Resistance to CYP17A1 Inhibition with Abiraterone in ...alone in men with CRPC previously treated with docetaxel (18). Although clinical studies of abiraterone have shown responses

Cancer Therapy: Preclinical

Resistance to CYP17A1 Inhibition with Abiraterone inCastration-Resistant Prostate Cancer: Induction ofSteroidogenesis and Androgen Receptor Splice Variants

Elahe A. Mostaghel1,3, Brett T. Marck4, Stephen R. Plymate3,4, Robert L. Vessella5, Stephen Balk6,Alvin M. Matsumoto3,4, Peter S. Nelson1,2,3, and R. Bruce Montgomery3,4

AbstractPurpose: Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor

growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in

reducing tumor androgens is not known, nor havemechanisms contributing to abiraterone resistance been

established.

Experimental Design: We treated human CRPC xenografts with abiraterone and measured tumor

growth, tissue androgens, androgen receptor (AR) levels, and steroidogenic gene expression versus

controls.

Results: Abiraterone suppressed serum PSA levels and improved survival in two distinct CRPC

xenografts: median survival of LuCaP35CR improved from 17 to 39 days (HR ¼ 3.6, P ¼ 0.0014) and

LuCaP23CR from14 to 24 days (HR¼ 2.5, P¼ 0.0048). Abiraterone strongly suppressed tumor androgens,

with testosterone (T) decreasing from 0.49 � 0.22 to 0.03 � 0.01 pg/mg (P < 0.0001), and from 0.69 �0.36 to 0.03 � 0.01 pg/mg (P ¼ 0.002) in abiraterone-treated 23CR and 35CR, respectively, with

comparable decreases in tissue DHT. Treatment was associated with increased expression of full-length

AR (ARFL) and truncated AR variants (ARFL 2.3-fold, P¼ 0.008 and ARdel567es 2.7-fold, P¼ 0.036 in 23 CR;

ARFL 3.4-fold, P ¼ 0.001 and ARV7 3.1-fold, P ¼ 0.0003 in 35CR), and increased expression of the

abiraterone target CYP17A1 (�2.1-fold, P ¼ 0.0001 and P ¼ 0.028 in 23CR and 35CR, respectively) and

transcript changes in other enzymes modulating steroid metabolism.

Conclusions: These studies indicate that abiraterone reduces CRPC growth via suppression of intra-

tumoral androgens and that resistance to abiraterone may occur through mechanisms that include

upregulation of CYP17A1, and/or induction of AR and AR splice variants that confer ligand-independent

AR transactivation. Clin Cancer Res; 17(18); 5913–25. �2011 AACR.

Introduction

Prostate cancer develops resistance to serum androgensuppression in essentially all patients with advanced dis-ease (1). Upregulated androgen receptor (AR) expressionand autonomous synthesis of androgens by neoplastic

prostate epithelium (either de novo from cholesterol orthrough metabolism of adrenal precursors) are importantcontributors to castration-resistant prostate cancer (CRPC)growth (2–5). Tissue androgens such as dihydrotestoster-one (DHT) may be maintained via the "classical" pathwayof steroidogenesis proceeding through dehydroepiandros-terone (DHEA), or through a "back-door" pathway using5a-reduced steroid precursors as the primary source ofDHT (6). In addition to upregulated expression of full-length AR (ARFL), the generation of constitutively active ARvariants by differential transcript splicing of the ligand-binding domain has been described (2, 7–12). These ARsplice variants have ligand-independent activity, andARv567es can also function by enhancing the response ofARFL to low ligand concentrations (10, 13).

Abiraterone is a novel agent designed to suppress growthof CRPC by inhibiting CYP17A1, a rate limiting enzyme ofsteroidogenesis (14). Potential sites of action include anyorgan capable of elaborating androgens, including testis,adrenal gland, or prostate cancer tissue. Positive results in

Authors' Affiliations: Divisions of 1Clinical Research and 2Human Biol-ogy, Fred Hutchinson Cancer Research Center; 3Department of Medicine,University of Washington; 4Geriatric Research, Education and ClinicalCenter, VA Puget Sound Health Care System; 5Department of Urology,University of Washington, Seattle, Washington; and 6Beth Israel Deacon-ess Medical Center, Boston, Massachusetts

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

Corresponding Author: Elahe A.Mostaghel, Division of Clinical Research,Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D4-100,Seattle, WA 91809. Phone: 206-667-3506; Fax: 206-667-5456; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-0728

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 5913

Research. on April 2, 2020. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 1, 2011; DOI: 10.1158/1078-0432.CCR-11-0728

http://clincancerres.aacrjournals.org/

phase I and II clinical trials, both before and after the use ofdocetaxel (15–17), have led to phase III studies of abir-aterone, showing a survival advantage over prednisonealone in men with CRPC previously treated with docetaxel(18).

Although clinical studies of abiraterone have shownresponses in the majority of men with CRPC, the extentof PSA declines and measurable tumor regression are vari-able, and the effect of abiraterone in suppressing tumor (asopposed to serum) androgens in men with CRPC has notbeendetermined.As shown inmultiple studies, suppressionof serumandrogensdoesnot correlatewellwith suppressionof tissue androgens in men undergoing androgen depri-vation (4, 19, 20). Moreover, most patients treated withabiraterone ultimately suffer tumor progression and infor-mation delineating how resistance to abiraterone occurs islimited. Establishing the effect of abiraterone on tissueandrogens and gene expression is critical for determiningwhether the clinical activity of abiraterone correlates withsuppression of tissue androgens and for identifying poten-tial mechanisms of resistance to abiraterone treatment.

To address these issues, we treated CRPC xenografts withabiraterone to determine the effects on tumor growth,tissue androgen concentrations, and tumor gene expres-sion. Our results indicate that alterations in pathways ofandrogen metabolism and AR expression are mechanismsof molecular adaptation in response to abiraterone treat-ment, and consequently represent attractive targets for newtherapeutic strategies.

