And Bicalutamide-Resistant Prostate Cancer Chengfei Liu1, Cameron M

Published OnlineFirst May 12, 2017; DOI: 10.1158/1535-7163.MCT-16-0912

Small Molecule Therapeutics Molecular Cancer Therapeutics Niclosamide and Bicalutamide Combination Treatment Overcomes Enzalutamide- and Bicalutamide-Resistant Prostate Cancer Chengfei Liu1, Cameron M. Armstrong1, Wei Lou1, Alan P. Lombard1, Vito Cucchiara1, Xinwei Gu1, Joy C. Yang1, Nagalakshmi Nadiminty1, Chong-xian Pan2,3,4, Christopher P. Evans1,3, and Allen C. Gao1,3,4

Abstract

Activation of the androgen receptor (AR) and its splice variants lutamide combination treatment not only suppressed AR and is linked to advanced prostate cancer and drives resistance to AR variants expression and inhibited their recruitment to the antiandrogens. The roles of AR and AR variants in the develop- PSA promoter, but also signiﬁcantly induced apoptosis in ment of resistance to androgen deprivation therapy (ADT) and bicalutamide- and enzalutamide-resistant CWR22Rv1 and bicalutamide treatment, however, are still incompletely under- C4-2B MDVR cells. In addition, combination of niclosamide stood. To determine whether AR variants play a role in bicaluta- with bicalutamide inhibited the growth of enzalutamide-resis- mide resistance, we developed bicalutamide-resistant LNCaP cells tant tumors. In summary, our results demonstrate that AR (LNCaP-BicR) and found that these resistant cells express signif- variants, particularly AR-V7, drive bicalutamide resistance and icantly increased levels of AR variants, particularly AR-V7, both at that targeting AR-V7 with niclosamide can resensitize bicalu- the mRNA and protein levels. Exogenous expression of AR-V7 in tamide-resistant cells to bicalutamide treatment. Furthermore, bicalutamide-sensitive LNCaP cells confers resistance to bicalu- combination of niclosamide with bicalutamide inhibits enza- tamide treatment. Knockdown of AR-V7 in bicalutamide- and lutamide resistant tumor growth, suggesting that the combi- enzalutamide-resistant CWR22Rv1, enzalutamide-resistant C4- nation of niclosamide and bicalutamide could be a potential 2B (C4-2B MDVR), and LNCaP-BicR cells reversed bicalutamide cost-effective strategy to treat advanced prostate cancer in resistance. Niclosamide, a potent inhibitor of AR variants, signif- patients, including those who fail to respond to enzalutamide icantly enhanced bicalutamide treatment. Niclosamide and bica- therapy. Mol Cancer Ther; 16(8); 1521–30. 2017 AACR.

Introduction CRPC (7, 8). Although both abiraterone and enzalutamide provide signiﬁcant beneﬁt to CRPC patients, resistance occurs Androgen signaling through the androgen receptor (AR) plays frequently. In addition, both abiraterone and enzalutamide an important role not only in maintaining the function of therapies are quite expensive. According to recent cost-effec- the prostate, but also in promoting the development of castra- tiveness analysis, abiraterone treatment cost about $123,400/ tion-resistant prostate cancer (CRPC). A common treatment quality-adjusted life year compared with placebo, and the cost modality for prostate cancer is androgen deprivation, which is of enzalutamide is $437,600/quality-adjusted life year com- achieved either by surgical or medical castration (1). The non- pared with abiraterone (9). The cost of these two drugs makes steroidal antiandrogen bicalutamide is often used in combination treating patients with metastatic CRPC (mCRPC) an extremely with androgen-deprivation therapy (2–4). Although bicaluta- expensive process, and cost-effective therapeutic strategies are mide treatment initially exhibits favorable responses, prostate in urgent need. cancers eventually become refractory and develop resistance to Bicalutamide is a nonsteroidal antiandrogen drug used in the bicalutamide and progress to CRPC (5, 6). Enzalutamide, abir- treatment of prostate cancer and is effective at blocking AR activity aterone, docetaxel, and cabazitaxel are standard treatments for and tumor growth in androgen-responsive prostate cancer (10, 11). Although the TERRAIN and STRIVE clinical trials revealed enzalutamide had better outcomes compared with bica- 1Department of Urology, University of California Davis, California. 2Department lutamide in CRPC patients (12), bicalutamide at a dose of 150 3 of Medicine, University of California Davis, California. UC Davis Comprehensive mg/day is regularly prescribed in men with locally advanced 4 Cancer Center, University of California Davis, California. VA Northern California nonmetastatic prostate cancer either as a monotherapy or an Health Care System, Sacramento, California. adjuvant to surgical or medical castration (13). The mechanisms Note: Supplementary data for this article are available at Molecular Cancer associated with bicalutamide resistance are still incompletely Therapeutics Online (http://mct.aacrjournals.org/). understood. To date, it is known that AR mutations and coacti- Corresponding Author: Allen C. Gao, University of California Davis, Research 3 vators such as TIF2 can confer resistance to (14, 15). Reduced AR Building, Suite 1300, 4645 2nd Avenue, Sacramento, CA 95817. Phone: 916-734- binding and hypersensitivity to low levels of endogenous andro- 8718; Fax: 916-734-8714; E-mail: [email protected] gens are also associated with bicalutamide resistance (16). Alter- doi: 10.1158/1535-7163.MCT-16-0912 native mRNA splicing or genome rearrangements generate trun- 2017 American Association for Cancer Research. cated and constitutively active AR variants which have been

