AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and ...enzyme synthesizing intratumoral testosterone (T) and 5a-dihydrotestosterone (DHT), the enzyme represents a promis-ing

Small Molecule Therapeutics

AKR1C3 Inhibitor KV-37 Exhibits AntineoplasticEffects and Potentiates Enzalutamide inCombination Therapy in ProstateAdenocarcinoma CellsKshitij Verma1, Nehal Gupta1, Tianzhu Zang2, Phumvadee Wangtrakluldee2,Sanjay K. Srivastava1,3, Trevor M. Penning2, and Paul C. Trippier1,4

Abstract

Aldo-keto reductase 1C3 (AKR1C3), also known as type 517 b-hydroxysteroid dehydrogenase, is responsible for intra-tumoral androgen biosynthesis, contributing to the develop-ment of castration-resistant prostate cancer (CRPC) and even-tual chemotherapeutic failure. Significant upregulation ofAKR1C3 is observed in CRPC patient samples and derivedCRPC cell lines. As AKR1C3 is a downstream steroidogenicenzyme synthesizing intratumoral testosterone (T) and 5a-dihydrotestosterone (DHT), the enzyme represents a promis-ing therapeutic target to manage CRPC and combat the emer-gence of resistance to clinically employed androgen depriva-tion therapy. Herein, we demonstrate the antineoplasticactivity of a potent, isoform-selective and hydrolyticallystable AKR1C3 inhibitor (E)-3-(4-(3-methylbut-2-en-1-yl)-3-(3-phenylpropanamido)phenyl)acrylic acid (KV-37), whichreduces prostate cancer cell growth in vitro and in vivo and

sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward theantitumor effects of enzalutamide. Crucially, KV-37 does notinduce toxicity in nonmalignant WPMY-1 prostate cellsnor does it induce weight loss in mouse xenografts. Moreover,KV-37 reduces androgen receptor (AR) transactivation andprostate-specific antigen expression levels in CRPC cell linesindicative of a therapeutic effect in prostate cancer. Combina-tion studies of KV-37 with enzalutamide reveal a very highdegree of synergistic drug interaction that induces significantreduction in prostate cancer cell viability via apoptosis, result-ing in >200-fold potentiation of enzalutamide action in drug-resistant 22Rv1 cells. These results demonstrate a promisingtherapeutic strategy for the treatment of drug-resistant CRPCthat invariably develops in prostate cancer patients followinginitial treatment with AR antagonists such as enzalutamide.Mol Cancer Ther; 17(9); 1833–45. �2018 AACR.

IntroductionProstate cancer is the third (1) leading causeofmortality among

American men and has the highest incidence of all cancersreported in the United States (2). Surgical removal of the prostateby radical prostatectomy and radiotherapy to resect the localizedtumor are standard therapeutic interventions (3). The treatmentof advanced andmetastatic forms of prostate cancer relies heavilyon androgen deprivation therapy (ADT) that involves surgical(orchiectomy) and/or chemical castration (4). Chemical agents

such as gonadotropin-releasing hormone agonists [Leuprolide(5), goserelin (6), and buserelin (7)] and antiandrogens (bicalu-tamide; ref. 8) retard prostate cancer cell proliferation and lead toremission. After an initial response to ADT that results in castratelevels of circulating androgens, the tumor adapts, giving rise to amore aggressive and fatal disease known as castration-resistantprostate cancer (CRPC), which is characterized by molecularchanges that include an increase in the expression of androgen-synthesizing enzymes and reactivation of androgen signaling(9, 10). Such adaptations can lead to relapse despite the presenceof circulating castrate levels of androgens (11, 12).

Abiraterone acetate (AA) was approved by the FDA for CRPCtreatment in 2011 and acts by inhibiting P450c17 (13), anupstream enzyme in the steroid biosynthetic pathway, and iseffective in CRPC where tumor progression is dependent onintracrine androgen synthesis that consequently activates ARsignaling (14, 15). However, to prevent hypertensive crisis dueto accumulation of desoxycorticosterone (DOC) in the adrenalgland, coadministration with prednisone is imperative (16).Enzalutamide (ENZ) is another clinically used therapeutic thatexerts potent androgen receptor (AR) antagonistic activity byinhibiting AR nuclear translocation, coactivator recruitment, andAR binding to androgen response elements (ARE; ref. 17). Thechemotherapeutic is effective up to stage 3 CRPC where prolifer-ation is dependent on AR activation that occurs even in theabsence of an androgen ligand (15). After an initial response toAA or ENZ, resistance develops rapidly due to increases in

1Department of Pharmaceutical Sciences, Texas Tech University Health SciencesCenter, School of Pharmacy, Amarillo, Texas. 2Center of Excellence in Environ-mental Toxicology, Department of Systems Pharmacology and TranslationalTherapeutics, Perelman School of Medicine, University of Pennsylvania, Phila-delphia, Pennsylvania. 3Department of Immunotherapeutics and Biotechnology,Texas Tech University Health Sciences Center, Abilene, Texas. 4Center forChemical Biology, Department of Chemistry and Biochemistry, Texas TechUniversity, Lubbock, Texas.