Materials and Methods

LuCaP human prostate cancer xenograftsThe establishment and maintenance of the LuCaP23

and 35 xenografts from lymph node metastases of 2 indi-viduals with CRPC as a component of the University of

Washington Rapid Autopsy program has been previouslydescribed (21, 22). The AR has been sequenced andcodes for a wild-type protein in both xenografts. In eugo-nadal hosts, these lines produce serum PSA, regress inresponse to castration, and subsequently regrow as castra-tion-resistant (CR), PSA-producing variants that wereutilized in the present studies. The CR variant of LuCaP35has previously been termed LuCaP35V, but for consis-tency the CR variants of both LuCaP23 and LuCaP35 arenow designated CR. All experiments involving animalswere done in accordance with protocols approved by theUniversity of Washington Institutional Animal Care UseCommittee.

Castrate male CB-17 SCID mice (Charles River Labora-tories) were implanted subcutaneously with 20mm3 piecesof LuCaP23CR or LuCaP35CR tumors. When tumorsreached 250 to 300 mm3 [length � (width2)/2] mice(n ¼ 46 and n ¼ 28 LuCaP23CR and LuCaP35CRtumor-bearing mice, respectively) were randomly assignedto vehicle control (5% benzyl alcohol, 95% safflower oilintraperitoneal injection) or abiraterone treatment (0.5mmol/kg/d in vehicle) daily for 21 days following enroll-ment (23). Unanticipated toxic or off target effects were notreported in the preclinical studies evaluating this dose(which is �10-fold the oral dose in clinical studies, dueto differences in absorption and bioavailability). Serumwas collected by retro-orbital bleeding at interval timepoints for determination of PSA. Tumors from a subsetof mice in each cohort were harvested at early time pointsof treatment (tumor size of �500mm3; range 7–21 days).When the remaining tumors reached approximately 1,000mm3 in size, animals were euthanized according to institu-tional protocol and xenografts harvested and flash frozenfor determination of tissue androgens and extraction oftotal RNA. Serum PSA was measured using the AbbottAxSYM immunoassay system (Abbot Laboratories). Abir-aterone was kindly provided by Cougar Biotechnology.Four mice bearing LuCaP35CR tumors survived for fol-low-up beyond day 40, whereas all mice bearingLuCaP23CR tumors reached the endpoint and were sacri-ficed by day 40 (except one at day 42).

Steroid measurementsAndrogen levels were determined by mass spectrometry

(MS) using methods we have recently described (24). Thisprocedure resulted in a lower limit of quantitation of 1 pgper sample for testosterone and DHT, respectively. Intra-assay coefficients of variation generated using humanserum for high-, mid-, and low-range samples were3.5%, 3.1%, and 3.8% for testosterone and 6.3%, 4.3%,and 15.8% for DHT, respectively.

RNA isolation and quantitative real time-PCRRNA was isolated and prepared for quantitative real time

PCR (qRT-PCR) as previously described (5). cDNA wasgenerated in a random-primed reverse transcription reac-tion, and qRT-PCR reactions were done in triplicate usingan Applied Biosystems 7900 sequence detector with 5 ng of

Translational Relevance

Abiraterone is a novel CYP17A1 inhibitor recentlyshown to extend the survival of men with castration-resistant prostate cancer (CRPC). However, many menultimately progress and mechanisms of resistance toabiraterone treatment in vivo have not been elucidated.We show in two xenograft models of CRPC that theclinical response to abiraterone is accompanied bymarked suppression of tumor androgen levels, andidentify increased expression of AR, ligand-independentAR splice variants, and the steroidogenic transcriptome(including the CYP17A1 target gene) as potentialmechanisms of adaptation to CYP17A1 blockade. Thesedata suggest that resistance can potentially be targetedby using higher dose levels of abiraterone or combina-tions with potent AR antagonists currently in develop-ment and show that more effective suppression of ARsignaling remains an important means of effectivelytreating advanced prostate cancer.

Mostaghel et al.

Clin Cancer Res; 17(18) September 15, 2011 Clinical Cancer Research5914




cDNA, 1 mmol/L of each primer pair and SYBR Green PCRmaster mix (Applied Biosystems). Primers (SupplementaryData 1) were designed using the Web-based primer designservice Primer3 from the Whitehead Institute for Biome-dical Research (http://www.steverozen.net/papers/rozen-and-skaletsky-2000-primer3.pdf), except for AKR1C2 andAKR1C3 (25) and RODH4, DHRS9 (NT-3alpha HSD), and17BHSD10 (26) for which previously published primersequences were used. Specificity of amplification wasassessed based on melting point of the dissociation curve.In certain cases, 2.5 mL of amplified product (total reactionvolume 10 mL) was also run on a 2% agarose gel to assessfor product size (compared with positive control) and thepresence of extraneous bands (using a Hamamatsu digitalcamera with acquisition software from LabWorks).

Statistical analysisThe effect of abiraterone on tumor growthwas quantified

using the following linear mixed effects model:

Volumeij ¼ m þ b1Dayij þ b2Treatmentijþ b3Dayij � Treatmentij þ bi þ eij

where Volumeij is log-transformed tumor volume mea-surement j for mouse i, Dayij is day of measurement,Treatmentij indicates treatment group (0 ¼ none and 1¼ Abiraterone), bi is a mouse-specific independent andnormally distributed random effect with mean 0 andvariance sb

2, and eij is an independent and normallydistributed error term with mean 0 and variance se

2.Median tumor volume trajectories and 95% confidencebands were derived for mice in each treatment group basedon mean predictions of empirical quantiles of 1,000 boot-strap replicates generated from the original model and refitusing identical model structures. Progression-free survival(PFS) in vehicle-control and abiraterone-treated (Abi-T)mice (defined as tumor size < 1,000 mm3) was determinedvia Kaplan–Meier analysis with comparison of curves usingtheMantel–Haenszel log-rank test. For analysis of qRT-PCRdata, the mean cycle threshold (Ct) for each gene wasnormalized to expression of the housekeeping geneRPL13A in the same sample (DCt). Unpaired 2 sample ttests were used to compare mean DCt’s for each genebetween vehicle-treated controls and abiraterone-treatedtumors. The values of P < 0.05 were considered significant.The fold change was calculated by the DDCt method (fold¼2DDCt).