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associated with enzalutamide and abiraterone resistance in late- resulting chromatin preparations were analyzed by PCR using stage prostate cancer patients (17–22). However, the role of AR primers spanning either the proximal or the distal enhancer AREs variants in the development of resistance to bicalutamide has not of the PSA promoter (24, 26). Isotype-matched IgG was used as been well studied. control. In the current study, we found that bicalutamide-resistant prostate cancer cells express higher levels of AR variants than Western blot analysis bicalutamide-responsive cells. Overexpression of AR-V7 confers Whole-cell protein extracts were resolved on SDS–PAGE and resistance to bicalutamide and targeting AR-V7 with niclosamide, proteins were transferred to nitrocellulose membranes. After a potent inhibitor of AR-V7 (23, 24), can resensitize bicalutamide- blocking for 1 hour at room temperature in 5% milk in PBS/ resistant cells to bicalutamide treatment. Furthermore, combina- 0.1% Tween-20, membranes were incubated overnight at 4 C tion of niclosamide with bicalutamide inhibits enzalutamide- with the indicated primary antibodies [AR (441, sc-7305, mouse resistant tumor growth, suggesting that the combination of niclo- mAb, 1:1,000 dilution, Santa Cruz Biotechnology); AR-V7 samide with bicalutamide could provide a potential cost-effective (AG10008, mouse monoclonal antibody, 1:1,000 dilution, pre- a strategy to treat advanced prostate cancer that fails to respond to cision antibody); Tubulin (T5168, monoclonal anti- -tubulin enzalutamide therapy. antibody, 1:5,000 dilution, Sigma-Aldrich)]. Tubulin was used as loading control. Following secondary antibody incubation, Materials and Methods immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Millipore). Reagents and cell culture LNCaP and CWR22Rv1 cells were obtained from the ATCC. All Luciferase assay experiments with cell lines were performed within 6 months of LNCaP cells were transfected with pGL3-PSA6.0-Luc reporters receipt from ATCC or resuscitation after cryopreservation. ATCC along with AR-V7 as indicated in the figures in charcoal-stripped uses short tandem repeat (STR) profiling for testing and authen- FBS (CS-FBS) conditions. Cell lysates were subjected to luciferase tication of cell lines. C4-2B cells were kindly provided and assays with the Luciferase Assay System (Promega) as described authenticated by Dr. Leland Chung, Cedars-Sinai Medical Center previously (23). (Los Angeles, CA). LNCaP, C4-2B, and CWR22Rv1 were maintained in RPMI1640 media supplemented with 10% FBS, 100 Cell growth assay U/mL penicillin, and 0.1 mg/mL streptomycin. LNCaP AR-V7 Cells were treated with the indicated compounds and total cell cells were stably transfected with AR-V7 plasmid and maintained numbers were counted and the cell survival rate [(treatment group in 300 mg/mL G418 RPMI1640 medium. LNCaP cells were cell number/control group cell number) 100%] was calculated incubated with increasing concentrations of bicalutamide after 3 or 5 days. (1–40 mmol/L) over 12 months to generate bicalutamide-resistant LNCaP cells (LNCaP-BicR) and stored for further analysis. Paren- Clonogenic assay tal LNCaP cells were passaged alongside the bicalutamide-treated Cells were plated at equal density (2,000 cells/dish) and treated cells as an appropriate control. C4-2B MDVR cells were main- with the indicated compounds in 100-mm dishes for 14 days. The tained in 20 mmol/L enzalutamide containing medium as colonies were rinsed with PBS before staining with 0.5% crystal described previously (25). All cells were maintained at 37Cin violet/4% formaldehyde for 30 minutes and the number of a humidified incubator with 5% carbon dioxide. Niclosamide was colonies was counted. purchased from Sigma. Cell death ELISA CWR22Rv1, C4-2B MDVR, and LNCaP-BicR cells were seeded Plasmids and cell transfection on 12-well plates (1 105 cells/well) in RPMI1640 media 5 For siRNA transfection, cells were seeded at a density of 1 10 containing 10% FBS and treated with DMSO or 0.5 mmol/L 5 cells per well in 12-well plates or 3 10 cells per well in 6-well niclosamide for 3 days. Mono- and oligonucleosomes in the plates and transfected with siRNA (Dharmacon) targeting the AR- cytoplasmic fraction were measured by the Cell Death Detection V7 sequence (GUAGUUGUGAGUAUCAUGA) or a control ELISA Kit (Roche, catalog. no. 11544675001) as described pre- sequence (CTTACGCTGAGTACTTCGA) using Lipofectamine viously (27). Briefly, cells were collected and homogenized in 400 2000 (Invitrogen). Cells were transiently transfected with AR- mL of incubation buffer. The wells were coated with anti-histone V7 plasmids using Attractene (Qiagen). antibodies and incubated with the lysates, horseradish peroxidase–conjugated anti-DNA antibodies, and the substrate. Absor- Chromatin immunoprecipitation assay bance was measured at 405 nm. DNA–AR protein complexes were cross-linked inside the cells by the addition of 1% formaldehyde. Whole-cell extracts were Real-time quantitative RT-PCR prepared by sonication, and an aliquot of the cross-linked DNA– Total RNA was extracted using TRIzol reagent (Invitrogen). protein complexes was immunoprecipitated by incubation with cDNAs were prepared after digestion with RNase-free RQ1 DNase antibodies against AR (AR-C19; Santa Cruz Biotechnology) or AR- (Promega). The cDNAs were subjected to real-time reverse tran- V7 (AG10008, precision antibody) overnight at 4C with rotation. scription-PCR (RT-PCR) using Sso Fast Eva Green Supermix (Bio- Chromatin–antibody complexes were isolated from solution by Rad) according to the manufacturer's instructions and as incubation with protein A/G agarose beads for 1 hour at 4C with described previously (28). Each reaction was normalized by rotation. The bound DNA–protein complexes were washed and coamplification of actin. Triplicates of samples were run on eluted from beads with elution buffer (1% SDS and 0.1 mol/L default settings of Bio-Rad CFX-96 real-time cycler. Primers used NaHCO3), crosslinking was reversed, and DNA was extracted. The for RT-PCR were: AR-full length: forward (F)-AAG CCA GAG CTG

