostate-specific antigen (PSA) positive [4–6]. These observa-tions suggest that the AR signal is still active inhormone-refractory prostate cancer cells, and thereforeAR is a potential therapeutic target for hormone-refractory prostate cancer.

In a recent review, Pienta and Bradley [7] suggestedthree major mechanisms of androgen refractoriness:overexpression of AR, mutations in AR genes, andandrogen-independent activation of AR. AR over-expression leads to hypersensitivity to very low levelsof androgen in patients treated with hormonal thera-pies [8–10]. Mutations in the ligand-binding domain ofAR create loss of ligand specificity and so anti-androgens act as agonists for the mutated ARs [11–15]. The androgen-refractory cells with androgen-independent activation of AR are termed ‘‘outlaw.’’In outlaw tumors, ARs are activated by androgen-independent pathways, such as deregulated cytokinesand growth factors, including IL-6, IGF, and EGF [16–22].

Although several hormone-refractory prostate can-cer cell lines with AR overexpression [10,23,24] or ARmutation [15,25] have been established, only a fewhormone-refractory prostate cancer cell lines using theoutlaw pathway have been established.

In this study, we isolated a bicalutamide-resistantand androgen-independent prostate cancer cell line(LNCaP-CS10) and demonstrated its outlaw pathwayboth in vitro and in vivo.

MATERIALSANDMETHODS

Cell Culture

The androgen-dependent human prostate cancercell line LNCaP was obtained from ATCC andmaintained in 10% FCS medium consisting ofRPMI1640 (Sigma) and 10% FCS (Japan Bioserum) at378C in 5% CO2. The LNCaP-CS10 cells were main-tained in 10% DCC medium consisting of phenol-red-free RPMI1640 (Invitrogen) and 10% charcoal/dextran-treated FCS (DCC-FCS; Hyclone) in the pres-ence of 10 mM bicalutamide.

Cell ProliferationAssays

Androgen-depleted cells were prepared by preincu-bation in 10% DCC medium for 3 days and the cellswere then plated onto a 96-well poly-D-lysine-coatedplate (Beckton Dickinson) at 1� 104 cells/well(LNCaP) or 5� 103 cells/well (LNCaP-CS10) in 10%DCC medium. After overnight incubation at 378C in 5%CO2, R1881 (NEN Life Science) or bicalutamide was

added. After a further 9 days of incubation at 378C in5% CO2, the number of cells remaining was counted byDNA quantity assay (FluoReporter Blue FluorometricdsDNA Quantitation Kit, Invitrogen).

Quantif|cation of Cellular PSA

After androgen depletion for 3 days, the cellswere plated onto a 24-well poly-D-lysine-coated plate(Beckton Dickinson) at 2� 105 cells/well in 10% DCCmedium, incubated overnight, and R1881 or bicaluta-mide were added. After 2 days of incubation, the cellswere lysed with ice-chilled 1� cell lysis buffer (CellSignaling) supplemented with Complete ProteaseInhibitor Cocktail Tablets (Roche Diagnostics). Aftercentrifugation at 14,000 rpm for 10 min at 48C, PSA inthe supernatant was quantified using PSA ELISA assay(E-plate Eiken PSA; Eiken Chemical).

KnockdownExperimentsWithAR siRNA

LNCaP and LNCaP-CS10 cells were transfected withAR siRNA (Santa Cruz Biotechnology) or scramblesiRNA (Invitrogen) using Hyperfect (Qiagen) and thenplated onto a 96-well poly-D-lysine-coated plate at5� 103 cells/well in 10% FCS medium (LNCaP) or 10%DCC medium (LNCaP-CS10), respectively. At 6 hr,3 days, and 6 days posttransfection, the cell counts werequantified as described above. In order to confirm ARknockdown in the transfected cells, the cells wereplated onto a 24-well poly-D-lysine-coated plate at4� 104 cells/well; intracellular PSA and AR werequantified by AR ELISA assay (Active Motif) after3 days.

