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The Prostate 65:287^298 (2005) Role of Androgen Receptor in the Progression of Human ProstateTumor Cells to Androgen Independence and Insensitivity John M Kokontis, Stephen Hsu, Chih-pin Chuu, Mai Dang, Junichi Fukuchi, Richard A Hiipakka, and Shutsung Liao* Ben May Institute for Cancer Research and the Department of Biochemistryand Molecular Biology, The University of Chicago,Chicago, Illinois BACKGROUND Various studies have implicated the androgen receptor (AR) in the progression of androgen-dependent human prostate cancer cells to androgen-independent and androgen-insensitive phenotypes, but the exact role of AR in progression is unclear METHODS To mimic the clinical situation and test the role of AR in progression, we cultured androgen-dependent LNCaP 104-S prostate tumor cells in the presence of the antiandrogen Casodex (bicalutamide) to derive resistant (CDXR) clones In a second step, we cultured CDXR cells in the presence of the androgen R1881, which generated androgen- and Casodexinsensitive (IS) cells These cells were then characterized with regard to AR function and the effect of ectopic AR expression or AR knockdown on androgen sensitivity RESULTS CDXR cells showed increased AR expression and transcriptional activity CDXR cell proliferation was unaffected by Casodex but was repressed by androgen in vitro and in vivo IS cells, on the other hand, had greatly reduced AR expression and activity compared to CDXR cells Knockdown of AR expression in CDXR cells produced cells that were insensitive to androgen Conversely, re-expression of AR in IS cells regenerated cells that were repressed by androgen Knockdown of AR expression in 104-S cells produced cells that remained stimulated by androgen, while overexpression of AR in 104-S cells generated an androgen-repressed phenotype but did not confer androgen-independent growth CONCLUSIONS Increased AR expression determines whether prostate cancer cells are repressed by androgen, but is not required for androgen independence These results may have implications for anti-AR therapy for prostate cancer Prostate 65: 287–298, 2005 # 2005 Wiley-Liss, Inc KEY WORDS: prostate cancer; progression; androgen; androgen receptor; LNCaP INTRODUCTION Progression of prostate tumors that are dependent upon androgen for survival and growth to tumors that are androgen-independent and -insensitive remains incompletely understood at the molecular level Antiandrogen or androgen deprivation therapy of prostate cancer, pioneered by Charles Huggins [1], is initially effective in repressing prostate tumor growth However, endocrine therapy rarely succeeds in killing all tumor cells and progression of androgen-dependent tumor cells to androgen-independent cells occurs often Several mechanisms have been identified that may participate in the transition of prostate tumor cells ß 2005 Wiley-Liss, Inc Abbreviations: CS-FBS, charcoal-stripped fetal bovine serum; AR, androgen receptor; 5a-DHT, 5a-dihydrotestosterone; R1881, 17bhydroxy-17a-methylestra-4,9,11-trien-3-one; IPTG, isopropyl thiogalactoside; PSA, prostate specific antigen; CDXR, Casodex-resistant; IS, androgen-insensitive This study is dedicated to the memory of Dr Charles B Huggins Grant sponsor: NIH (to SL); Grant numbers: CA58073, AT00850; Grant sponsor: Robert Earp Trust *Correspondence to: Shutsung Liao, The Ben May Institute for Cancer Research, The University of Chicago, Box MC6027, 5841 S Maryland Ave., Chicago, Illinois 60637 E-mail: sliao@huggins.bsd.uchicago.edu Received November 2004; Accepted 21 April 2005 DOI 10.1002/pros.20285 Published online 13 July 2005 in Wiley InterScience (www.interscience.wiley.com) 288 Kokontis et al to clinical hormone-independence These include androgen receptor (AR) gene amplification, AR mutation, and bypass of androgenic activation of AR or of AR signaling itself for cell survival and proliferation (for reviews, see refs [2–4]) We reported previously that clonally derived androgen-dependent LNCaP 104S cells, after long-term androgen deprivation in vitro, can give rise to 104-R1 and 104-R2 cells, the growth of which is repressed by androgen [5–8] We demonstrated that these hormone independent cells exhibited increased AR expression and transcriptional activity and their proliferation was repressed by low concentrations of androgen The 5a-reductase inhibitor finasteride and the antiandrogen Casodex (bicalutamide) blocked the repressive effects of testosterone Androgen-repressed LNCaP 104-R1 cells can revert to an androgen-stimulated phenotype when treated with androgen in vitro [7] and a similar phenomenon is observed in LNCaP 104-R1 cells grown as tumors in castrated athymic mice [8] In this report, we sought to clarify the role of AR in the progression of LNCaP 104 tumor cells from hormone-dependence to hormone-independence Recent reports have demonstrated sustained and heightened AR expression, function and sensitivity to androgen in hormone-independent or recurrent prostate cancer cells [9–16] Therefore, it was of interest to establish whether continued and elevated AR expression was necessary for the hormone-independent growth we observed in the LNCaP 104 progression model For this purpose we derived new androgenindependent and -repressed clonal sublines from androgen-dependent 104-S cells by selecting for growth in the presence of Casodex We extended the progression model by re-exposing Casodex-resistant (CDXR) cells to androgen to generate cells (IS) that were completely androgen and antiandrogen-insensitive We then studied the effect of enforced reduction or re-expression of AR in these cells and in progenitor 104S cells Our results indicate that elevated AR expression mediates androgenic repression of cell growth but is not responsible, at least by itself, for androgenindependent growth MATERIALS AND METHODS Materials The LNCaP 104-S, 104-R1, and 104-R2 sublines and the AN-21 anti-AR polyclonal antibody were described previously [5,7] A polyclonal anti-prostate specific antigen (PSA) antibody was from DAKO (Glostrup, Denmark), a monoclonal anti-p27Kip1 antibody was from Transduction Laboratories/BD Biosciences (Lexington, KY), a monoclonal anti-actin antibody was from Chemicon (Temecula, CA) R1881 (17b-hydroxy-17a-methylestra-4,9,11-trien-3-one) was from Perkin Elmer/NEN Life Science Products (Boston, MA) Casodex1 (ICI 176,334; (2RS)-40 -cyano-3-(4fluorophenylsulfonyl)-2-hydroxy-2-methyl-30 -(trifluoromethyl)-propionanilide; bicalutamide) was from AstraZeneca Pharmaceuticals (Wilmington, DE) Cell Culture and Generation of Casodex-Resistant and Androgen-Insensitive Clones InVitro LNCaP 104-S, 104-R1, and 104-R2 cells were passaged and maintained as described previously [7] For the selection of Casodex-resistant (CDXR) cells,  106 LNCaP 104-S cells were plated in DMEM supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS) and mM Casodex, and were grown for weeks Six colonies were amplified into clonal sublines called CDXR1 through CDXR6, which were used in subsequent characterization of proliferative behavior and AR expression For the generation of androgeninsensitive (IS) cells, the CDXR1, CDXR2, and CDXR3 sublines were cultured in DMEM supplemented with 10% CS-FBS and 10 nM R1881 for a period of weeks before replating Cell Cycle Distribution Analysis Cell cycle distribution analysis on LNCaP 104-S, 104R1, CDXR, or IS cells was performed as described previously [7] One day after plating, cells were treated with R1881 or Casodex and grown for an additional days with a change of medium on day After overnight fixation at À208C in 70% ethanol/30% phosphatebuffer saline (PBS), cells were treated with 0.1 mg/ml RNAse A in PBS for 30 at 378C After pelleting, cells were stained with 50 mg/ml propidium iodide in PBS Cell cycle profiles and distributions were determined by flow cytometric analysis of  104 cells using the CellQuest program (v 3.1f) on a BD Facscan flow cytometer (BD Biosciences, San Jose, CA) Cell cycle distribution analysis was performed using ModFit LT software (Verity Software House, Topsham, ME) Fluorometric Cell Growth Assay For measurement of cell growth, the DNA fluorometric method of Rago et al [17] was used Cells (5  103/well) were plated in 96-well plates The next day cells were treated, wells per treatment, with R1881 at 0.1 or 10 nM, Casodex at mM, tetracyline at mg/ml, or isopropyl-b-D-thiogalactopyranosideside (IPTG) at mg/ml and grown for additional days with a medium change on day after plating After Role of Androgen Receptor in Progression removal of growth medium, 100 ml of water was added and the plates were frozen at À908C until assay Cells were thawed and 100 ml of a solution containing 20 mg/ml Hoechst 33258, 2M NaCl, 10 mM Tris HCl pH 7.4, and mM EDTA was added After thorough mixing, plates were read in a Wallac 1420 fluorometric plate reader using a 355/460 nm excitation/emission filter Growth of CDXR3 and IS3 Tumors in Athymic Mice Six to eight-week old castrated male athymic BALB/ c mice (NCI-Frederick) were injected subcutaneously with  106 CDXR3 or IS3 cells suspended in 0.5 ml of Matrigel (BD Bioscience) Four weeks after injection, half of tumor-bearing mice were implanted