Exploiting AR-Regulated Drug Transport to Induce Sensitivity to … · Cell Death and Survival...

Cell Death and Survival

Exploiting AR-Regulated Drug Transport toInduce Sensitivity to the Survivin Inhibitor YM155Michael D. Nyquist1, Alexandra Corella1, John Burns2, Ilsa Coleman1, Shuai Gao3,4,Robin Tharakan1, Luke Riggan1, Changmeng Cai3,4, Eva Corey5, Peter S. Nelson1,5,6,and Elahe A. Mostaghel6,7

Abstract

Androgen receptor (AR) signaling is fundamental to prostatecancer and is the dominant therapeutic target in metastaticdisease. However, stringent androgen deprivation therapy regi-mens decrease quality of life and have been largely unsuccessfulin curtailing mortality. Recent clinical and preclinical studieshave taken advantage of the dichotomous ability of AR signal-ing to elicit growth-suppressive and differentiating effects byadministering hyperphysiologic levels of testosterone. In thisstudy, high-throughput drug screening identified a potent syn-ergy between high-androgen therapy and YM155, a transcrip-tional inhibitor of survivin (BIRC5). This interaction was medi-ated by the direct transcriptional upregulation of the YM155transporter SLC35F2 by the AR. Androgen-mediated YM155-

induced cell death was completely blocked by the overexpres-sion of multidrug resistance transporter ABCB1. SLC35F2expression was significantly correlated with intratumor andro-gen levels in four distinct patient-derived xenograft models, andwith AR activity score in a large gene expression dataset ofcastration-resistant metastases. A subset of tumors had signif-icantly elevated SLC35F2 expression and, therefore, may iden-tify patients who are highly responsive to YM155 treatment.

Implications: The combination of androgen therapy withYM155 represents a novel drug synergy, and SLC35F2 mayserve as a clinical biomarker of response to YM155. Mol Cancer Res;15(5); 521–31. �2017 AACR.

IntroductionProstate cancer is the most frequently diagnosed cancer in men

and the second leading cause of male cancer mortality (1). Theandrogen receptor (AR) is a master regulator of prostate devel-opment and the maintenance of prostate epithelial cell viabilityand secretory activity. The AR-directed transcriptional programalso serves to maintain the survival of prostate cancer and is thefocal point for therapeutic strategies in localized diseasewhere it iscombined with radiation treatment, and for advanced diseasewhere AR pathway suppression remains first-line therapy. Thephysiologic role of the AR changes as prostate cells undergotumorigenesis and progression (2). Notably, as AR signalingpromotes cellular differentiation and can suppress proliferation,

early oncogenic events, including MYC gain, loss of tumor sup-pressors such as PTEN and TP53, and ERG overexpression, allowtransforming cells to uncouple the suppressive functions of ARsignalingwhile benefiting from growth andmetabolic advantages(3–5).

Although suppressing AR activity through ligand reduction inthe form of androgen deprivation therapy (ADT) is initiallyeffective inmetastatic prostate cancer, disease progression, termedcastration-resistant prostate cancer (CRPC), inevitably manifestsafter 2 to 3 years. This occurs due to the reactivation of AR throughAR amplification, ARmutations, intratumoral synthesis of andro-gens, and the production of AR splice variants (6). The relation-ship between AR and prostate cancer further evolves upon pro-gression to CRPC through genomic (7), cistromic (8), and tran-scriptional (9) alterations. Because of the maintenance of ARactivity, survival benefits are achieved by retargeting AR signalingwith next-generation AR-directed therapeutics, althoughresponses are generally measured in months rather than years(10, 11). Although resistance is again often accompanied bypersistent AR activity, prolonged and effective AR suppressioncan induce epithelial-to-mesenchymal transition (12–14), acqui-sition of stem-like cell characteristics (13, 15–17), and transdif-ferentiation into anAR-null neuroendocrine phenotype. There arealso significant complications and quality-of-life issues that arisewith long-term ADT (18, 19). For these reasons, there has been alongstanding interest in the discovery of therapeutic modalitiesthat may acutely synergize with AR-directed therapy to improvelong-term outcomes in men with advanced prostate cancer.

AR activity can be dichotomous in action by promoting pros-tate cancer growth under normal circumstances and retardingprostate cancer growth when overstimulated with excessive

1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle,Washington. 2Virginia Mason Medical Center, Seattle, Washington. 3Center forPersonalized Cancer Therapy, University of Massachusetts Boston, Boston,Massachusetts. 4Hematology-Oncology Division, Beth Israel Deaconess MedicalCenter, Harvard Medical School, Boston, Massachusetts. 5Department of Urol-ogy, University of Washington, Seattle, Washington. 6Division of Oncology,Department of Medicine, University of Washington, Seattle, Washington. 7Clin-ical Research Division, Fred Hutchinson Cancer Research Center, Seattle,Washington.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Elahe A. Mostaghel, University of Washington, 1100Fairview Avenue N, P.O. Box 19024, Seattle, WA 98109. Phone: 206-667-2746;Fax: 206-667-2917; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0315-T

�2017 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 521

on March 9, 2021. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 24, 2017; DOI: 10.1158/1541-7786.MCR-16-0315-T

http://mcr.aacrjournals.org/

androgens. AR signaling induces cell-cycle arrest, in part, byupregulating negative regulators of the cell cycle (20, 21). AR isalso a licensing factor for DNA replication, and excessive andro-gens prevent AR cycling and relicensing, resulting in cell-cycleblock (22, 23). In addition, AR can recruit TOP2B to sites of activetranscription, resulting in double-strand DNA breaks (24).

The observation that prostate cancer cells can adjust to toomuch or too little AR signaling over time has led to the develop-ment of an approach termed bipolar androgen therapy thatrapidly cycles androgen levels to maximize the suppressive ben-efits of ADT while attempting to prevent prostate cancers fromadapting to a static low androgen environment (25, 26). Thisapproach has been evaluated in phase I and II clinical trials (27,28). Furthermore, the use of sustained high-dose androgens orbipolar therapy has also been evaluated to exploit potentialsynergies with other therapeutics such as ionizing radiation(29). In this study, we sought to determine whether thegrowth-suppressive effects of high-dose androgen, or other cel-lular activities regulated through AR, would synergize with anti-cancer drugs to further inhibit proliferation or induce prostatecancer cell death.

Materials and MethodsCell culture and drug screen

The LNCaP cell line (ATCC, CRL-1740) was cultured inRPMI1640, no phenol red (GIBCO, cat. no. 11835030) with10% FBS (GIBCO, cat. no. 10437-02). VCaPs (ATCC, CRL-2876) were cultured in DMEM/F-12, no phenol red (GIBCO, cat.no. 21041025) with 10% FBS. Cell lines were lineage and myco-plasma validated by DDC (DNA Diagnostic Center) Medical.Cells were cultured for no more than 20 passages from thevalidated stocks. Drug screening was carried out at Quellos HighThroughput Screening Core (University of Washington, Seattle,WA). The epigenetics, apoptosis, and stem cell modifier screeninglibraries were obtained from SelleckChem. LNCaPswere plated in384-well plates at 2,500 cells per well in 50 mL of complete media(10% FBS) with either 10 mmol/L enzalutamide, 10 nmol/LR1881, or DMSO control using a PerkinElmerWellmate Dispens-er. They were incubated at 37�C in 5% CO2 overnight. The nextday, the compound libraries were added using a CyBio CyBi-WellVario outfitted with tip washing stations and a 384-headequippedwith apin tool using50-nL slottedpins (V&PScientific).Fifty nanoliters of compounds dissolved in 100%DMSO(1,000�concentrated) were adsorbed onto the pins and washed off intocell assaywells containing 50 mL of completemedia (0.1%DMSOfinal). Plates were then incubated for 72 to 75 hours at 37�C, 5%CO2. Cells were harvested with CellTiter-Glo (Promega, cat. no.G7572) according to the manufacturer's protocol and read withPerkinElmer Envision Multi-label Plate Reader outfitted with aplate stacker and ultrasensitive luminescence detection. Viabilitysignalwas blank subtracted andnormalized toDMSOcontrol andplotted using Microsoft Excel and Tibco Spotfire.