Preparation ofNuclear,Cytoplasmic,and Total Fractions

Cells were plated onto six-well poly-D-lysine-coatedplates (Beckton Dickinson) at 3� 105 cells/well in 10%DCC medium. After 3 days of incubation, 10 mMbicalutamide was added and the plates were incubatedfor another 24 hr. Then nuclear and cytoplasmicfractions were prepared using NE-PER Nuclear andCytoplasmic Extraction Reagents (Pierce). Total frac-tion was prepared as described above. AR was detectedby Western blot analysis using antibody to AR (N-20;Santa Cruz Biotechnology) and an ECLþ Westernblotting analysis system (Amersham). Immunodetec-tions of c-Jun and IkBa (H-79 and C-15, respectively;Santa Cruz Biotechnology) were used as the loadingcontrol of nuclear and cytoplasmic/total fraction.

Animal Experiments

Five-week-old male C.B-17/Icr-scid Jcl severe com-bined immune-deficient (SCID) mice purchased from

The Prostate

458 Ishikura et al.

CLEA Japan were maintained on a 12:12 hr light/darkcycle (lights on at 7 a.m.) with constant temperature20–268C and humidity (35–75%). Food and water wereavailable ad libitum. For xenograft experiments, 2� 106

cells of LNCaP or LNCaP-CS10 were subcutaneouslyinoculated with 50% phenol-red-free Matrigel (BDBiosciences) into the flank region of intact or surgicallycastrated mice. The tumor size was measured by caliperand expressed as cubic millimeters using the formula0.5� length�width2. Tumorigenicity was evaluatedaccording to the number of mice bearing palpabletumors (>50 mm3). Sensitivity of LNCaP or LNCaP-CS10 xenograft models to bicalutamide was evaluatedas tumor growth inhibition. When the tumor sizereached 110–370 mm3 (LNCaP) or 90–270 mm3

(LNCaP-CS10), the animals were randomized andorally administered either bicalutamide (10 or100 mg/kg) or the drug vehicle (5% gum arabic). Thedrug was administered in 4–5 cycles of 5 days on,2 days off, and tumor size and plasma PSA levels weremeasured at the designated time points.

RESULTS

Establishmentof theAndrogen-Independent,Bicalutamide-Resistant LNCaP-CS10 Cell Line

To establish a total androgen blockade therapy-resistant prostate cancer cell line with outlaw pathwayactivation, LNCaP cells were incubated in 10% DCCmedium in the presence of 10 mM bicalutamide. After a4-month incubation, the surviving cells in the andro-gen-depleted media in the presence of bicalutamidewere selected and designated as LNCaP-CS10.

LNCaP-CS10 exhibited faster growth and higherPSA production in the androgen-depleted mediumcompared with LNCaP (4.9- and 8.3-fold, respectively,Fig. 1A,B). The growth of LNCaP-CS10 in the andro-gen-depleted medium was comparable to that ofparental LNCaP cells in the presence of 0.1 nM R1881.Although LNCaP-CS10 grew in an androgen-inde-pendent manner, R1881 treatment further stimulatedthe cell growth and PSA production of LNCaP-CS10(Fig. 1A,B). Bicalutamide showed agonist activity andinduced cell growth and PSA production in a dose-dependent manner in LNCaP-CS10 cells but did notstimulate growth or PSA production of LNCaP cells atany concentration (Fig. 1C,D).

Characterization of theOutlawPathwayof CS10 InVitro

To confirm the outlaw mechanism of LNCaP-CS10,we first analyzed the sequence of AR cDNA in LNCaP-CS10 by a direct sequence method using PCR-amplifiedAR cDNA. However, LNCaP-CS10 cells did not

express a new alteration of AR sequence in comparisonto LNCaP (data not shown). Next, we examined thelevel of AR expression and subcellular localization inLNCaP-CS10 cells and the effect from bicalutamide. ARprotein expression in LNCaP-CS10 was similar to thatin LNCaP (Fig. 1E). The bicalutamide treatment did notaffect the total or cytoplasmic AR protein level in eitherLNCaP or LNCaP-CS10 cells (Fig. 1E, data not shown).On the other hand, the level of nuclear AR in theLNCaP-CS10 cells was increased by bicalutamide butnot in the LNCaP cells (Fig. 1E).

To examine the involvement of the AR signal on theandrogen independence of LNCaP-CS10 cells, AR wassuppressed with AR-specific siRNAs. The knockdowneffect of these AR siRNAs was confirmed by measuringAR protein levels with ELISA (Fig. 2A,B). The siRNAsinhibited the growth and PSA production of LNCaPcells in the 10% FCS medium (Fig. 2C,E). And, in theandrogen-depleted medium, the AR siRNAs alsoinhibited the growth and PSA production of LNCaP-CS10 cells (Fig. 2D,F).