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Niclosamide plus Bicalutamide Overcomes Resistance

TGC AGA TGA, reverse (R)-TGT CCT GCA GCC ACT GGT TC; AR- Results V1: F-AAC AGA AGT ACC TGT GCG CC, R-TGA GAC TCC AAA Bicalutamide-resistant prostate cancer cells express AR variants CAC CCT CA; AR-V3: F-TGG ATG GAT AGC TAC TCC GG, R-GTT Numerous reports show that enzalutamide and abiraterone CAT TCT GAA AAA TCC TTC AGC; AR-V7: F-AAC AGA AGT ACC treatment induce expression of AR variants, particularly AR-V7, in TGT GCG CC, R-TCA GGG TCT GGT CAT TTT GA; AR-V9: F-TGC prostate cancer cells (20, 21, 23, 29). But, to date, the effect of GCC AGC AGA AAT GAT TG, R-GCA GCT GCT CAG GTA AGT TG; bicalutamide treatment on AR variants expression in prostate PSA: F-GCC CTG CCC GAA AGG, R-GAT CCA CTT CCG GTA ATG cancer cells or patients is unknown. To address this issue, we CA; FKBP5: F-AGA ACC AAA CGG AAA GGA GA, R-GCC ACA TCT generated bicalutamide-resistant cells (LNCaP-BicR) by chroni- CTG CAG TCA AA; UBE2C: F-TGG TCT GCC CTG TAT GAT GT, R- cally treating LNCaP cells with bicalutamide for more than 6 AAA AGC TGT GGG GTT TTT CC; Myc: F-TGA GGA GAC ACC months. As shown in Fig. 1A, after 12 months of continuous GCC CAC, R-CAA CAT CGA TTT CTT CCT CAT C; Actin: F-AGA culture in media containing bicalutamide, LNCaP-BicR, cells ACT GGC CCT TCT TGG AGG, R-GTT TTT ATG TTC CTC TAT exhibited increased resistance to bicalutamide compared with GGG. LNCaP parental cells. These results were confirmed by clonogenic assay (Fig. 1B and C). Next, we examined the expression of AR In vivo tumorigenesis assay variants in LNCaP parental and LNCaP-BicR cells. LNCaP-BicR 6 CWR22Rv1 cells (4 10 ) were mixed with Matrigel (1:1) cells express higher levels of AR variant mRNAs than LNCaP fl – andinjectedsubcutaneouslyintothe anks of 6 7weeksold parental cells, including AR-V1, AR-V3, AR-V7, and AR-V9 (Fig. male SCID mice. Tumor-bearing mice (tumor volume around 1D). The levels of AR-V7 protein expression were confirmed by – 3 n ¼ 50 100 mm ) were randomized into four groups ( 5per Western blot analysis (Fig. 1E). LNCaP-BicR cells express signif- group) and treated as follows: (i) vehicle control (5% Tween 80 icantly higher levels of AR-V7 protein compared with LNCaP and 5% ethanol in PBS, intraperitoneally), (ii) bicalutamide parental cells, particularly in CS-FBS conditions. We also exam- (25 mg/kg, orally), (iii) niclosamide (25 mg/kg, intraperitone- ined the location of AR-V7 expression in LNCaP-BicR cells. As þ ally), (iv) bicalutamide (25 mg/kg, orally) niclosamide (25 shown in Fig. 1F, AR-V7 was highly expressed in the nucleus in mg/kg, intraperitoneally). Tumors were measured using cali- LNCaP-BicR cells. Full-length AR was also upregulated in LNCaP- pers twice a week and tumor volumes were calculated using BicR cells compared with the parental LNCaP cells. Collectively, 2 length width /2. Tumor tissues and vital organs were har- these results demonstrated that bicalutamide-resistant LNCaP vested after 3 weeks of treatment. All the animal experiments cells express higher levels of AR and AR variants than parental were performed in accordance with the approved guidelines. cells. The experimental animal protocol was approved by the Insti- tutional Animal Care and Use Committee (IACUC), University of California, Davis, and satisfied all University and NIH rules Overexpression of AR-V7 confers resistance to for the humane use of laboratory animals. bicalutamide treatment Previous studies revealed that AR-V7 confers resistance to enzalutamide and abiraterone (21, 29, 30). To determine whether IHC overexpression of AR-V7 also confers resistance to bicalutamide in Tumors and vital organs were fixed by formalin- and paraffin- bicalutamide-resistant LNCaP-BicR cells, we knocked down AR- embedded tissue blocks were dewaxed, rehydrated, and blocked V7 by transfecting siRNA specific to AR-V7, and then treated the for endogenous peroxidase activity. Antigen retrieval was per- cells with 20 mmol/L bicalutamide for 3 days and cell numbers formed in sodium citrate buffer (0.01 mol/L, pH 6.0) in a were counted. As shown in Fig. 2A, downregulation of AR-V7 microwave oven at 1,000 W for 3 minutes and then at 100 W expression resensitized LNCaP-BicR cells to bicalutamide treat- for 20 minutes. Nonspecific antibody binding was blocked by ment. The effects of AR-V7 knockdown by AR-V7 siRNA were incubation in 10% FBS in PBS for 30 minutes at room temper- validated by Western blot analysis (Fig. 2B). To further examine ature. Slides were then incubated with anti–Ki-67 (at 1:500; whether AR-V7 drives bicalutamide resistance, we generated AR- NeoMarker), or anti-AR-V7 (at 1:250; AG10008, precision anti- V7–overexpressing LNCaP AR-V7 cells by stably expressing AR-V7 body) at room temperature for 30 minutes. Slides were then in LNCaP cells (Fig. 2C). LNCaP-neo and LNCaP AR-V7 cells were washed and incubated with biotin-conjugated secondary anti- treated with different concentrations of bicalutamide for 3 days bodies for 30 minutes, followed by incubation with avidin DH- and cell numbers were determined. Two independent LNCaP AR- biotinylated horseradish peroxidase complex for 30 minutes V7–stable cell lines exhibited resistance to bicalutamide treatment (Vectastain ABC Elite Kit, Vector Laboratories). The sections were in a dose-dependent manner compared with LNCaP-neo cells developed with the diaminobenzidine substrate Kit (Vector Lab- (Fig. 2D). Collectively, these results suggest that overexpression of oratories) and counterstained with hematoxylin. Nuclear staining AR-V7 confers resistance to bicalutamide. cells were scored and counted in 5 different vision areas. Images Both CWR22Rv1 and C4-2B MDVR cells are resistant to enza- were taken with an Olympus BX51 microscope equipped with lutamide and bicalutamide and express higher levels of AR-V7 DP72 camera. than cells that respond to enzalutamide or bicalutamide treatment (21, 23). We have previously shown that overexpression of Statistical analysis AR-V7 drives resistance to enzalutamide in CWR22Rv1 and C4-2B All data are presented as means SD of the mean. Statistical MDVR cells (23), and downregulation of AR-V7 resensitizes these analyses were performed with Microsoft Excel analysis tools. cells to enzalutamide treatment. To test whether downregulation Differences between individual groups were analyzed by one- of AR-V7 in CWR22Rv1 and C4-2B MDVR cells can resensitize way ANOVA followed by the Scheffe procedure for comparison these enzalutamide-resistant cells to bicalutamide treatment as of means. P < 0.05 was considered statistically significant. well, we knocked down AR-V7 by transfecting AR-V7 siRNA to