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

K. Verma and N. Gupta contributed equally to this article.

Corresponding Author: Paul C. Trippier, Texas Tech University Health SciencesCenter, 1300 S. Coulter Street, Amarillo, TX 79106. Phone: 806-414-9245; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-17-1023

�2018 American Association for Cancer Research.

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intratumoral androgen biosynthesis (18), AR overexpression, ARmutation that makes the receptor ligand promiscuous, or theappearance of AR splice variants that makes the AR constitutivelyactive in the absence of the ligand-binding domain (15, 19, 20).Due to tumor heterogeneity and the emergence of adaptive path-ways of androgen biosynthesis (21), combined with frequentmutations inAR and androgen biosynthetic enzymes (10, 22), theprognosis for patients that have advanced to CRPC remains bleak.Thus, there is an urgent need for novel therapeutics that can delayor reverse the inevitable emergence of resistance.

Type 5 17b-hydroxysteroid dehydrogenase, also known asaldo–keto reductase 1C3 (AKR1C3), acts downstream in thesteroidogenesis pathway and plays a pivotal role in the patho-genesis and progression of CRPC by catalyzing the conversion ofweak androgen precursors to the potent AR ligands: testosterone(T) and 5a-dihydrotestosterone (5a-DHT; ref. 23). Also known asprostaglandin (PG) F synthase, AKR1C3 catalyzes the conversionof PGD2 to11b-PGF2a andPGF2aprostanoids (24). An increase inPGF2a leads to activation of the FP receptor and consequentlyinduces proliferation and radiation resistance in prostate cancercells (25).

Clinically, AKR1C3has been shown to be themost upregulatedsteroidogenic enzyme inCRPCpatients (26). Apart from inducinga CRPC phenotype, AKR1C3 also mediates resistance to ENZ andAA by providing a source of intratumoral androgens, and thisresistance can be surmounted somewhat by indomethacin(INDO), a known AKR1C3 inhibitor that also inhibits COXisozymes (27, 28). As the enzyme acts in the final steps of theandrogen synthesis pathway in the prostate (16), AKR1C3 inhi-bitors would not cause the accumulation of DOC in the adrenalgland andwould not have to be coadministered with prednisone.These properties make AKR1C3 an attractive target for managingCRPC disease progression as well as countering therapeutic resis-tance. The related isoforms AKR1C1 and AKR1C2 share highhomology with AKR1C3, function to eliminate DHT, and shouldnot be inhibited (29). Numerous AKR1C3 inhibitors with diversescaffolds are known; however, none have made their way intothe clinic (30–32). Considerable interest has developed in the roleof AKR1C3 in a variety of cancers of the breast (33), lung (34), andcolon (35).

Herein, we report on the actions of a novel, potent, isoform-selective and metabolically stable AKR1C3 inhibitor, KV-37(Fig. 1A; refs. 36–38), in androgen-sensitive prostate cancer celllines and a xenograft model. We have previously reportedAKR1C3 inhibitors that act synergistically with etoposide anddaunorubicin as chemotherapeutic agents in a panel of leukemiacell lines (38). The AKR1C3 inhibitor KV-37 exerts high syner-gistic drug interaction in combinationwithENZ,which is superiorto that seen with INDO. Mechanistic studies reveal that KV-37causes apoptotic cell death in ENZ-resistant prostate cancer celllines with a consequent reduction in PSA levels, and in vivo studiesdemonstrate >50% reduction in tumor growth without observ-able toxicity.

Materials and MethodsCell culture and reagents

22Rv1 and LNCaP cells were purchased from the AmericanType Culture Collection (ATCC in 2016) and cultured in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin, and0.1 mg/mL streptomycin (39). LNCaP1C3 cells overexpressing

AKR1C3 were generated by stable transfection of AKR1C3plasmid as previously described (12). WPMY-1 cells were pur-chased from the ATCC (2017) and maintained in DMEM mediasupplementedwith 5%FBS, 100U/mLpenicillin, and 0.1mg/mLstreptomycin. All cell lines were authenticated via short tandemrepeat analysis and tested for mycoplasma using the MycoAlertmycoplasma detection kit as per the manufacturer's instructions(Texas cancer cell repository) in May 2017, showing no contam-ination. Cell line use was limited to passage nine or lower. Whereindicated, cells were also cultured in charcoal-stripped (CSS)media prepared by supplementing RPMI 1640 without phenolred with charcoal-stripped FBS. All cells were maintained at 37�Cin a humidified incubator with 5% carbon dioxide. ENZ orMDV3100 (catalog no. 50-101-3979) and INDO (catalog no.AAA1991006) were purchased from Fisher Scientific. Stock solu-tions (200mmol/L) of ENZ, INDO, baccharin,KV-32, andKV-37were prepared in DMSO and were serially diluted for cell culturetreatments to a final concentration range of 1 to 200 mmol/L,maintaining the final DMSO concentration at less than 1%.

