Cancer Research The Bioreductive Prodrug PR-104A Is ... · and PR-104A (0–150 μmol/L). Reactions...

Published OnlineFirst February 9, 2010; DOI: 10.1158/0008-5472.CAN-09-3237

Therapeutics, Targets, and Chemical Biology
Cancer
Research

The Bioreductive Prodrug PR-104A Is Activated under AerobicConditions by Human Aldo-Keto Reductase 1C3

Christopher P. Guise1, Maria R. Abbattista1, Rachelle S. Singleton1, Samuel D. Holford1, Joanna Connolly1,Gabi U. Dachs2, Stephen B. Fox3, Robert Pollock4, Justin Harvey4, Parry Guilford4,Fernando Doñate5, William R. Wilson1, and Adam V. Patterson1

Abstract

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PR-104, currently in phase II clinical trials, is a phosphate ester pre-prodrug which is converted in vivo to itscognate alcohol, PR-104A, a prodrug designed to exploit tumor hypoxia. Bioactivation occurs via one-electronreduction to DNA crosslinking metabolites in the absence of oxygen. However, certain tumor cell lines acti-vate PR-104A in the presence of oxygen, suggesting the existence of an aerobic nitroreductase. Microarrayanalysis identified a cluster of five aldo-keto reductase (AKR) family members whose expressions correlatedwith aerobic metabolism of PR-104A. Plasmid-based expression of candidate genes identified aldo-keto reduc-tase 1C3 as a novel nitroreductase. AKR1C3 protein was detected by Western blot in 7 of 23 cell lines andcorrelated with oxic PR-104A metabolism, an activity which could be partially suppressed by Nrf2 RNAiknockdown (or induced by Keap1 RNAi), indicating regulation by the ARE pathway. AKR1C3 was unableto sensitize cells to 10 other bioreductive prodrugs and was associated with single-agent PR-104 activityacross a panel of 9 human tumor xenograft models. Overexpression in two AKR1C3-negative tumor xenograftmodels strongly enhanced PR-104 antitumor activity. A population level survey of AKR1C3 expression in 2,490individual cases across 19 cancer types using tissue microarrays revealed marked upregulation of AKR1C3 in asubset including hepatocellular, bladder, renal, gastric, and non–small cell lung carcinoma. A survey of normaltissue AKR1C3 expression suggests the potential for tumor-selective PR-104A activation by this mechanism.These findings have significant implications for the clinical development of PR-104. Cancer Res; 70(4); 1573–84.©2010 AACR.

Introduction

Bioreductive prodrugs are designed to provide targetedrelease of toxins in tumors. Enzymatic addition of one (1e−)or two (2e−) electrons initiates the formation of DNA reactivespecies. 2e− reduction of quinone-based prodrugs (e.g., mito-mycin C, apaziquone) could provide tumor selectivity becauseof the elevated expression of enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) in some tumors (1). Nitrohe-terocyclic compounds are rarely substrates for 2e− reduction,with the notable exception of tretazicar (CB 1954), an aziridi-

ffiliations: 1Auckland Cancer Society Research Centre,Medical Sciences, The University of Auckland, Auckland,and; 2Department of Pathology, University of Otago,rch, New Zealand; 3Department of Pathology, PeterCancer Centre, East Melbourne, Victoria, Australia; 4Pacificchnology, Ltd., Dunedin, New Zealand; and 5Proacta, Inc.,California

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

ding Author: Adam V. Patterson, Auckland Cancer Societyentre, The University of Auckland, Private Bag 92019, Auck-Zealand. Phone: 64-9373-7599, ext. 86941; Fax: 64-9373-il: [email protected].

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nyl dinitrobenzamide substrate for both NQO1 and NQO2 (2).More typically, nitroreduction in human tissues occurs via 1e−

addition in a process that can be inhibited by molecular ox-ygen. This redox relationship serves to restrict net metabo-lism to hypoxic cells which, together with the absence ofoxygen-insensitive (2e−) nitroreduction, underlies the utilityof nitro compounds as hypoxia imaging agents (e.g., PIMO,F-MISO, EF5, and FAZA; ref. 3) and as hypoxic cytotoxins(e.g., CI-1010, PR-104, TH-302, NLCQ-1, and KS119W; ref. 4).Clinical development of this class of agents reflects the prev-alence of hypoxia in solid tumors, the recognition of its neg-ative effect on treatment outcome and the strong associationswith malignant progression (5, 6).PR-104 is a water-soluble phosphate ester that is hydro-

lyzed rapidly in vivo to the corresponding alcohol PR-104A,a bioreductive prodrug (7). PR-104A is a dinitrobenzamidemustard that undergoes nitro reduction to hydroxylaminePR-104H and amine PR-104M, which are DNA cross-linkingmetabolites able to diffuse locally and kill neighboring cells.Cytochrome P450 reductase (POR) is an important 1e− oxido-reductase that accounts for the majority (∼60%) of anaerobicPR-104A activation in anoxic SiHa cells in vitro (8). Back-oxidation of the initial nitro radical is an efficient process,with 50% inhibition by as little as 0.1 μmol/L of oxygen (9).Consistent with this oxygen-sensitive mechanism, PR-104Ais 5 to 120 times more cytotoxic under anoxia for all neoplastic