We examined the phosphorylation status of AKTand ERK and the protein level of IL-6Ra and performeda comparative genomic hybridization analysis andgene-expression profiling of in vitro cultured LNCaP-CS10. No apparent differences between LNCaP-CS10and LNCaP were observed for pAKT and pERK proteinlevels or copy number changes in PTEN genes, butoverexpression of the IL-6Ra gene and protein wasfound in LNCaP-CS10 (data not shown). To investigatewhether IL-6 axis activation plays a role in themechanism of outlaw activation in LNCaP-CS10, weanalyzed the effect of anti-IL-6 neutralizing antibodyand anti-IL-6Ra neutralizing antibody on the growth ofLNCaP-CS10 in the absence of androgen. We foundthat the cell growth of LNCaP-CS10 was not affectedby anti-IL-6 antibody or anti-IL-6Ra antibody in theabsence of androgen (data not shown).

Lastly, to analyze the expression of downstreamfactors for AR signaling in LNCaP-CS10 cells, the levelsof gene expression of cyclin-dependent kinase (CDK)-1, CDK-2, CDK-4, cyclin A2, and cyclin B1 wereexamined. No apparent differences between LNCaP-CS10 and LNCaP were found for these factors (data notshown).

Tumorigenicity andBicalutamide Resistance ofLNCaP-CS10Cells in Castrated SCIDMice

In order to confirm the outlaw mechanism ofLNCaP-CS10 in vivo, we inoculated castrated andnon-castrated SCID mice with LNCaP-CS10 and theparental LNCaP cells. In the LNCaP-CS10-inoculatedmice, palpable tumors were detected in 90% (9/10) ofnon-castrated mice and 90% (9/10) of castrated mice

The Prostate

Prostate CancerWithActivatedOutlawPathway 459

The Prostate

Fig. 1. Effects of androgen andbicalutamide ongrowth,PSAproduction, nuclear localization ofAR in LNCaP-CS10 cells.A: Cellgrowth ofLNCaPandLNCaP-CS10cellswasanalyzedin thepresenceofR1881after9daysof treatment.B: IntracellularPSAlevelsofLNCaPandLNCaP-CS10 cells were measured in the presence of R1881. C: Cell growth of LNCaP and LNCaP-CS10 cells was analyzed in the presence ofbicalutamide after 9days of treatment.D: Intracellular PSAlevels of LNCaP andLNCaP-CS10 cellsweremeasuredin thepresence ofbicaluta-mide.Alldata are expressed as themeanþ SDof triplicate determinations.E:Nuclear localization andexpressionofARwere determinedbyWesternblottingofARagainstnuclearandtotalfractions,respectively,preparedfromLNCaPandLNCaP-CS10cellsinthepresenceorabsenceofbicalutamide.

460 Ishikura et al.

The Prostate

Fig. 2. EffectsofARsiRNAonandrogen-independentgrowthofLNCaP-CS10cells.A:LNCaPcellsweretransientlytransfectedwithtwoARsiRNAsor scramble siRNA.IntracellularARproteinlevelsweredeterminedbyELISAandareexpressedas thepercentageof thecontrol (non-siRNA).B:LNCaP-CS10cellswere transiently transfectedwithtwoARsiRNAsor scramblesiRNA.IntracellularARproteinlevelsweredeter-minedby ELISA and are expressed as the percentage of the control (non-siRNA).C: LNCaP cells were transiently transfectedwith two ARsiRNAsor scramble siRNA.Cell countondays0 and6 after transfectionwasmeasuredandgrowthis expressedas thepercentage ofday0.D:LNCaP-CS10 cells were transiently transfected with two AR siRNAs or scramble siRNA.Cell count on days 0 and 6 after transfectionwasmeasured andgrowth is expressed as thepercentage of day 0.E: LNCaPcellswere transiently transfectedwith twoAR siRNAs or scramblesiRNA.IntracellularPSAlevelsweredeterminedbyELISA.F:LNCaP-CS10cellswere transiently transfectedwithtwoARsiRNAsor scramblesiRNA.IntracellularPSAlevelsweredeterminedbyELISA.Alldata areexpressedas themean� SDof the triplicatedeterminations.