Results

Abiraterone inhibits the growth of castrationresistant prostate cancersProstate cancers progressing in the setting of castration

maintain or reactivate the gene expression program regu-lated by the AR (27). In studies designed to identifymechanisms responsible for AR signaling in CRPC, wepreviously showed that tissue levels of T and DHT inLuCaP23CR and LuCaP35CR xenograft tumors grown incastrate mice are similar to tumor levels measured in

isogenic variants passaged in noncastrate mice (5). Todetermine whether CYP17A1 activity contributes to tumorgrowth and the maintenance of tumor androgen levels,we treated cohorts of castrate mice bearing LuCaP23CRor LuCaP35CR xenografts with the CYP17A1 inhibitor,abiraterone.

Treatment with abiraterone led to a rapid decline inserum PSA over the first 10 days of treatment in micebearing either LuCaP23CR or LuCaP35CR tumors(Fig. 1A and B). Abiraterone also had significant effectson tumor growth, with more rapid median growth per dayin control versus Abi-T tumors (Fig. 2A and C; LuCaP23CR:7.4% (95% CI: 6.2–8.0%) vs. 5% per day (95% CI: 3.0%–6.8%), P ¼ 0.0001; and LuCap35CR: 4.8% (95% CI: 3.9–5.2%) vs. 2.5% per day (95%CI: 1.2%–3.7%), P < 0.0001).Accordingly, abiraterone treatment resulted in statisticallysignificant improvements in PFS (defined as tumor size <1,000 mm3), and median survival (MS) in both xenografts(Fig. 1B andD). Themedian survival of LuCap23 improvedfrom 14 to 24 days [HR for survival 2.5 (95% CI: 1.6–11.2)], whereas the median survival of LuCaP35 improvedfrom 17 to 39 days [HR 3.6 (95% CI: 2.3–34.6)]. Interest-ingly, serum PSA levels at tumor progression remained lowin mice bearing LuCaP23CR tumors, although they beganto rise in mice bearing LuCaP35CR tumors. These observa-tions are consistent with a number of xenograft modelsshowing that coordinate regulation of tumor growth andPSA expression is not necessarily universal (28).

Abiraterone treatment reduces tumor androgen levelsin castration resistant prostate cancers

To determine the effect of abiraterone on tumor andro-gen levels, we measured levels of T and DHT in tumorsresected at different time points during and after abira-terone treatment. At early time points during therapy (day7 for LuCaP23 and days 7–21 for LuCaP35), abirateroneresulted in nearly complete suppression of T (Fig. 3A andB) and marked suppression of DHT (Fig. 3C and D) inboth LuCaP23CR and LuCap35CR tumors, respectively(P < 0.0001 compared with controls for all comparisons).Levels of T (Fig. 3A) and DHT (Fig. 3C) remained sup-pressed at later time points in the majority of LuCaP23CRtumors. This included abiraterone-resistant (Abi-R)tumors that recurred within the 21 day treatment period(Abi-R), and tumors which recurred after therapy hadbeen completed (Abi-T). In contrast, androgen levels inAbi-T LuCaP35 tumors (all of which recurred after day21) showed a trend toward reconstitution, with a smallbut statistically significant increase in T (P ¼ 0.032,Fig. 3B) and a trend toward increase in DHT (P ¼0.058, Fig. 3D) compared with tumors resected earlierwhile on therapy (d7-21). Notably, tumor androgenlevels in the abiraterone treatment arms were correlatedwith serum PSA levels in both LuCa23CR and 35CR (r ¼0.7168, P < 0.0001; and r ¼ 0.9163, P < 0.0001; forPearson correlations with tumor DHT, respectively), con-sistent with biologic activity associated with androgenlevels in the recurrent tumors.

Resistance to CYP17A1 Inhibition in Castration-Resistant Prostate Cancer

www.aacrjournals.org Clin Cancer Res; 17(18) September 15, 2011 5915




Abiraterone treatment alters the expression of full-length and splice-variant forms of the AR

Castration resistant prostate tumors frequently expresselevated levels of ARFL and, as recently reported, increasesin expression of AR splice variants that confer constitutive

ligand-independent activity (2, 7–11, 12). To determinewhether castration resistant tumors recurring after abira-terone treatment show further alterations in AR expression,we quantitated mRNAs encoding ARFL, and the ARdel567es

and ARV7 variants recently identified in CRPC metastases

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Figure 1. Changes in serum PSAlevels in LuCaP23CR andLuCaP35CR prostate cancerxenografts in response totreatment with abiraterone.Castrate male SCID mice wereimplanted subcutaneously withLuCaP23CR or LuCaP35CRtumors and randomly assigned tovehicle control or abirateronetreatment for 21 days. Serum PSAcurves for control (blue) or Abi-Tmice (red) are shown for individualmice bearing LuCaP23CR (A) andLuCaP35CR (B) xenografts. Thesegment of the y-axis denoted bythe heavy bar in each graph isenlarged and presented in theadjacent panels on an expandedy-axis to more clearly show thedecline in serum PSA over the firstapproximately 10 to 15 days afterinitiation of treatment.

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Figure 2. Tumor growth andsurvival in LuCaP23CR andLuCaP35CR prostate cancerxenografts treated withabiraterone. Castrate maleSCID mice were implantedsubcutaneously with LuCaP23CR(A and B) or LuCaP35CR (C and D)tumors and randomly assigned tovehicle control or abirateronetreatment for 21 days. Mediantumor volume trajectories with95% confidence bands for control(blue) or Abi-T mice (red) bearingthe LuCaP23CR (A) andLuCaP35CR (C) xenografts.Kaplan–Meier plots of PFS(defined as tumor size < 1,000mm3) in control or Abi-T micebearing the LuCaP23CR (B) andLuCaP35CR (D) xenografts.P values for curve comparisonsgenerated using the Mantel–Haenszel log-rank test.

Mostaghel et al.