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cell lines tested and PR-104 has striking antitumor activity inhuman tumor xenograft models when combined with antican-cer agents that spare hypoxic cells (7). These properties haveled to the clinical evaluation of PR-104 (10).Despite clear evidence for hypoxia-selective activation, the

oxygen concentration dependence of PR-104A cytotoxicity inSiHa cultures shows an asymptote at high oxygen levels (9),consistent with a residual oxygen-independent mechanism,with metabolism (8) and DNA crosslinking (11), being dueto aerobic bioreduction to PR-104H/M, as for the hypoxicmechanism. In addition, xenograft growth inhibition follow-ing single-agent PR-104 treatment seems to track with aero-bic sensitivity of the corresponding cell lines in vitro (7).Taken together, these observations suggest the existence ofan oxygen-independent 2e− metabolic pathway.In a previous study we tested whether NQO1 might be re-

sponsible for this aerobic activation of PR-104A. Cellularsensitivity to PR-104A weakly correlated with NQO1 enzy-matic activity in a panel of eight cell lines, but overexpressionof the enzyme showed that NQO1 itself is not a PR-104A re-ductase (8). This led us to suggest that NQO1 is coordinatelyregulated with an unknown oxidoreductase responsible for2e− reduction of PR-104A. Given the NQO1 gene is regulatedby nuclear factor erythroid 2-related factor 2 (Nrf2; refs. 12,13), other 2e− oxidoreductases regulated by the Keap1-Nrf2-ARE antioxidant response pathway would be likelycandidates.Here, we identify candidate aerobic PR-104A reductases by

correlating gene expression with aerobic PR-104A reduction.By expressing the most highly correlated of these genesin HCT116 cells, we show that the PR-104A reductase isaldo-keto reductase 1C3 (AKR1C3), a ketosteroid reductase(14) not previously described as a nitroreductase. We confirmthat AKR1C3 is a bona fide oxygen-insensitive PR-104A re-ductase using purified enzyme, that it does not activate otherbioreductive prodrugs, and that it contributes to the activityof PR-104 in human tumor xenografts. In a tissue microarray(TMA) survey of AKR1C3 expression in surgical samples,we show that AKR1C3 is highly expressed in some humantumors.

Materials and Methods

Compounds. PR-104 was supplied by Proacta, Inc. PR-104A, PR-104H, and tetradeuterated derivatives were synthe-sized, purified, and stored as described previously (15, 16).Sources of other compounds are in Supplementary Table S1.Cell lines, cytotoxicity assays, and PR-104A metabolism.

Cells were maintained in culture as described (7, 17) with<6 mo cumulative passage from sources (see SupplementaryTable S2). Antiproliferative and clonogenic survival assayswere performed as previously described (7, 17) and measure-ment of metabolism of PR-104A to hydroxylamine PR-104Hand amine PR-104M by liquid chromatography-tandem massspectrometry (LC/MS/MS) as before (11).RNA expression microarray and data analysis. Micro-

array-based RNA expression profiles covering 38,500 probes(Affymetrix HG-U133 Plus 2.0) were obtained for cultures of

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23 human tumor cell lines. RNA was purified using Trizol-chloroform extraction (Invitrogen) and RNeasy columns(Qiagen). Both RNA purification and Affymetrix array ana-lysis were conducted according to the manufacturers'instructions. The raw data are available from ArrayExpress.6

Using the “GO” and “hgu133plus2” packages from Biocon-ductor (18), we searched for all descendants of the nodeGO:0016491 (oxidoreductase activity) to obtain a list ofprobes corresponding to 370 oxidoreductase gene productswhich were analyzed by unsupervised hierarchical clustering.To identify candidate PR-104A reductases, we calculated thecorrelation coefficient (Kendall τ) between gene expressionand aerobic PR-104A metabolism (to PR-104H/M) across allprobe sets; correlation P values were adjusted using the falsediscovery rate correction (Limma's empirical Bayes adjusted;ref. 19) for multiple comparisons.Candidate gene expression. Plasmids encoding sequence-

confirmed open reading frames for AKR 1B1, 1B10, 1C1, 1C2,1C3, 1C4, and NQO1 were purchased (SupplementaryTable S3) and cloned into the Gateway compatible vectorF527-V5, constructed from pcDNA6.2V5DEST (Invitrogen)and a modified version of the pEFIRES-P plasmid (20).F527-V5 provides the transcription of a bicistronic mRNAwhich encodes the oxidoreductase and pac (puromycin resis-tance) open reading frames, the former harboring an occultCOOH-terminal V5-tag inducible by infection with AdV5 ex-pressing a TAG suppressor tRNA (Tag-on-Demand, Invitro-gen). HCT116 and H1299 cells were transfected usingFugene6 and pooled stable populations selected with puro-mycin as previously described (21). Functional activity ofclones was established by incubating cells with the fluoro-genic AKR1C probe coumberone (ref. 22; 5 μmol/L, 37°C,3 h) using a fluorescent platereader (Molecular DevicesSpectraMax M2; Ex/Em 385/510 nm) and standard curves ofauthentic reduced coumberone.Western immunoblot analysis. Cell lysates were prepared

