Critical Role of AKR1C2 As a Pre-Receptor Regulator of Androgen Receptor Signaling

Research Article

Impaired Dihydrotestosterone Catabolism in Human Prostate Cancer: Critical Role of AKR1C2 as a Pre-Receptor Regulator of Androgen Receptor Signaling

Qing Ji,1 Lilly Chang,2 Frank Z. Stanczyk,2,3 Murad Ookhtens,1 Andy Sherrod,4 and Andrew Stolz1

Departments of 1Medicine, 2Obstetrics and Gynecology, 3Preventive Medicine, and 4Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California

Abstract cornerstone of medical management since Huggins first showed We previously reported the selective loss of AKR1C2 and symptomatic response and regression of prostate cancer after AKR1C1 in prostate cancers compared with their expression in androgen elimination (2, 3). Epidemiologic studies have also paired benign tissues. We now report that dihydrotestosterone implicated life-long exposure to androgens as a contributing factor (DHT) levels are significantly greater in prostate cancer for prostate cancer development due to increased cellular tumors compared with their paired benign tissues. Decreased proliferation (4, 5). Dihydrotestosterone (DHT) is the key catabolism seems to account for the increased DHT levels as ligand for the androgen receptor (AR) in the prostate and is locally a expression of AKR1C2 and SRD5A2 was reduced in these synthesized predominately from circulating testosterone by 5 - tumors compared with their paired benign tissues. After 4 h of steroid reductase type II (SRD5A2; ref. 6). The clinical importance incubation with benign tissue samples, 3H-DHT was predom- of this pathway was amply shown in a successful chemopreventive inately catabolized to the 5A-androstane-3A,17B-diol metab- trial in which blocking the prostatic conversion of circulating olite. Reduced capacity to metabolize DHT was observed in testosterone to DHT by finasteride, a SRD5A2 inhibitor, led to a tumor samples from four of five freshly isolated pairs of tissue significant 24.8% reduction of the incidence of prostate cancer in samples,which paralleled loss of AKR1C2 and AKR1C1 those treated with the inhibitor compared with the control group expression. LAPC-4 cells transiently transfected with AKR1C1 (7). Thus, in situ hormone synthesis plays an essential role in the and AKR1C2,but not AKR1C3,were able to significantly intracellular availability of DHT to interact with AR. inhibit a dose-dependent,DHT-stimulated proliferation, Numerous studies have focused on the in situ synthesis of which was associated with a significant reduction in the prostatic DHT and the molecular mechanism of AR activation concentration of DHT remaining in the media. R1881- and function (8, 9). However, little emphasis has been placed stimulated proliferation was equivalent in all transfected on the importance of DHT catabolism in the prostate and what cells,showing that metabolism of DHT was responsible for role it might play in regulating the intracellular pool of this the inhibition of proliferation. PC-3 cells overexpressing critical androgen. In the prostate, DHT is predominately a a h AKR1C2 and,to a lesser extent,AKR1C1 were able to signif- metabolized to the weak androgen 5 -androstane-3 ,17 -diol a a a icantly inhibit DHT-dependent androgen receptor reporter (3 -diol) by 3 -hydroxysteroid dehydrogenase (3 -HSD) type activity,which was abrogated by increasing DHT levels. We III encoded by AKR1C2 (10, 11). We originally identified this speculate that selective loss of AKR1C2 in prostate cancer protein by its high affinity for bile salts as the human bile acid a promotes clonal expansion of tumor cells by enhancement of binder, whereas others purified it as a 3 -HSD or a dihydrodiol androgen-dependent cellular proliferation by reducing DHT dehydrogenase (12–14). Subsequent studies revealed that metabolism. [Cancer Res 2007;67(3):1361–9] AKR1C2 is one of four highly related AKR1C subfamily members that have unique substrate specificity and tissue distribution, Introduction despite sharing >84% sequence identity (10, 15). In the human prostate, three of these family members are expressed (11), and Prostate cancer is the second leading cause of cancer-related they have the following enzymatic activities pertinent to deaths among men and has a major effect on the health of older androgen metabolism: AKR1C2, 3a-HSD; AKR1C1, 3h-HSD with Americans (1). Androgens are essential for prostate cancer minor 3a-HSD (97% identity; ref. 16); and AKR1C3, 17h-HSD development, and their elimination and blockade remain the type V (84% identity). DHT is predominately metabolized to 3a- diol by AKR1C2 and 3h-diol by AKR1C1, whereas AKR1C3 can convert androstendione to testosterone but has little 3a-HSD Note: Supplementary data for this article are available at Cancer Research Online activity for DHT (10, 17). (http://cancerres.aacrjournals.org/). We previously observed the selective reduction of AKR1C2 and The radio-HPLC analyses were conducted by the Cell Biology Core, and the real- AKR1C1, but not AKR1C3, gene expression in tumor samples time PCR analysis was done in the Molecular Biology Core of the University of Southern California Research Center for Liver Diseases (National Institutes of compared with their paired benign tissue (11). Our findings have Diabetes, Digestive and Kidney Diseases grant P30-DK48522). All tissue procurements now been extended to establish that loss of these genes is were done by the University of Southern California/Norris Comprehensive Cancer f 3 Center Translational Pathology Core Facility. associated with 2-fold reduced ability to catabolize H-DHT to Requests for reprints: Andrew Stolz, Hoffman Medical Research, Room 101A, 3a-diol, its major metabolite, in freshly isolated tumors compared 2011 Zonal Avenue, Los Angeles, CA 90033. Phone: 323-442-2699; Fax: 323-442-5425. with their paired benign tissues. Furthermore, using prostate E-mail: [email protected]. I2007 American Association for Cancer Research. cancer cell lines, we have found that increased AKR1C2 or AKR1C1, doi:10.1158/0008-5472.CAN-06-1593 but not AKR1C3, could inhibit DHT-stimulated growth and AR www.aacrjournals.org 1361 Cancer Res 2007; 67: (3). February 1, 2007