Inhibition of testosterone production by AKR1C3 inhibitorsSystems (200 mL) containing 100 mmol/L potassium

phosphate (pH 7.0), 0.9 mmol/L NADPH, 1.2 to 40 mmol/L4-androstene-3,17-dione (D4-AD; containing 2.6 nmol/L [3H]-4-androstene-3,17-dione), KV-37 (0–10 mmol/L), and 0.3 to10 mmol/L inhibitor plus 1.65 mmol/L AKR1C3 were incubatedat 37�C, and samples were prepared as previously reported (40).Quantification of the individual steroid analytes was achieved byintegrating the radioactivity corresponding to each peak in theradiochromatogram and representing the radioactivity as a per-centage of the total corrected cpm for the organic fraction fromwhich it was derived.

Stability testing of AKR1C3 inhibitorsBaccharin,KV-37,anda related ester analogueKV-32 (2mmol/L

solution in DMSO) were incubated with male CD-1 murine liverS9 fractions containing a NADPH-generating system for 0 to 240minutes at 37�C. Reactions were quenched with 0.5 mL (1:1) ofacetonitrile (ACN) containing 0.2% formic acid and 100 ng/mLinternal standard (IS; final conc. ¼ 50 ng/mL). Samples werevortexed for 15 seconds, incubated at room temperature for 10minutes, and spun for 5 minutes at 2,400 rpm. The supernatant(0.9 mL) was then transferred to an Eppendorf tube, clarified bycentrifugation for 5 minutes at 13,200 rpm at 4�C, and trans-ferred to an HPLC vial for analysis by Qtrap 4000 mass spec-trometry using the following parameters: Ion Source/Gas Para-meters: curtain gas (CUR) ¼ 35, collisionally activated disso-ciation (CAD) ¼ medium, ionspray voltage (IS) ¼ 4,500,temperature (TEM) ¼ 600, nebulizing gas GS1 ¼ 70, dryinggas GS2 ¼ 70. Buffer A: dH2O þ 0.1% formic acid; Buffer B:ACNþ 0.1% formic acid; flow rate 1.5 mL/min; column AgilentC18 XDB column, 5 mm packing 50 � 4.6 mm size; 0–1 min97% A, 1–2.5 min gradient to 99% B, 2.5–3.5 min 99% B, 4–4.1 min gradient to 97% A, 4.1–4.5 min 97% A; IS: Warfarin (inMeOH, transition 307.3 to 160.9). Ion transitions followedwere 363.1 [M-H] to 186.9 for baccharin and KV-32, and 362.1[M-H] to 318.1 for KV-37.

Cell viability assaysCells were seeded at a density of 10,000 cells/well in 96-well

plates and were incubated in either normal media (24 hours) or

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CSS media (48 hours). Treatments with ENZ, INDO, KV-37, orcombinations of ENZ and KV-37 were made with or without10 nmol/L D4-AD (AKR1C3 substrate) and incubated at theindicated timepoints (24, 48, 72, and 96hours). For pretreatmentexperiments, cells were treated with KV-37 for 24 hours followedby the addition of ENZ and incubated for a further 72 hours. Cellviability was determined by the MTS tetrazolium dye assay asdescribed previously (38). No intrinsic absorbance was notedwith either KV-37 or ENZ in the MTS assay.

Inhibition of testosterone production in LNCaP1C3 cellsLNCaP1C3 cells (1.5 � 106 cells per well) were placed in 2 mL

of phenol red–free RPMI, 5% CD-FBS, 2 mmol/L L-glu, and 1%

P/S. Cells were incubated with DMSO, or 10 or 30 mmol/L KV-37for 30 minutes at 37�C. After the preincubation, 100 nmol/L 3HD4-AD final concentration was added to the wells and cells wereincubated for 24 hours at 37�C. Cell media were transferred intolabeled borosilicate glass tubes after 48 hours and extracted with2 mL ethyl acetate. Samples were vortexed for 30 seconds andplaced in a –20�C freezer for 1 hour to separate the phases. Thefrozen samples were allowed to thaw at room temperature for 10minutes and centrifuged for 20 minutes before the organic phasewas extracted and the process repeated. The 4 mL organic layerextract was then dried under vacuum for radiochromatography.The aqueous fractions were dried under vacuum for 30minutes toevaporate any leftover organic solvent residue. Note that 1%

Figure 1.