29 days after tumor inoculation (Fig. 3B). Incontrast, palpable LNCaP tumors were detected in80% (8/10) of non-castrated mice and no tumorswere found in castrated mice 29 days after tumorinoculation (Fig. 3A). In addition, the growth rate ofLNCaP-CS10 tumors was similar in both castrated andnon-castrated mice, whereas the growth rate of LNCaPwas lower in castrated mice than in non-castrated mice(Fig. 3C,D).

To confirm the bicalutamide resistance of LNCaP-CS10 cells in vivo, we administered bicalutamide to theLNCaP-CS10 xenograft model in castrated SCID mice.The tumor growth of LNCaP-CS10 was not inhibited by10 or 100 mg/kg bicalutamide in the castrated mice(Fig. 4B). The serum PSA level in LNCaP-CS10 tumorswas slightly increased by the administration of 10 or100 mg/kg bicalutamide (Fig. 4D). On the other hand,in non-castrated mice, the growth and the serum PSAlevel of parental LNCaP were inhibited to a castrationlevel by the administration of 10 or 100 mg/kgbicalutamide (Fig. 4A,C).

DISCUSSION

We have established an ‘‘outlaw’’ cell line LNCaP-CS10 that is androgen independent and AR dependentfrom androgen-dependent LNCaP cells after a 4-monthincubation in an androgen-depleted medium supple-mented with bicalutamide. The androgen independ-ence of LNCaP-CS10 was confirmed by its growthunder androgen-depleted conditions. The parentLNCaP cell line showed no growth under the sameconditions (Fig. 1A). Furthermore, LNCaP-CS10 pro-duced PSA without androgen (Fig. 1B), indicating thatthe AR signal was still active.

According to recent studies, the AR signal plays apivotal role in a large segment of hormone-refractoryprostate cancers [4–6]. The mechanisms of hormonerefractoriness include AR overexpression, AR muta-tion, and ligand-independent activation of AR coinedthe outlaw pathway [7]. To understand the mechanismfor hormone refractoriness of LNCaP-CS10, we ana-lyzed the level of AR expression and the sequence of

The Prostate

Fig. 3. Tumorigenicity of LNCaP-CS10 in non-castrated and castrated SCIDmice.A: LNCaP cells were inoculated into non-castrated andcastratedmaleSCIDmice(n¼10/group)andthemicewithpalpable tumors (>50mm3)werescored.B:LNCaP-CS10cellswereinoculatedintonon-castrated and castrated male SCID mice (n¼10/group) and the mice with palpable tumors (>50mm3) were scored.C: LNCaP cellswere inoculated into non-castrated and castratedmale SCIDmice (n¼10/group) and the tumorsweremeasured.D: LNCaP-CS10 cellswereinoculatedintonon-castratedandcastratedmale SCIDmice (n¼10/group) and the tumorsweremeasured.Alldata represent themean� SE(C,D).

462 Ishikura et al.

AR cDNA. LNCaP-CS10 did not show overexpressionof AR or a new alteration of the AR sequence incomparison to LNCaP (Fig. 1E, data not shown).However, the experiments with AR siRNAs revealedoutlaw activation that stimulated the growth and PSAproduction of LNCaP-CS10 cells (Fig. 2D,F).

Our tumorigenicity study results demonstrate thatLNCaP-CS10 cells formed palpable tumors not only innon-castrated SCID mice but also in castrated SCIDmice. The high tumor take rate and the ability to grow incastrated mice indicate that the LNCaP-CS10 cell line issuitable for evaluating new therapeutics for hormone-refractory prostate cancer (Fig. 3B,D).

To determine whether LNCaP-CS10 cells showbicalutamide resistance in vivo, we inoculated cas-trated SCID mice with LNCaP-CS10 cells. Growth and

serum PSA level of LNCaP-CS10 tumors were notinhibited by 10 or 100 mg/kg bicalutamide, whereastumor growth and the serum PSA level in LNCaPxenografts were suppressed by 10 or 100 mg/kgbicalutamide (Fig. 4). We consider the reason theLNCaP-CS10 tumor growth-inducing (agonist) activityof bicalutamide detected in the in vitro assay was notdetected in this xenograft model to be the low level ofadrenal gland-derived androgen remaining in thecastrated mice. From these lines of evidence, LNCaP-CS10 was designated an ‘‘outlaw’’ hormone-refractoryprostate cancer cell line.

