p300 regulates androgen receptor–independent expression ......histone acetyltransferase activity...

p300 Regulates Androgen Receptor–Independent Expression of

Prostate-Specific Antigen in Prostate Cancer Cells Treated

Chronically with Interleukin-6

Jose D. Debes,1Barbara Comuzzi,

2Lucy J. Schmidt,

1Scott M. Dehm,

1

Zoran Culig,2and Donald J. Tindall

1

1Departments of Urology, Biochemistry, and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota and2Department of Urology, Innsbruck Medical University, Innsbruck, Austria

Abstract

Prostate cancer is the most frequent non–skin cancer in men.Although the mechanisms involved in the progression ofprostate cancer are not entirely understood, androgenreceptor has been shown to play an important role. Androgenreceptor is expressed in both early and late-stage prostatecancer. Also, androgen-regulated pathways are thought to beactive as evidenced by elevated levels of prostate-specificantigen (PSA). In addition, several androgen receptor co-activators and cytokines are involved in prostate cancerprogression. In this regard, we have shown previously thatthe coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), acytokine involved in late-stage prostate cancer. In this study,we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronicallywith IL-6. We found that p300 but not CREB-binding proteininduced activation of PSA in these cells and that the histoneacetyltransferase activity of p300 was critical. This effect wasindependent of the presence of androgens or antiandrogens.Moreover, we found markedly reduced levels of androgenreceptor in these cells and p300 transfection did not affectthose levels, suggesting that the p300 effect on PSA could bebypassing the androgen receptor. Transfection with exogenousandrogen receptor showed minimal response of PSA toandrogens but higher response to p300. We found similareffects of p300 on the androgen response element III, whichmediates the androgen receptor–dependent activation of PSA.Finally, we showed that p300 alone regulates expression of theendogenous PSA gene in the IL-6–treated cells. These findingsreveal a new insight in the progression of prostate cancer,suggesting that coactivators, such as p300, play more impor-tant roles in late-stage prostate cancer, and could regulateandrogen-dependent genes in the absence or with very lowlevels of androgen receptor. (Cancer Res 2005; 65(13): 5965-73)

Introduction

Prostate cancer is the second leading cause of cancer-relateddeath in men (1). The majority of prostate cancers are androgen-dependent at diagnosis, thus, they will respond to androgen-

ablation therapy. However, most tumors will relapse in anandrogen-independent manner following this therapy (2). Cur-rently, there is no successful treatment for the androgen-independent stage of prostate cancer. The androgen receptor isthought to play an important role in both androgen-dependentand -independent prostate cancer because expression of theandrogen receptor protein is present at both stages. Moreover,the androgen receptor is thought to be active as evidenced bylevels of the androgen-regulated protein, prostate-specific antigen(PSA), in sera of patients with prostate cancer (3). Indeed, theandrogen receptor is important for the proliferation of prostatecancer cells and is expressed after progression from androgen-dependent to -independent disease (4, 5).Generally, the androgen receptor is activated by androgens in a

ligand-dependent manner. However, in prostate cancer, theandrogen receptor can be activated in a nonandrogenic fashion.Some of the proposed mechanisms by which this occurs are basedon androgen receptor mutations, androgen receptor amplification,alterations of growth factors and/or cytokines, and differentialregulation of androgen receptor coactivators (6). A cytokine thatseems to play a major role in prostate cancer progression isinterleukin 6 (IL-6). Elevated levels of IL-6 are found in the sera ofpatients with prostate cancer when compared with non–prostatecancer patients, and IL-6 levels are even higher in patients withandrogen-independent prostate cancer when compared to thosewith androgen-dependent disease (7, 8).IL-6 can induce androgen-independent activation of the

androgen receptor and can promote or inhibit prostate cancercell proliferation depending on the context of the cell milieu (9–11).In addition, one study showed that IL-6 can inhibit androgenreceptor activation (12).In order to better understand the role of IL-6 in late-stage prostate

cancer, we have attempted to mimic the environment found in late-stage prostate cancer by growing LNCaP cells (which are prostatecancer cells that express androgen receptor) in the presence of IL-6for an extended period of time (over 40 passages). Interestingly, thesecells (LNCaP-IL-6+) have higher proliferation rates than their sistercells, LNCaP-IL-6� (LNCaP cells of the same passage but without IL-6 treatment; ref. 13). Moreover LNCaP-IL-6+ cells developed largertumors than parental cells when inoculated into nude mice, in-dicating that these cells represent a more aggressive phenotype (14).The mechanisms related to the IL-6-mediated regulation of the

androgen receptor involve coactivators of the androgen receptor.p300 and its homologous protein, CREB-binding protein (CBP), areubiquitous coactivators that interact with the androgen receptorand are involved in tumorigenesis (15–17). Both p300 and CBP playimportant roles in the androgen-dependent activation of the an-drogen receptor (18, 19). p300 alters chromatin structure via its