(the latter also separately reported as ARV3; refs. (2, 7–12).Compared with controls, LuCaP23CR showed no changesin AR expression at early time point of therapy (Abi d7,Fig. 4A, C, and E), but showed significant changes at latertime points. Specifically, both Abi-R and Abi-T LuCaP23CRtumors showed increased expression of ARFL (Abi-R 2.7-fold, P < 0.0001; Abi-T 2.3-fold, P ¼ 0.008; Fig. 4A) and ofARdel567es (Abi-R 2.2-fold, P < 0.026; Abi-T 2.7-fold, P ¼0.036, Fig. 4C) with no change in ARV7 (Fig. 4E).LuCaP35CR tumors showed increases in ARFL expres-

sion at both early and late time points after treatment(Abi d7-21 2.5-fold, P ¼ 0.028; and Abi-T 3.4-fold, P ¼0.001, Fig. 4B). AR variant expression was also altered,with changes in ARdel567es trending toward significance(Abi d7-21, 7.6-fold, P ¼ 0.078; and Abi-T 5.2-fold, P ¼0.073, Fig. 4D), and ARV7 significantly increased in Abi-Ttumors (3.1-fold, P ¼ 0.0003, Fig. 4F). Notably, themagnitude of full length and AR variant expression ishigher at baseline in LuCaP23CR versus LuCaP35CRtumors (ARFL 1.8-fold higher, P ¼ 0.0265; ARdel567es

255-fold, P < 0.0001; ARV7 2.8-fold, P ¼ 0.0001). It ispossible a difference in baseline AR levels may facilitatethe more rapid growth rate observed in LuCaP23CR andthe ability of this tumor to recur in a setting of ongoingligand suppression.

Abiraterone treatment alters the expression of AR-regulated genes and transcripts encodingsteroidogenic enzymes

The ultimate objective of CYP17A1 inhibition by abir-aterone is suppression of intratumoral androgens withconcomitant inhibition of AR-mediated signaling. Wetherefore determined the impact of abiraterone on tumoralexpression of androgen-regulated genes. We also evaluatedexpression of steroidogenic genes throughout the androgenbiosynthetic pathway, as alteration in steroidogenic activitymight mitigate the impact of abiraterone on intratumoralandrogen levels.

Expression of androgen regulated genesConsistent with decreases in tumor androgen levels

associated with abiraterone treatment, both LuCap23CRand LuCap35CR show significant decreases in expressionof androgen-regulated genes (e.g., NKX3.1, FKBP5,TMPRSS2; Table 1). Interestingly, tissue PSA expressionwas not altered by abiraterone treatment in LuCaP23CR(Fig. 5A), remaining highly expressed despite the rapidand sustained suppression of serum PSA levels associatedwith abiraterone treatment (Fig. 1A). In contrast, treat-ment with abiraterone was clearly associated with inhi-bition of tissue PSA expression in LuCaP35CR (Fig. 5B),

Figure 3. Tumor testosterone (top)and DHT levels (bottom) in controland Abi-T LuCaP23CR (A, C) andLuCaP35CR (B, D) xenografts.Androgen levels in Abi-Txenografts were evaluated bymass spectrometry in tumorsresected at early (days 7–21) orlate time points after therapy.Abi-R tumors recurred (defined asprogression to > 1,000 mm3) andwere resected during the 21-dayperiod of abiraterone treatment.Abi-T tumors recurred and wereresected after the completion ofabiraterone treatment. Thisoccurred between days 24 to 42for LuCap23CR tumors, andbetween days 29 to 67 forLuCaP35CR (none of whichrecurred during the abiraterone-treatment period). P valuesrepresent unpaired 2-sided t testsbetween the indicated groups.The values of P < 0.05 wereconsidered significant. OneP value in panel D (in italics) isincluded as trending towardstatistical significance (P < 0.10).

AAA BBB

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P < 0.0001P < 0.0001P < 0.0001 P = 0.002P = 0.002P = 0.002

P < 0.0001P < 0.0001P < 0.0001 P = 0.032P = 0.032P = 0.032P < 0.0001P < 0.0001P < 0.0001P < 0.0001P < 0.0001P < 0.0001

P < 0.0001P < 0.0001P < 0.0001

P < 0.0001P < 0.0001P < 0.0001

P = 0.0003P = 0.0003P = 0.0003 P = 0.005P = 0.005P = 0.005P = 0.058P = 0.058P = 0.058P < 0.0001P < 0.0001P < 0.0001

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particularly at the early time points (d7-21) when tumorswere under active treatment. Moreover, tissue and serumPSA levels were directly correlated in Abi-T LuCaP35CRtumors (Pearson correlation r ¼ 0.8226; P ¼ 0.0006)but not in LuCaP23CR. These observations suggestthat androgen-mediated effects on serum PSA levelsmay reflect alterations in release/secretion of PSAwith or without associated changes in tumoral PSAtranscription.

Expression of genes required for androgen synthesisAbi-T tumors showed increased expression of numerous

genes throughout the androgen biosynthetic pathway(Fig. 6). Both LuCaP23CR and LuCaP35CR tumorsresponded to abiraterone treatment with increased expres-sion of the target gene CYP17A1 (Fig. 5C, LuCaP23CR Abi-R 1.7-fold, P¼ 0.0002; Abi-T 2.1-fold, P¼ 0.0001; Fig. 5D,LuCaP35CR Abi-T 2.1-fold, P ¼ 0.0278). The expression ofAKR1C3 and HSD17B3, 2 key enzymes mediating conver-

sion of adrenal androgen intermediates to T, was alsoincreased in both LuCaP23CR and LuCap35CR tumorscompared with controls (Table 1). Of note, expressionchanges in Abi-R LuCaP23CR tumors recurring while ontherapy were generally similar in direction and magnitudeto changes observed in Abi-T tumors recurring after com-pletion of therapy (Abi-T), suggesting the effects of abir-aterone were sustained beyond the immediate treatmentinterval.