Table 1. DHT levels compared with relative expression fold changes of AKR1Cs, SRD5A1, SRD5A2, KRT8, and SM1 in paired human prostate tumor tissues versus prostate benign tissues

Sample code Diagnosis and Ages AKR1C2 AKR1C1 SRD5A2 Gleason grade (mean F SD) (mean F SD) (mean F SD)

1 BM-PDPA/3+4 61 À36.7 F 2.7 À11.7 F 1.2 À2.8 F 0.3 2 BM-PDPA/3+4 61 À12.9 F 1.2 2.1 F 0.4 À12.8 F 1.2 3 BMPA/3+3 55 1.6 F 0.2 À4.7 F 0.3 À6.3 F 0.5 4 BPDPA/4+5 74 À4.8 F 0.4 À10.8 F 0.9 1.2 F 0.1 5 PA/4+3 67 À16.3 F 1.2 2.8 F 0.5 À8.2 F 0.6 6 PA/3+3 57 À77.8 F 5.2 À14.3 F 1.2 ND 7 BPDPA/4+5 74 6.4 F 0.4 16.9 F 1.4 1.5 F 0.1 8 BM-PDPA/3+4 55 À31.2 F 0.3 À1.2 F 0.4 À2.1 F 0.2 9 PA/3+4 56 À24.8 F 2.1 À26.4 F 2.2 À1.5 F 0.1 10 BM-PDPA/3+4 76 À21.4 F 1.9 À3.5 F 0.3 À10.2 F 0.2 11 BM-PDPA/3+4 66 À16.8 F 1.7 À49.7 F 4.2 À2.1 F 0.2 12 BM-PDPA/3+4 67 À53.4 F 4.9 6.4 F 0.5 À5.3 F 0.2 13 BM-PDPA/3+4 58 1.5 F 0.2 1.8 F 0.2 1.7 F 0.1

NOTE: DHT levels in paired human prostate tumors tissues versus prostate benign tissues were measured and compared with relative gene expression fold changes of AKR1Cs, SRD5A1, SRD5A2, KRT8, and SM1. Positive values of gene expression fold changes refer to increased expression in tumors whereas negative values represent decreased expression in tumors compared with paired benign tissues. P is the outcome of a paired t test with two tails. Abbreviations: NA, not available in both normal and cancer tissues; ND, not detectable in both normal and cancer tissues; PA, prostatic adenocarcinoma; BMPA, bilateral moderate prostatic adenocarcinoma; BPDPA, bilateral poorly differentiated prostatic adenocarcinoma; BM-PDPA, bilateral multifocal to poorly differentiated prostatic adenocarcinoma; PT, prostate tumor; PN, prostate normal tissue; W, weight.

signaling, which was associated with reduced DHT levels in the proliferation and AR function. Thus, androgen catabolism, in media. In contrast, no inhibition in proliferation or AR reporter addition to synthesis, can indirectly regulate the activity of AR and activity was found when using the nonmetabolizable androgen thereby provide new therapeutic targets for the treatment of R1881, confirming that catabolism can modify DHT-stimulated prostate cancer.

Figure 1. Representative histology of paired tumor and benign appearing prostatic tissue samples. Prostatic tissue slices were processed, stained with H&E, and reviewed to assess tumor stage and homogeneity of resected tissue samples. Histology of two representative pairs of tumor (T) and benign-appearing (N) tissue samples are shown.

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Table 1. DHT levels compared with relative expression fold changes of AKR1Cs, SRD5A1, SRD5A2, KRT8, and SM1 in paired human prostate tumor tissues versus prostate benign tissues (Cont’d)

SRD5A1 KRT8 SM1 DHT in PT DHT in PN DHT PT/PN (mean F SD) (mean F SD) (mean F SD) (ng/g W) (ng/g W) fold changes

1.3 F 0.1 1.3 F 0.2 À1.5 F 0.1 7.42 6.29 1.18 1.8 F 0.1 1.2 F 0.3 À1.1 F 0.1 11.3 7.24 1.56 À1.6 F 0.1 1.3 F 0.1 À1.7 F 0.1 8.34 5.32 1.57 À2.6 F 0.2 À1.1 F 0.2 1.7 F 0.1 3.68 2.35 1.57 1.4 F 0.1 1.4 F 0.4 1.2 F 0.3 6.10 5.91 1.03 2.9 F 0.3 À1.1 F 0.3 À1.9 F 0.1 10.7 6.15 1.74 À1.9 F 0.1 1.4 F 0.1 À1.9 F 0.1 6.59 4.88 1.35 ND À1.3 F 0.3 À1.7 F 0.1 1.72 1.56 1.10 1.2 F 0.1 À1.3 F 0.1 1.3 F 0.2 À F À F À F F F 2.6 0.2 1.4 0.1 1.7 0.1 PP== 0.012 0.012 1.42 0.25 (mean SD) À3.1 F 0.3 À1.2 F 0.2 1.3 F 0.1 1.8 F 0.2 NA NA 1.3 F 0.1 1.3 F 0.2 1.5 F 0.6