Isobolographic analyses and dose–response curvesLNCaP or VCaP cells were plated into 96-well plates at 5,000

cells/well in complete growth media (10% FBS). The next day, aone-fourth dose dilution series of YM155 was added in 25 mLcomplete media following a one-fifth R1881 dose dilution seriesor a one-third enzalutamide dose dilution series also in 25 mL incomplete growth media. Cells were incubated for 5 days at 37�C,5%CO2, then harvested with 30 mL/well CellTiter-Glo (Promega,

cat. no. G7572). LNCAP and VCAP cells were plated as describedfor isobolographic analysis except either 10 nmol/L R1881 or 2.5nmol/L testosterone was added and cultured for 5 days unlessotherwise noted. IC50 valueswere generatedwithGraphPadPrism6 and evaluated with the extra sum of squares F-test applying a Pvalue of 0.05 (n ¼ 4).

Western blotsWestern blots were run on NuPAGE 4% to 12% Bis-tris gels

(Thermo Fisher Scientific, cat. no. NP0321) with MOPS SDSbuffer (Thermo Fisher Scientific, cat. no. NP0001) then trans-ferred to PVDF membranes (Thermo Fisher Scientific, cat. no.LC2002) for SLC35F2 or nitrocellulose membranes (ThermoFisher Scientific, cat. no. LC2000) for other proteinswithNuPAGEtransfer buffer (Thermo Fisher Scientific, cat. no. NP0006). Mem-branes were blocked in TBS with 0.1% Tween-20, 5% milk, and2.5% BSA. The antibodies used were SLC35F2 (Proteintech, cat.no. 25526-1-AP), GAPDH (Cell Signaling Technology, cat. no.2118), ATG5 (Cell Signaling Technology, cat. no. 8540), Beclin(Cell Signaling Technology, cat. no. 3495), gH2AX (Cell SignalingTechnology, cat. no. 9718), PARP (Cell Signaling Technology, cat.no. 9542), BIRC5 (Abcam, cat. no. ab76424), and caspase 3 (CellSignaling Technology, cat. no. 9662).

qRT-PCR and RNA-seqGene expression microarray data were analyzed as described

previously (30, 31). Expression across primary prostate tumors (n¼ 11; ref. 32) and CRPC tumor samples (n ¼ 171; ref. 30) wasplotted in GraphPad Prism version 7.00 (GraphPad Software)using normalized, log2-transformedmicroarray signal intensities.The AR activity score was determined by the expression of a 20-gene signature (33) and calculated as described previously (34).Briefly, the activity score is defined as the sum of the expression Z-scores converted to a percent. Pearson correlation coefficient of ARactivity and SLC35F2 expression (normalized, log2 signal inten-sity) in patient CRPC tumors (n¼ 171)was assessed using the cor.test function in R. qRT-PCR was performed Power Sybr Green(Applied Biosystems, cat. no. 4367659) and run on a Bio-RadCFX384 real-time system according to the manufacturer's recom-mendation. Primers used were RPL13A_F-CCTGGAGGAGAA-GAGGAAAGAGA, Hs_RPL13A_R-TTGAGGACCTCTGTGTATTT-GTCAA, Arv567es_F TGCTGGACACGACAACAA, Arv567es_RGCAGCTCTCTCGCAATCA SLC35F2_F-AGGCAAACTCTTCAC-CTGGAAT, and SLC35F2_R-TCTGAAGCATGGGGGTGTTC.

ChIP-seq and ChIP-qPCRPreviously published chromatin immunoprecipitation

sequencing (ChIP-seq) data for AR were obtained from thefollowingGene-ExpressionOmnibus andSequenceReadArchive:LNCaP-1F5 vehicle reps 1-3 (GSM973815-GSM973817), LNCaP-1F5 DHT reps 1-3 (GSM973818-GSM973820; ref. 35); LNCaPethanol (GSM353643), LNCaP R1881 (GSM353644; ref. 36);LNCaP vehicle (GSM696839), LNCaP R1881-stimulated(GSM696840; ref. 37). Raw reads were aligned to hg19 withbowtie (38). Reads that had more than a single alignment weresuppressed. Data were visualized with IGV (39).

AR-directed ChIP-qPCRwas performed as described previously(40). Briefly, LNCaP cells in 5%CSSmediumwere pretreatedwithenzalutamide for 2hours and followedby10nmol/L ofDHT for 4hours. ChIP was performed using anti-AR antibody (Santa CruzBiotechnology, 816�). TheqPCRanalysiswas carriedout using the

Nyquist et al.

Mol Cancer Res; 15(5) May 2017 Molecular Cancer Research522




SYBR Green method on the QuantStudio 3 Real-Time PCR system(Applied Biosystems). The primers used were SLC35F2_AR1_F:50-AGAGAATCGTCCTTCAGAACC, SLC35F2_AR1_R:50-GGACTGA-GCACAAACAAACC, SLC35F2_AR2_F:50-GGTCACTACCAAATG-AACTGATCATG, SLC35F2_AR2_R:50-AGTAGATAAGAAGGCTGA-CACCTG, SLC35F2_AR3_F:50-GTTGAACTAACAGAGGTTTCAG,SLC35F2_AR3_R:50-GATATGAATCAATACGGGCTGGCAC.

Overexpression and knockdown vectorsThe SLC35F2 ORF was obtained from a previously published

ORF library (41) and expressed using the pLX304 (Addgene,plasmid no. 25890) lentivirus backbone. The ABCB1 expres-sion vector was constructed from pHAMDRwt (Addgene, plas-mid no. 10957) by PCR amplifying out ABCB1 using theprimers ABCB1_F-gccaccATGGATCTTGAAGGGGACCGCAAT-GG and ABCB1_R-TCACTGGCGCTTTGTTCCAGCCTGGACand cloning it into PCR8-GW-TOPO (Thermo Fisher Scientific,cat. no. K250020); then, a gateway reaction was used to cloneinto the pL6.3/V5 lentivirus (Thermo Fisher Scientific,V53306). The SLC35F2 shRNAs were the GIPZ Open Biosys-tems Human shRNAmirs V3LHS_377258 and V2LHS_154477.

LuCaP human prostate cancer xenograftsAnimal studies were carried out in strict accordance with NIH

guidelines and with protocols approved by the Fred Hutch-inson Center and the University of Washington InstitutionalAnimal Care and Use Committees. All surgeries were performedunder isoflurane anesthesia, and all efforts were made tominimize suffering. Five different LuCaP patient-derived xeno-graft (PDX) models established as part of the University ofWashington tissue bank (42, 43) were used (LuCaP 23, LuCaP35, LuCaP 96, LuCaP 86.2, and LuCaP 136). All lines expressthe wild-type AR and secrete PSA. Intact 6- to 8-week-old male

C.B-17 SCID mice (Charles River Laboratories) were implantedsubcutaneously with 30-mm3 tumor pieces. When tumorsreached an average of 100 mm3, a subset of mice were castrated(Cx). Tumor volume was determined by the following formula(long and short axis lengths in mm): long � (short2)/2. Tumorsfrom a subset of mice in each cohort were harvested at days 7and 21 after castration, and the rest of the animals werefollowed and sacrificed until tumors exceeded 1,000 mm3 (endof study) or sacrificed if animals became compromised. Xeno-grafts were harvested and flash frozen for the determination oftissue androgens and extraction of total RNA.