Overall, genes required for androgen biosynthesis wereexpressed in both LuCaP23CR and LuCaP35CR tumors,and observed changes in expression are consistent with anupregulated capacity for androgen biosynthesis. As mightbe anticipated, the relative magnitude of steroidogenicgene induction in CRPC xenografts, before and afterabiraterone treatment, is lower than that observed inclinical studies of CRPC tissues, in which CRPC metas-tases were compared with primary, untreated prostatetumors (5).

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rian

tA

Rd

el56

7es

vari

ant

norm

aliz

ed d

CT

AR

FL

norm

aliz

ed d

CT

Abid7

Abid7-21

Abi-Rd11-21

Abi-Td24-42

Abi-Td29-67

Control Control

norm

aliz

ed d

CT

norm

aliz

ed d

CT

Abid7

Abid7-21

Abi-Rd11-21

Abi-Td24-42

Abi-Td29-67

Control Control

norm

aliz

ed d

CT

norm

aliz

ed d

CT

Abid7

Abid7-21

Abi-Rd11-21

Abi-Td24-42

Abi-Td29-67

Figure 4. Expression of full-lengthand truncated AR splice variants inLuCaP23CR and LuCaP35CRtumors treated with abirateronecompared with vehicle control.Transcript levels for ARFL (A andB), the AR7 splice variant (C andD), and the ARdel567es splicevariant (E and F) were measuredby qRT-PCR in frozenLuCaP23CR (A, C, E) andLuCaP35CR (B, D, F) tumors.White circles denote vehicletreated controls. Black circlesdenote tumors resected while onabiraterone treatment, either atearly time points (d7-21) or atAbi-R regrowth. Gray circlesdenote Abi-T tumors whichrecurred and were resected aftercompletion of abirateronetreatment. Fold changes arecalculated from the difference inmean DCt's between abirateronetreated and vehicle treatedcontrols (DDCt method;fold ¼ 2DDCt). P values from2 sample t tests. The values ofP < 0.05 were consideredsignificant. The P values in panel D(in italics) are included as trendingtoward statistical significance(P < 0.10). n.s., not significant.

Mostaghel et al.





Expression of genes mediating prereceptor regulationof DHT levelsTumor androgen levels reflect the sum total of activity in

both androgen biosynthetic and androgen catabolic path-ways. The back-conversion of 3a-diol to DHT, which canbe mediated by enzymes with 3 alpha-hydroxysteroidreductase (3a-HSD) activity, has primarily been attributedto RL-HSD, although RODH4, RDH5, HSD17B10, andDHRS9 can also mediate this reaction and are expressedto varying degrees in prostate tissue (29, 30). We thereforeexaminedwhether genes involved in prereceptor regulationof DHT levels via back conversion of 3-a-androstanediol(3-a-diol) to DHT were also altered by abiraterone treat-ment. Interestingly, changes in expression of oxidativegenes mediating back conversion of 3a-diol to DHT inabiraterone-treated tumors were mixed, with increases ingenes such as DHRS9 in LuCaP23CR, and RDH5 inLuCaP35, but significant decreases in expression of RL-HSD in both LuCaP23CR and LuCaP35CR (Table 1).Themarked suppression of RL-HSD in response to ligand

inhibitionwith abiraterone is consistentwith a recent reportshowing suppression of RL-HSD levels in CWR22 PCaxenografts in response to castration (30), and may explainthe relatively low levels of androgens measured in Abi-Ttumors despite general induction of genesmediating andro-gen biosynthesis. Notably, tumor DHT levels were stronglycorrelated with RODH4 (r ¼ 0.7348, P < 0.0001) and RL-HSD (r ¼ 0.6431, P ¼ 0.0005) in LuCaP23CR, and withRODH4 (r¼ 0.6211, P¼ 0.0235), 17BHSD10 (r¼ 0.6332,P¼ 0.0202), DHRS9 (r¼ 0.7815, P¼ 0.0016) and RL-HSD(r ¼ 0.6329, P ¼ 0.0202) in LuCaP35CR (Pearson productcorrelations). These data suggest that despite an overalldecrease in expression of genes such as RL-HSD, androgenlevels in abiraterone-treated tumors are, nevertheless, spe-cifically associated with expression of genes inhibiting cat-abolism of DHT.As tissue DHT levels in Abi-T LuCaP35CR tumors ranged

from approximately 0.1 to >4 pg/mg (Fig. 4D), we alsodetermined whether these differences in androgen recon-stitution were related to changes in the steroidogenictranscriptome. Notably, Abi-T LuCaP35CR tumors thatrecurred with high versus low tissue androgens (definedas tissue DHT above or below the median of �0.5 pg/mg)showed 2.5-fold higher PSA (P ¼ 0.007), 2.9-fold higherCYP17A1 (P¼ 0.033), markedly higher HNF4A (97-fold, P¼ 0.020, a CYP17A1 cofactor), and 8.2 and 5.4 higherlevels of genes involved in preventing DHT catabolism (P¼0.011 and P¼ 0.005 for RL-HSD andDHRS9, respectively),consistent with a critical role for genes involved in bothandrogen synthesis and prereceptor regulation of DHTcatabolism in determining tissue androgen levels. (Statis-tically significant differences in expression of AR or ARvariants were not observed, data not shown).

Discussion

Clinical studies with abiraterone have shown strikingresponses in men with CRPC, though not all patients

respond and most patients eventually develop resistance.Although abiraterone is known to inhibit CYP17A1 in vitro,the effectiveness of abiraterone in suppressing intratumoralandrogens has not been established, and it remains unclearwhich CRPC phenotypes and genotypes are susceptible toCYP17A1 inhibition and how tumors develop resistance.Using 2 distinct CRPC models, we show that treatmentwith abiraterone significantly inhibited tumor growth,serum PSA, and intratumoral androgen levels, supportingthe hypothesis that abiraterone’s primary mechanism ofaction is through effects on tissue androgens. Furthermore,both CRPCmodels responded to CYP17A1 inhibition withmechanisms that maintain AR signaling. This includedupregulated expression of ARFL and ligand-independentAR variants, as well as induction of steroidogenic genes(including the target gene, CYP17A1), several of whichshowed strong correlations withDHT levels in the recurrenttumors. Thus, in the setting of tumor progression onabiraterone, the rationale for focusing further therapeuticefforts on more potent AR antagonists and agents suppres-sing AR ligands remains strong.