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Donald J. Tindall, Departments of Urology, Biochemistry,and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest,Rochester, MN 55905. Phone: 507-284-8139; Fax: 507-284-2384; E-mail: [email protected].

I2005 American Association for Cancer Research.

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histone acetyltransferase activity by weakening histone-DNA inter-actions, thus inducing nucleosome displacement and favoringaccess of different transcription factors to the DNA template (20).We have shown previously that p300mediates the androgen-independent activation of the androgen receptor by IL-6 in prostate cancercells and that the histone acetyltransferase activity of p300 is re-quired in this context (21). In that study, we showed that althoughp300 alone is not sufficient to induce androgen receptor activation, itis a necessary component of the IL-6 pathway because p300 sup-pression impeded androgen receptor activation by IL-6. Moreover,we found that IL-6 increases the proliferation of prostate cancercells and that the silencing of p300 through small interfering RNAreduces the effect, indicating the importance of this coactivatorfor the androgen-independent proliferation of prostate cancer cells(17). In addition, we have shown that protein levels of p300 correlatewith prostate cancer progression following surgical treatment ofthe tumor (17).In the present study, we investigated the role of p300 in prostate

cancer cells treated long-term with IL-6 (LNCaP-IL-6+). We foundthat p300 plays a differential role in the regulation of androgenreceptor–dependent genes in these cells. Moreover, we show thatp300 can induce activation of PSA in what seems to be an androgenreceptor–independent manner in IL-6-treated cells. These findingsreveal a new insight into the regulation of androgen receptor–dependent genes in prostate cancer.

Materials and Methods

Cell culture. LNCaP cells were purchased from the American Type

Culture Collection (Rockville, MD). LNCaP-IL-6+ cells were maintained in

RPMI 1640 (Celox, St. Paul, MN) containing 5% fetal bovine serum(Biosource International, Rockville, MD) and 5 ng/mL of IL-6 (R&D Systems,

Minneapolis, MN). LNCaP-IL-6� cells were derived from the same stock of

cells as LNCaP-IL-6+ and maintained in RPMI 1640 containing 5% fetal

bovine serum for an equivalent number of passages. All cells used in ourexperiments were cultured for >100 passages in the presence of IL-6.

Transfections and luciferase assays. LNCaP-IL-6� and -IL-6+ cells were

plated (2.5 � 105 in six-well plates) and 24 hours later transfected with

plasmids at quantities described in the figure legends using the Gene PorterTransfection System (GTS, Inc., San Diego, CA). Plasmids containing full-

length p300 (p300), mutant histone acetyltransferase–negative derivative

(p300-HAT), PSA promoter (PSA-prom), androgen response elements (hK2-

ARE), androgen receptor, and CBP have been described previously (19, 22–24). Empty vectors PGL3 and pcDNA3.1 were purchased from Promega

(Madison,WI) and Invitrogen (Carlsbad, CA), respectively. Twenty-four hours

following transfection, cells received fresh corresponding medium or thatcontaining charcoal-stripped serum (to remove androgens). Steady-Glo

Luciferase Assays (Promega) were done according to the manufacturer’s

instructions. Transfection efficiency was monitored by cotransfection with a

plasmid containing green fluorescence protein (Promega), and visualizedwith a Zeiss fluorescent microscope at 499 nm. Transfection efficiencies off40%were obtained routinely. In early experiments, cotransfection of renilla

constructs were used to normalize values. However, after finding constant

transfection efficiency, experiments were done with the firefly luciferasereporter alone.