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A B 120 Figure 1. 100 Chronic bicalutamide treatment in 80 LNCaP parental LNCaP cells increases expression of AR LNCaP - * parental and AR variants. A, LNCaP parental or 60 LNCaP-BicR LNCaP-BicR cells were treated with 40 different concentrations of bicalutamide in RPMI 1640 media 20 BicR LNCaP - containing 10% FBS, and total cell Cell survival rate (%) 0 numbers were counted after 3 days. 0 10 20 50 10 20 B–C, The clonogenic ability of LNCaP µ Bicalutamide (µmol/L) Bicalutamide ( mol/L) parental and LNCaP-BicR cells treated with 10 mmol/L and 20 mmol/L * bicalutamide was analyzed. Colonies C D were counted, and results are 14 presented as means SD of 2 800 ** experiments performed in duplicate. 700 12 AR-V1 D, LNCaP parental cells and LNCaP- 600 10 BicR cells were cultured in RPMI 1640 500 AR-V3 400 8 media containing 10% FBS for 3 days, LNCaP parental AR-V7 total RNA was extracted and AR-V1, 300 6 LNCaP BicR AR-V3, AR-V7, AR-V9, and full-length 200 AR-V9 expression 4 AR mRNA levels were analyzed by Colony numbers 100 Relative mRNA AR-FL 0 2 qRT-PCR. E, LNCaP parental cells and 0210 0 LNCaP-BicR cells were cultured in Bicalutamide (µmol/L) 0 media containing 10% FBS or 10% CS- LNCaPLNCaP-BicR FBS for 3 days, the total cell lysates were harvested and analyzed by E F Western blotting using antibodies Cytoplasm Nucleus against AR-V7. F, LNCaP parental cells FBS CSS and LNCaP-BicR cells were cultured in LNCaP- LNCaP- LNCaP- LNCaP- media containing 10% CS-FBS for 3 parental BicR LNCaP- LNCaP- LNCaP- LNCaP- parental BicR days, the cells were harvested for parental BicR parental BicR AR-V7 preparation of cytosolic and nuclear fractions and analyzed by Western AR-V7 AR-FL blotting using antibodies against AR- V7, AR, RNA polymerase II, or Tubulin. AR-FL AR Variants The expression of RNA polymerase II and tubulin was used as markers for AR Variants Pol II the integrity of the nuclear and cytosolic fractions, respectively. , P < 0.05. Tubulin Tubulin