Although specific mechanisms driving induction of alter-native AR splicing have not been established, generation ofAR splice variants following suppression of tumor andro-gens by abiraterone is consistent with the castration-mediated induction of AR splice variants observed incastration sensitive prostate cancer models (10, 12, 13).Interestingly, studies of testosterone replacement in thissetting (either in vitro, or when given within days ofcastration in vivo) have been shown to inhibit castration-associated increases in AR variant expression (10, 12).However, we did not observe lower levels of full lengthor variant AR expression in those tumors with higher levelsof androgens at recurrence (data not shown). These obser-vations suggest that factors regulating the initial inductionof AR splice variant expression could differ from thosemaintaining variant expression at later time points ofrecurrent growth. Moreover, these observations show thatcertain tumors may simultaneously engage or accrue multi-ple resistance pathways directed at preserving the AR axis.

The molecular alterations occurring in CRPC tumorsfollowing abiraterone treatment suggest tumor-specificmethods of addressing resistance, either through optimiz-ing steroidogenic blockade or by inhibiting AR signaling.Although substantially suppressed, androgen levelsremained detectable in Abi-T tumors. Importantly, a 2-to 3-fold increase in expression of ARFL is known to renderlow androgen levels physiologically relevant in promotingAR driven growth (2), suggesting clinical treatment of Abi-Rpatients with more stringent ligand inhibition may be ofbenefit. More complete reduction of steroidogenesis mightbe achieved through enhancing local concentrations ofCYP17A1 inhibitors, or targeting transcriptional activationof the enzyme. CYP17A1 is regulated by SF-1 and othercofactors (31), and its regulatory domains containmultiplecAMP responsive elements, providing several targets formodulation of enzyme expression, such as using phospho-diesterase inhibitors (32). Combining CYP17A1 blockade






Tab

le1.

Cha

nges

inAR

andsteroidog

enic

gene

expressionin

Abi-TCRPC

xeno

grafts

LuCaP

23CR

LuCaP

35CR

Tim

epointofrese

ction

Early

(d7)

Abiraterone

a

resistan

t(d11

-21)

Abiraterone

b

trea

ted

(d24

-42)

Early

(d7-21

)Abiraterone

b

trea

ted

(d29

-67)

Gen

ean

dfunc

tion

Fold

cPd

Fold

PFo

ldP

Fold

PFo

ldP

AR

ARFL

2.7

P<0.00

012.3

0.00

82.5

0.02

83.4

0.00

1ARdel567es

2.2

0.02

62.7

0.03

67.6

0.07

85.2

0.07

3ARV7

3.1

0.00

03AR-reg

ulated

gen

esPSA

�3.4

0.00

3�2

.00.02

3

NKX3.1

�1.2

0.02

6�1

.90.03

8�1

.60.03

6FK

BP5

�1.6

0.00

4�3

.0P<0.00

01�2

.7P<0.00

01�3

.80.00

2TM

PRSS2

�1.3

0.00

5�1

.6P<0.00

01Cho

lesterolimport

andproce

ssing

SCARB1

�1.5

0.04

2

LDLR

�3.0

0.00

1�1

.90.00

3LIPE

4.5

P<0.00

017.1

P<0.00

01SCAP

1.8

0.00

32.6

P<0.00

011.3

0.02

4CYP11

A1

1.3

0.02

21.2

0.03

11.6

0.00

03STA

R1.5

0.00

21.5

0.01

8STA

RD3

1.8

0.00

32.4

P<0.00

01STA

RD4

�1.6

P<0.00

01�1

.40.00

7�2

.60.00

41.6

0.05

7STA

RD5

1.5

0.00

51.7

0.00

61.8

0.00

3CYP17

A1an

dco

factors

CYP17

A1

1.6

0.02

21.7

0.00

022.1

0.00

012.2

0.02

8

HNF1

A1.7

0.00

41.8

0.00

32.1

0.00

3HNF4

A�1

.70.01

22.7

0.01

5NR5A

1(SF-1)

1.3

0.02

1.3

0.04

1.5

0.01

0CYB5A

1.4

0.00

01POR

1.6

P<0.00

012.1

P<0.00

01�1

.50.00

021.2

0.04

3DAX1

2.8

0.03

2.0

0.04

3DUSP

1.7

0.02

6�3

.1P<0.00

01Conv

ersionof

C-19steroids

toTan

dDHT

HSD3B

1

HSD3B

21.5

0.05

01.7

0.06

3HSD17

B3

1.3

0.00

61.4

0.00

32.2

0.00

1AKR1C

31.6

0.08

61.9

0.00

11.6

0.05

35.2

P<0.00

01SDR5A

1�1

.40.00

4SDR5A

21.9

0.02

83.5

P<0.00

014.5

P<0.00

01SDR5A

31.2

0.03

81.4

P<0.00

011.7

P<0.00

01

(Con

tinue

don

thefollo

wingpag

e)

Mostaghel et al.





Tab

le1.

Cha

nges

inAR

andsteroidog

enic

gene

expressionin

Abi-TCRPC

xeno

grafts

(Con

t'd)

LuCaP

23CR

LuCaP

35CR

Tim

epointofrese

ction

Early

(d7)

Abiraterone

a

resistan

t(d11

-21)

Abiraterone

b

trea

ted

(d24

-42)

Early

(d7-21

)Abiraterone

b

trea

ted

(d29

-67)

Gen

ean

dfunc

tion

Fold

cPd

Fold

PFo

ldP

Fold

PFo

ldP

CYP19

A1.7

0.00

92.5

0.00

12.5

0.03

7Prerece

ptor

regulation

ofDHT

AKR1C

24.2

P<0.00

013.9

0.00

13.3

0.00

01

HSD17

B10

RODH

4�2

.90.00

0�4

.8P<0.00

01�5

.9P<0.00

01�1

.80.02

4RDH5

2.8

0.00

01RLH

SD

(HSD17

B6)