Steroid measurementsMethods for the determination of steroids in tissue samples by

mass spectrometry were as previously reported (44). Briefly,frozen tissues were weighed, added to 60�C water containingdeuterated internal standards, heated to 60�C for 10minutes, andhomogenized using a tissue homogenizer (Precellys; Bertin);supernatant was extracted twice with hexane [ethyl acetate(80:20, v/v)], and the organic layer was dried (SpeedVac; ThermoFisher Scientific), derivatized with 0.025 mol/L hydroxylaminehydrochloride for 24 hours at room temperature to form oximes,and quantified using liquid chromatography electrospray-ioni-zation tandemmass spectrometry. The lower limit of detection forsteroids in tissue was 0.49 pg/sample (0.02 pg/mg) for DHT andtestosterone.

ResultsThe survivin/BIRC5 inhibitor YM155 synergizes with androgentherapy to suppress prostate cancer cell proliferation

The LNCaP prostate cancer cell line grows maximally in 10%FBS containing media, and growth is repressed when androgens

Figure 1.

YM155 synergizes with high-androgentherapy to suppress prostate cancerviability.A,Design of high-throughputscreen in LNCaP grown in 10% FBSmedia supplemented with either 1nmol/L R1881, 10mmol/L enzalutamide(ENZ), or DMSO. B, AverageCellTiter-Glo luciferase signal(viability) for all control wells on drugscreen 96 hours after cell seeding.C and D, Inhibitory concentration for50% viability (IC50) plotted for variousdoses of R1881 (C) and (D)enzalutamide.

AR-Regulated Drug Transport in Prostate Cancer

www.aacrjournals.org Mol Cancer Res; 15(5) May 2017 523




are added (Supplementary Fig. S1). To identify drugs capable ofenhancing the activity of AR inhibition or AR activation, wescreened a library of 145well-characterized pharmacologic agentsthat impair cancer cell proliferation or survival using the andro-gen-sensitive prostate cancer cell line LNCaP (Fig. 1A). Briefly, weplated LNCaP cells in standard growthmedia containing 10%FBSwith either the AR antagonist enzalutamide, the synthetic andro-gen R1881, or DMSO vehicle. After 24 hours, a 14-point range ofconcentrations of each library compound was applied to theplates and incubated for an additional 72 hours, at which point,cell viability was quantitated. Strikingly, in control cultures withno other added drugs, R1881 suppressed cell proliferation aspotently as the enzalutamide treatment (Fig. 1B). No compoundssynergized with enzalutamide under these conditions. However,YM155, which suppresses transcription of the antiapoptotic pro-tein survivin/BIRC5, displayed a supra-additive effect withR1881.

To validate the synergy between R1881 and YM155, wemeasured YM155 IC50 values in response to varying R1881concentrations. These data demonstrated a potent synergisticinteraction between R1881 and YM155 in LNCaP cells withR1881 increasing LNCaP sensitivity to YM155 from IC50 24nmol/L without R1881 to 3.85 nmol/L with an R1881 con-centration of 160 pmol/L (Fig. 1C). Synergy with YM155 wasnot achieved with enzalutamide (Fig. 1D). Surprisingly, theVCaP prostate cancer cell line, which has amplification of AR,was initially more sensitive to YM155 but not further sensitizedto YM155 by the addition of androgen (Fig. 1C).

Themechanism by which YM155 is reported to elicit cell deathis controversial and varies based on cell line, time, and YM155concentration (45). However, YM155 is reported to suppressBIRC5 expression, increase DNA damage, and induce autophagy(45). Given that BIRC5 is involved in DNA repair (46), and DNAdamage can induce autophagy (47), we sought to determine theaction of YM155-mediated cellular effects before significant cellloss and secondary effects occurred. To this end, we performedWestern blots on LNCaP cells preincubated for 48 hours with 10nmol/LR1881or vehicle control and subsequently dosedwith 0.5or 1 mmol/L of YM155 for 24 hours (in contrast to the prolifer-

ation studies carried out at 96 hours). R1881 alone suppressedBIRC5 levels, consistent with androgen-induced G1 cell-cyclearrest (Fig. 2A; ref. 21). YM155 and R1881 coordinately sup-pressed BIRC5 levels (Fig. 2A). Modest DNA damage (gH2AX)was observed at the higher dose of YM155 with R1881 (Fig. 2A).Reductions in gH2AX and cleaved PARP1 were observed in AR-suppressed cells in the control and low-dose YM155 groups,consistent with reduced replicative stress (48) and DNA repairsupportive effects of AR signaling (Fig. 2A; ref. 49). In addition,there was no change in the levels of autophagy (as measured byBeclin and AT5 levels) and negligible changes in apoptosis (asmeasured by cleaved caspase-3 and PARP1) between the YM155and control groups (Fig. 2A).

To discount the possibility that the effects we observed arespecific to R1881, a potent synthetic androgen, we performeda dose–response curve of LNCaP viability to YM155 with aphysiologic dose of testosterone (2.5 nmol/L). The addition oftestosterone also increased LNCaP sensitivity to YM155 (Fig.2B). To determine the time dependence of androgen andYM155 synergy, we preincubated LNCaPs with 10 nmol/LR1881 or vehicle for 24 hours and then determined YM155IC50 values after 24, 48, and 72 hours (Fig. 2C). Both thevehicle and R1881 groups increased sensitivity to YM155 withlonger incubation times, consistent with reported observationsthat efficacy of YM155 increases with time (50). Given thatR1881 potently suppresses BIRC5 without inducing apoptosisand does not induce DNA damage under these experimentalconditions (Fig. 2A), these data suggest the mechanism bywhich androgen therapy sensitizes cells to YM155 is mediatedthrough AR transcriptional activity rather than through geno-toxicity or loss of BIRC5.

The membrane transport protein SLC35F2 is regulated by ARactivation

A recent study demonstrated that YM155 is transported bySLC35F2, a member of the solute carrier group of membranetransport proteins (51). Therefore, one possiblemechanismof theR1881-YM155 synergy is through transcriptional upregulation of

Figure 2.

High-dose androgens suppress BIRC5 levels and increase YM155-mediated DNA damage. A, Western blots for BIRC5 (survivin), gH2AX, Beclin, ATG5,caspase-3, and PARP1 on LNCaP treated for 24 hours with 0.5 or 1 mmol/L YM155 with and without a 48-hour preincubation with 10 nmol/L R1881.B, LNCaP viability (CellTiter-Glo) in response to YM155 with or without 2.5 nmol/L testosterone (T; error bars ¼ SD; n ¼ 4). C, YM155 IC50 values of LNCaPpreincubated 24 hours with 10 nmol/L R1881 or vehicle control then exposed to a dose range of YM155 for 24, 48, and 72 hours (error bars ¼ 95%confidence interval, n ¼ 4).

Nyquist et al.





SLC35F2 by AR. To establish a transcriptional relationship, weperformed qRT-PCR to measure SLC35F2 transcript levels inLNCaP and VCaP cells cultured with 10 nmol/L R1881 or ethanolvehicle for 48 hours. In cells treated with 10 nmol/L R1881,transcript levelswere approximately 5-fold higher than the vehiclecontrol in LNCaP and approximately 4-fold higher in VCaP (Fig.3A). Importantly, a similar increase was not seen with 10 nmol/Lenzalutamide, suggesting transcriptional upregulation ofSLC35F2 is not simply associated with cell-cycle arrest, whichoccurs in response to either R1881 or enzalutamide (Fig. 3A). Tocompare expression levels across multiple prostate cancer celllines, we plotted the RNA-seq counts per million (CPM) ofSLC35F2 after exposure to 1 nmol/L R1881 or vehicle control

for 24 hours. The prostate cancer cell lines used were androgen-sensitive lines LNCaP and VCaP, castration-resistant lines C42,C42B, LNCAP-ABL, and VCaP-AI, and androgen-independentlines 22Rv1 and E006. As expected, SLC35F2 transcription levelswere regulated by androgens with the exception of the androgen-independent lines (Fig. 3B).