KV-37 induces potent antitumoractivity in prostate cancer cell lines.A, Structure of KV-37. Percentagecell viability of 22Rv1 cells cultured in(B) normal media, (C) CSS media, and(D) normal media supplemented with10 nmol/L D4-androstenedione, and(E) CSS media supplemented with10 nmol/L D4-androstenedione.Percentage cell viability of LNCaP cellscultured in (F) normal media and (G)CSS media. H, IC50 values of KV-37determined in 22Rv1 and LNCaP cells.I, Comparative expression of AKR1C3in prostate cancer cell lines and normalprostate cells (left) and overexpressionof AKR1C3 in 22Rv1 cells when culturedin CSS media (right). Data shown asmean � SD of at least threeindependent experiments, n ¼ 6. NM,normal media.

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glacial acetic acid aqueous solution (25 mL) was added to thesamples to adjust the pH to 6.5, followed by addition of 18 mL of25U/mL Escherichia coli b-glucuronidase. Samples were placed in awater bath at 37�C for 24 hours. Samples were then extracted asbefore. All samples were resuspended in 100 mL of ethyl acetateand vortexed for 30 seconds to mix. Using 10 mL capillary tubes,each sample was spotted onto a multi-channel UNIPLATE 20 �20 cm TLC plate and analyzed as described before (32). The peakof testosterone observed in LNCaP cells represented the basalformation of testosterone that was independent of AKR1C3,because LNCaP cells are AKR1C3 null. In the presence of10mmol/LAKR1C3 inhibitor, the amountof testosterone formedin the LNCaP1C3 cells was identical to the basal level in LNCaPcells showing complete inhibition of AKR1C3-mediated testos-terone production.

Apoptosis assayAnnexin V/FITC apoptosis assay was performed using a kit and

according to the manufacturer's instructions (BD Biosciences,catalog no. 556547). Cells were seeded at a density of 0.2 �106 cells/well in 24-well plates and incubated in CSS media for48 hours followed by treatment with KV-37 for 48 and 72 hours.For pretreatment experiments, cells were treated with KV-37 for24 hours followed by the addition of ENZ and incubated for afurther 72 hours. After appropriate treatments, the samples wereanalyzed by flow cytometery (Accuri C6).

Western blottingWhole cell lysates were prepared using 4% (w/v) CHAPS in

urea-tris buffer. Protein was quantified and electrophoresed asdescribed previously (41). The membranes were probed withprimary antibodies overnight against AR (Cell Signaling Technol-ogy; #5153P, rabbit mAb), PSA (Cell Signaling Technology;#5877S, rabbit mAb), AKR1C3 (Sigma; #A6229, mouse mAb),C-PARP (Cell Signaling Technology; #5625S, Rabbit mAb),C-caspase3 (Cell Signaling Technology; #9661S, rabbit mAb),and actin (Sigma; #A5441, mouse mb) followed by incubationwith anti-mouse (Sigma; #SAB4600224) or anti-rabbit(Perkin Elmer; #NEF812001EA) secondary antibody (1:2,000)horseradish peroxidase conjugate for 2 hours and detected bychemiluminescence (42). All the antibodies from Cell SignalingTechnology were diluted 1:1,000, AKR1C3 (1:500), and actin(1:2,000). Bands were quantified by densitometry using ImageJsoftware.

Tumor xenograft and pharmacokinetic studyAll animal experiments were carried out according to approved

Institutional Animal Care and Use Committee protocols at theUniversity of Texas Southwestern Medical Center (Dallas, TX).Eighteen 6- to 8-week-old female NOD-SCID mice (low circulat-ing testosterone) were implanted with 4 � 106 22Rv1 cells in avolume of 0.1 mL in the left flank. When tumor volume reachedapproximately 100 mm3 (day 13), mice were randomly dividedinto two groups. One group was administered 20 mg/kg KV-37QD intraperitoneally, and the other group was given vehiclecontrol (10% DMSO, 20% PEG400, 0.5% tween 80, 69.5%carbonate buffer, pH ¼ 9.2). Tumor volumes were measuredtwice a week with Vernier calipers, and tumor volume wascalculated as (L � W2) � 3.14)/6 (41). On day 34, mice werehumanely euthanized, and tumors were collected, weighed, andfrozen in liquid nitrogen after taking pictures. Tumors were lysed

for Western blot analysis. Whole blood was collected in anEppendorf tube with ACD anti-coagulant for plasma separationfor pharmacokinetic evaluation of KV-37.