�4.3

0.00

3�2

6.6

P<0.00

01�2

6.2

P<0.00

01�2

7.3

P<0.00

01�4

.80.00

1

DHRS9

(HSD17

B13

)3.7

0.00

017.5

P<0.00

01�6

.30.00

03

Glucu

ronide

form

ation

UGDH

1.7

0.00

62.3

P<0.00

01�1

.60.01

6

UGT2

B15

4.6

P<0.00

019.3

P<0.00

015.1

0.00

2UGT2

B17

2.6

0.00

013.6

P<0.00

01UGT2

B17

isdeleted

inLu

CaP

35CR

aAbira

terone

resistan

ttumorsrecu

rred

(size1,00

0mm3)

andwererese

cted

while

onab

iraterone

.bAbira

terone

trea

tedtumorsrecu

rred

(size1,00

0mm3)

afterthe21

daytrea

tmen

tperiod.

cFo

ldch

ange

from

differen

cein

mea

nDC

t's(DDC

tmetho

d;fold

¼2D

DCt )be

twee

nco

ntrolsan

dAbi-Ttumorsin

theindicated

grou

ps(early,res

istant,o

rtreated

).Fo

ldch

ange

sfor

gene

swhich

wereno

tstatistic

ally

sign

ifica

ntareom

itted

.dPva

lues

from

2sa

mplettes

ts.T

heva

lues

ofP�0.05

wereco

nsidered

sign

ifica

nt.G

enes

with

theva

lues

ofP<0.10

wereco

nsidered

tren

dingtowardsign

ifica

nceiffold

chan

gewas

also

1.5or

more(sho

wnin

italics).






with inhibitors of other critical components of the pathwaysuch as HSD3B1 or SRD5A1/2 could also offset adaptiveupregulation of CYP17A1 (33).

Data regarding expression of C terminal truncated ARsplice variants in CRPC continues to emerge, and will be acritical area of investigation asmore potent ligand synthesisand AR inhibitors become utilized in the treatment ofCRPC. AR splice variants may act by potentiating activityof ARFL and mediating constitutive AR transactivation (10,13). Thus, increased expression of AR splice variants in Abi-T tumors may be an important biomarker of resistance andtarget for therapy. Incorporation of potent AR inhibitors,such as MDV3100, or agents targeting the AR N-terminaldomain, such as EPI-001, could be utilized for tumorsadapting to CYP17A1 inhibition via induction of ARFL

and/or AR splice variants lacking the ligand-bindingC-terminal domain (13, 34–36). Studies to delineatewhether sequential or concurrent use of these agents withabiraterone can improve tumor growth inhibition and/orsuppress adaptation in xenograft models will be importantto provide rationale for human studies evaluating theseagents in the treatment of clinical disease.

The response to abiraterone in our study is most likelydue to suppression of de novo intratumoral steroidogenesis(due to the reported lack of CYP17A1 in rodent adrenalglands; refs. 37, 38), whereas in human studies theresponse to abiraterone may reflect inhibition of bothadrenal and/or intratumoral CYP17A1 activity. Impor-tantly, the proposed mechanism driving clinical activity

of abiraterone in both scenarios is a decrease in intratu-moral androgens (whether from suppression of adrenalsteroidogenesis, intratumoral steroidogenesis, or both).Our observations confirm this expected mechanism ofactivity. Furthermore, increases in expression of ARFL andligand-independent AR splice variants following abirater-one treatment are likely to be clinically relevant mechan-isms of resistance regardless of whether tumoral CYP17A1activity is present.

Another consideration for translation of our results tothe clinical setting is that men with CRPC are likely to betreated with abiraterone plus prednisone (or dexametha-sone) rather than abiraterone alone. In castrate men treatedwith abiraterone, glucocorticoids are primarily used toinhibit pituitary-mediated secretion of ACTH (inducedby adrenal CYP17A1 inhibition) which can exacerbate sideeffects of mineralocorticoid excess, but may potentiallyinhibit tumor growth directly (39). ACTH may also driveclinically significant increases in production of androgenicprecursors by the adrenal gland, as evidenced by PSAdeclines following the addition of dexamethasone inmen who had progressed on abiraterone (15). Thus, theinclusion of prednisone in men with CRPC is likely toaccentuate any decrease in tumor androgens caused byabiraterone, and may accentuate the types of tumoralresponses observed in the Abi-T xenografts.

Our hypothesis that tumoral androgens in these xeno-graft studies are derived from de novo steroidogenesis isconsistent with previous observation that a subset of CRPC

A B

C D

LuCaP23 CR

0

–2

–4

–6

–12

–14

–16

–18

–20

–12

–14

–16

–18

–20

0

–2

–4

–6

LuCaP35 CR

Control

2.1x, P = 0.0001

1.7x, P = 0.0002

1.6x, P = 0.0220

–2.0x, P = 0.023

2.2x, P = 0.028

–3.4x, P = 0.003

Control

Tis

sue

PS

Ano

rmal

ized

dC

TC

YP

17A

1no

rmal

ized

dC

T

Tis

sue

PS

Ano

rmal

ized

dC

TC

YP

17A

1no

rmal

ized

dC

T

Abid7

Abid7-21

Abi-Rd11-21

Abi-Td24-42

Abi-Td29-67

Control ControlAbid7

Abid7-21

Abi-Rd11-21

Abi-Td24-42

Abi-Td29-67

Figure 5. Expression of PSA andCYP17A1 in LuCaP23CR andLuCaP35CR tumors treated withabiraterone comparedwith vehiclecontrol. Transcript levels for PSA(A and B) and CYP17A1 weremeasured by qRT-PCR in frozenLuCaP23CR (A, C) andLuCaP35CR (B, D) tumors. Whitecircles denote vehicle treatedcontrols. Black circles denotetumors resected while onabiraterone treatment, either atearly time points (d7-21) or atAbi-R regrowth. Gray circlesdenote Abi-T tumors whichrecurred and were resected aftercompletion of abirateronetreatment. Fold changes arecalculated from the difference inmean DCt's betweenabiraterone-treated andvehicle-treated controls (DDCt

method; fold ¼ 2DDCt). P valuesfrom 2 sample t tests. The valuesof P < 0.05 were consideredsignificant.