Materials and Methods Medical Institute, University of California at Los Angeles, Los Angeles, CA). Both cell lines were cultured in phenol red–free RPMI 1640 with 10% fetal Prostate tissue processing and hormone measurements. After Institu- bovine serum (FBS; Invitrogen, Carlsbad, CA). Permanently transfected tional Review Board approval, 13 freshly isolated and intact human PC-3 cell lines expressing AKR1C1, AKR1C2, or AKR1C3 were developed as prostatectomy samples (primary cancers) were immediately cut into 5- or described (11) and used to assess the effects of AKR1C family members on 6-mm slices, and visibly apparent tumors in the posterior zone of the prostate the DHT- and AR-dependent activation of a mouse mammary tumor virus were removed with 6-mm punch biopsy or a 5-mm punch biopsy (MMTV) promoter. For these studies, 4% charcoal/dextran–treated FBS if tumor was limited. Benign-appearing tissue was isolated from anterior or (HyClone, Logan, UT) was used with DHT or R1881 treatments. central zone with a 6-mm punch biopsy. Tumors were staged by standard Western blot. AKR1C expression was monitored by Western blot with histology, and the purity of tumor and benign-appearing tissue samples was a1850 antiserum that recognizes each AKR1C family member as previously confirmed by reviewing tissue surrounding punch biopsy sites yielding highly described (11, 20). enriched tumor and benign tissue samples minimally contaminated with Quantitative real-time PCR. Relative expression of AKR1Cs, SRD5A1, tumor. Qualified freshly isolated samples were used for RNA isolation, DHT and SRD5A2 was done using a gene-specific real-time PCR as described 3 measurement, or incubations with H-DHT for monitoring of DHTmetabolism. (11, 20), in which their expression in paired tissues were compared with DHT measurement. Prostate tissue samples were homogenized in 0.1 RNase P and reported as mRNA fold change by dividing expression levels in 3 mol/L phosphate buffer (pH 7.4; assay buffer); internal standard ( H-DHT) tumor by its paired normal expression level. Primer/probe sequences for was added to follow procedural losses; and the homogenate was real-time PCR are listed in Table S1. Total RNA was isolated using RNeasy centrifuged. Steroids in the supernatant were then extracted with ethyl Midi kit (Qiagen, Valencia, CA) as described (11, 20). cDNA libraries for acetate/hexane (3:2, v/v). After evaporating the organic solvents, the residue Taqman quantitative real-time PCR were made using the Omniscript kit was reconstituted in isooctane and applied on a Celite partition (Qiagen) primed with random hexamers (Applied Biosystems, Foster City, chromatography column using ethylene glycol as a stationary phase and CA). Statistical analysis was used to compare gene expression profile, DHT toluene in isooctane as the mobile phase. The solvents in the eluted levels, and DHT metabolism as previously described (11, 20, 21). fractions [10% (v/v) toluene in isooctane] containing DHT were evaporated; Cell proliferation assay. Nine micrograms of pSVL-AKR1C1, pCED4- the residue was reconstituted in assay buffer; and DHT was quantified by a AKR1C2, or pcDNA3.1(+)AKR1C3 encoding the corresponding AKR1C sensitive and specific RIA (18). This method was also used in cellular family members in combination with 3 Ag of CMV-eGFP-c2 plasmid (kindly proliferation studies. provided by Dr. Debbie Johnson, Keck School of Medicine, University of DHT metabolism. Fresh pairs of prostate tumor and benign tissue Southern California) were transiently transfected into LAPC-4 cells at 80% samples were minced in 2 mL of RPMI 1640 supplemented with 100 Amol/L to 90% confluence plated in 10-cm dishes as previously described (20). NADP(H). A total of 0.08 ACi (124 Ci/mmol; final concentration, 320 pmol/L) Following transfection, batches of cells were allowed to recover for 24 h and 3 of H-DHT (Perkin-Elmer Life Science, Boston, MA) was added to 2 mL of then grown in RPMI 1640 supplemented with 4% charcoal/dextran–treated the incubation media. Tissue and media were extracted following 4 h of FBSand 1 nmol/L DHT. Following recovery, a portion of the cells was incubation in a shaking water bath maintained at 37jC. DHT metabolism applied onto a Becton Dickinson FACSCalibur (BD Biosciences, Rockville, was terminated by adding the extraction solvent mixture ethyl acetate/ MD) to assess transfection efficiency. Pools of transfected cells were then hexane (3:2, v/v), and the residue was reconstituted in methanol. 3H-DHT seeded onto six-well plates and maintained in RPMI 1640 supplemented and its metabolites were well resolved by the reverse-phase high- with 4% charcoal/dextran–treated FBS; 3 mL of cell culture medium were performance liquid chromatography (HPLC) method of O’Donnell et al. changed daily and supplemented with 1 or 10 nmol/L DHT or R1881, and (19) employing a Waters Spherisorb ODS-2 column (5 mm, 250 mm Â 4 mm). cells were counted for three consecutive days as previously described (20). A Shimadzu LC-10AT HPLC system was used in series with an IN/US hRAM Real-time PCR was used to document gene expression levels of AKR1C 2B detector to monitor and quantify the radioactivity by continuously family members 2 days after the transfection. mixing 1 mL/min of the effluent with 3 mL/min of ScintiVerse E scintillation Effect of AKR1Cs on DHT-dependent AR transactivation. PC-3 cell cocktail (Fisher, Tustin, CA). lines expressing different amounts of AKR1Cs, along with a stable, vehicle Cell culture. The PC-3 cell line was purchased from the American Type vector–transfected PC-3 cell line used as a control, were transiently Culture Collection (Rockville, MD), and the LAPC-4 cell line was kindly transfected with 2 Ag pMMTV-Luc (22), 0.2 Ag pCMV-hAR expression provided by Dr. Sawyers (Department of Medicine and Howard Hughes plasmid (kindly provided by Dr. Gerhard A. Coetzee, Keck School of www.aacrjournals.org 1363 Cancer Res 2007; 67: (3). February 1, 2007