Pharmacokinetic evaluation of KV-37For Standards, 98 mL of blank plasma was added to an Eppen-

dorf and spikedwith 2mL of IS (Warfarin). ForQCs, 98.8mL blankplasma was added to an Eppendorf and spiked with 1.2 mL of IS.Note that 100 mL of plasma was mixed with 200 mL of methanolcontaining 0.15% formic acid and 37.5 ng/mL IS (IS final conc.¼25 ng/mL). The samples were vortexed for 15 seconds, incubatedat room temperature for 10minutes, and spun at 13,200 rpm in astandard microcentrifuge. The supernatant was then analyzed byLC-MS/MS as described above to test the stability of the AKR1C3inhibitors.

Statistical analyses and quantification of degree of synergismExperiments were repeated at least thrice, and the statistical

significance was calculated using the Student t test. A P valueof 80%

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inhibition of testosterone production is blocked and the residualtestosterone produced is that seen in untransfected LNCaP cells(Supplementary Fig. S2).

KV-37 exhibits excellent plasma stability above that of theparent and other derivatives

Baccharin undergoes rapid hydrolysis to yield the inactivephenol drupanin (AKR1C3 IC50 ¼ 15 mmol/L; ref. 37). In orderto compare the hydrolytic stability of lead compounds, baccharinand its derivatives were subjected to incubation with mouse S9fractions in the presence of phase I cofactors from 0 to 240minutes. Aliquots were withdrawn and analyzed by LC-MS atvarious time points. As expected, baccharin and a related esteranalogue (KV-32) were readily hydrolyzed (SupplementaryFig. S3). The amide-based inhibitor KV-37 displayed remarkablestability (t1/2 ¼ >240 minutes).

AKR1C3 inhibitor KV-37 exhibits greater reduction of prostatecancer cell viability compared with baccharin

Baccharin and its analogue KV-37 were screened for antineo-plastic effects in a panel of human prostate cancer cell lines(LNCaP, 22Rv1, and LNCaP1C3). Baccharin consistently dis-played no toxicity among all the cell lines tested, up to concen-trations as high as 100 mmol/L (Supplementary Fig. S4). This is inagreement with previous studies (38). However, treatment withKV-37 demonstrated a greater dose and time-dependent reduc-tion in cell viability in androgen-dependent 22Rv1 and LNCaPcell lines. The antineoplastic effect was greater in 22Rv1 cells dueto the higher expression of AKR1C3 as compared with the LNCaPcell line (Fig. 1B–H). For 22Rv1 cells, a significant decline in IC50values (P ¼ 0.0015,

Figure 2.

Pretreatment with KV-37 sensitizes prostate cancer cell lines to the antitumor effects of ENZ. Percentage cell viability of prostate cancer cellswhen pretreated with KV-37 for 24 hours followed by ENZ treatment at indicated concentrations and time points in (A) 22Rv1 cells cultured in normalmedia and (B and C) 22Rv1 cells cultured in CSS media, (D) 22Rv1 cells cultured in normal media supplemented with 10 nmol/L D4-androstenedioneand (E and F) 22Rv1 cells cultured in CSS media supplemented with 10 nmol/L D4-androstenedione, and (G) LNCaP cells cultured in normalmedia and (H) LNCaP cells cultured in CSS media. I, Quantification of the degree of synergism. CI and DRI values for KV-37 and ENZ combinationtreatments in prostate cancer cells. CI and DRI indices generated using CompuSyn software. Data shown as mean � SD of at least threeindependent experiments, n ¼ 6.

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increasing doses of KV-37. After 48- and 72-hour time periodsafter treatment, the cells were harvested and stained with AnnexinV/PI to measure apoptotic cell percentage. The percentage of theapoptotic cell population increasedwithKV-37 concentration in adose- and time-dependent manner (Fig. 4A; SupplementaryFig. S7). Consistent with the results from the cytotoxicity screen-ing, the effectwas significantly greater in the LNCaP1C3 cell line ascompared with the 22Rv1 cells at both time points (P ¼

level of AKR1C3 was noted in 22Rv1 cells at 48 hours, whereasafter 72-hour AKR1C3 expression increased, both changes wereobserved at 25 and 50 mmol/L KV-37 concentrations (Fig. 5A). InLNCaP1C3 cells, a decrease in AKR1C3 expression was noted atonly 50 mmol/L concentration at 48 hours. On the contrary, nochange in AKR1C3 expressionwas observed in the LNCaP1C3 cellline at all concentrations tested at 72 hours after KV-37 exposure

(Fig. 5B). Because AKR1C3 serves as a coactivator for AR (46), itwas prudent to measure AR levels after treatment. We observedthat expression of AR complements AKR1C3 levels andwas foundto be decreased after 48 hours of treatment, whereas at 72 hours,no change in AR levels was observed in both 22Rv1 andLNCaP1C3 cell lines. By contrast, a concentration-dependentdecline in PSA levels was observed with KV-37 in both cell lines

Figure 4.