The androgen-induced increase in SLC35F2 transcript levelswas accompanied by increases in SLC35F2 protein levels inLNCaP cells (Fig. 3C). We then compared levels of SLC35F2protein across a dose range of R1881 in LNCaP cells. In concor-dance with the 160 pmol/L of R1881 at which the maximumresponse to YM155 was achieved (Fig. 1C), SLC35F2 levels weredramatically increased at 100 pmol/L R1881 (Fig. 3D). Of note,

Figure 3.

SLC35F2 is regulated by AR signaling. A, qRT-PCR showing relative fold change of SLC35F2 in LNCaP and VCaP exposed to 10 mmol/L enzalutamide (ENZ), vehicle(veh), 10 nmol/L R1881 (error bars ¼ SD, n ¼ 6). B, RNA-seq CPM of prostate cancer cell lines cultured with 1 nmol/L R1881 or vehicle for 24 hours, each induplicate. C,Western blot analysis of SLC35F2 on LNCaP exposed to 10 mmol/L enzalutamide, 10 nmol/L R1881, or ethanol vehicle. D,Western blots of SLC35F2 andBIRC5 in response to a dose range of R1881 in LNCaP. E, Western blot analysis of SLC35F2 with or without a blocking peptide of SLC35F2 and GAPDH onVCaP and LNCaP cultured with 10 nmol/L R1881 for 48 hours. F, Western blot of SLC35F2 on VCaP cultured with 10 nmol/L R1881 or vehicle for 48 hours and5 mg/mL cycloheximide (CHX) or 5 mmol/L MG132 for 24 hours (� , P < 0.05).






SLC35F2 upregulation corresponded with the downregulation ofBIRC5 (Fig. 3D).

Despite the androgen-mediated increase of SLC35F2 transcriptlevels in VCaP, SLC35F2 protein levels were barely detectable inVCaP cells cultured for 48 hours with 10 nmol/L R1881 (Fig. 3E).Aband close to the correct size of 45 kD for SLC35F2was observedby Western blot analysis in VCaP but was not masked by aSLC35F2-blocking peptide, in contrast with AR-regulated productobserved in LNCaP cells, which was masked by the blockingpeptide (Fig. 3E). However, a faint band corresponding toSLC35F2 was detected in VCaP cells. Notably, SLC35F2 did notappreciably increase or decrease in VCaP cells incubated with thetranslational inhibitor cycloheximide or the proteasome inhibitorMG132, respectively (Fig. 3F). Taken together, we hypothesizethat the lack of synergy between R1881 and YM155 in VCaP is dueto the inefficient translation of additional SLC35F2 transcripts.

To provide further evidence that SLC35F2 transcription isdirectly regulated by the AR, we evaluated AR ChIP-seq data fromthree published studies of LNCaP cells andone studyof VCaP cellsevaluating AR-binding sites throughout the genome. Three peaks,termed AR1-3, were observed in intron 1 of SLC35F2 underandrogen exposure but not the androgen-depleted control con-dition (Fig. 4A). We designed PCR primers recognizing theseregions and performed qPCR on LNCaP lysates following ARcrosslinking and immunoprecipitation. Each peak demonstrateda substantial and significant increase in product following andro-gen treatment relative to either no androgen control or exposureto enzalutamide (Fig. 4B).

SLC35F2 and ABCB1 expression determines cellular responseto YM155 exposure

To further examine SLC35F2-mediated YM155 sensitivity, wegenerated SCL35F2 knockdown and overexpression cell lines andmeasured viability in response to a range of YM155 doses (Fig.5A). IC50 values of YM155 shifted from 46.5 nmol/L in GFP-control LNCaP cells treated with vehicle to 679 pmol/L in LNCaPSCL35F2-overexpressing cells treated with vehicle (�68-foldchange; Fig. 5B). The YM155 IC50 only shifted from 679 to363 pmol/L (�2-fold) when 10 nmol/L R1881 was added to

SLC35F2-overexpressing cells, whereas the IC50 values shiftedfrom 46.5 to 4.35 nmol/L when 10 nmol/L R1881 was added toGFP control cells. In agreement, VCaP sensitivity to YM155increased from 14.8 nmol/L in the GFP cells with vehicle to1.49 nmol/L when SLC35F2 was overexpressed. Consistent withthe lack of androgen-induced changes in SLC35F2 protein levelsin VCaP cells, IC50 values did not shift in VCaP cells dosedwith 10nmol/L R1881 or vehicle (Fig. 5C; Supplementary Fig. S2A).

Conversely, the reduction of SLC35F2 by shRNAs decreased thesensitivity of LNCaP cells to YM155, with IC50 values going from35.8 nmol/L with the nontargeting shRNA to 267 nmol/L forSLC35F2-directed shRNA#7 and 100 nmol/L for shRNA#8 (Fig.5D; Supplementary Fig. S2B and S2C). VCAPs displayed a similarshift in IC50 values following SLC35F2 suppression (Fig. 5E;Supplementary Fig. S2D). YM155 has also been reported to beeffluxed by the ABCB1 transporter with a consequent resistance todrug treatment (52, 53). Strikingly, ABCB1 overexpression ren-dered LNCaP cells completely resistant to YM155 regardless ofandrogen levels, suggesting the ratio of SLC35F2 to ABCB1expression in tumor cells is an important determinant of sensi-tivity to YM155 (Fig. 5F).

SLC35F2 expression correlates with AR activity in metastaticprostate cancer

Identifying the patient population most likely to respond toYM155 is crucial for studies designed to establish YM155 as atargeted therapy for prostate cancer. To this end, we compared theexpression of SLC35F2with ametric of in vivoAR activity, which isbased on a panel of AR-regulated genes not including SLC35F2(30, 31).We analyzed SLC35F2 transcript levels and calculated anAR activity score for 171 CRPC metastases by gene expressionmicroarrays (30). SLC35F2 expressionwas significantly correlatedwithAR activity score (r¼0.62,P<0.001; Fig. 6A). To examine thedistribution of SLC35F2 expression across various states of pros-tate cancer progression, we compared microarray signal intensi-ties for SLC35F2 in primary prostate tumors (n¼ 11; ref. 32) andCRPC tumors (n ¼ 171; Fig. 6B; ref. 30). Notably, SLC35F2 wasbroadly expressed in all progression states and spanned a partic-ularly large range of expression in CPRC. A subset of tumors had

Figure 4.

The membrane transport protein SLC35F2 is regulated by AR activation. A, ChIP-seq histograms of AR binding to the SLC35F2 locus from published datasets.Black arrows, peaks selected for validation. B, ChIP-qPCR of peaks "AR1-3" in LNCaPs cultured in CSS with DHT, enzalutamide (ENZ), or ethanol vehiclecontrol (EtOH).

Nyquist et al.





very high levels of SLC35F2 expression, suggesting that a subset ofpatients harbor tumors that may be highly responsive to YM155treatment.