in radioimmunoprecipitation assay buffer and 10 to 20 μg ofprotein was loaded on SDS-PAGE gels (4–12% gradient gelsor 12% gels), transferred, blocked and probed with pri-mary antibodies (Supplementary Table S4) and detectedusing chemiluminescent ECL detection (Supersignal,Thermoscientific).RNAi knockdown of Nrf2 and Keap1 mRNA. Stealth short

interfering RNA (siRNA; Invitrogen) against Nrf2 (HSS107128)were tested in A549 and H460, or against Keap1 (HSS114801)in HCT116 and MDA231 cells. Cells (106/10 cm dish) weretransfected with 10 nmol/L of siRNA and LipofectAMINERNAiMAX transfection reagent (Invitrogen), harvested 72 hlater by trypsinization, and immunoblotted and assayed forPR-104A metabolites as above.Kinetics of PR-104A reduction by recombinant AKR1C3.

Recombinant AKR1C3 was diluted to 2 μmol/L in potassiumphosphate buffer [100 mmol/L (pH 7.4), 37°C] containingNADPH (100 μmol/L), bovine serum albumin (0.5 mg/mL),

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Aerobic Activation of PR-104A by AKR1C3


and PR-104A (0–150 μmol/L). Reactions (400 μL) weremonitored by absorbance (340 nm, Agilent 8453E diode arrayspectrophotometer) and initial rates determined. Productswere analyzed with an Agilent LC/MS (7), following incuba-tion for 10 min in the same (aerobic) reaction mix or underanoxic conditions (Coy anaerobic chamber).Antitumor activity in human tumor xenografts. Animal

studies were approved by the University of Auckland AnimalEthics Committee and international guidelines were fol-lowed. Tumors were grown subcutaneously in the flank offemale NIH-III or CD1 nude mice (Charles River Laborato-ries) by inoculating 5 × 106 cells in 100 μL αMEM. To evalu-ate clonogenic survival, tumors (800–1500 mm3) weredissected 24 h after single dose PR-104 and plated as de-scribed (7). Tumor growth delay experiments were under-taken as described previously (7). Median time for tumorsto increase in volume 4-fold (RTV4) was determined, andtumor growth inhibition (TGI) was calculated as the per-centage of increase in RTV4 for treated over control (statis-tical difference tested by Mann-Whitney U test usingSigmaStat v3.5).Immunostaining for AKR1C3 and hypoxia in xenografts.

Formalin-fixed tumors were sectioned (5 μm) and immuno-stained using anti-AKR1C3 monoclonal antibody (ref. 23;Sigma) and visualized with the EnVision Dual Link-HRP/DAB kit (Dako). For detection of hypoxia, mice were dosedi.p. with 60 mg/kg of pimonidazole (Hypoxyprobe-1 kit;Chemicon Int) 90 min before removing tumors. To determinethe pimonidazole adducts, half of each tumor was formalin-fixed for immunofluorescence microscopy and the other halfdissociated enzymatically for flow cytometry according tothe manufacturer's instructions.AKR1C3 expression in human tumor TMA. TMAs were

sourced as shown in Supplementary Table S5. A total of3,932 individual cores representing 19 cancer types (2,490cases) were analyzed across 38 TMAs with an average of207 cores per disease (median, 174; range, 31–452). Method-ology optimization was carried out and cross-validatedagainst paired frozen samples by Western blot. Slides wereimmunostained for AKR1C3 as for tumor xenografts andcores were scored for staining intensity and proportion ofAKR1C3-positive neoplastic cells by a certified pathologist(S.B. Fox) using a semiquantitative measure on a 7-pointscale ranging from negative (score 0) to diffuse strong stain-ing (score 6). This measure was applied to the neoplastic cellelement of the tumors within the TMAs and the epithelialelements within the normal TMA. An unrelated series of nor-mal tissue, NSCLC and breast cancer sections were evaluatedindependently by Mosaic Laboratories, LLC.

Results

PR-104A is activated by an oxygen-independent processin a subset of cultured cell lines. As reported previously forSiHa cells (8), the flavoenzyme inhibitor diphenyliodoniumprevented the anoxic cytotoxicity of PR-104A in A549 cells(Fig. 1A). However, it had no effect on the aerobic cytotoxi-city of PR-104A. In contrast, ketoconazole (identified from a

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chemical inhibitor screen; Supplementary Fig. S1), blockedaerobic but not anoxic cytotoxicity (Fig. 1A), suggesting thatdifferent enzymes mediate the activation of PR-104A underthese two conditions. To distinguish metabolism-dependentfrom intrinsic cell sensitivity, inhibition of aerobic cellproliferation by PR-104A and its cytotoxic metabolite PR-104H were compared across 12 cell lines (Fig. 1B). A consis-tent ∼20-fold PR-104 A/H deactivation ratio was found forseven cell lines (r2 = 0.96), whereas in five cell lines (A549,H460, SiHa, 22Rv1, and HT29) PR-104A showed anomalouslyhigh toxicity. Notably, these five cell lines were recentlyshown to have high rates of aerobic reduction of PR-104A(11). Taken together, these results suggest variable ex-pression of an aerobic PR-104A reductase in human tumorcell lines.Aerobic PR-104A metabolism correlates with AKR family