Medicine, University of Southern California) together with 5 ng Renilla quarters of the patients had a relative loss of AKR1C2 and/or luciferase plasmid pRL-SV40 (Promega, Madison, WI) as described AKR1C1 in their tumor samples. As AKR1C1 and AKR1C2 are previously (20). Transiently transfected cells were then allowed to recover equivalent expressed in both stroma and epithelium, relative for 36 h in RPMI 1640 with 4% charcoal/dextran–treated FBS. Cells were enrichment of either component in tumor samples could not then washed three times with PBSand treated for 16 h with DHT or R1881 account for the observed loss of AKR1C1 or AKR1C2 gene in 4% charcoal/dextran–treated FBS. Luciferase activity was measured using the Luminoskan Ascent Machine (Labsystems, Franklin, MA) with the Dual expression. These findings are in close agreement with our prior Luciferase Activity kit (Promega). The ratio of firefly to Renilla luciferase findings (11). Relative reduction of SRD5A2 expression (>4-fold), activity was normalized to total protein to compare promoter activity from but not SRD5A1, in prostate tumors were found in 5 of 13 paired different cell lines. Luciferase activity of control PC-3 cells transfected with samples, consistent with previous studies (29). The average DHT vehicle vector was arbitrarily defined as 100 F SD units. Experiments were levels detected in our paired prostate tissues were comparable with repeated three times in triplicate. previously reported values. When changes in DHT levels were compared within individual pairs, a 42% higher DHT levels was detected on average in prostate tumors compared with their paired Results benign tissue samples (P < 0.05). As SRD5A2 expression was DHT levels in paired prostate cancer tumor and benign decreased, we reasoned that increased DHT synthesis was unlikely tissue samples. Little is known about the cellular localization of to account for the greater DHT levels in these samples. AKR1C1 and AKR1C2 proteins in the prostate as their high amino Reduced DHT metabolism in prostate tumor tissues com- acid sequence homology (97% identity) prevents differentiating paredwithpairedbenigntissues.We developed in vitro each other from AKR1C3, which shares 86% sequence identity. We incubations of freshly harvested prostatic tissues to assess if loss have previously developed an antiserum that cross-reacts with of AKR1Cs was associated with altered metabolism of exogenously AKR1C1 and AKR1C3 but not AKR1C2 (20). In contrast, AKR1C3- supplied 3H-DHT by using a radio-reverse phase HPLC (11, 19). To specific polyclonal antisera and a monoclonal antibody are assess the importance of AKR1Cs in the metabolism of DHT in available as its sequence significantly diverges from the other benign tissue, the non–substrate-competitive inhibitors indometh- family members at its COOH-terminal end. In normal prostate acin (100 Amol/L) or tolmetin (200 Amol/L) were added to the tissue, Fung et al. immunolocalized AKR1C3 using a monoclonal media. We (31) and Steckelbroeck et al. (16) have previously used antibody on endothelial, perineural, smooth muscle cells, and these agents to inhibit the enzymatic activity of AKR1Cs within cells stromal cells with minimal staining in the epithelium (23, 24). In to determine their relative contribution to either androgen contrast, El-Alfry et al. localized AKR1C3 predominately on the metabolism or intracellular distribution and transport of bile salts basal epithelium and luminal cells using a polyclonal AKR1C3- by primary hepatocytes. In two independent benign tissue samples, specific antiserum, which was confirmed by in situ hybridization indomethacin inhibited the loss of 3H-DHT by >3-fold in each case (25, 26). Another report using the same antiserum also showed a (data not shown). In one sample, the AKR1C inhibitor tolmetin was similar immunohistochemical staining pattern (27). To identify also as effective at inhibiting the loss of 3H-DHT, thereby confirming which populations of prostatic cells expressed AKR1C1 and that the AKR1Cs are required for the rapid catabolism of 3H-DHT. AKR1C2, transcriptome analysis of four highly enriched popula- DHT metabolism in five pairs of prostate tissue samples was tions of luminal, basal, stromal, and endothelial cells from the then quantified using this radio-HPLC, and results were compared prostate was queried using the SCGAP Urologic Epithelial Stem with their relative AKR1C1 and AKR1C2 gene expression. The 3H- 5 Cells Project web site (28). Relative expression profile from the DHT data were normalized to tissue weight to allow cross- most abundant to least revealed the following expression patterns: comparisons of changes in the relative radioactivities of 3H-DHT AKR1C1, stromal, luminal > endothelial >> basal; AKR1C2, stromal, and its metabolites with those of the endogenous DHT levels. luminal > endothelial > basal; and AKR1C3, basal, endothelial > Within individual pairs, results from tumors were compared with stromal > luminal cells. These studies indicate that AKR1C1 and their corresponding benign tissues, which were assigned a value of AKR1C2 transcripts are equivalently expressed in the stromal and 100. As shown in Fig. 2A, greater 3H-DHT retention was found in acinar components. four of the five prostate tumors compared with their paired benign Highly enriched tumors with corresponding paired benign tissue tissues, and as a group, significantly greater 3H-DHT retention was samples were harvested and confirmed by histologic evaluation, as found in these tumors compared with their paired benign tissues illustrated in Fig. 1. Table 1 lists the pathologic diagnosis, DHT (P < 0.05). As the average relative retention of 3H-DHT was 2-fold levels, and the relative changes in gene expression responsible for greater in tumors compared with their corresponding benign either DHT catabolism (AKR1C2 and AKR1C1) or DHT synthesis tissues (P < 0.05), isotope dilution within their respective (SRD5A1 and SRD5A2) in these paired prostate samples, compared endogenous intracellular pool of DHT (Table 1) could not account with RNase P, whose expression is unchanged in prostate cancer for this significantly greater retention of 3H-DHT. Moreover, as (11). Relative enrichment of stromal or epithelial content for each shown in Fig. 2B, the radioactivity associated with the 3a-diol sample was broadly assessed by comparing the relative expression metabolite in the tumors consistently accounted for a lower profile of the human keratin 8 gene (KRT8), a recognized marker fraction (54 F 15%) of the combined radioactivity present in the for the epithelial compartment, and the human myosin heavy elution of DHT and its metabolites compared with benign tissues polypeptide 11, smooth muscle (SM1), a stromal-specific marker (P < 0.01). As shown in Fig. 2C, production of the 3h-diol metabolite (29, 30). In these paired samples, relative expression of stromal was also reduced in tumors, and the 3h-diol metabolite only or epithelial markers was closely matched. Approximately three constituted f20% of all the DHT metabolites present in the incubation from benign tissues. In Fig. 2D, relative changes in AKR1C2 and AKR1C1 expression in tumors compared with paired benign tissues paralleled their reduction in DHT metabolism. No 5 http://scgap.systemsbiology.net/. correlation was found between DHT metabolism and the expression