SLC35F2 expression correlates with androgen levels inxenograft models of prostate cancer

Because SLC35F2 is regulated by androgens, and previousclinical trials of YM155 in prostate cancer were performed inmenwith castrate levels of testosterone (54), we evaluated the rela-tionship between SLC35F2 expression and intratumor androgenlevels in AR-positive PDX models before and after castration.Levels of testosterone andDHT in LuCaP96 andLuCap23prostateadenocarcinoma PDX models were substantially decreased atdays 7 and 21 after castration (Fig. 6C and F) but partiallyrecovered upon progression to castration-resistant disease, con-sistent with previous reports of increased tumoral androgensynthesis leading to elevated levels of androgens in castration-resistant tumors (55). Concordantly, tumor SLC35F2 mRNAlevels decreased upon castration and increased in castration-

resistant tumors, although not necessarily to levels present intumors from intact mice (Fig. 6D and G). Importantly, weobserved strong correlations between SLC35F2 expression andtumor testosterone and/or DHT levels in both LuCaP96 (testos-terone r ¼ 0.456, P ¼ 0.003; Fig. 6E) and LuCaP23 (DHT r ¼0.459, P ¼ 0.009; Fig. 6H), as well as in two additional PDXmodels (LuCaP35 DHT r¼ 0.483, P¼ 0042; LuCap136 DHT r¼0.327, P ¼ 0.08, Supplementary Fig. S3; data for all modelssummarized in Supplementary Table S1), suggesting that exog-enous administration of high-dose androgens has the potentialfor further regulating SLC35F2 expression. In contrast, SLC35Fexpression was not associated with tumor androgen levels in theLuCaP86.2 PDXmodel. Thismodel is known to harbor a genomicAR rearrangement resulting in high-level expression of the exon-skipping androgen-independent AR splice variant ARV567 (56).Notably, we found that SLC35F2 expression was strongly corre-lated with ARV567 expression (r¼ 0.745, P¼ 0.0004), consistentwith the ability of ligand-independentARvariants to stimulate theexpression of AR target genes (57).

Figure 5.

SLC35F2 and ABCB1 expressiondetermine response to YM155. A,YM155 dose–response curve forLNCaPs transduced with SLC35F2 orGFP overexpression vectors. B, IC50

values plotted for curves in A. C, SameasB for VCaPs.D, YM155 IC50 values ofLNCaPs transduced with shRNAvectors to SLC35F2 (shSLC) comparedwith a nontargeting control (NTC)vector. E, Same as D for VCaP. F,YM155 dose–response curves areplotted for LNCaPs overexpressingABCB1 with and without 10 nmol/LR1881 [error bars, SD (A) and95% confidence interval (B–E);� , P < 0.05, n ¼ 4].






DiscussionBIRC5/survivin is a critical mediator of cancer cell growth,

survival, DNA repair, and therapy resistance (58). Althoughpreclinical studies have repeatedly demonstrated the relevanceof BIRC5 as a therapeutic target, clinical efforts to exploit thesefindings have largely been unsuccessful (59, 60). YM155 wasoriginally developed to suppress the transcription of BIRC5 (50).Subsequent studiesmeasuring the efficacy of YM155 inmore than100 human cancer cell lines and xenografts found YM155 topotently cause cell death across a broad spectrum of cancers withlow systemic toxicity in xenografts (61–63). YM155 inhibitsBIRC5 by perturbing transcription factor–DNA interactions ofSp-1 (64), ILF3 (65), p50 (66), and NONO (67). However, the

anticancer effects of YM155 are unlikely to be due to BIRC5suppression alone (68). Other targets related to cell survival, suchas Mcl1, Bcl-2, and Bcl-xl, have also been reported (66).

A phase I pharmacokinetic study of YM155 on 41 patients,including 9 hormone- and docetaxel-refractory patients, deter-mined YM155 was very well tolerated with very few grade 3 and 4toxicities. Two CPRC patients had PSA responses, in addition toone complete and two partial responses in 3 patients with non–Hodgkin lymphoma. A subsequent phase II study in 35 progres-sing, hormone- and docetaxel-refractory, prostate cancer patientsfound modest single-agent activity of YM155, with 25% ofpatients achieving prolonged stable disease of �18 weeks (54).Importantly, eligibility requirements for the trial required castratelevels of serum testosterone (�50 ng/mL). It is possible that the

Figure 6.

SLC35F2 expression correlates with AR activity and androgen levels in CRPC. A, Pearson correlation plot comparing AR activity and SLC35F2 expressionin 171 CRPC samples. B, Waterfall plot of microarray signal intensities for SLC35F2 in primary tumors (n ¼ 11), and CRPC (n ¼ 171). C, Intratumoral testosteroneand DHT levels in the LuCaP 96 PDX model in intact mice, at 7 and 21 days postcastration (Cx), and after castration-resistant (CR) regrowth. D, qRT-PCR fortumoral SLC35F2 expression in LuCaP 96 xenograft samples shown in C. E, Spearman correlation comparing intratumoral testosterone levels withnormalized SLC35F2 mRNA expression in LuCaP 96 tumors. F, Intratumoral androgen levels in LuCaP 23. G, qRT-PCR for tumoral SLC35F2 expression in LuCaP 23.H, Spearman correlation comparing normalized SLC35F2 levels and intratumoral DHT in LuCaP 23. Student t tests comparing SLC35F2 levels in intact andcastrate mice.

Nyquist et al.





drug was inefficiently absorbed by tumor cells due to a loss of AR-mediated SLC35F2 expression. If so, our data suggest YM155 andrelated compounds may be more effective in the context ofintermittent ADT or high-dose testosterone cycles. Intriguingly,our data showing a correlation between tumor androgen levelsandSLC35F2expression also suggests YM155maybe effective in asubset of patients that progress on ADT with elevated levels ofintratumoral androgens caused by aberrant expression of ste-roidogenic enzymes (69).

SLC transporters are among the least studied protein families,and many receptors such as SLC35F2 are considered "orphan"with unknown substrates and physiologic roles (70, 71). How-ever, there is an evolving conception of SLC transporters asmediators of intertissue communication or "remote sensing" ofmetabolites, signaling molecules, and morphogenic compounds(70). A growing body of literature onmodel–organism knockoutsand human genetic diseases reveals complexity in how transpor-ters are regulated and function in the development of an organism(72, 73). For example, both developing and mature tissues haveselective SLC expression profiles, sensitizing, or insulating cellsfrom signals in their environment (72, 74). It is tempting tospeculate that SLC transport profiles change with developmentaland progression states in cancer, potentially presenting targets ofopportunity. In support, it has been reported that SLC35F2 ismore highly expressed in non–small cell lung cancers than insurrounding tissue (75).

Although controversial, it is increasingly appreciated that drugtransport by passive diffusion across a membrane is often negli-gible and instead mediated by transmembrane proteins thatnormally transport metabolites (76). Furthermore, drugsdesigned with "metabolite-likeness" (77) have a greater chanceof achieving standards of efficacy, such as Lipinski's rule of five(78), suggesting an important role for SLC transporters in druguptake. Several important cancer drugs have known transporters.SLC22A1/SLC22A2 transports daunorubicin (79), imatinib (80),cisplatin, and oxaliplatin (81, 82), and SLC22A16 transportsdoxorubicin (83). Further research on SLC transporter regulationand their substrates may lead to precision medicine–guidedtherapies with high antitumor subtype efficacies and low systemictoxicities.

In addition to the potential prostate cancer growth-suppressiveeffects and quality-of-life benefits of high-testosterone therapy,androgens may selectively sensitize prostate cancer to YM155.Future studies are needed to evaluate the use of YM155 with high-testosterone therapies. For example, it may be optimal to treatpatients with cycles of synergistic therapeutic combinations byalternating testosterone/YM155 with ADT and docetaxel (84) orPI3K/AKT inhibitors (85). Structure–activity relationship analysison YM155 examined the determinants of its selective lethality totransformed cells and derived several dioxonaphthoimidazoliumanalogues with similar potency (68). Future work is required todetermine whether molecules derived from YM155 have similarefficacy and transport mechanisms (66). Finally, it is unclear whatother factors regulate SLC35F2 gene expression or how protein

levels are being regulated. The VCaP cell line does not upregulateSLC35F2 protein levels to the same extent as LNCaPs with andro-gens, despite similarly upregulating the transcript.One of the greatchallenges in the study of SLC transporters is their apparentfunctional redundancy and observed compensatory regulation(70). The mechanism by which cells and tissues coordinatelyregulate SLC transport profiles is largely unknown. Moreover, theregulation and functional significance of SLC transporters incancers as well as their relevance to chemotherapeutics is increas-ingly appreciated yet still poorly understood (86).