with pellets containing 20 mg testosterone propionate Pellets were replaced with fresh pellets after 30 days Tumor growth was determined weekly by caliper measurement Tumor volume was calculated by the formula (length  width  depth)/2 Real-Time PCR Total RNA was isolated using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) Contaminating DNA was removed using DNase I (DNA-free, Ambion, Austin, TX) cDNA was synthesized from mg total RNA using an Omniscript RT synthesis kit (Qiagen, Valencia, CA) For real-time PCR, a QuantiTect Probe PCR kit (Qiagen) and a Prism 7700 cycler (Applied Biosystems, Foster City, CA) were used in a dual-labeled probe protocol The following primer and probe sequences, selected using the Primer Express program (Applied Biosystems), were used for AR real-time PCR: forward primer, 50 -CGCCCCTGATCTGGTTTTC; reverse primer, 50 -TTCGGACACACTGGCTGTACA; FAM-labeled probe, 6FAM-50 -TGAGTACCGCATGCACAAGCTCCG-30 -TAMRA The primer and FAM-labeled probe sequences used for PSA realtime PCR were described by Gelmini et al [18] A Ribosomal RNA Control kit (Applied Biosystems) was used to normalize transcript levels among samples RNA Interference for AR Knockdown Complementary 64-base oligonucleotides were synthesized (Integrated DNA Technologies, Inc., Coralville, IA) containing a 19-base C-terminal AR sequence (50 GCACTGCTACTCTTCAGCA) in inverted repeat orientation [19] After annealing, the 64-mer duplex was inserted into BglII/HindIII-digested pH1RP RNAi expression vector [20] After transfection, G418-resistant 104-S and CDXR3 colonies were expanded and 289 screened for reduced AR expression by Western blot analysis For tetracycline-induced knockdown of AR, a 19 bp TETO2 operator sequence was introduced immediately 30 of the TATA box of the H1 promoter by PCR amplification [21] The 64-base AR RNAi construct was then inserted into the BglII/HindIII-digested pH1RP-TETO2 vector Prior to transfection with the pH1RP-TETO2-AR vector, 104-S and CDXR3 cells were stably transfected with the pcDNA6/TR vector (Invitrogen) for TET repressor expression Blasticidin S-resistant colonies were screened for TET repressor expression using an anti-TET repressor antibody (MoBiTec; Goettingen, Germany) AROverexpression Overexpression of AR was achieved by infecting IS cells with the pLNCX2 retrovirus (BD-Clontech, Palo Alto, CA) carrying a kb BglII human AR cDNA fragment derived from the pSG5-hAR vector [22] Retrovirus was generated using the Phoenix-ampho packaging cell line (G Nolan, Stanford University) IPTG-induced AR expression in 104-S cells was carried out using the 30 SS (Stratagene) and pLNXRO2 [23] vectors using methods described previously [7] RESULTS Progression of LNCaP104 -S Cells to CDXR Cells During a 3-week exposure of androgen-dependent 104-S cells to mM Casodex in medium supplemented with CS-FBS, most cells stopped proliferating and detached from the dishes Parallel cultures of these cells, fixed and stained with 4,6-diamidino-2-phenylindole (DAPI), did not show typical apoptotic nuclear condensation and fragmentation Discrete colonies soon appeared at low frequency Direct counting of colony number and Poisson estimation of colony frequency by tallying growth verses non-growth in plated aliquots of known cell number showed that CDXR cells were present in the 104-S cell population at a frequency of about in 1.4  105 cells at passage Six colonies chosen at random were amplified into sublines, designated CDXR1 through CDXR6 A diagram showing the lineages of cells we have derived from the LNCaP 104-S clone is shown in Figure 1a Proliferation of CDXR cells, as measured by the percentage of cells in S phase, was independent of and repressed by androgen as well as insensitive to Casodex, similar to 104-R1 cells (Fig 1b) CDXR cells treated with androgen accumulated in the G1 phase of the cell cycle (data not shown) The percentage of sub-G1 apoptotic cells under all conditions was negligible Although Casodex did not 290 Kokontis et al Fig a: Derivation of androgen-independent and -insensitive sublines from LNCaP 104 -S cells after androgen (A) deprivation (top) and combined androgen deprivation/Casodex treatment followedby androgenre-exposure (bottom) b:Percentage of LNCaP 104 -S, 104 -R1 or CDXR cells in S phase determined by flow cytometry of propidium iodide-stained cells Cells were incubated in medium containing ethanol vehicle (control), 0.1 nM R1881 and/or mM Casodex (CDX) for 96 hr prior to trypsinization and fixation Values represent the mean Ỉ standard error derived from three to fiveindependentexperiments.AsterisksindicateP value