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Reduced AKR1C2 and Impaired DHT Metabolism in Prostate Cancer profile of SRD5A1 and SRD5A2 (data not shown). Three other pairs AKR1C2 were significantly reduced to, respectively, f80% and 30% of samples processed using TLC chromatography also showed a of that remaining in the media of LAPC-4 cells transfected with similar retention of 3H-DHT in tumor samples relative to their paired AKR1C3 or vector controls. As R1881-stimulated proliferation was benign tissues (data not shown). Taken together, these results not reduced by AKR1C1 and AKR1C2, our findings confirm that indicate that metabolism of DHT to 3a-diol was significantly inhibition of DHT-dependent proliferation was not due to impaired in tumors compared with their paired benign tissues. metabolism of another substrate or some other unknown function Inhibition of DHT-dependent growth of LAPC-4 cells by of AKR1C1 or AKR1C2, such as inhibition of the cell cycle. The expression of AKR1C1 and AKR1C2. Similar to the prostate significant reduction in DHT levels after 4 h implicates reduction in cancer cell lines PC-3, DU-145, and LNCaP (11), LAPC-4 cells minimally expresses AKR1C1 and AKR1C2, in contrast to AKR1C3 (data not shown). LAPC-4 cells were chosen for these studies because they express a wild-type AR, in contrast to the AR expressed in LNCaP cells that harbors a mutation in the ligand- binding domain, which permits binding by other class of steroids (32). LAPC-4 cells were individually transfected with AKR1C family members to determine if they can modify androgen-stimulated cellular proliferation. Transfection efficiencies for AKR1C1, AKR1C2, and AKR1C3 expression plasmids in LAPC-4 cells were, correspondingly, 43%, 44%, and 38% for these studies. LAPC-4 cells were transiently transfected with AKR1C1, AKR1C2,orAKR1C3 expression plasmids, which resulted in >5,000-fold increased in AKR1C gene expression, and their growth in response to 1 or 10 nmol/L DHT was compared with LAPC-4 cells transfected with comparable amount of empty vector. In Fig. 3, transfection with pSVL-AKR1C1 (A) or pCED4-AKR1C2 (B) significantly reduced dose-dependent DHT-stimulated proliferation compared with vehicle vectors. However, no inhibition was observed when the nonmetabolizable androgen R1881 was used to stimulate cellular proliferation. In addition, proliferations of nontransfected LAPC-4 cells by R1881 treatments were comparable with LAPC-4 cells transfected with vehicle vector or expression plasmids, which were also observed with DHT treatments (data not shown). AKR1C2 was more effective at inhibiting DHT-stimulated proliferation compared with AKR1C1-transfected LAPC-4 cells. Transfection with AKR1C2 significantly suppressed 1 and 10 nmol/L DHT- stimulated cellular proliferation (P < 0.01), whereas AKR1C1- transfected cells were only able to significantly reduce 1 nmol/L DHT-dependent proliferation (P < 0.05). In contrast, as shown in Fig. 3C, cells transfected with pcDNA3.1(+)AKR1C3 showed no inhibition of DHT- or R1881-stimulated growth. As shown in Fig. 3D, DHT levels in LAPC-4 cells transfected with AKR1C1 and