In summary, we demonstrated that YM155 synergizes withendogenously achievable levels of androgens to eliminate pros-tate cancer cells. This is due to the upregulation of YM155transporter SLC35F2 by AR transcriptional activity. However,YM155-mediated cell death can be counteracted by high ABCB1levels. Furthermore, SLC35F2 expression correlates with AR activ-ity in CRPC tumors. Interestingly, someCRPCs express high levelsof SLC35F2 despite lower AR activity scores (Fig. 6A), suggestingSLC35F2 could be used as a biomarker for YM155 susceptibility.Given that YM155 is well tolerated and synergizes with varioustherapies (45), YM155 may be an effective and well-toleratedcotherapy for prostate cancer.

Disclosure of Potential Conflicts of InterestP.S. Nelson is a consultant/advisory board member for Astellas and Janssen.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.D. Nyquist, P.S. Nelson, E.A. MostaghelDevelopment of methodology: M.D. Nyquist, L. Riggan, E.A. MostaghelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.D. Nyquist, J. Burns, R. Tharakan, P.S. Nelson,E.A. MostaghelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Corella, J. Burns, I. Coleman, S. Gao, C. Cai,E.A. MostaghelWriting, review, and/or revision of the manuscript: M.D. Nyquist, J. Burns,E. Corey, P.S. Nelson, E.A. MostaghelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Burns, E. Corey, P.S. Nelson, E.A. MostaghelStudy supervision: P.S. Nelson, E.A. Mostaghel

AcknowledgmentsThe authors thank Steven Plymate, MD, for discussions.

Grant SupportThis work was supported by the NIH: Pacific Northwest Prostate Cancer

SPORE grant no. P50 CA097186 (to E.A. Mostaghel, E. Corey, and P.S.Nelson), P01 CA163227 (to E.A. Mostaghel, E. Corey, and P.S. Nelson), R21CA194798 (to E. Corey), U.S. Department of Defense awards W81XWH-15-1-0319 (to E.A. Mostaghel), W81XWH-16-1-0206 (to M.D. Nyquist), andthe Lucas Foundation.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 22, 2016; revisedDecember 3, 2016; acceptedDecember22, 2016; published OnlineFirst May 1, 2017.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin

2016;66:7–30.2. Mills IG. Maintaining and reprogramming genomic androgen receptor

activity in prostate cancer. Nat Rev Cancer 2014;14:187–98.

3. Antony L, Van Der Schoor F, Dalrymple SL, Isaacs JT. Androgen receptor(AR) suppresses normal human prostate epithelial cell proliferation viaAR/b-catenin/TCF-4 complex inhibition of c-MYC transcription. Prostate2014;74:1118–31.






4. Chen Y, Chi P, Rockowitz S, Iaquinta PJ, Shamu T, Shukla S, et al. ETSfactors reprogram the androgen receptor cistrome and prime prostatetumorigenesis in response to PTEN loss. Nat Med 2013;19:1023–9.

5. Abeshouse A, Ahn J, Akbani R, Ally A, Amin S, Andry CD, et al. Themolecular taxonomy of primary prostate cancer. Cell 2015;163:1011–25.

6. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance toandrogen receptor inhibitors in prostate cancer. Nat Rev Cancer 2015;15:701–11.

7. Robinson D, Van Allen EM,Wu Y-M, Schultz N, Lonigro RJ, Mosquera J-M,et al. Integrative clinical genomics of advanced prostate cancer. Cell2015;161:1215–28.

8. Pomerantz MM, Li F, Takeda DY, Lenci R, Chonkar A, Chabot M, et al. Theandrogen receptor cistrome is extensively reprogrammed in human pros-tate tumorigenesis. Nat Genet 2015;47:1346–51.

9. Wang Q, Li W, Zhang Y, Yuan X, Xu K, Yu J, et al. Androgen receptorregulates a distinct transcription program in androgen-independent pros-tate cancer. Cell 2009;138:245–56.

10. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al.Abiraterone and increased survival in metastatic prostate cancer. N Engl JMed 2011;364:1995–2005.

11. Scher HI, Fizazi K, Saad F, Taplin M-E, Sternberg CN, Miller K, et al.Increased survival with enzalutamide in prostate cancer after chemo-therapy. N Engl J Med 2012;367:1187–97.

12. Liu X, Chen X, Rycaj K, ChaoH,DengQ, Jeter C, et al. Systematic dissectionof phenotypic, functional, and tumorigenic heterogeneity of human pros-tate cancer cells. Oncotarget 2015;6:23959–86.

13. Rycaj K, Cho EJ, Liu X, ChaoH-P, Liu B, Li Q, et al. Longitudinal tracking ofsubpopulation dynamics and molecular changes during LNCaP cell cas-tration and identification of inhibitors that could target the PSA-/locastration-resistant cells. Oncotarget 2016;7:14220–40.

14. Sun Y, Wang BE, Leong KG, Yue P, Li L, Jhunjhunwala S, et al. Androgendeprivation causes epithelial-mesenchymal transition in the prostate:implications for androgendeprivation therapy. Cancer Res 2012;72:527–36.

15. Bishop JL, Davies A, Ketola K, Zoubeidi A. Regulation of tumor cellplasticity by the androgen receptor in prostate cancer. Endocr Relat Cancer2015;22:R165–82.

16. Bishop JL, Sio A, Angeles A, Roberts ME, Azad A, Chi KN, et al. PD-L1 ishighly expressed in enzalutamide resistant prostate cancer. Oncotarget2015;6:234–42.

17. Seiler D, Zheng J, LiuG,Wang S, Yamashiro J, Reiter RE, et al. Enrichment ofputative prostate cancer stem cells after androgen deprivation: upregula-tion of pluripotency transactivators concurs with resistance to androgendeprivation in LNCaP cell lines. Prostate 2013;73:1378–90.

18. Cary KC, Singla N, Cowan JE, Carroll PR, Cooperberg MR. Impact ofandrogen deprivation therapy onmental and emotional well-being inmenwith prostate cancer: analysis from the CaPSURETM registry. J Urol2014;191:964–70.

19. Zejnullahu K, Arevalo MG, Ryan CJ, Aggarwal R. Approaches to minimizecastration in the treatment of advanced prostate cancer. Urol Oncol2016;34:368–74.

20. Kim YC, Chen C, Bolton EC. Androgen receptor-mediated growth sup-pression of HPr-1AR and PC3-Lenti-AR prostate epithelial cells. PLoS One2015;10:1–31.

21. Roediger J,HessenkemperW, Bartsch S,ManvelyanM,Huettner SS, Liehr T,et al. Supraphysiological androgen levels induce cellular senescence inhuman prostate cancer cells through the Src-Akt pathway. Mol Cancer2014;13:214.

22. D'Antonio JM, Vander Griend DJ, Isaacs JT. DNA licensing as a novelandrogen receptor mediated therapeutic target for prostate cancer. EndocrRelat Cancer 2009;16:325–32.

23. Vander Griend DJ, Litvinov IV, Isaacs JT. Stabilizing androgen receptor inmitosis inhibits prostate cancer proliferation. Cell Cycle 2007;6:647–51.