regulation. To identify candidate PR-104A oxidoreductases,we examined gene expression by microarray profiling andcompared these data with aerobic reduction of PR-104Ato PR-104H/M, adding a further 11 cell lines to the above12 cell line panels to increase statistical power. Unsupervisedhierarchical clustering of oxidoreductase gene expressionidentified two distinct groups, defined largely by membersof the AKR superfamily and other Keap1/Nrf2/ARE regulatedgenes such as NQO1 (“Nrf2 cluster”; Fig. 1C; SupplementaryFigs. S2 and S3). Using an LC/MS/MS assay to quantify PR-104A reduction (Fig. 1D) we showed large differences be-tween the same cell lines (160-fold range) with the fastmetabolizers associated with the “Nrf2 cluster.” A principalcomponent analysis, accounting for false discovery (adjustedP value < 0.1), identified 20 probes positively correlatedwith PR-104H/M formation (Supplementary Table S6), 8 ofwhich corresponded to five members of the AKR family(1C1, 1C2, 1C3, 1C4, and 1B10).Aerobic PR-104A nitroreduction is mediated by AKR1C3.

These five AKR1 candidates, along with AKR1B1 andknown aerobic nitroreductase, NQO1, were expressed byplasmid transfer into HCT116 cells and stable populationstested for aerobic PR-104A metabolism. Only AKR1C3transfectants were able to generate PR-104H/M (Fig. 2A),despite all the candidate reductases being immunodetectedfollowing induced translation of a cryptic COOH-terminalV5-tag (Fig. 2B). Expression of AKR1C3, AKR1B10, andNQO1 was confirmed independently using monoclonalantibodies and an AKR1C1 polyclonal with cross-reactivityfor other AKR1C family members (Fig. 2B; antibody detailsSupplementary Table S4). All AKR1C family transfectantsreduced the fluorogenic substrate coumberone (22),confirming the functional expression of these enzymes(Fig. 2C). We next confirmed that AKR1C3 itself is a 2e−

PR-104A reductase by demonstrating that purified recom-binant AKR1C3 catalyses NADPH-dependent formation ofPR-104H in the presence or absence of oxygen (Fig. 2D).The reaction followed Michaelis-Menten kinetics with an ap-parent Km of 20.6 ± 2.6 μmol/L and Kcat of 0.800 ± 0.025 min−1

(Supplementary Fig. S4).Evaluation of AKR1C3 expression in the 23 cell line panel

by Western blotting (Fig. 3A) showed highly variable protein

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Figure 1. Evidence for an aerobic PR-104A reductase in some human tumor cell lines. A, clonogenic survival curves of A549 cells following a 1-h exposureto PR-104A with and without 100 μmol/L of diphenyliodonium (DPI) or ketoconazole (KTZ), which were added 2 h before PR-104A. Values are mean ± rangefor duplicate samples. B, antiproliferative potency following 4 h of aerobic exposure to PR-104A or PR-104H. Values are mean ± SEM for three experiments.The solid line is the regression through the circles. C, gene expression profile of a panel of 23 human tumor cell lines by unsupervised hierarchicalclustering analysis (Cluster3.0) of probes associated with representative descendents of the gene ontology term “oxidoreductase activity” (GO:0016491), i.e.,GO:0004997, GO:0016616, and GO:0016651. D, aerobic metabolism of PR-104A (100 μmol/L, 1 h) in a panel of 23 human tumor cell lines, by LC/MS/MSquantitation of active metabolites PR-104H and PR-104M. Values are means and error bars show the SEM for total metabolites from two to eight experiments.

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levels; linear regression of PR-104H/M formation (Fig. 1D)versus AKR1C3/actin (from Fig. 3A) showed a highlysignificant correlation (r2 = 0.83; P < 0.001), which was stron-ger than the correlations with AKR1B10/actin (r2 = 0.27; P =0.011) or NQO1/actin (r2 = 0.17; P = 0.053).AKR1C3 expression is regulated in part by Nrf2. Given

the discordant relationship between NQO1 and AKR1C3levels across the 23 cell line panel in vitro (r2 = 0.19), we evalu-ated the role of the Nrf2 in the expression of AKR1C3. RNAinterference of Keap1, a specific Nrf2 repressor, by siRNAtransfection in MDA231 cells induced AKR1C3 protein levels5.7-fold (Fig. 3B) and aerobic PR-104A metabolism 7.0-fold(Fig. 3B), with attendant 4.5-fold induction of an ARE-luciferasereporter (Supplementary Fig. S5A). In analogous experiments,Nrf2 siRNA transfection of A549, in which Nrf2 is constitutivelyactivated by a Keap1 point mutation (24, 25), suppressed