KV-37 induces apoptosis in prostate cancer cells alone and potentiates the degree of apoptosis when combined with ENZ. A, Dose- and time-dependentincrease in apoptotic cell population after treatment with KV-37 in indicated cell lines as analyzed by Annexin V/PI costaining. B, Increase in apoptotic cellpopulation after treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in indicated cell linesanalyzed 72 hours after ENZ exposure by Annexin V/PI costaining.C,Western blot showing an increase in C-caspase3 and C-PARP levels after treatment withKV-37at indicated concentration and time points in prostate cancer cells. D, Western blot showing a greater increase in C-caspase3 and C-PARP levels with a 24-hourpretreatment combination of KV-37 and ENZ in prostate cancer cells analyzed 72 hours after ENZ exposure. Data shown as mean � SD of at least threeindependent experiments, n ¼ 3. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001; and ns, nonsignificant.

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at 48- and 72-hour treatments. Such findings signify a beneficialtherapeutic outcome inprostate cancer after treatmentwithKV-37(Fig. 5A and B).

Combination of KV-37 with ENZ downregulates AR expressionthat increases after ENZ treatment

Todetermine themechanismof synergistic interactionbetweenKV-37 and ENZ, Western blot analysis was performed on AR,AKR1C3, and PSA. In contrast to previous reports that indicateAR degradation in 22Rv1 cells (47), treatment of 25 mmol/LENZalone increasedAR expression inboth22Rv1 andLNCaP1C3cells signifying drug resistance. By contrast, a combinationtreatment of 25 mmol/L ENZ with 10 mmol/L KV-37 reduced theexpression to less than control levels (Fig. 5C and D). Consistentwith inhibitor treatment alone, the combination produced nochange in AKR1C3 expression levels. However, the combinationof 10 mmol/L KV-37 with 25 mmol/L ENZ completely abrogatesPSA expression in 22Rv1 cells and reduced PSA to controllevels in LNCaP1C3 cells. These data suggest apoptotic celldeath as a primary mechanism of synergistic antitumor effectbetween the two compounds leads to a decline in AR andPSA expression.

Antitumor effect and initial pharmacokinetics of KV-37 in a22Rv1 xenograft model

Treatment with KV-37 at 20 mg/kg per day in female miceharboring 22Rv1 xenografts significantly (P ¼ 0.0003) inhibited

tumor volume by greater than 50% and tumor weight by 35%(P ¼ 0.0179; Fig. 6A–C) as compared with vehicle-treatedcontrols. No significant change in body weight was observed(Fig. 6D), indicative of a nontoxic effect. On day 34, mice wereeuthanized and blood collected at 10, 120, and 360minutes afterKV-37 administration at 20 mg/kg and pharmacokinetic para-meters analyzed. The peak plasma concentration (Cmax) wasdetermined to be 86.5 mg/mL, and Tmax, AUC, Vz/F, and CL/Fwere calculated (Fig. 6E). Further, Western blot analyses of thetumor samples were conducted that showed no significant upre-gulation of AKR1C3 expression in KV-37–treated tumors ascompared with nontreated controls (Fig. 6F).

INDO displays diminished cell viability reduction and a weaksynergistic drug action compared with the more potentinhibitor KV-37

INDO (AKR1C3 Ki ¼ 8 mmol/L for the E.NADPH.Indometh-acin complex) has been shown to reverse ENZ resistance inprostate cancer models (27). The dose–response curve of INDOshowed a weak inhibition of cell viability at 96-hour incubation,IC50 ¼ 112.8 mmol/L (Supplementary Fig. S8A). Further, we alsoadministered INDOas a 24-hour pretreatment to LNCaP1C3 cellsfollowed by ENZ exposure for 72 hours. A moderate synergisticdrug action was observed (CI ¼ 0.32; DRI ¼ 6.4; SupplementaryFig. S8B) as comparedwith the very high degree of synergismwithKV-37 under identical conditions (CI¼ 0.14; DRI¼ 24.8). Thesefindings further bolster the concept that AKR1C3 inhibition by

Figure 5.

Molecular changes in AR signaling following KV-37 treatment. Treatment with KV-37 alone at indicated concentration and time points in (A) 22Rv1 and (B)LNCaP1C3 cells cultured in CSS media. Treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 andENZ in (C) 22Rv1 cells and (D) LNCaP1C3 cells cultured in CSS media analyzed 72 hours after ENZ exposure.