24. Haffner MC, Aryee MJ, Toubaji A, Esopi DM, Albadine R, Gurel B, et al.Androgen-induced TOP2B-mediated double-strand breaks and prostatecancer gene rearrangements. Nat Genet 2010;42:668–75.

25. Denmeade SR, Isaacs JT. Bipolar androgen therapy: The rationale for rapidcycling of supraphysiologic androgen/ablation in men with castrationresistant prostate cancer. Prostate 2010;70:1600–7.

26. Isaacs JT, D'Antonio JM, Chen S, Antony L, Dalrymple SP, Ndikuyeze GH,et al. Adaptive auto-regulation of androgen receptor provides a paradigm

shifting rationale for bipolar androgen therapy (BAT) for castrate resistanthuman prostate cancer. Prostate 2012;72:1491–505.

27. Schweizer MT, Antonarakis ES, Wang H, Ajiboye AS, Cao H, Luo J, et al.Effect of bipolar androgen therapy for asymptomatic men with castration-resistant prostate cancer: results from a pilot clinical study. Sci Transl Med2015;7:269ra2.

28. Schweizer MT,WangH, Luber B, Nadal R, Spitz A, Rosen DM, et al. Bipolarandrogen therapy for men with androgen ablation na€�ve prostate cancer:results from the phase II BATMAN study. Prostate 2016;76:1218–26.

29. Hedayati M, Haffner MC, Coulter J, Raval RR, Zhang Y, Zhou H, et al.Androgen deprivation followed by acute androgen stimulation selectivelysensitizes AR-positive prostate cancer cells to ionizing radiation. ClinCancer Res 2016;22:3310–9.

30. Kumar A, Coleman I, Morrissey C, Zhang X, True LD, Gulati R, et al.Substantial interindividual and limited intraindividual genomic diversityamong tumors from men with metastatic prostate cancer. Nat Med2016;22:369–78.

31. Mostaghel EA, Geng L, Holcomb I, Coleman IM, Lucas J, True LD, et al.Variability in the androgen responseof prostate epithelium to5a-reductaseinhibition: implications for prostate cancer chemoprevention. Cancer Res2010;70:1286–95.

32. Roudier MP, Winters BR, Coleman I, Lam HM, Zhang X, Coleman R, et al.Characterizing the molecular features of ERG-positive tumors in primaryand castration resistant prostate cancer. Prostate 2016;76:810–22.

33. HieronymusH, Lamb J, Ross KN,PengXP,ClementC, RodinaA, et al. Geneexpression signature-based chemical genomic prediction identifies a novelclass of HSP90 pathway modulators. Cancer Cell 2006;10:321–30.

34. Carver BS, Chapinski C, Wongvipat J, Hieronymus H, Chen Y, Chan-darlapaty S, et al. Reciprocal feedback regulation of PI3K and androgenreceptor signaling in PTEN-deficient prostate cancer. Cancer Cell 2011;19:575–86.

35. Sahu B, Laakso M, Pihlajamaa P, Ovaska K, Sinielnikov I, Hautaniemi S,et al. FoxA1 specifies unique androgen and glucocorticoid receptor bindingevents in prostate cancer cells. Cancer Res 2013;73:1570–80.

36. Yu J, Yu J, Mani R-S, Cao Q, Brenner CJ, Cao X, et al. An integrated networkof androgen receptor, polycomb, and TMPRSS2-ERG gene fusions inprostate cancer progression. Cancer Cell 2010;17:443–54.

37. Choudhary V, Kaddour-Djebbar I, Lakshmikanthan V, Ghazaly T, Thang-jam GS, Sreekumar A, et al. Novel role of androgens in mitochondrialfission and apoptosis. Mol Cancer Res 2011;9:1067–77.

38. LangmeadB, Trapnell C, PopM, Salzberg S. Ultrafast andmemory-efficientalignment of short DNA sequences to the human genome. Genome Biol2009;10:R25.

39. Robinson JT, Thorvaldsd�ottir H, Winckler W, GuttmanM, Lander ES, GetzG, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24–6.

40. Wang Q, Carroll JS, Brown M. Spatial and temporal recruitment ofandrogen receptor and its coactivators involves chromosomal loopingand polymerase tracking. Mol Cell 2005;19:631–42.

41. Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, et al. A publicgenome-scale lentiviral expression library of human ORFs. Nat Methods2011;8:659–61.

42. Morrissey C, Roudier MP, Dowell A, True LD, Ketchanji M, Welty C, et al.Effects of androgen deprivation therapy and bisphosphonate treatment onbone in patients withmetastatic castration-resistant prostate cancer: resultsfrom the University ofWashington Rapid Autopsy Series. J BoneMiner Res2013;28:333–40.

43. Corey E,Quinn JE, Buhler KR,Nelson PS,Macoska JA, True LD, et al. LuCaP35: Anewmodel of prostate cancer progression to androgen independence.Prostate 2003;55:239–46.

44. Kalhorn TF, Page ST, Howald WN, Mostaghel EA, Nelson PS. Analysis oftestosterone and dihydrotestosterone from biological fluids as the oximederivatives using high-performance liquid chromatography/tandem massspectrometry. Rapid Commun Mass Spectrom 2007;21:3200–6.

45. Rauch A, Hennig D, Sch€afer C, Wirth M, Marx C, Heinzel T, et al. Survivinand YM155: How faithful is the liaison? Biochim Biophys Acta 2014;1845:202–20.

46. V�equaud E, Desplanques G, J�ez�equel P, Juin P, Barill�e-Nion S. Survivincontributes to DNA repair by homologous recombination in breast cancercells. Breast Cancer Res Treat 2016;155:53–63.

47. Rodriguez-Rocha H, Garcia-Garcia A, Panayiotidis MI, Franco R. DNAdamage and autophagy. Mutat Res 2011;711:158–66.

Nyquist et al.





48. BonnerWM, Redon CE, Dickey JS, Nakamura AJ, SedelnikovaOA, Solier S,et al. gH2AX and cancer. Nat Rev Cancer 2008;8:957–67.

49. Schiewer MJ, Knudsen KE. Linking DNA damage and hormone signalingpathways in cancer. Trends Endocrinol Metab 2016;27:216–25.

50. Nakahara T, Takeuchi M, Kinoyama I, Minematsu T, Shirasuna K, Matsu-hisa A, et al. YM155, a novel small-molecule survivin suppressant, inducesregression of established human hormone-refractory prostate tumor xeno-grafts. Cancer Res 2007;67:8014–21.

51. Winter GE, Radic B, Mayor-Ruiz C, Blomen VA, Trefzer C, Kandasamy RK,et al. The solute carrier SLC35F2 enables YM155-mediated DNA damagetoxicity. Nat Chem Biol 2014;10:768–73.

52. Lamers F, Schild L, Koster J, Versteeg R, Caron HN, Molenaar JJ. TargetedBIRC5 silencing using YM155 causes cell death in neuroblastoma cells withlow ABCB1 expression. Eur J Cancer 2012;48:763–71.

53. Iwai M, Minematsu T, Li Q, Iwatsubo T, Usui T. Utility of P-glycoproteinand organic cation transporter 1 double-transfected LLC-PK1 cells forstudying the interaction of suppressant, with P-glycoprotein. Drug MetabDispos 2011;39:2314–20.

54. Tolcher AW, Quinn DI, Ferrari A, Ahmann F, Giaccone G, Drake T, et al. Aphase II study of YM155, a novel small-molecule suppressor of survivin, incastration-resistant taxane-pretreated prostate cancer. Ann Oncol 2012;23:968–73.

55. Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, HiganoCS, et al. Maintenance of intratumoral androgens in metastatic prostatecancer: a mechanism for castration-resistant tumor growth. Cancer Res2008;68:4447–54.