ARE reporter activity (Supplementary Fig. S5B), AKR1C3 andNQO1 expression, and aerobic PR-104A metabolism (Fig. 3B).Similar results were obtained with H460 cells (SupplementaryFig. S5C). However, Keap1 siRNA increased NQO1 but notAKR1C3 (or PR-104A reduction) in HCT116 cells (Supplemen-tary Fig. S5D). These results suggest a role for Nrf2 in inductionof AKR1C3 expression, but that additional cell line–specific fac-tors result in differential regulation relative to NQO1.AKR1C3 expression sensitizes cells to PR-104A but not to

other bioreductive drugs. To test whether AKR1C3 ex-pression enhances the cytotoxicity of PR-104A, we isolatedtwo clones from the pool of HCT116 cells transfected withAKR1C3. Clonogenic survival curves showed clone no. 1 tobe 10-fold more sensitive to PR-104A than the parentalcells under aerobic conditions, and was further sensitized(44-fold) under anoxia (Fig. 3C). Using aerobic IC50 assays,

Figure 2. AKR1C3 reduces PR-104A to its cytotoxic metabolites under aerobic conditions. A, aerobic metabolism of PR-104A in HCT116 pools expressingcandidate reductases, determined by LC/MS/MS assay for PR-104H and PR-104M. B, detection of AKR enzymes and NQO1 in stably transfectedHCT116 cell pools by Western blotting. COOH-terminal V5 tags were transiently expressed using an adenoviral encoded TAG suppressor tRNA(Tag-on-Demand, Invitrogen; multiplicity of infection 50, 24 h). C, demonstration of functional expression of AKR1C enzymes using a fluorogenic probe(coumberone, 5 μmol/L, 4 h). Values are mean ± SD for two experiments. D, representative high-performance liquid chromatography chromatogramsdemonstrating reduction of PR-104A (100 μmol/L) to PR-104H by purified recombinant AKR1C3 (2 μmol/L) with 100 μmol/L of NADPH for 10 min underboth aerobic and anoxic conditions.




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we compared the sensitivity of clone no. 1 and no. 2, relativeto the parental line, to PR-104A and 10 other bioreductiveagents including 6 other nitro compounds, 3 quinones anda tertiary amine N-oxide (Fig. 3D). This confirmed the ∼10-fold sensitization to PR-104A, and showed that this activationby AKR1C3 is unique among the bioreductive drugs tested.



AKR1C3 expression is a major determinant of PR-104activity in human tumor xenografts. A panel of nine celllines were grown as subcutaneous solid tumors in nude mice.Expression of AKR1C3, measured by Western blot andimmunohistochemistry (Fig. 4A), was broadly similar tothat in culture. Tumor cell survival determined by ex vivo

Figure 3. Expression of AKR1C3, its regulation by Keap1/Nrf2, and role in PR-104A cytotoxicity. A, AKR1C3, AKR1B10, and NQO1 in human cancercell lines by Western blotting. Cell lines are shown in rank order of aerobic metabolism of PR-104A to PR-104H/M (Fig. 1D). B, induction ofAKR1C3 in (NQO1 mutant) MDA231 cells by Keap1 siRNA and suppression of AKR1C3 and NQO1 in A549 cells by Nrf2 siRNA by Western blotting,with associated changes in PR-104A reduction to PR-104H/M in the same cell populations. Mean ± SEM for triplicate determinations. C, clonogenicsurvival curves of HCT116 wild-type and AKR1C3-overexpressing cells (clone no. 1) exposed to PR-104A for 2 h under aerobic and hypoxicconditions. Mean ± SD for two experiments. D, IC50 ratios of bioreductive prodrugs following 4-h aerobic exposure of HCT116 wild-type and HCT116AKR1C3 (clone nos. 1 and 2).

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clonogenic assay 24 hours after single-dose PR-104 showed atrend to greater activity in tumors with high AKR1C3 levels(Mann-Whitney, P = 0.063; Fig. 4B). However, there was awide range of sensitivities among the AKR1C3-positive tu-mors, leading us to ask whether the extent of hypoxia alsoinfluenced PR-104 sensitivity. Pimonidazole binding, as ahypoxia marker, showed similar trends by flow cytometryand immunostaining (Fig. 4C; see Supplementary Fig. S6for a quantitative comparison of the two assays), but didnot correlate with the activity of PR-104. Indeed, some ofthe least hypoxic tumors (H460 and SiHa) were the most sen-sitive to PR-104, whereas the most hypoxic (H1299 andA2780) were relatively insensitive. However, these relation-ships may be confounded by cell line differences in intrinsicsensitivity to the PR-104H/M active metabolites. To more



clearly identify the role of AKR1C3 in PR-104 monotherapy,we compared xenografts grown from AKR1C3-overexpres-sing H1299 transfectants and parental cells by tumor growthdelay (Fig. 4D). Parental tumors showed modest growth inhi-bition (TGI, 77%; P = 0.03), possibly due to the hypoxic acti-vation of PR-104 with attendant metabolite redistribution(bystander effect). In contrast, AKR1C3-expressing H1299 xe-nografts regressed following PR-104 treatment with a TGI of314% (P < 0.01; Fig. 4D). Hypoxic fraction was similar be-tween these two models (Fig. 4D; data not shown); this estab-lishes a major role for AKR1C3 expression in the response ofthis NSCLC tumor model to PR-104. Similar experimentswith an isogenic pair of HCT116 xenografts differing inAKR1C3 expression corroborated this observation (Supple-mentary Fig. S7).