both CWR22Rv1 and C4-2B MDVR cells, and then treated the cells with or without 20 mmol/L bicalutamide for 3 or 5 days with 20 mmol/L bicalutamide for 3 days and cell numbers were and total cell numbers were counted. As shown in Fig. 3A, counted. As shown in Fig. 2E, knockdown of AR-V7 resensitized 20 mmol/L bicalutamide had no effect on cell growth and enzalutamide-resistant CWR22Rv1 and C4-2B MDVR cells to 0.5 mmol/L niclosamide had some degree of growth inhibition bicalutamide. The effects of AR-V7 knockdown were confirmed in both CWR22Rv1 and C4-2B MDVR cells. However, combi- by the Western blot analysis (Fig. 2F). These results suggest that nation treatment of niclosamide with bicalutamide further bicalutamide can be combined with AR-V7–targeting agents to inhibited cell growth. The results were confirmed by clonogenic treat advanced prostate cancer cell resistance to either enzaluta- assay. As shown in Fig. 3B, combination of bicalutamide mide or bicalutamide. with niclosamide significantly inhibited colony formation in CWR22Rv1 and C4-2B MDVR cells. These results suggest that Combination of niclosamide with bicalutamide overcomes bicalutamide can be combined with niclosamide to effectively treatment resistance to enzalutamide in vitro treat enzalutamide-resistant prostate cancer cells. In previous studies, we identified niclosamide as a potent To further examine niclosamide and bicalutamide combina- inhibitor of AR-V7, and demonstrated that niclosamide can tion effects in enzalutamide-resistant prostate cancer cells, cell reverse enzalutamide resistance in advanced prostate cancer apoptosis was examined by cell death ELISA. CWR22Rv1 and C4- cells (23). Having demonstrated that downregulation of AR-V7 2B MDVR cells were treated with 0.5 mmol/L niclosamide with or can resensitize enzalutamide-resistant CWR22Rv1 and C4-2B without 20 mmol/L bicalutamide for 3 days and then the cell MDVR cells to bicalutamide treatment, we next determined if lysates were collected and subjected to cell death ELISA. Bicalu- niclosamide, which targets AR-V7, could resensitize enzaluta- tamide did not induce apoptosis in these two cell lines and mide-resistant cells to bicalutamide treatment. CWR22Rv1 and niclosamide slightly induced apoptosis. The combination treat- C4-2B MDVR cells were treated with 0.5 mmol/L niclosamide ment significantly induced cell death (Fig. 3C). Cell survival genes

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Niclosamide plus Bicalutamide Overcomes Resistance

A LNCaP-BicR B * 120 + + − − sicontrol 100 − − + + si AR-V7 − + − + Bicalutamide (20 µmol/L) 80 sicontrol AR-V7 60 si AR-V7 40 AR-FL AR variants 20 Cell survival rate (%) 0 Tubulin DMSO Bica 20 µmol/L

C D 120

100

80 − + − + − + Bicalutamide (20 µmol/L) AR-V7 60 * AR-FL 40 LNCaP neo AR variants 20 LNCaP AR-V7 #2

Cell survival rate (%) LNCaP AR-V7 #4 Tubulin 0 50 10 20 Bicalutamide (µmol/L)

E F sicontrol* si AR-V7 CWR22Rv1 C4-2B MDVR 120 * + + − − + + − − sicontrol 100 − − + + − − + + si AR-V7 80 − + − + − + − + Bicalutamide (20 µmol/L) 60 AR-V7 40 20 AR-FL

Cell survival rate (% ) 0 AR variants

Tubulin DMSO DMSO

Bica 20 µmol/L Bica 20 µmol/L CWR22Rv1 C4-2B MDVR

Figure 2. AR-V7 is involved in bicalutamide resistance. A–B, LNCaP-BicR cells were transiently transfected with AR-V7 siRNA, and then treated with 20 mmol/L bicalutamide for 3 days. AR-V7 and AR expression were examined by Western blot. Total cell numbers were counted, and cell survival rate (%) was calculated. C, LNCaP-neo, LNCaP AR-V7 #2, and #4 clones were treated with 20 mmol/L bicalutamide for 3 days, the total cell lysates were harvested and analyzed by Western blotting using antibodies against AR and AR-V7. D, LNCaP-neo, LNCaP AR-V7 #2, and #4 clones were treated with 20 mmol/L bicalutamide in media containing FBS, and cell numbers were counted after 3 and 5 days. Results are presented as means SD of 3 experiments performed in duplicate. E–F, CWR22Rv1 and C4-2B MDVR cells were transiently transfected with AR-V7 siRNA, and then treated with 20 mmol/L bicalutamide for 3 days. Total cell numbers were counted, and cell survival rate (%) was calculated. AR-V7 and AR expression were examined by Western blot. , P < 0.05. Bica, Bicalutamide. downstream of AR and AR variants such as c-Myc, CyclinD1 and with bicalutamide can overcome resistance to enzalutamide survivin were also examined. Combination of niclosamide and treatment in vitro. bicalutamide signiﬁcantly inhibited cell survival gene expression. To explore the combinatory effects of niclosamide and bica- The levels of c-Myc, Cyclin D1, survivin, Bcl-2, and Bcl-xl expres- lutamide on androgen signaling, CWR22Rv1, C4-2B MDVR, and sion were reduced and cleaved caspase activation and cleaved LNCaP-BicR cells were treated with DMSO, 0.5 mmol/L niclosa- PARP expression were increased (Fig. 3D). In summary, combi- mide, 20 mmol/L bicalutamide, or their combination for 3 days nation of niclosamide and bicalutamide treatment signiﬁcantly and total cell lysates were collected and subjected to Western blot. induced apoptosis in enzalutamide-resistant cell lines. Taken As shown in Fig. 4A, bicalutamide treatment had no effect on AR- together, the above results show that combination of niclosamide V7 expression, niclosamide reduced the AR-V7 expression and the

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CWR22Rv1 C4-2B MDVR

120 ) 50 A ) 4 4 100 40 80 * DMSO 30 µ 60 * * Bica 20 mol/L 20 Nic 0.5 µmol/L 40 * * Combination 10 20 * Cell number (×10 Cell number (×10 0 0 053 053 Days Days Figure 3. Niclosamide and bicalutamide CWR22Rv1 C4-2B MDVR B treatment suppresses enzalutamide- * resistant cell growth and induces 800 apoptosis in vitro. A, CWR22Rv1 cells 700 and C4-2B MDVR cells were treated

CWR22Rv1 600 with 0.5 mmol/L niclosamide with or 500 without 20 mmol/L bicalutamide in 400 media containing FBS, and cell 300 numbers were counted after 3 and 5 200 days. Results are presented as means