Figure 2. DHT metabolism in short-term incubations is impaired in prostate cancer. A, relative amounts of 3H-DHT retained in five individual pairs of benign and prostate cancer samples after 4 h of incubation. 3H-DHT radioactivity quantified by radio-HPLC was initially expressed per gram of tissue, which was then normalized for each pair as the ratio of values in tumors divided by paired benign tissue sample, which was designated as 100. Significantly higher ratio 2.0 F 0.7 (mean F SD; P < 0.05) of relative 3H-DHT levels were retained in all the combined prostate tumors compared with their paired benign tissue samples. B, relative 3H-3a-diol values in tumors were compared with their corresponding paired benign tissue samples, which were normalized to 100. In all tumor samples, 3H-3a-diol values contributed a significantly (P < 0.01) lower percentage (54 F 15%) of the combined radioactivity of 3H-DHT and its metabolites compared with their paired benign tissue samples, showing a relative reduction in DHT metabolism by tumors. C, relative 3H-3h-diol values in tumors were compared with their corresponding paired benign tissue samples, which were normalized to 100. In two of the samples, 3H-3h-diol was below levels of detection. The amount of DHT metabolized to 3H-3h-diol was significantly (P < 0.05) lower in the combined tumor samples compared with their paired benign tissues sample. D, changes in gene expression of AKR1C2 and AKR1C1 in tumors were divided by their corresponding relative expression in paired benign tissue samples and defined as mRNA fold change. Negative number represents a decreased expression in tumor compared with paired benign tissue sample. Bars, SD. www.aacrjournals.org 1365 Cancer Res 2007; 67: (3). February 1, 2007

Figure 3. Transient expression of AKR1C2 or AKR1C1 reduces DHT- stimulated proliferation of LAPC-4 cells. Proliferative response of LAPC-4 cells to DHT or a nonmetabolizable androgen (R1881) were determined in cells transiently transfected with AKR1C1, AKR1C2, AKR1C3, or empty vector plasmid. A common legend for (A), (B), and (C) are shown above depicting the concentration of DHT or R1881 with or without the AKR1Cs expression plasmids used for that Panel. A, 1 or 10 nmol/L DHT or R1881 significantly increased cellular proliferation. Proliferations of nontransfected LAPC-4 cells by R1881 were comparable with LAPC-4 cells transfected with vehicle vector or AKR1C expression plasmids. Transient transfection of AKR1C1 plasmid was only able to significantly suppress 1 nmol/L DHT-stimulated cellular proliferation (P < 0.05 significant level) compared with corresponding vehicle vector control cells. AKR1C1 was unable to significantly suppress proliferation of either concentration of R1881 or 10 nmol/L DHT. B, transient transfection of AKR1C2 was able to significantly inhibit 1 or 10 nmol/L DHT-dependent cellular proliferation (P < 0.01) compared with vehicle vector control cells. Comparable proliferation rates were also observed in cells transfected with AKR1C2 and treated with either 1 or 10 nmol/L R1881. The ability of AKR1C2 to inhibit proliferation of both 1 and 10 nmol/L DHT compared with AKR1C1 suggests greater catalytic activity than AKR1C1 for DHT. C, transfection with AKR1C3 was unable to inhibit either 1 or 10 nmol/L DHT-dependent proliferation, confirming that DHT is a poor substrate for AKR1C3. No inhibition of R1881- dependent proliferation was also observed. D, AKR1C1 and AKR1C2 transfected cell respectively showed f80% and 30% of the relative content of DHT remaining in the media after 4 h of incubation in vehicle vector– or AKR1C3-transfected cells.