Figure 4. Expression of AKR1C3 sensitizes human tumor xenografts to PR-104 in vivo. A, detection of AKR1C3 in human tumor xenografts byWestern blotting (three pooled tumors) and representative immunohistochemistry. B, clonogenic cell kill of human tumor xenografts 24 h afterPR-104 monotherapy (348 mg/kg, i.p.). C, assessment of hypoxia in tumor xenografts by pimonidazole binding, assessed by flow cytometry(mean ± SEM for five tumors) and immunostaining (quantified in Supplementary Fig. S4), with H&E staining of parallel sections. D, inhibition of growthof parental or AKR1C3-overexpressing H1299 tumors by PR-104 monotherapy (550 mg/kg/dose, on day 0, 4, 8; seven to eight tumors/group). Images showpimonidazole binding (green) and AKR1C3 immunostaining for representative tumors.




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AKR1C3 expression in human tumor surgical samples isheterogeneous. AKR1C3 was evaluated by immuno-histochemistry using TMAs. A scoring system, illustratedin Fig. 5A, gave higher ranking to uniform over focalstaining with scores of 4, 5, and 6 considered “positive.”Expression of AKR1C3 was present in most tumor types(Fig. 5B). HCC showed the highest frequency of positivecores with most (58%) staining strongly in all cells (score 6).Other disease types with >50% positive cores included blad-der, renal, and gastric carcinomas. A summary of all scores



is shown in Supplementary Table S7. Examination of sub-types of lung carcinoma showed marked heterogeneity, withexpression restricted to NSCLC whereas small cell lung car-cinoma was negative; expression was also present in a highproportion (54%) of lung tumor metastases (Fig. 5C). Toconfirm these TMA observations, a series of lung and breastcancer tissue sections were analyzed by an independentlaboratory using a separate optimized staining protocoland scoring criteria (see Supplementary Methods); 48%(10 of 21) of NSCLC and 14% (3 of 21) breast cancers were

Figure 5. Expression levels of AKR1C3 by immunohistochemistry of human tumor TMAs. A, illustration of scoring system. B, frequency of AKR1C3staining for 2,700 tumors across 19 tumor types. C, frequency of AKR1C3 staining (score >3) for lung cancer subtypes. D, representative stainedTMA showing AKR1C3 expression in liver cancer. Alternate rows are duplicate cores from the same tumor (LVC1501; 36 cases, duplicate cores) andnormal tissue (MBO661; 33 tissues, duplicate cores). Tissue are 1, adrenal; 2, bladder; 3, bone marrow; 4, eye; 5, breast; 6, cerebellum; 7,cerebral cortex; 8 fallopian tube; 9, esophagus; 10, stomach; 11, small intestine; 12, colon; 13, rectum; 14, heart; 15, kidney; 16, liver; 17, lung;18, ovary; 19, pancreas; 20, parathyroid; 21, pituitary; 22, placenta; 23, prostate; 24, skin; 25, spinal cord; 26, spleen; 27, muscle; 28, testis; 29, thymus; 30,thyroid; 31, tonsil; 32, cervix; and 33, endometrium.

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classified as highly positive for AKR1C3 (SupplementaryTables S8 and S9, respectively).AKR1C3 expression in normal tissues. A survey of 33 nor-

mal tissue TMA cores identified small intestine and kidneyas containing moderate numbers of cells with strongAKR1C3 immunoreactivity (score 4), with mixed stainingseen in the liver core (score nos. 5 and 4; Fig. 5D). Occasionalweak positivity (score 1) was seen in bone marrow cell TMAs(morphology undetermined). Notably, the intensity ofnormal tissue staining was substantially less than that seenwith positive neoplasia samples, as illustrated by a side-by-side macro comparison with a set of HCC cores (Fig. 5D).Independently, 23 normal tissue sections were analyzedand showed strong positive AKR1C3 staining in seven tis-sues; stomach, small intestine, colon, pancreas, kidney,uterus and ovary, with weak/diffuse staining in the majorityof liver cells (Table 1). Most specimens showed bothnuclear and cytoplasmic staining but adrenal and livershowed cytoplasmic staining only. Thus, the full section his-



topathology analysis was broadly consistent with the TMAscoring.

Discussion

The observation that certain human neoplastic cell linesare able to convert the bioreductive prodrug PR-104A to itscytotoxic metabolites under oxygenated conditions led us toseek the identity of the putative aerobic oxidoreductase(s).Microarray analysis of 23 cell lines highlighted a cluster offive AKR family 1 members (1B10, 1C1, 1C2, 1C3, and 1C4)coordinately upregulated in a manner that correlatedwith oxic PR-104A metabolism (compare Figs. 1D and 3A).Plasmid-based cel lular expression confirmed AKRfamily 1 member C3 (AKR1C3) as a functional PR-104Anitroreductase. Recombinant AKR1C3, a ketosteroid reduc-tase better known for its role in the pre-receptor regulationof steroid hormones and prostaglandins (14, 26), was able tocatalyze NADPH-dependent reduction of PR-104A to the

Table 1. Pathology review of AKR1C3 immunohistochemical staining in normal tissues