Colony number s 100 SD of 3 experiments performed in C4-2B MDVR 0 duplicate. B, Clonogenic assays were DMSO Bica 20 µmol/L Nic 0.5 µmol/L Combination performed in CWR22Rv1 and C4-2B MDVR cells, colony pictures were taken DMSO C under microscope and the number of Combination colonies was counted. Results are CWR22Rv1 Bica 20 Nicµmol/L 0.5 µmol/L 1 * presented as means SD of 2 0.8 experiments performed in duplicate. D CWR22Rv1 C4-2B MDVR C, CWR22Rv1 cells and C4-2B MDVR 0.6 m − + − + − + − + Bica (20 µmol/L) cells were treated with 0.5 mol/L niclosamide with or without 20 mmol/L 0.4 * − − + + − − + + Nic (0.5 µmol/L) bicalutamide in media containing FBS Absorbance

(450 nmol/L) 0.2 Cleaved-PARP for 3 days, cell lysates were subjected 0 to cell death ELISA. Results are Cleaved-caspase-7 presented as means SD of 3 experiments performed in duplicate. Control c-Myc D, CWR22Rv1 cells and C4-2B MDVR m Combination cells were treated with 0.5 mol/L Bica 20 Nicµmol/L 0.5 µmol/L CyclinD1 niclosamide with or without 20 mmol/L bicalutamide in media containing FBS 1.8 C4-2B MDVR Survivin for 3 days, total cell lysates were 1.6 * collected and subjected to Western 1.4 Bcl-2 blot. , P < 0.05. Bic, Bicalutamide; Nic, 1.2 Niclosamide. 1 Bcl-xl 0.8 * 0.6 Absorbance (450 nmol/L) 0.4 Bax 0.2 0 Tubulin

Control

Combination Bica 20 Nicµmol/L 0.5 µmol/L

combination further inhibited both AR and AR-V7 protein expres- the AR-V7 transcriptional activity, combined niclosamide and sion. Knockdown of AR-V7 expression using AR-V7 siRNA sup- bicalutamide further inhibited both DHT and AR-V7 induced AR pressed PSA luciferase activity in C4-2B MDVR cells, and combi- transcriptional activity. We also examined the full-length AR and nation of AR-V7 siRNA and bicalutamide further reduced PSA AR-V7 target genes by niclosamide and bicalutamide combina- luciferase activity (Supplementary Fig. S1). To examine if niclo- tion treatment in C4-2B MDVR cells. As shown in supplementary samide combined with bicalutamide could affect AR and AR-V7 Fig. S2, bicalutamide treatment suppressed full-length AR target transcriptional activity, LNCaP cells were transiently transfected genes, such as PSA, NKX3.1 and FKBP5 but not AR-V7 target genes, with PSA luciferase plasmid with or without AR-V7 or 1 nmol/L such as UBE2C and Myc. However, niclosamide treatment sup- DHT and then treated with 0.5 mmol/L and 1 mmol/L niclosamide pressed the expression of both full-length AR and AR-V7 target with or without 20 mmol/L bicalutamide overnight. As shown genes, and the niclosamide and bicalutamide combination fur- in Fig. 4B, both niclosamide and bicalutamide signiﬁcantly ther suppressed the expression of these target genes. These results inhibited DHT induced PSA activity. While niclosamide inhibited were conﬁrmed in a C4-2 AR-V7 stable clone by ChIP assay. As

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Niclosamide plus Bicalutamide Overcomes Resistance

A CWR22Rv1C4-2B MDVR LNCaP BicR − + − + − + − + − + − + Bicalutamide (20 µmol/L) − − + + − − + + − − + + Niclosamide (0.5 µmol/L) AR-V7

AR-FL Figure 4. Niclosamide and bicalutamide AR variants combination treatment suppresses Tubulin AR and AR variants signaling in enzalutamide-resistant prostate cancer. A, CWR22Rv1, C4-2B MDVR, and LNCaP-BicR cells were treated B LNCaP PSA-E/P-LUC m * with 0.5 mol/L niclosamide with or * without 20 mmol/L bicalutamide in * 4,500 media containing FBS for 3 days, DMSO total cell lysates were collected and 4,000 20 µmol/L Bicalutamide subjected to Western blot. B, LNCaP 3,500 cells were transiently transfected 3,000 with PSA-E/P luciferase with 0.5 mg 2,500 * * AR-V7 plasmid or control plasmid for RLU 2,000 24 hours, cells were then treated with 1,500 DMSO, 0.5 mmol/L niclosamide, 20 mmol/L bicalutamide, or their 1,000 combination with or without 1 nmol/L 500 DHT overnight and luciferase activity 0 was measured. Results are presented 000.5 1 0.5101 0.5 Niclosamide (µmol/L) as means SD of 3 experiments performed in duplicate. C, C4-2 AR- Vector 1 nmol/L DHT AR-V7 V7 cells were treated with DMSO, m m 0.5 mol/L niclosamide, 20 mol/L C C4-2 AR-V7 bicalutamide, or their combination for 2 days. Recruitment of AR to AREs − + − + − + − + Bicalutamide (20 µmol/L) in the PSA promoter was analyzed by − − + + − − + + Niclosamide (0.5 µmol/L) ChIP assay. , P < 0.05. IP: AR (C19) IP: AR-V7 AR E IP: IgG IP: IgG