To investigate the mechanism of the resistance ofLNCaP-CS10, we analyzed the effects of bicalutamideon growth and PSA production. To our surprise,bicalutamide induced the growth and PSA production

The Prostate

Fig. 4. EffectofbicalutamideongrowthofLNCaP-CS10xenografttumorsandserumPSAlevelsincastratedmaleSCIDmice.A:LNCaPcellswere inoculated into non-castratedmale SCIDmice.The tumor-bearingmicewere orally administeredbicalutamide (0,10, and100mg/kg) orcastrated(n¼ 5/group)withadministrationof thedrug for fivecyclesof5daysonand2daysoff.Tumor sizewasmeasured.B:LNCaP-CS10cellswere inoculated into castratedmale SCIDmice.The tumor-bearingmicewere orally administeredbicalutamide (0,10, and100mg/kg, n¼ 5/group) with administration for four cycles of 5 days on and 2 days off.Tumor size was measured.C: LNCaP cells were inoculated into non-castratedmaleSCIDmice.Thetumor-bearingmicewereorallyadministeredbicalutamide(0,10,and100mg/kg)orcastrated(n¼ 5/group)withadministrationof thedrug for fivecyclesof5daysonand2daysoff.SerumPSAlevelsweremeasured.D:LNCaP-CS10cellswereinoculatedintocastratedmaleSCIDmice.The tumor-bearingmicewere orallyadministeredbicalutamide (0,10, and100mg/kg,n¼ 5/group)withadministra-tion for fourcyclesof5daysonand2daysoff. SerumPSAlevelsweremeasured.Alldatarepresent themean� SE.

Prostate CancerWithActivatedOutlawPathway 463

of LNCaP-CS10 (Fig. 1C,D). The results indicate thatbicalutamide acted as an agonist in LNCaP-CS10 cellsbut not in LNCaP cells. And induction of nuclear AR bybicalutamide was more notable in LNCaP-CS10 cellsthan in LNCaP cells (Fig. 1E), although the total ARprotein level was not increased in the cells. Bicaluta-mide is reported to translocate the transcriptionallyinactive AR to the nuclei of LNCaP cells [26,27]. But, thefact that bicalutamide acted as an agonist on growthand PSA production in LNCaP-CS10 suggests that theinduction activity of bicalutamide on AR nucleartranslocation contributed to the AR agonist activity ofbicalutamide in LNCaP-CS10.

Various mechanisms of hormone refractoriness inoutlaw tumors have been suggested such as activationof AR by deregulated cytokines and growth factors,including IL-6, IGF-1, EGF, KGF, through the JAK–STAT, Akt, MAPK pathways in the absence ofandrogen [20,28–32]. To investigate the mechanism ofoutlaw activation in LNCaP-CS10, we first analyzedAKT and ERK status but found no apparent differencebetween LNCaP-CS10 and LNCaP for pAKT or pERK(data not shown). Next, our comparative genomichybridization analysis did not reveal any of the copynumber changes in the PTEN gene known to beinvolved in the AKT pathway regulator and frequentlydeleted in prostate cancer cells [33,34]. In addition,gene-expression profiling and Western blottingshowed an overexpression of the IL-6Ra gene andprotein in LNCaP-CS10. But the cell proliferation assayrevealed that the anti-IL-6 neutralizing antibody andanti-IL-6Ra neutralizing antibody did not affect thegrowth of LNCaP-CS10 in the absence of androgen,indicating that IL-6 axis activation is not a mechanismof the outlaw activation of LNCaP-CS10 (data notshown).

Lastly, we investigated the downstream of ARsignaling in LNCaP-CS10 cells. The mitotic signalingof the AR is thought to ultimately target the cell-cyclemachinery [35]. Androgen stimulates the expression ofthe cell-cycle genes, CDK-1, CDK-2, and CDK-4, andincreases the levels of cyclin A and cyclin B1 mRNAs[36,37]. Therefore, we analyzed the expression ofandrogen-regulated CDKs and cyclins using gene chipanalysis under an androgen-depleted condition butfound no apparent differences between LNCaP-CS10and LNCaP for CDK-1, CDK-2, CDK-4, cyclin A2, andcyclin B1 (data not shown). Further study is necessaryto elucidate the exact mechanism of the refractorinessof LNCaP-CS10.