Tissue Type
Staining of distinctive tissue element
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Staining of other cell types

Cells staining at each intensity (%)
Positive (%) End
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SCL 2+ SCL 1+ SCL 0
Adrenal
0 0 5 C 95 5 1+ 1+ NS 3+ 1+ NS NS Cerebellum 0 0 5 N 95 5 1+ 1+ NS 0 B NS NS Cerebrum 0 0 25 CN 75 25 1+ 0 NS 0 0 NS NS Esophagus 0 0 0 100 0 0 2+ 0 2+ 0 2+ NS Colon 15 NC 40 NC 30 CN 15 85 3+ 3+ 0 3+ 0 2+ NS Heart 0 0 0 100 0 0 0 NS 0 0 NS NS Kidney 40 NC 40 NC 10 CN 10 90 3+ 1+ 0 0 1+ NS NS Liver 1 C 10 C 84 C 5 95 3+ 0 0 0 0 0 NS 1 Lung 0 0 0 100 0 0 1+ 0 0 0 3+ NS 2 Ovary 0 50 NC 40 CN 10 90 2+ 2+ 0 2+ 0 NS NS Pancreas 30 NC 20 NC 40 CN 10 90 3+ 3+ 0 1+ 1+F NS NS Pituitary 0 0 0 100 0 0 0 NS 0 0 NS NS Prostate 0 0 0 100 0 0 3+ 0 0 0 1+ NS Skeletal Muscle 0 0 0 100 0 0 3+ 0 0 0 NS 1+ Skin 0 0 0 100 0 0 3+ NS 3+ 0 2+ NS Small Bowel 80 NC 10 NC 10 CN 0 100 3+ 3+ 0 3+ 0 1+ NS Spleen 0 0 1 NC 99 1 1+ 1+ 0 0 0 1+ NS Stomach 50 NC 10 NC 39 CN 1 99 3+ 1+ 0 0 0 1+ NS Salivary Gland 1 NC 1 NC 1 NC 97 3 3+ 2+ 0 0 2+ 0 NS 3 Testis 0 0 3 CN 97 3 1+ 2+ 0 2+ 0 NS NS 4 Thymus 0 10 NC 50 NC 40 60 2+ 1+ 0 0 0 2+ NS Thyroid 0 0 0 100 0 0 2+ 0 0 0 NS NS Uterus 0 50 N 40 N 10 90 2+ 3+ 2+ 0 0 NS NS
NOTE: Pathologists comments: 1. Strong cytoplasmic staining in bile duct epithelium; 2. (Staining in) alveolar macrophages; 3.(Staining in) large duct; 4. (Staining in) Leydig cells.Abbreviations: SCL, subcellular location; N, nuclear; C, cytoplasmic; NC, nuclear < cytosolic; CN, cytosolic < nuclear; Max SI,maximum staining intensity; End, endothelia; Smo, smooth muscle; Fib, fibroblast; Str, stroma; Inf, inflammatory cells; Ner, nerve.

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hydroxylamine metabolite PR-104H in the presence ofoxygen (Fig. 2). Western blotting of the cell line panel shownthat AKR1C3 was expressed in 30% (7 of 23) of cell lines andis correlated with elevated aerobic PR-104A reduction.Expression of NQO1, a prototypic reporter gene for theKeap1-Nrf2-ARE pathway (12), was only weakly correlatedwith AKR1C3 expression, although AKR1C3 has recently beenreported to be regulated by this pathway (27). Keap1 knock-down induced AKR1C3 protein with attendant elevation ofPR-104A metabolism in MDA-231 cells (Fig. 3B), de-monstrating that regulation of AKR1C3 by the Nrf2/Keap1pathway is possible. In support, constitutive AKR1C3 ex-pression in A549 and H460 cells was sensitive to knockdownof Nrf2 activity (as confirmed by ARE-luciferase activity),leading to reduced PR-104A metabolism, and implicatingthe AKR1C3-positive phenotype as being associated withthe known Keap1 mutations in these cell lines (24, 28).Notably, biallelic loss at the Keap1 locus is common in lungcancer cell lines with attendant nuclear accumulation ofNrf2 (24, 29), and expression of this AKR1 gene cluster(including AKR1C3) has been proposed as a biomarker ofNrf2 activation (27). However, Keap1 siRNA induced NQO1but not AKR1C3 expression in HCT116 cells, indicatingthat the two aerobic reductases are differentially regulatedin certain contexts.In vitro, PR-104A displays hypoxia-selective toxicity