Input Input

shown in Fig. 4C, both bicalutamide and niclosamide reduced the weight (Fig. 5D). To determine whether niclosamide alone or recruitment of full-length AR to the PSA promoter, however, only in combination with bicalutamide represses AR-V7 expression niclosamide significantly reduced the recruitment of AR-V7 to the and tumor proliferation in vivo, representative tumor samples PSA promoter and combination treatment further reduced the were analyzed by IHC for AR-V7 and Ki67. As shown in Fig. 5E, recruitment. Taken together, these results show that combination niclosamide inhibited AR-V7 and Ki67 expression while com- of niclosamide with bicalutamide suppresses AR-V7 expression bination treatment further decreased the levels of their expres- and transcriptional activity and induces cell growth inhibition sion. These results suggest that niclosamide can improve bica- and apoptosis. lutamide treatment, and combination of niclosamide with bicalutamide can be used to treat enzalutamide-resistant pros- Combination of niclosamide with bicalutamide suppresses tate cancer. tumor growth of enzalutamide-resistant prostate cancer To examine the treatment toxicity in each group, histopath- To test whether niclosamide plus bicalutamide can overcome ologic examination of the vital organs was performed. Overall, resistance to enzalutamide in vivo, xenografts generated from no significant pathological changes were noted in any group. enzalutamide-resistant CWR22Rv1 cells were treated with vehi- AsshowninFig.5F,allthevitalorganswerenormalafter3 cle, bicalutamide, niclosamide, or their combination for 3 weeks of treatment; the livers did not show any vacuolar weeks as described in the methods. As shown in Fig. 5A–C, changes, and there was no sign of inflammation at the renal CWR22Rv1 cells were resistant to bicalutamide treatment with pelvis in single treatment or combination treatment group. In tumor volumes comparable with those in the vehicle treated conclusion, both the niclosamide and combination treatment control group. Niclosamide alone decreased the tumor volume were well tolerated. while combination of niclosamide and bicalutamide synergis- tically decreased CWR22Rv1 tumors, indicating that niclosa- Discussion mide plus bicalutamide could overcome enzalutamide resistance and restore sensitivity of CWR22Rv1 xenografts to bica- In this study, we demonstrated that AR-V7 expression confers lutamide treatment in vivo.Thetreatmentdidnotaffectanimal resistance to bicalutamide and that downregulation of AR-V7

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Liu et al.

ABCD 25

) 3,000 1.6 3 1.4 20 2,500 1.2 Control 15 Control 1 2,000 0.8 * 10 Bicalutamide Bicalutamide 0.6 1,500 * 25 mg/kg 0.4 5

* Body weight (g) Niclosamide 0.2 1,000 * Niclosamide Tumor weight (g) 0 0 25 mg/kg Combination 500 * Combination Tumor volume (mm 0 0 3 7 10 14 17 21 Days after treatment

E 600 F Control Bica Nic Combination 500 Control Bicalutamide Niclosamide Combination 400 300 * 200 Heart 100 * Ki67 0 Ki67–Positive cells Liver AR-V7

350 Lung 300 250 200 150 Kidney

H/E 100 * 50 * 0 AR-V7–Positive cells Spleen

Figure 5. Niclosamide and bicalutamide treatment suppressed enzalutamide-resistant tumor growth. A, Mice bearing CWR22Rv1 xenografts were treated with vehicle control, bicalutamide (25 mg/kg orally), niclosamide (25 mg/kg intraperitoneally), or their combination for 3 weeks. Tumor volumes were measured twice weekly. B–C, Tumors were photographed and weighed. D, Body weight for each group was calculated. E, IHC staining of Ki-67 and AR-V7 in each group was performed and quantiﬁed. F, Hematoxylin and eosin (H&E) staining of vital organs from each group was performed. , P < 0.05. Bic, Bicalutamide; Nic, Niclosamide.

expression resensitizes bicalutamide and enzalutamide resistance other mechanisms involved in bicalutamide resistance have been cells to bicalutamide treatment. In addition, combination of discovered. The CAMK2N1 gene, an inhibitor of CaMKII (calci- bicalutamide with niclosamide, a potent inhibitor of AR-V7, um/calmodulin-dependent protein kinase II), has been shown to suppresses tumor growth of enzalutamide-resistant prostate can- induce bicalutamide resistance and is inversely correlated with AR cer in vitro and in vivo. These results indicate the potential ther- in prostate cancer (38). TIF2/glucocorticoid receptor-interacting apeutic efficacy of niclosamide in combination with bicalutamide protein (TIF2/GRIP1/SRC2) is a member of the steroid receptor to treat advanced prostate cancer that is resistant to enzalutamide. coactivator (SRC/p160) family and has also been found to con- Bicalutamide has been widely used as the first line treatment for tribute to bicalutamide resistance (15). In the present study, we prostate cancer that has failed androgen deprivation therapy. provide evidence that AR variants, particularly AR-V7, confer Although initially effective, resistance frequently occurs, most resistance to bicalutamide treatment. Overexpression of AR-V7 likely due to structural changes within the AR or modifications in bicalutamide-sensitive LNCaP cells confers resistance to bica- in androgen-dependent signaling cascades. AR mutation has been lutamide, while knockdown AR-V7 in bicalutamide-resistant cells linked to anti-androgen resistance. Mutations in the ligand-bind- resensitizes them to bicalutamide treatment. Chronic bicaluta- ing domain that alter ligand specificity and sensitivity frequently mide treatment of LNCaP cells significantly increased expression increase after androgen ablation. The AR F877L mutation has of several AR variants including AR-V1, AR-V7, and AR-V9. Among been found in enzalutamide-resistant patients and might allow the identified AR variants, AR-V7, which is encoded by contiguous for enzalutamide acting as an AR antagonist, especially when this splicing of AR exons 1/2/3/CE3, has been well studied mainly due mutation co-occurs with the T878A mutation in prostate cancer to its prevalence in prostate cancer samples (17–19). AR-V7 is a cells (31–33). Prolonged enzalutamide or apalutamide treatment constitutively active, ligand independent transcription factor that might reposition the drug to eliminate steric clashes that pro- can induce castration-resistant cell growth in vitro and in vivo moted helix 12 terminus (H12) dislocation in AR (34). The T878A (19, 39, 40). AR-V7 also may heterodimerize with full-length and H875Y AR mutants have been reported to be activated by androgen receptor (AR-FL) in an androgen-independent manner flutamide leading to anti-androgen withdrawal syndrome (41). Recent studies have linked AR alternative splicing, particu- (35–37). Similarly, bicalutamide is an agonist to the W741L larly AR-V7, to the development of enzalutamide, abiraterone mutant AR in prostate cancer (14). In addition to AR mutations, and docetaxel resistance (21, 29, 42). Clinical studies suggest that