LNCaP-CS10 showed characteristics different fromother bicalutamide-resistant cell lines previouslyreported. LNCaP-abl, which was derived fromLNCaP and established after long-term culture in anandrogen-depleted medium, showed AR overexpres-

sion and did not have a newly altered AR sequence[23]. And LNCaP-abl cells showed hypersensitivity toandrogen and induction by bicalutamide on cellgrowth. LNCaP-CDXRs established in an androgen-depleted medium with 5 mM bicalutamide showedAR overexpression [24]. And LNCaP-CDXR cellgrowth was unaffected by bicalutamide but wasrepressed by androgen. LNCaP-BICs, established inan androgen-depleted medium with 10 pM R1881 and1 mM bicalutamide for 3 months, did not show ARoverexpression or alteration of the AR sequence andits cell growth was unaffected by bicalutamide orR1881. Thus, LNCaP-BICs were designated as AR by-pass models [38]. On the other hand, LNCaP-CS10did not show AR overexpression but cell growth wasinduced by both bicalutamide and R1881. KUCaPwas established by transplanting liver metastatictissue into male SCID mice [15]. Tissue samples wereobtained from a patient who was treated with totalandrogen blockade using bicalutamide. This cell linehas W741C mutation in the ligand-binding domain ofAR. LNCaP-cxDs established in an androgen-depleted medium with 1 or 0.1 mM bicalutamide haveW741C or W741L mutation of AR in addition to theT877A mutation [25]. In KUCaP and LNCaP-cxDsmodels, bicalutamide acts as an agonist as a result ofthe mutations. On the other hand, LNCaP-CS10 doesnot have these new mutations.

In conclusion, we established a bicalutamide-resist-ant cell line, LNCaP-CS10, with outlaw pathwayactivation and is applicable to xenograft experiments.LNCaP-CS10 is useful for the elucidation of the outlawpathway mechanisms of hormone refractoriness andthe evaluation of the efficacy of new therapeutics forhormone-refractory prostate cancer.

ACKNOWLEDGMENTS

The authors thank Mr. Kazutaka Tachibana for thepurification of bicalutamide from Casodex1 tablets,Dr. Hiroshi Sakamoto for his critical reading of themanuscript.

REFERENCES

1. Jemal A, Center MM, Ward E, Thun MJ. Cancer occurrence.Methods Mol Biol 2009;471:3–29.

2. Taplin ME, Balk SP. Androgen receptor: A key molecule in theprogression of prostate cancer to hormone independence. J CellBiochem 2004;91 (3):483–490.

3. Arai Y, Akaza H, Deguchi T, Fujisawa M, Hayashi M, Hirao Y,Kanetake H, Naito S, Namiki M, Tachibana M, Usami M, OhashiY. Evaluation of quality of life in patients with previouslyuntreated advanced prostate cancer receiving maximum andro-gen blockade therapy or LHRHa monotherapy: A multicenter,randomized, double-blind, comparative study. J Cancer Res ClinOncol 2008;134 (12):1385–1396.

The Prostate

464 Ishikura et al.

4. Roudier MP, True LD, Higano CS, Vesselle H, Ellis W, Lange P,Vessella RL. Phenotypic heterogeneity of end-stage prostatecarcinoma metastatic to bone. Hum Pathol 2003;34 (7):646–653.

5. Shah RB, Mehra R, Chinnaiyan AM, Shen R, Ghosh D, Zhou M,Macvicar GR, Varambally S, Harwood J, Bismar TA, Kim R,Rubin MA, Pienta KJ. Androgen-independent prostate cancer isa heterogeneous group of diseases: Lessons from a rapid autopsyprogram. Cancer Res 2004;64 (24):9209–9216.

6. Gregory CW, He B, Johnson RT, Ford OH, Mohler JL, French FS,Wilson EM. A mechanism for androgen receptor-mediatedprostate cancer recurrence after androgen deprivation therapy.Cancer Res 2001;61 (11):4315–4319.

7. Pienta KJ, Bradley D. Mechanisms underlying the developmentof androgen-independent prostate cancer. Clin Cancer Res2006;12 (6):1665–1671.