Western blot analysis. Cells were washed once with PBS and lysed in

cold radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCL (pH 7.4),1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, and 1 mmol/L

EDTA], plus Complete Protease Inhibitor (Roche, Indianapolis, IN). Western

blotting was done using antibodies to androgen receptor (N-20, 441), PSA

(A67-B/E3), both from Santa Cruz Biotechnology (Santa Cruz, CA) andandrogen receptor (Biogenex, The Hague, the Netherlands). Immunode-

tection of ERK-2 (D-2, Santa Cruz Biotechnology) was used as a control for

Figure 1. RNA and protein levels aredifferentially expressed in LNCaP-IL-6�and -IL-6+ cells. A, mRNA extracted fromcells was subjected to reversetranscription PCR in order to obtain cDNA.cDNA was amplified with primers specificto either p300 or CBP using real-timePCR. Yellow line, LNCaP-IL-6� cells;green line, -IL-6+ cells. Amplificationof glyceraldehyde-3-phosphatedehydrogenase (control) was identicalbetween the two cell lines (data notshown). B, nuclear protein extraction ofLNCaP-IL-6� and -IL-6+ cells wereanalyzed by Western blot using antibodiesto either p300 or CBP. h-Actin was used asa loading control. The data isrepresentative of three experiments.

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Cancer Res 2005; 65: (13). July 1, 2005 5966 www.aacrjournals.org



equal protein loading. Nuclear and cytoplasmic protein extraction reagentswere purchased from Pierce (Rockford, IL).

p300 and CREB-binding protein Western blots. Nuclear extracts wereprepared from 2 � 106 cells according to the manufacturer’s instructions.

Cell passages 100 and higher were used. Samples for Western analysiswere prepared with addition of NuPage sample buffer (Invitrogen) and

boiled for 10 minutes at 70jC. Nuclear fractions were loaded onto a 3% to

8% Tris-acetate gel, and Western analysis was carried out as described

previously. The monoclonal anti-CBP antibody (C-1, Santa CruzBiotechnology) and the polyclonal anti-p300 (C-20, Santa Cruz Biotech-

nology) were diluted 1:100. Western blots for h-actin (Chemicon

International, Hofheim/TS, Germany) were done as a control for equal

protein loading.Immunocytochemistry. Cells were plated on coverslips in six-well

plates (1 � 104). After 24 hours, immunocytochemistry was done using

primary antibodies to p300 (C-20) or androgen receptor (441, both SantaCruz Biotechnology), followed by incubation with fluorescent secondary

antibodies (Molecular Probes, Eugene, OR). Cells were counterstained with

bis-benzimide (Sigma, St. Louis, MO), and images were visualized with an

LSM510 confocal microscope (Carl Zeiss, Inc., Oberkochen, Germany). Cellswere transfected with p300 24 hours before immunocytochemistry as

described in the figures.

Real-time PCR. RNA was isolated from cells using Trizol (Invitrogen). A

two-step real-time PCR was done using cDNA prepared from RNA using aFirst Strand cDNA Synthesis Kit (Roche) and a SYBR Green PCR Master Mix

(Applied Biosystems, Foster City, CA) on an ABI PRISM 7700 SDS

instrument following the manufacturer’s instructions. Both forward andreverse primers were used at a final concentration of 900 nmol/L. PCR

products (100-150 bp) were electrophoresed in 1.2% agarose gels to check

for nonspecific amplification. The fold-change in expression levels (using

glyceraldehyde-3-phosphate dehydrogenase as control) was determined bya comparative CT method using the formula 2�DDCT; where CT is the

threshold cycle of amplification.

Conventional PCR. RNA was isolated and cDNA prepared as described

above. cDNA was amplified for up to 30 cycles with the GC-RICH PCRSystem (Roche) following the manufacturer’s instructions. PCR products

were electrophoresed on a 1.5% agarose gel. h-Actin (Promega) primers

were used as controls.Plasmid construction. sPSA, a luciferase reporter construct harboring

the minimal core enhancer and promoter regions of the PSA gene (25) was a

gift from Dr. Leland Chung (University of Virginia). S-All, containing the PSA

core enhancer with androgen response elements V, IV, III, and IIIA replacedwith GAL4-binding sites (26) was a gift from Dr. Michael Carey (University

of California at Los Angeles). The PSA enhancer core region was amplified

from S-All with the primers FWD (5V-CAAGATGATATCTCTCTCAG) and

REV (5V-GGGTGACTCGAGCAGTCTAGGTGG). The PCR product wassubsequently digested with EcoRV/Xho I and ligated to EcoRV/Xho I-

digested sPSA, thus generating mutant sPSA. All plasmids were sequenced

to verify integrity.