across all cell lines tested (hypoxic/aerobic cytotoxicityratio of 5–120; ref. 7). However, the role of hypoxia is lessapparent for PR-104 monotherapy of tumors in which hy-poxic cells are a geometrically constrained minority pop-ulation (Fig. 4C) and metabolite diffusion is mandatoryfor single-agent activity. Consequently, tumor xenograftsensitivity to PR-104 monotherapy (measured by clonogenicsurvival; Fig. 4B) seems to be strongly, but not exclusively,influenced by the more uniform presence of AKR1C3(Fig. 4A) rather than the heterogeneous patterning of hypoxia(Fig. 4C), suggesting that the bystander cell killing effect isnot in itself sufficient to exploit tumor hypoxia in a mono-therapy context. This postulate is supported by severallines of evidence, including the modest growth delayachieved with PR-104 treatment of parental H1299 NSCLCxenografts (TGI, 77%), despite the large hypoxic fraction(62% pimonidazole-positive), and an ability of H1299 cellsto generate PR-104A metabolites 35-fold more efficientlyunder hypoxia (11). Even in this hypoxia and hypoxicmetabolism–rich setting, expression of AKR1C3 is necessaryfor substantial tumor control (TGI, 314%; Fig. 4D). Thissupports our original observation that aerobic cell sensitivitypredicts in vivo activity as measured by tumor growth delay(7), leading to the conclusion that AKR1C3 expression inindividual tumors may be an important determinant indefining the most responsive patient population. However,given that Nrf2 regulates a plethora of gene products asso-ciated with drug resistance (12, 30–32), one possibility isthat cellular resistance mechanisms may concurrently op-pose AKR1C3-dependent sensitivity to PR-104. Nevertheless,it is evident that AKR1C3-rich cell lines such as A549 andH460 are sensitive in vitro and in vivo (7), despite constitu-



tive upregulation of multiple Nrf2-regulated cytoprotectivegenes (24, 28, 30) indicating that, on balance, a PR-104sensitive phenotype prevails.Whether AKR1C3 can be clinically exploited by PR-104

will depend in part on its expression in tumors relative tonormal tissues. Immunohistopathology of normal tissuesshowed the expression of AKR1C3 in a subset of epithelialcells in the stomach, gastrointestinal tract, pancreas,liver, and kidney (Table 1), broadly consistent with literaturefindings (23, 33–35). Some endothelial cell expression wasalso evident. Of potential concern is the immunodetectionof AKR1C3 in a minority of bone marrow cells, particularlyin light of the recent report that CD34+ human myeloidprogenitor cells are positive for AKR1C3 mRNA (36). Thisobservation may be associated with the myelotoxic effectsof PR-104 in humans (10). A population analysis of AKR1C3expression in tumor cores (2490 cases) showed that it isstrongly and frequently upregulated in some carcinomas(Fig. 5B). Across 19 tumor types, the highest frequency ofstrongly positive biopsies was in HCC, with many otherpositive tumor types including bladder, renal, gastric, cer-vix, colon, and NSCLC (Fig. 5B; Supplementary Table S8).Overexpression of AKR1C3 has been documented in carci-nomas of the breast (23, 37, 38), prostate (39–41), endome-trium (42, 43), and kidney (44). Surprisingly, the prostateand breast carcinoma cores did not score highly in ourscreen (see Supplementary Fig. S8 for validation and Sup-plementary Table S9 for independent laboratory analysis ofadditional breast cancer tissue samples), suggesting theseTMAs (Supplementary Table S5) may be sourced fromearly stage disease. Overall, the immunohistochemical anal-ysis shows that the intensity of AKR1C3 expression isstrikingly elevated in certain tumors relative to normaltissues (Fig. 5D). The distribution of AKR1C3 overexpres-sion has prompted the evaluation of PR-104 in HCC andNSCLC (ClinicalTrials.gov identifier NCT00862082 andNCT00862134, respectively). Collectively, these data indi-cate that AKR1C3 is a novel target for PR-104, andAKR1C3 detection may identify individual patients withPR-104–sensitive tumors.

Disclosure of Potential Conflicts of Interest

W.R. Wilson is a stockholder and consultant to Proacta, Inc. A.V. Pattersonis a consultant to Proacta, Inc. The other authors disclosed no potential con-flicts of interest.

Acknowledgments

We thank Proacta, Inc. for PR-104, Graham Atwell and Prof. William Dennyfor PR-104A and tetradeuterated standards, Kashyap Patel for PR-104H,Dianne Ferry and Yongchuan Gu for assistance with bioanalytical methods,Sophie Syddall for construction of the F527-V5 vector, Dan Li forpreparation of high titer Tag-on-Demand adenovirus, Susan Pullen for cellgrowth inhibition experiments, Wouter van Leeuwen for RNA isolation fromcultured cell lines, Dr. Jeffrey Smaill for synthesis of coumberone, Dr.Christopher Squire for recombinant AKR1C3, Prof. David Ross for the gift ofanti-human NQO1 monoclonal antibody, and Helen Morrin for curating tissuesfor the Cancer Society NZ tissue microarray.

Cancer Research





Grant Support

Health Research Council of New Zealand, Program grant 08/103(C.P. Guise, G.U. Dachs, W.R. Wilson, and A.V. Patterson) and Proacta, Inc.(M.R. Abbattista, R. Pollock, J. Harvey, P. Guilford, S. Fox, F. Doñate).



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

Received 8/31/09; revised 11/10/09; accepted 11/30/09; publishedOnlineFirst 2/9/10.

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Cancer Research



2010;70:1573-1584. Published OnlineFirst February 9, 2010.Cancer Res Christopher P. Guise, Maria R. Abbattista, Rachelle S. Singleton, et al. Conditions by Human Aldo-Keto Reductase 1C3The Bioreductive Prodrug PR-104A Is Activated under Aerobic

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