8. Visakorpi T, Hyytinen E, Koivisto P, Tanner M, Keinanen R,Palmberg C, Palotie A, Tammela T, Isola J, Kallioniemi OP. Invivo amplification of the androgen receptor gene and progres-sion of human prostate cancer. Nat Genet 1995;9 (4):401–406.

9. Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL,Visakorpi T. Amplification and overexpression of androgenreceptor gene in hormone-refractory prostate cancer. Cancer Res2001;61 (9):3550–3555.

10. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R,Rosenfeld MG, Sawyers CL. Molecular determinants of resist-ance to antiandrogen therapy. Nat Med 2004;10 (1):33–39.

11. Zhao XY, Malloy PJ, Krishnan AV, Swami S, Navone NM, PeehlDM, Feldman D. Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutatedandrogen receptor. Nat Med 2000;6 (6):703–706.

12. Shi XB, Ma AH, Xia L, Kung HJ, de Vere White RW. Functionalanalysis of 44 mutant androgen receptors from human prostatecancer. Cancer Res 2002;62 (5):1496–1502.

13. Taplin ME, Rajeshkumar B, Halabi S, Werner CP, Woda BA,Picus J, Stadler W, Hayes DF, Kantoff PW, Vogelzang NJ, SmallEJ. Androgen receptor mutations in androgen-independentprostate cancer: Cancer and Leukemia Group B Study 9663.J Clin Oncol 2003;21 (14):2673–2678.

14. Hara T, Nakamura K, Araki H, Kusaka M, Yamaoka M.Enhanced androgen receptor signaling correlates with theandrogen-refractory growth in a newly established MDA PCa2b-hr human prostate cancer cell subline. Cancer Res 2003;63(17):5622–5628.

15. Yoshida T, Kinoshita H, Segawa T, Nakamura E, Inoue T,Shimizu Y, Kamoto T, Ogawa O. Antiandrogen bicalutamidepromotes tumor growth in a novel androgen-dependentprostate cancer xenograft model derived from a bicalutamide-treated patient. Cancer Res 2005;65 (21):9611–9616.

16. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J,Hittmair A, Bartsch G, Klocker H. Androgen receptor activationin prostatic tumor cell lines by insulin-like growth factor-I,keratinocyte growth factor, and epidermal growth factor. CancerRes 1994;54 (20):5474–5478.

17. Gioeli D, Ficarro SB, Kwiek JJ, Aaronson D, Hancock M, CatlingAD, White FM, Christian RE, Settlage RE, Shabanowitz J, HuntDF, Weber MJ. Androgen receptor phosphorylation. Regulationand identification of the phosphorylation sites. J Biol Chem2002;277 (32):29304–29314.

18. Ueda T, Mawji NR, Bruchovsky N, Sadar MD. Ligand-independent activation of the androgen receptor by interleu-kin-6 and the role of steroid receptor coactivator-1 in prostatecancer cells. J Biol Chem 2002;277 (41):38087–38094.

19. Debes JD, Schmidt LJ, Huang H, Tindall DJ. p300 mediatesandrogen-independent transactivation of the androgen receptorby interleukin 6. Cancer Res 2002;62 (20):5632–5636.

20. Gregory CW, Whang YE, McCall W, Fei X, Liu Y, Ponguta LA,French FS, Wilson EM, Earp HS III. Heregulin-inducedactivation of HER2 and HER3 increases androgen receptortransactivation and CWR-R1 human recurrent prostate cancercell growth. Clin Cancer Res 2005;11 (5):1704–1712.

21. Culig Z, Steiner H, Bartsch G, Hobisch A. Interleukin-6regulation of prostate cancer cell growth. J Cell Biochem 2005;95 (3):497–505.

22. Ponguta LA, Gregory CW, French FS, Wilson EM. Site-specificandrogen receptor serine phosphorylation linked to epidermalgrowth factor-dependent growth of castration-recurrent pros-tate cancer. J Biol Chem 2008;283 (30):20989–21001.

23. Culig Z, Hoffmann J, Erdel M, Eder IE, Hobisch A, Hittmair A,Bartsch G, Utermann G, Schneider MR, Parczyk K, Klocker H.Switch from antagonist to agonist of the androgen receptorbicalutamide is associated with prostate tumour progression in anew model system. Br J Cancer 1999;81 (2):242–251.