56. Li Y, Hwang TH, Oseth LA, Hauge A, Vessella RL, Schmechel SC, et al. ARintragenic deletions linked to androgen receptor splice variant expressionand activity in models of prostate cancer progression. Oncogene2012;31:4759–67.

57. NyquistMD, Li Y, Hwang TH,Manlove LS, Vessella RL, Silverstein KA, et al.TALEN-engineered AR gene rearrangements reveal endocrine uncouplingof androgen receptor in prostate cancer. Proc Natl Acad Sci U S A 2013;110:17492–7.

58. Antonio Cheung CH, Huang CC, Tsai FY, Lee JYC, Cheng SM, Chang YC,et al. Survivin - biology and potential as a therapeutic target in oncology.Onco Targets Ther 2013;6:1453–62.

59. Groner B, Weiss A. Targeting Survivin in cancer: novel drug developmentapproaches. BioDrugs 2014;28:27–39.

60. Altieri DC. Targeting survivin in cancer. Cancer Lett 2013;332:225–8.61. Mehta A, Zhang L, BoufraqechM, Liu-Chittenden Y, Zhang Y, Patel D, et al.

Inhibition of survivin with YM155 induces durable tumor response inanaplastic thyroid cancer. Clin Cancer Res 2015;21:4123–32.

62. Nakahara T, Kita A, Yamanaka K, Mori M, Amino N, Takeuchi M, et al.Broad spectrum and potent antitumor activities of YM155, a novel small-molecule survivin suppressant, in a wide variety of human cancer cell linesand xenograft models. Cancer Sci 2011;102:614–21.

63. Wang Q, Chen Z, Diao X, Huang S. Induction of autophagy-dependentapoptosis by the survivin suppressant YM155 in prostate cancer cells.Cancer Lett 2011;302:29–36.

64. Cheng Q, Ling X, Haller A, Nakahara T, Yamanaka K, Kita A, et al.Suppression of survivin promoter activity by YM155 involves disruptionof Sp1-DNA interaction in the survivin core promoter. Int J Biochem MolBiol 2012;3:179–97.

65. Nakamura N, Yamauchi T, Hiramoto M, Yuri M, Naito M, Takeuchi M,et al. Interleukin enhancer-binding factor 3/NF110 is a target ofYM155, a suppressant of survivin. Mol Cell Proteomics 2012;11:M111.013243.

66. Ho SH, Ali A, Chin TM, Go ML. Dioxonaphthoimidazoliums AB1 andYM155 disrupt phosphorylation of p50 in the NF-kB pathway. Oncotarget2016;7:11625–36.

67. Yamauchi T, Nakamura N, HiramotoM, Yuri M, YokotaH, NaitouM, et al.Sepantroniumbromide (YM155) induces disruption of the ILF3/p54(nrb)complex, which is required for survivin expression. Biochem Biophys ResCommun 2012;425:711–6.

68. Ho S-HS, SimM-Y, YeeW-LS, Yang T, Yuen S-PJ, GoM-L. Antiproliferative,DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: a structure–activity relationship study.Eur J Med Chem 2015;104:42–56.

69. Mostaghel EA, Marck BT, Plymate SR, Vessella RL, Balk S, Matsu-moto AM, et al. Resistance to CYP17A1 inhibition with abirateronein castration-resistant prostate cancer: induction of steroidogenesisand androgen receptor splice variants. Clin Cancer Res 2011;17:5913–25.

70. Nigam SK. What do drug transporters really do? Nat Rev Drug Discov2014;14:29–44.

71. C�esar-Razquin A, Snijder B, Frappier-Brinton T, Isserlin R, Gyimesi G,Bai X, et al. A call for systematic research on solute carriers. Cell2015;162:478–87.

72. Song Z. Roles of the nucleotide sugar transporters (SLC35 family) in healthand disease. Mol Aspects Med 2013;34:590–600.

73. HedigerMA, Cl�emencon B, Burrier RE, Bruford EA. The ABCs ofmembranetransporters in health and disease (SLC series): introduction. Mol AspectsMed 2013;34:95–107.

74. Nishimura M, Suzuki S, Satoh T, Naito S. Tissue-specific mRNA expressionprofiles of human solute carrier 35 transporters. Drug Metab Pharmaco-kinet 2009;24:91–9.

75. Wang J. Highly expressed SLC35F2 in non-small cell lung cancer isassociated with pathological staging. Mol Med Rep 2011;4:1289–93.

76. Kell DB,Oliver SG.Howdrugs get into cells: tested and testable predictionsto help discriminate between transporter-mediated uptake and lipoidalbilayer diffusion. Front Pharmacol 2014;5:231.

77. Kell DB. Implications of endogenous roles of transporters for drugdiscovery: hitchhiking and metabolite-likeness. Nat Rev Drug Discov2016;15:143.

78. Leeson P. Drug discovery: chemical beauty contest. Nature 2012;481:455–6.

79. Andreev E, Brosseau N, Carmona E, Mes-Masson A-M, Ramotar D. Thehuman organic cation transporter OCT1 mediates high affinity uptake ofthe anticancer drug daunorubicin. Sci Rep 2016;6:20508.

80. Thomas J, Wang L, Clark RE, Pirmohamed M. Active transport of imatinibinto and out of cells: implications for drug resistance. Transport2004;104:3739–45.

81. Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui K. Cisplatin and oxali-platin, but not carboplatin and nedaplatin, are substrates for humanorganic cation transporters (SLC22A1-3 andmultidrug and toxin extrusionfamily). J Pharmacol Exp Ther 2006;319:879–86.

82. Zhang S, Lovejoy KS, Shima JE, Lagpacan LL, Shu Y, Lapuk A, et al. Organiccation transporters are determinants of oxaliplatin cytotoxicity. Cancer Res2006;66:8847–57.

83. Okabe M, Unno M, Harigae H, Kaku M, Okitsu Y, Sasaki T, et al. Char-acterization of the organic cation transporter SLC22A16: a doxorubicinimporter. Biochem Biophys Res Commun 2005;333:754–62.

84. Sweeney CJ, Chen Y-H, Carducci M, Liu G, Jarrard DF, Eisenberger M, et al.Chemohormonal therapy inmetastatic hormone-sensitive prostate cancer.N Engl J Med 2015;373:737–46.

85. Thomas C, Lamoureux F, Crafter C, Davies BR, Beraldi E, Fazli L, et al.Synergistic targeting of PI3K/AKT pathway and androgen receptor axissignificantly delays castration-resistant prostate cancer progression in vivo.Mol Cancer Ther 2013;12:2342–55.

86. Li Q, Shu Y. Role of solute carriers in response to anticancer drugs. Mol CellTher 2014;2:15.






2017;15:521-531. Published OnlineFirst January 24, 2017.Mol Cancer Res Michael D. Nyquist, Alexandra Corella, John Burns, et al. the Survivin Inhibitor YM155Exploiting AR-Regulated Drug Transport to Induce Sensitivity to

Updated version

10.1158/1541-7786.MCR-16-0315-Tdoi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2017/01/24/1541-7786.MCR-16-0315-T.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/15/5/521.full#ref-list-1

This article cites 86 articles, 18 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/15/5/521.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/15/5/521To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-16-0315-T

http://mcr.aacrjournals.org/content/suppl/2017/01/24/1541-7786.MCR-16-0315-T.DC1

http://mcr.aacrjournals.org/content/15/5/521.full#ref-list-1

http://mcr.aacrjournals.org/content/15/5/521.full#related-urls

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/15/5/521


Exploiting AR-Regulated Drug Transport to Induce Sensitivity to … · Cell Death and Survival...

Documents

Transcript of Exploiting AR-Regulated Drug Transport to Induce Sensitivity to … · Cell Death and Survival...