DHT levels as the major cause for reduce proliferation of all LAPC- As shown in Fig. 4B, increasing AKR1C2 significantly reduced AR- 4 cells present in the wells transfected with AKR1C1 or AKR1C2 dependent MMTV reporter activity in a dose response fashion with plasmids. a single DHT concentration at the P < 0.01 level but had no effect Inhibition of DHT-dependent AR activation by AKR1C2. To on R1881-dependent activation. Conversely, increasing concentra- assess if AKR1C2 could directly block DHT-dependent activation of tions of DHT were able to overcome the inhibition of the reporter an AR-driven reporter, PC-3 cells stably expressing AKR1C1, activation in PC-3 cells permanently expressing the lowest level of AKR1C2, or AKR1C3 were transfected with a MMTV promoter AKR1C2 in Fig. 4C. Taken together, these data show that AKR1C2 that harbors a hormone-responsive element along with an AR can influence DHT-dependent gene expression and signaling by expression plasmid (33). No luciferase activity was detectable in the promoting DHT metabolism. absence of AR or DHT (data not shown). In Fig. 4A, AKR1C2 significantly reduced AR- and DHT-dependent MMTV reporter activity (P < 0.01) to a greater extent than AKR1C1, although more Discussion AKR1C1 was expressed than AKR1C2. Note that no change in Prostate cancer and tissue DHT. In prostate cancer cell luciferase activity was found in cells overexpressing AKR1C3, lines, androgens activate survival pathways that block cell death confirming that DHT is a poor substrate for this family member. mediated by either tumor necrosis factor-a or Fas activation

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Figure 4. Effects of AKR1Cs on DHT-dependent activation of AR-dependent MMTV promoter. DHT- and AR-dependent reporter activity was determined with PC-3 cell lines stably expressing AKR1C1, AKR1C2, or AKR1C3 and were compared with cells stably transfected with vehicle vector. A, expression of AKR1C2 was able to significantly reduce AR-dependent MMTV reporter activity by DHT (P < 0.01). AKR1C1 was also able to reduce AR-dependent MMTV reporter activity (P < 0.05) but not to the same extent as AKR1C2. AKR1C3 was unable to reduce DHT-dependent activation of the MMTV promoter. B, increased expression of AKR1C2 in PC-3 cells was able to significantly inhibit AR-dependent MMTV reporter activity with 100 pmol/L DHT (P < 0.01) compared with vehicle vector control. No inhibition of reporter activity was observed when R1881 was used. C, in PC-3 cells expressing the least amount of AKR1C2, increasing concentrations of DHT were able to significantly overcome the inhibition of AR-dependent reporter activation.