24. Kokontis JM, Hsu S, Chuu CP, Dang M, Fukuchi J, Hiipakka RA,Liao S. Role of androgen receptor in the progression of humanprostate tumor cells to androgen independence and insensitiv-ity. Prostate 2005;65 (4):287–298.

25. Hara T, Miyazaki J, Araki H, Yamaoka M, Kanzaki N, Kusaka M,Miyamoto M. Novel mutations of androgen receptor: A possiblemechanism of bicalutamide withdrawal syndrome. Cancer Res2003;63 (1):149–153.

26. Tomura A, Goto K, Morinaga H, Nomura M, Okabe T, Yanase T,Takayanagi R, Nawata H. The subnuclear three-dimensionalimage analysis of androgen receptor fused to green fluorescenceprotein. J Biol Chem 2001;276 (30):28395–28401.

27. Masiello D, Cheng S, Bubley GJ, Lu ML, Balk SP. Bicalutamidefunctions as an androgen receptor antagonist by assembly of atranscriptionally inactive receptor. J Biol Chem 2002;277 (29):26321–26326.

28. Chen T, Wang LH, Farrar WL. Interleukin 6 activates androgenreceptor-mediated gene expression through a signal transducerand activator of transcription 3-dependent pathway in LNCaPprostate cancer cells. Cancer Res 2000;60 (8):2132–2135.

29. Lee SO, Lou W, Hou M, de Miguel F, Gerber L, Gao AC.Interleukin-6 promotes androgen-independent growth inLNCaP human prostate cancer cells. Clin Cancer Res 2003;9(1):370–376.

30. Wallner L, Dai J, Escara-Wilke J, Zhang J, Yao Z, Lu Y, Trikha M,Nemeth JA, Zaki MH, Keller ET. Inhibition of interleukin-6 withCNTO328, an anti-interleukin-6 monoclonal antibody, inhibitsconversion of androgen-dependent prostate cancer to anandrogen-independent phenotype in orchiectomized mice.Cancer Res 2006;66 (6):3087–3095.

31. Malinowska K, Neuwirt H, Cavarretta IT, Bektic J, Steiner H,Dietrich H, Moser PL, Fuchs D, Hobisch A, Culig Z. Interleukin-6stimulation of growth of prostate cancer in vitro and in vivothrough activation of the androgen receptor. Endocr RelatCancer 2009;16 (1):155–169.

32. Wen Y, Hu MC, Makino K, Spohn B, Bartholomeusz G, Yan DH,Hung MC. HER-2/neu promotes androgen-independentsurvival and growth of prostate cancer cells through the Aktpathway. Cancer Res 2000;60 (24):6841–6845.

33. Suzuki H, Freije D, Nusskern DR, Okami K, Cairns P, SidranskyD, Isaacs WB, Bova GS. Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancertissues. Cancer Res 1998;58 (2):204–209.

The Prostate

Prostate CancerWithActivatedOutlawPathway 465

34. Bertram J, Peacock JW, Fazli L, Mui AL, Chung SW, Cox ME,Monia B, Gleave ME, Ong CJ. Loss of PTEN is associated withprogression to androgen independence. Prostate 2006;66 (9):895–902.

35. Yamamoto A, Hashimoto Y, Kohri K, Ogata E, Kato S, Ikeda K,Nakanishi M. Cyclin E as a coactivator of the androgen receptor. JCell Biol 2000;150 (4):873–880.

36. Lu S, Tsai SY, Tsai MJ. Regulation of androgen-dependentprostatic cancer cell growth: Androgen regulation of CDK2,CDK4, and CKI p16 genes. Cancer Res 1997;57 (20):4511–4516.

37. Gregory CW, Johnson RT Jr, Presnell SC, Mohler JL, French FS.Androgen receptor regulation of G1 cyclin and cyclin-depend-ent kinase function in the CWR22 human prostate cancerxenograft. J Androl 2001;22 (4):537–548.

38. Hobisch A, Fritzer A, Comuzzi B, Fiechtl M, Malinowska K,Steiner H, Bartsch G, Culig Z. The androgen receptor pathwayis by-passed in prostate cancer cells generated afterprolonged treatment with bicalutamide. Prostate 2006;66 (4):413–420.

The Prostate

466 Ishikura et al.