KV-37 sensitizes ENZ-resistant prostate cancer cells to the che-motherapeutic, and is superior to INDO.

KV-37 exhibits no toxicity alone, or in combination with ENZ,in WPMY-1 prostate cells

The nonmalignant prostate stromal cell line WPMY-1 waschosen to evaluate the toxicity of KV-37. Consistent with the

observation that WPMY-1 cells are low expressers of AKR1C3, thereduction of cell viability induced by KV-37 was minimal with96-hour IC50 ¼ 80 mmol/L. At concentrations of 1, 10, and25 mmol/L of KV-37, those used in our study of prostate cancercells, no reduction in WPMY-1 cell viability was observed, anobservation consistent with no loss of body weight in micexenografts upon 20 mg/kg daily dosing. Further, pretreatment

Figure 6.

KV-37 induces tumor growth inhibition in prostate cancer xenografts. Reduction in (A) prostate tumor volume and (B) prostate tumor weight in 22Rv1murine xenografts after treatment with 20 mg/kg KV-37. C, Representative images showing a reduction in tumor size after treatment with 20 mg/kg KV-37. D,Mice body weight after KV-37 or vehicle treatment. E, Pharmacokinetic parameters determined in plasma after treatment with 20 mg/kg KV-37. F, Expressionof AKR1C3 in tumor lysates (left plot), and each blot indicates tumor lysate from individual mouse. Bar graph representation of quantified blots (right plot).

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with KV-37 for 24 hours followed by ENZ exposure for 72 hoursresulted in no loss of cell viability (Supplementary Fig. S8Cand S8D).

DiscussionProstate tumors resistant to AA and ENZ are characterized by

overexpression of steroidogenic enzymes where AKR1C3 isamong the most overexpressed (26, 48, 49). Thus, in situ orintratumoral androgen biosynthesis is a predominant factor oftherapeutic failure with second-line ADT (13, 48). Overexpres-sion of AKR1C3 by stable transfection in prostate cancer cellsimparted ENZ resistance which was overcome by AKR1C3knockdown by shRNA. INDO, a weak AKR1C3 inhibitor, wasalso able to rescue xenografts from ENZ and AA resistance(27, 28). Continuing our efforts to identify potent and selectiveAKR1C3 inhibitors, we synthesized and characterized KV-37, ananomolar inhibitor of AKR1C3 with >100-fold isoform selec-tivity and metabolic stability (38). We demonstrate that thesynergistic effects of KV-37 with ENZ in reducing prostatecancer cell viability are much greater than that seen with INDO(Supplementary Fig. S9) and that the effects of KV-37 aremediated by AKR1C3 inhibition.

We demonstrate that KV-37 inhibits the activity of AKR1C3 inprostate cancer cells by inhibiting the conversion of D4-AD totestosterone. Crucially, KV-37 exerts a preferential antineoplasticeffect in AKR1C3-overexpressing 22Rv1 and LNCaP1C3 cells ascompared with the low expressing LNCaP cell line and nonma-lignant WPMY-1 cells. Furthermore, culture of 22Rv1 cells in CSSmedia, which are devoid of androgens, upregulates AKR1C3expression further and results in greater susceptibility toKV-37--induced antineoplastic effects. Because the physiologicandrogen level in clinical castrate conditions is of the orderof 1 to 10 nmol/L, CSS was supplemented with 10 nmol/LD4-androstenedione to further mimic the CRPC phenotype(44). KV-37 displayed an equally robust inhibition of cellviability in such cultures. The 22Rv1 cell line is inherentlyresistant to ENZ, whereas LNCaP cells became resistant to ENZwhen AKR1C3 was stably expressed in this cell line. Pretreatmentwith KV-37 restored the sensitivity of 22Rv1 cells to ENZ andprovided a strong synergistic effect resulting in >200-fold reduc-tion in chemotherapeutic dose under conditions that mimic theCRPC disease phenotype. The strong synergistic effect was main-tained in LNCaP1C3 cells upon pretreatment with KV-37. Amoderate synergistic effect was also observed in these cells ifKV-37 was coadministered with ENZ. By contrast, as LNCaP cellsare low expressers of AKR1C3, only an additive effect wasobserved, an observation that may be ascribed to the generaltoxicity of KV-37 toward cancer cells as evidenced previously inleukemic cell lines (38).