(34), prevent etoposide-induced cell death (35), inhibit phospha- prostate cancer risk associated with functional polymorphism of tase and tensin homologue deleted on chromosome 10–induced SRD5A2 that increase catalytic activity (43). apoptosis (36), and abrogate the toxicity of FKHR (37). Androgen Role of AKR1C2 as a pre-receptor regulator of AR signaling. ablation, by both decreased testosterone synthesis and blockade Steady-state levels of intracellular DHT are maintained through a of AR, effectively reduces AR signaling with both shrinkage of balance between their local synthetic and catabolic rates. In LAPC- tumors and symptomatic relief. Ultimately, a so-called androgen- 4 cells, metabolism of DHT by AKR1C1 and AKR1C2 was sufficient independent state evolves in which prostate cancer becomes to inhibit DHT-stimulated proliferation, although a majority of cells progressively less responsive to androgen ablation (38). Numer- were not transfected. However, the significant reduction in DHT ous pathways participate in this process, including AR activation levels in the media for all cells exposed to the AKR1C1 or AKR1C2 in the absence of ligand and nongenomic effects of AR (38). expression plasmid is likely to have also reduced the DHT- Recent studies have implicated increased AR expression as a dependent proliferation of the nontransfected cells. In addition, no cause for androgen independence with increased sensitivity to low levels of androgens (9). In cell lines, expression of AR by itself is sufficient to induce androgen-independent features, and selective deletion of AR can reverse tumorigenicity of androgen- independent cells (9, 39). Prior epidemiologic and pathologic studies have carefully sought for correlations between androgen levels in either serum or within prostate cancer tumors and the risk of development or progression of prostate cancer (4, 40, 41). Mohler et al. reported changes in prostatic androgen levels in primary prostate cancer compared with recurrent disease as well as differences in prostatic androgens among different ethnic groups (40, 41). However, prior studies have failed to directly compare androgen levels in paired tissues, and none have correlated these changes in DHT levels with gene expression of AKR1C. Most studies have compared production of DHT from testosterone, whereas use of 3H-DHT in our experimental design allowed us to bypass the synthetic pathway to specifically focus on the ability of these samples to catabolize DHT. Kreig et al. also noted significantly Figure 5. Proposed model for increased AR signaling due to loss of AKR1C2 greater DHT levels and reduced 3a-diol levels in prostate cancer and AKR1C1 in prostate cancer. Circulating testosterone (T) diffuses into prostatic cells and is reduced by SRD5A2 to form DHT, the major ligand for the compared with nonpaired benign tissues (42). In our present AR. AKR1C2 is responsible for the majority of DHT catabolism and studies, DHT levels were found on average to be 42% higher in predominately reduces DHT to the weak androgen 3a-diol. AKR1C1 also catalyzes the stereospecific reduction of DHT to the weak androgen 3h-diol. tumors compared with paired benign tissues. Over prolonged 3h-diol is a ligand for ER-h, which promotes antiproliferative response in the periods of time, a proportional 42% increase in DHT binding to prostate. In paired prostate cancer samples, decreased expression of AKR1C2, AR could potentially cause cumulative proliferation and growth of AKR1C1, and SRD5A2 was observed with increased DHT levels. Our model predicts that reduced AKR1C2 expression in prostate cancer leads to reduced tumors. This would be consistent with the molecular epidemi- DHT catabolism, resulting in a greater retention of DHT, thereby enhancing ologic studies of Makridakis et al., who have reported increases in AR-dependent DHT signaling in prostate cancer. www.aacrjournals.org 1367 Cancer Res 2007; 67: (3). February 1, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research inhibition of proliferation was observed with R1881 treatment. (48, 49). From these studies, we conclude that increased DHT as a Data from promoter activity assay revealed that increasing AKR1C2 result of reduced DHT catabolism in prostate cancer would lead to expression reduced the AR-DHT driven MMTV promoter activity in cellular proliferation in an AR-dependent fashion. Our cellular PC-3 cell line. In our paired tissue samples, reduced metabolism of proliferation experiments showed that increased AKR1C2 expres- DHT was found to correspond with the loss of AKR1C2 and sion can reduce DHT-stimulated cell growth, thereby confirming AKR1C1 expression. As SRD5A2 expression was not increased in that increased metabolism of DHT can block the activation of AR. these prostate cancer samples, the loss of DHT metabolism In summary, catabolism of androgens by AKR1Cs can function associated with reduced AKR1C2 expression is probably the major as effective pre-receptor regulators of AR-dependent gene expres- mechanism for the observed increase in DHT levels in the tumor as sion by modulation of the essential ligand for AR. Figure 5 a result of enhanced retention. Of note, Bauman et al. recently illustrates our model for how reduction of AKR1C2 and possibly reported comparable levels of AKR1C2 transcripts in epithelial cell AKR1C1 metabolism of DHT in prostate cancer could promote AR lines established from normal and prostate cancer tissue samples signaling by increasing the intracellular DHT levels. According to (44). Potential explanations for these discrepancies include Steckelbroeck et al., AKR1C2 favors the reduction of DHT to the comparison of whole tissue expression levels with epithelial cell 3a-diol compared with the 3h-diol metabolites by a ratio of 20:1, lines established from tissue samples and variation in gene whereas AKR1C1 ratio of metabolites is 1:4. In addition, AKR1C2 expression profile in these established cell lines due to culture apparent Kcat activity for 3a-diol production is 2.81 compared with h conditions or lack of contact with other cell types. AKR1C1’s Kcat of 0.61 for 3 -diol production, which agrees with Currently, androgen ablation is the major treatment for our findings in Figs. 3 and 4 (16). Thus, AKR1C2 is responsible for hormone-sensitive prostate cancer and is achieved by inhibition reducing DHT to 3a-diol because of its catalytic efficiency and is of testicular testosterone synthesis with gonadotropin-releasing the major enzyme responsible for DHT catabolism. Although hormone receptor antagonists in combination with an AR ligand formation of 3h-diol from DHT was relatively minor compared antagonist (8). However, androgens synthesized from adrenal with 3a-diol, 3h-diol is a recognized ligand for estrogen receptor precursors are only reduced by 50% with these treatments, and h (ER-h), which promotes antiproliferative pathways (50). Loss of Mizokami et al. have reported elevated androst-5-ene-3h,17h-diol AKR1C1 resulting in decreased production of a potent ER-h levels after androgen ablation (45). This and other adrenal derived ligand would also provide a selective growth advantage for these androgens may be important contributors to AR-dependent gene tumor cells. As SRD5A2 expression is decreased in prostate expression (46). Indeed, Nishiyama et al. reported that prostatic cancer, we predict that a significant impairment in the catabolic DHT levels remained at 25% of pretreatment levels in patients pathway is responsible for the increased DHT levels that we undergoing androgen deprivation therapy, in sharp contrast to observed in tumor samples. We speculate that selective loss of serum levels of testosterone that were decreased by 93% (47). Based AKR1C2 and AKR1C1 in prostate cancer promotes clonal on the reasons mentioned above, in situ DHT catabolism mediated expansion of tumor cells by enhancement of androgen-depen- by AKR1C2 in the prostate could be developed as a potential dent cellular proliferation. adjuvant therapy to androgen ablation for hormone-sensitive prostate cancer by induction of AKR1C2 expression. Acknowledgments We speculate that the selective loss of AKR1C2 in prostate cancer Received 5/2/2006; revised 11/13/2006; accepted 12/1/2006. promotes clonal expansion of tumor cells by enhancement of Grant support: University of Southern California/Norris Comprehensive Cancer androgen-dependent cellular proliferation. Isaacs et al. have Center grant 5P30 CA14089-24, Robert E. and Mary R. Wright Foundation, and the emphasized the acquired remodeling of terminal epithelium from Margaret E. Early Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page being normally dependent on paracrine stimulation for growth to charges. This article must therefore be hereby marked advertisement in accordance being independent of these factors after malignant transformation with 18 U.S.C. Section 1734 solely to indicate this fact.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Impaired Dihydrotestosterone Catabolism in Human Prostate Cancer: Critical Role of AKR1C2 as a Pre-Receptor Regulator of Androgen Receptor Signaling

Qing Ji, Lilly Chang, Frank Z. Stanczyk, et al.

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