Results

In order to investigate the role of IL-6 in prostate cancer, LNCaPcells were grown in the presence of IL-6 (5 ng/mL) to mimic theenvironment found in late-stage prostate cancer. Levels of p300and its homologue CBP were assessed in these cells. We found thatmRNA levels of p300 and CBP, measured by real-time PCR,remained unchanged in LNCaP-IL-6+ cells when compared withLNCaP-IL-6� cells (Fig. 1A). Surprisingly, the protein levels of bothcoactivators were decreased in LNCaP-IL-6+ cells of high passage(>100; Fig. 1B). Considering the importance of these coactivatorsfor androgen receptor activity, we investigated the effect ofreintroducing p300 or CBP into the cells. LNCaP-IL-6+ and -IL-6� cells were cotransfected with expression constructs of eitherp300 or CBP together with a luciferase reporter constructcontaining an androgen receptor–responsive PSA promoter. In

parental LNCaP cells (LNCaP-IL-6�), CBP increased PSA promoteractivity, whereas p300 had no effect (Fig. 2A). In contrast, inLNCaP-IL-6+ cells, p300 substantially increased the activity of thePSA promoter, whereas CBP had no effect (Fig. 2B). Cellstransfected with an empty PGL3 vector exhibited no reporteractivity, thus verifying that the effect of p300 was mediated via thepromoter and not through the vector.Although the effect of CBP, as well as lack of effect of p300, on

androgen receptor transactivation in LNCaP cells has beenreported previously (19, 21), the ability of p300 to inducetransactivation of the androgen receptor after long-term treatment

Figure 2. p300 induces PSA promoter activity in LNCaP-IL-6+ cells but not inLNCaP-IL-6� cells. A, LNCaP-IL-6� cells (in six-well plates) were transfectedwith PSA-prom (2 Ag), PGL-3 vector (lane 1, 2 Ag; lane 2, 1 Ag), p300 (1 Ag),or CBP (1 Ag) as indicated. DNA content was kept constant by transfectionof pcDNA3.1 empty vector. Forty-eight hours after transfection, cells were lysed,and luciferase assays were done. B, the same experiment was done inLNCaP-IL-6+ cells as in (A). C, LNCaP-IL-6� or -IL-6+ cells were grown in5% RPMI without IL-6 for at least 5 days. Cells were transfected withPSA-prom (2 Ag) and p300 (1 Ag) or p300-HAT (1 Ag). Forty-eight hours aftertransfection, cells were lysed, and luciferase assays were done. Eachexperiment contained triplicates and was repeated at least thrice.

p300 and PSA Activation in Prostate Cancer




Cancer Research




with IL-6 was unexpected. p300 is a coactivator of the androgenreceptor, and thus is necessary, but not sufficient, to inducetranscriptional activity. In order to determine whether the presenceof exogenous IL-6 contributes to the p300 effect, LNCaP-IL-6+ cellswere depleted of IL-6 for 5 days and then cotransfected with p300along with the PSA-promoter. PSA promoter activity was increasedby p300 even in the absence of IL-6 in the medium (Fig. 2C),suggesting that the p300 effect is not dependent on exogenous IL-6.Moreover, treatment with additional IL-6 decreased the ability ofp300 to induce PSA-promoter activity (data not shown). Thisfinding suggests that the p300 effect is due to changes in the

phenotype of the cells after long-term IL-6 treatment or endo-genous production of IL-6 from the cells.p300 contains histone acetyltransferase activity, which could

alter chromatin structure and induce activation of a number oftranscription factors, including the androgen receptor. In order todetermine if the histone acetyltransferase activity of p300 isimportant for its effects on the PSA promoter activity, wecotransfected cells with a mutant p300 that lacks histoneacetyltransferase activity (p300-HAT). This mutant p300 failed toinduce PSA promoter activation in the presence (data not shown)or absence of IL-6 (Fig. 2C ), suggesting that the histoneacetyltransferase activity is important for the biological effect ofp300 in this context.In order to assess whether androgens contribute to the p300-

mediated induction of the PSA promoter, LNCaP-IL-6+ cells weregrown in charcoal-stripped serum, which removes androgens(normal serum level <1 pmol/L), and cotransfected with p300 andthe PSA-prom. The response of the PSA promoter to p300 was thesame whether cells were grown in normal serum (Fig. 3A , lane 2),or in charcoal-stripped serum (Fig. 3A , lane 4), indicating that theeffect of p300 is independent of the presence of androgens. Anothergroup of cells were treated with the antiandrogen bicalutamide(casodex; Fig. 3A , lane 5). Cells treated with bicalutamide exhibiteda marginal decrease of activation of the PSA promoter by p300,again supporting the concept that the p300 effect is independent ofandrogen-mediated pathways.The fact that treatment with antiandrogens did not alter the