Mostaghel et al.





metastases have increased levels of transcripts for CYP17A1and HSD3B1 (necessary for de novo synthesis) and thedemonstration by Locke and colleagues that CRPC xeno-grafts are capable of synthesizing DHT from acetate (5, 6,40). However, alternative androgenic precursors (of adre-nal or other origin)may also be present in the circulation aspotential substrates. Unfortunately, serum samples ade-quate to assess circulating androgen levels were not avail-able for analysis in this study. In addition, the duration ofabiraterone treatment in our study was 21 days. It ispossible that more prolonged treatment would haveresulted in amore robust induction of the adaptive changesalready observed. Finally, metastatic CRPC is characterizedby significant heterogeneity, while our conclusions reflectan analysis of only 2 CRPC phenotypes, and we had smallnumbers in some of the treatment groups. These limita-

tions can be addressed by evaluating diverse panels ofCRPC xenografts over a more prolonged time course oftherapy, and through studies of human tumor biopsiesobtained at abiraterone resistance.

In conclusion, our finding that abiraterone suppressesintratumoral androgens and inhibits CRPC growth sup-ports the hypothesis that tissue androgen levels are majorcontributors to AR signaling and mediators of CRPC pro-gression. Though hypothesized, this is the first demonstra-tion that the efficacy of abiraterone is related to its ability tosuppress tumor androgen levels and complements pre-viously published data from phase I studies regardingsuppression of serum androgens (15, 41). Our results alsoidentify potential mechanisms of adaptation to CYP17A1blockade, including increased expression of AR, AR splicevariants, and the steroidogenic transcriptome. Importantly,

Cholesterol

CYP11A1

CYP17A1

CYP17A1

HSD3B 1,2

SRD5A1,2 SRD5A1,2 SRD5A1,2 SRD5A1,2

HSD3B 1,2 HSD3B 1,2

HSD17B2

HSD17B3

HSD17B2

AKR1C3

HSD3B 1,2

hydroxylase

hydroxylase

CYP17A1hydroxylase

lyasePregnenolone

Progesterone 17α-OH Progesterone

5α-Pregnane3,20-dione

5α-Pregnane17α-ol-3,20-dione

5α-Pregnane3α-ol-20-one

5α-Pregnane3α-17α-diol-20-one

17α-OH Pregnenolone

DHEA-S

STS

DHEA

HSD17B3AKR1C3

5-Androstenediol

Androstenediol(3α-diol)

glucuronides

DHT

HSD17B3

UGT2B15

HSD17B6 (RL-HSD)HSD17B10 (ERAB)RODH4, RDH5(oxidative 3α-HSD)

UGT2B17

HSD17B2

AKR1C3

AKR1C2AKR1C2AKR1C2AKR1C2

Androstenedione

Androstenedione

Androsterone

Testosterone

CYP17A1

lyaseCYP17A1

lyase

(reductive 3α-HSD)(reductive 3α-HSD)

CYP17A1

Figure 6. The pathways of androgen biosynthesis. In the classical pathway (solid gray arrow), C21 precursors (pregnenolone and progesterone) are convertedto the C19 adrenal androgens DHEA and androstenedione (AED) by the sequential hydroxylase and lyase activity of CYP17A1. DHEA and AED areconverted to testosterone by a series of reactions involving the activity of HSD3B1 and 2, HSD17B3 and AKR1C3 enzymes. Testosterone is converted to thepotent androgen DHT by the activity of SRD5A1 and 2. Oxidative 3 a-HSD enzymes [including HSD17B6 (RL-HSD), HSD17B10, HSD17B13 (DHRS9),RODH4, and RDH5] can act to inhibit the prereceptor catabolism of DHT. An alternative pathway (short gray arrows) has also been proposed in which C21precursors are first acted upon by SRD5A and the reductive activity of AKR1C2, followed by CYP17A1, HSD17B3, and subsequent oxidation to DHT. Adaptedfrom ref. 42 with permission.






these adaptive mechanisms can potentially be targeted byusing higher dose levels of abiraterone or combinationswith potent AR antagonists currently in development.These data provide optimism that more effective suppres-sion of AR signaling will continue to be an importantmeans of effectively treating advanced prostate cancer.

Disclosure of Potential Conflicts of Interest

R.B. Montomery received commercial research grant from Cougar Bio-technology. The other authors disclosed no potential conflicts of interest.

Acknowledgments

We thank Holly Nguyen, Andrew Morgan, Jared Lucas, and Roger Cole-man for expert technical assistance; Roman Gulati for statistical support;

Cougar Biotechnology for providing abiraterone; and the Lucas Family fortheir generous support.

Grant Support

Prostate Cancer Foundation (Career Development Award to E.A. Mostagheland Synergy Award to S. Balk, R.B. Montgomery, and P.S. Nelson); DamonRunyon Cancer Research Foundation (Damon Runyon-Genentech ClinicalInvestigator Award CI-40-08 to E.A. Mostaghel); NIH [Career DevelopmentAward K23 CA122820 to E.A. Mostaghel; Pacific Northwest Prostate CancerSPORE P50 CA97186 (R.L. Vessella, P.S. Nelson); the Cancer Center SupportGrant to Fred Hutchinson Cancer Research Center (P30 CA015704 pilot to R.B.Montgomery)]; and the Veterans Affairs Research Service (S.R. Plymate).

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 March 18, 2011; revised July 12, 2011; accepted July 14, 2011;published OnlineFirst August 1, 2011.

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2011;17:5913-5925. Published OnlineFirst August 1, 2011.Clin Cancer Res Elahe A. Mostaghel, Brett T. Marck, Stephen R. Plymate, et al. Steroidogenesis and Androgen Receptor Splice VariantsCastration-Resistant Prostate Cancer: Induction of Resistance to CYP17A1 Inhibition with Abiraterone in

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