1528 Mol Cancer Ther; 16(8) August 2017 Molecular Cancer Therapeutics

Niclosamide plus Bicalutamide Overcomes Resistance

AR-V7 in circulating tumor cells from patients with CRPC is sible, but also would provide an economical cost benefit com- associated with resistance to enzalutamide and abiraterone (22, pared with current treatment strategies. 43). Several preclinical studies revealed that AR variants might In summary, our results demonstrate that AR variants, partic- play certain roles in taxane resistance which could be through ularly AR-V7, drive bicalutamide resistance. Targeting AR-V7 with blocking nuclear translocation via AR. AR-V7 lacks the hinge niclosamide can resensitize bicalutamide-resistant cells to bica- region that mediates the microtubule binding, which reduces the lutamide treatment. Furthermore, combination of niclosamide association of AR-V7 with microtubules (42, 44). Thus, targeting with bicalutamide inhibits enzalutamide-resistant tumor growth, AR-V7 could be a valuable strategy to treat CRPC patients. The suggesting that the combination of niclosamide with bicaluta- challenge to designing a drug that targets AR-V7 in patients is mide could provide a potential cost-effective strategy to treat mainly drug toxicity and clinical efficacy. The ideal strategy to advanced prostate cancer that fails to respond to enzalutamide target AR-V7 is to decrease AR-V7 expression level or suppress its therapy. A clinical trial of niclosamide plus bicalutamide to treat transcriptional activity by targeting the N-terminal domain. Our mCRPC patients is planned. previous studies revealed that niclosamide targets AR variants and enhances enzalutamide and abiraterone treatment (23, 24). In the Disclosure of Potential Conflicts of Interest present study, we demonstrated that niclosamide could be used to C. Liu, W. Lou, and A.C. Gao have ownership interest (including patents) resensitize bicalutamide-resistant cells via downregulation of AR- in Patent application covering the use of niclosamide. C.-x. Pan has ownership interest (including patents) in Pandomedx. C.P. Evans has received speakers V7 expression. Combination of niclosamide with bicalutamide bureau honoraria from, and is a consultant/advisory board member for Astellas. significantly suppressed AR and AR-V7 transcriptional activity and No potential conflicts of interest were disclosed by the other authors. blocked their recruitment to the PSA promoter. A key finding of our study is that combination of niclosamide Authors' Contributions with bicalutamide inhibits enzalutamide-resistant prostate cancer Conception and design: C. Liu, C.-x. Pan, C.P. Evans, A.C. Gao cell growth in vitro and tumors in vivo, suggesting that the com- Development of methodology: C. Liu, A.C. Gao bination of niclosamide with bicalutamide could be used to treat Acquisition of data (provided animals, acquired and managed patients, enzalutamide-resistant prostate cancer. This finding could have provided facilities, etc.): C. Liu, C.M. Armstrong, A.P. Lombard, C.P. Evans, fi A.C. Gao both clinical and economic signi cance toward the management Analysis and interpretation of data (e.g., statistical analysis, biostatistics, of mCRPC. Although enzalutamide is effective in the treatment of computational analysis): C. Liu, C.-x. Pan, C.P. Evans, A.C. Gao mCRPC, resistance eventually occurs. The recent PLATO trial of Writing, review, and/or revision of the manuscript: C. Liu, C.M. Armstrong, continued treatment with enzalutamide plus abiraterone and C.-x. Pan, C.P. Evans, A.C. Gao prednisone in chemotherapy-na€ve patients whose disease pro- Administrative, technical, or material support (i.e., reporting or organizing gressed on enzalutamide alone failed to show improvement in data, constructing databases): C. Liu, W. Lou, V. Cucchiara, X. Gu, J.C. Yang, N. Nadiminty progression-free-survival (PFS), highlighting an urgent need to Study supervision: C. Liu, C.-x. Pan, A.C. Gao identify methods of successfully treating enzalutamide-resistant tumors. Our preclinical data showing the ability to inhibit the Grant Support growth of enzalutamide-resistant tumors with niclosamide plus This work was supported in part by grants NIH/NCICA140468, CA168601, bicalutamide could pave the way for a novel strategy to address the CA179970 (A.C. Gao), DOD PC150229 (A.C. Gao), and U.S. Department of unmet needs of men with mCRPC who fail to respond to enza- Veterans Affairs, ORD VA Merits I01BX0002653 (A.C. Gao). lutamide treatment. Furthermore, bicalutamide is widely avail- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in able in most developed and developing countries throughout the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. world and due to the fact that its patent protection has expired, the drug is available in low-cost generic formulations. This makes the Received December 27, 2016; revised February 28, 2017; accepted April 27, niclosamide plus bicalutamide treatment not only widely acces- 2017; published OnlineFirst May 12, 2017.

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1530 Mol Cancer Ther; 16(8) August 2017 Molecular Cancer Therapeutics

Niclosamide and Bicalutamide Combination Treatment Overcomes Enzalutamide- and Bicalutamide-Resistant Prostate Cancer

Chengfei Liu, Cameron M. Armstrong, Wei Lou, et al.

Mol Cancer Ther 2017;16:1521-1530. Published OnlineFirst May 12, 2017.

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