Insights into the mechanism of apoptosis mediated by KV-37were obtained by measuring an increase in the apoptotic markersC-caspase3 and C-PARP, as well as a dose-dependent increase inAnnexin V/PI after KV-37 treatment. The observation that percentapoptotic cells were greater in LNCaP1C3 cells at any givenconcentration, compared with 22Rv1 cells, corroborates AKR1C3as the target ofKV-37. In combinationdrug treatments, 25mmol/LENZ was employed, as this corresponds to the minimum steady-state plasma concentration of 12 mg/mL of the drug, the physi-ological concentration achieved clinically in prostate cancerpatients at 150 mg/day dosing regimen (17). A subtherapeutic

concentration of KV-37 (10 mmol/L), which exerts no more than20% reduction in prostate cancer cell viability, was chosen for thedrug combination treatments. In combination drug treatments,Annexin V/PI staining demonstrated a significant increase in thepercentage of apoptotic cells compared with either treatmentalone, which was confirmed by an increase in C-caspase3 andC-PARP levels. These data indicate that apoptotic cell death is aprimary mechanism of the synergistic drug effect. Androgen-dependent gene expression was analyzed after KV-37 treatment,and a dose-dependent reduction in PSA levels was observed.Although the activity of AKR1C3 was inhibited by KV-37, theprotein expression levels either remained unchanged or slightlyincreased in 22Rv1 cells, after treatment, indicating a feedbackincrease in AKR1C3 expression. Interestingly, we observed anincrease in the AR expression levels after treatment with ENZalone in both 22Rv1 and LNCaP1C3 cell lines, which is one of thesignatures of drug resistance. A combination of ENZ with KV-37reduced AR expression to control levels or less. Further, treatmentwith KV-37 alone in 22Rv1 murine xenografts reduced tumorvolume by more than 50%, whereas no change in mouse bodyweightwas observed. In order to compare the degree of synergisticdrug interaction exhibited by the weak AKR1C3 inhibitor INDO(Ki ¼ 8 mmol/L) with KV-37 (Ki ¼ 3 mmol/L), pretreatmentexperiments with INDO were conducted in LNCaP1C3 cellsfollowed by ENZ treatment that revealed a moderate degree ofdrug synergism, much lower than that achieved with KV-37. Thisstudy serves as proof of concept that AKR1C3 inhibition has thepotential to overcome ENZ resistance and corroborates previousfindings (27). Further, we also analyzed the activity of KV-37alone and in combination with ENZ in the nonmalignant humanprostate stromal cell line WPMY-1. No reduction in cell viabilityup to 25 mmol/L was observed, whereas no cell viability reductionin combination experiments was noted, indicating selectivity tocells overexpressing AKR1C3.

In conclusion, we highlight the potential of KV-37 as acombination therapy with ENZ for CRPC because it retardsENZ-resistant prostate cancer cell growth and induces apopto-sis. Furthermore, it is conceivable from the results of this studythat the structurally novel AKR1C3 inhibitor KV-37 scaffold canbe developed as a stand-alone therapeutic for prostate cancermanagement.

Disclosure of Potential Conflicts of InterestK. Verma has ownership interest (including stock, patents, etc.) in a

provisional patent application describing KV-37. T.M. Penning reportsreceiving commercial research grant from Forendo and other commercialresearch support from Constellation, and is a consultant/advisory boardmember for Research Institute for Fragrance Materials, Markey CancerCenter, Mailman School of Public Health, Columbia U, and EOSHI, RutgersUniversity. P.C. Trippier has ownership interest (including stock, patents,etc.) in a provisional patent application describing KV-37. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K. Verma, N. Gupta, T.M. Penning, P.C. TrippierDevelopment ofmethodology:K. Verma,N.Gupta, T. Zang, P.Wangtrakluldee,S.K. Srivastava, T.M. Penning, P.C. TrippierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee,T.M. PenningAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee,S.K. Srivastava, T.M. Penning, P.C. Trippier






Writing, review, and/or revision of the manuscript: K. Verma, T. Zang,P. Wangtrakluldee, S.K. Srivastava, T.M. Penning, P.C. TrippierAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. ZangStudy supervision: K. Verma, S.K. Srivastava, T.M. Penning, P.C. Trippier

AcknowledgmentsWe are indebted to Drs. Noelle Williams and Sukesh Voruganti at the

University of Texas Southwestern Medical Center Preclinical PharmacologyCore for conducting in vivo pharmacokinetic studies and tumor measurements.

Financial support for thisworkwas providedby Texas TechUniversityHealthSciences Center (to P.C. Trippier).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 30, 2017; revised March 1, 2018; accepted June 4, 2018;published first June 11, 2018.

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2018;17:1833-1845. Published OnlineFirst June 11, 2018.Mol Cancer Ther Kshitij Verma, Nehal Gupta, Tianzhu Zang, et al. Adenocarcinoma CellsPotentiates Enzalutamide in Combination Therapy in Prostate AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and

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AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and ...enzyme synthesizing intratumoral testosterone (T) and 5a-dihydrotestosterone (DHT), the enzyme represents a promis-ing