effects of p300 raised the question of whether the androgenreceptor was mediating the p300 induction of PSA activity. Inorder to determine this, we measured androgen receptor mRNAlevels by real-time PCR and confirmed the results with semiquan-titative PCR. We found that the mRNA levels of androgen receptorwere considerably lower in LNCaP-IL-6+ cells compared withLNCaP-IL-6� cells (Fig. 3B). Furthermore, LNCaP-IL-6+ cellsshowed undetectable levels of androgen receptor protein(Fig. 3C) by Western blot, and very low levels of androgen receptorwere detected by immunocytochemistry (Fig. 3D). These resultswere surprising because activation of the PSA promoter usuallyrequires androgen receptor activity. To exclude any possibility thatthe antibody was not recognizing the androgen receptor protein,these experiments were repeated with three different antibodiesagainst the androgen receptor.The finding that LNCaP-IL-6+ cells have decreased expression of

the androgen receptor suggests that either p300-mediated activationis independent of androgen receptor or that p300 is inducingtranscription of PSA at very low levels of androgen receptor. Todetermine whether the p300 effects involve the androgen receptor,luciferase reporter experiments were repeated in cells cotransfectedwith androgen receptor. When androgen receptor was transfectedinto the LNCaP-IL-6+ cells, there was no increase in PSA promoteractivity by the synthetic androgen mibolerone (Fig. 3E). However,

Figure 3. p300 induces PSA promoter activity in an androgen-independent manner in cells expressing nondetectable levels of androgen receptor protein.A, LNCaP-IL-6+ cells (in six-well plates) were transfected with PSA-prom (2 Ag) and p300 (1 Ag). DNA content was kept constant by transfection of pcDNA3.1empty vector. Twenty-four hours after transfection, medium was changed to that containing charcoal-stripped serum (CSS), and cells in lane 5 were treated with10 Amol/L casodex (CX). After 24 hours, cells were lysed and luciferase assays were done. B, RNA was extracted from LNCaP-IL-6� and -IL-6+ cells. RNA wasconverted to cDNA, and amplified (using androgen receptor–specific primers) through real-time PCR (left) or conventional PCR and electrophoresed in a 1.5% agarosegel (right). C, protein lysates were analyzed by Western blot using an androgen receptor–specific antibody. Total ERK-2 antibody was used as a loading control.Figure represents whole cell extract. D, cells were subjected to immunocytochemistry using a specific androgen receptor antibody. E, LNCaP-IL-6+ cells weretransfected with PSA-prom (2 Ag), p300 (1 Ag), androgen receptor (1 Ag) as indicated. DNA content was kept constant by transfection of pcDNA3.1 empty vector. After24 hours, medium was changed to that with CSS, and cells in lane 4 were treated with 1nmol/L mibolerone (Mib). Twenty-four hours later, cells were lysed and luciferaseassay was done. Experiments were repeated at least thrice with the exception of (D ), which was repeated twice.

Figure 4. p300 induces activation of the androgen response element promoterin the presence of androgen receptor. A, LNCaP-IL-6� cells (in six-well plates)were transfected with androgen response element (1.5 Ag), androgen receptor(1 Ag), p300 (2 Ag), as indicated. DNA content was kept constant with pcDNA3.1.Twenty-four hours after transfection, cells received fresh medium containingcharcoal-stripped serum. Cells in lane 5 received 1 nmol/L mibolerone (Mib).After 24 hours, luciferase assays were done. B, LNCaP-IL-6+ cells weresubjected to the same procedure as in (A). Data represent an average of threeexperiments.





when androgen receptor was cotransfected together with p300,there was a marked increase of the PSA promoter activity (Fig. 3E).The increase in activity was higher when p300 was cotransfectedwith androgen receptor than that found with p300 alone (Fig. 3E).This finding suggests a mechanism by which, after long-termtreatment with IL-6, androgen-dependent genes in prostate cancercells become less responsive to androgen but become moreresponsive to coactivators like p300.The activation of androgen-dependent genes in both the normal

and malignant prostate is mediated by androgen responseelements (24, 25). In particular, the promoters of the androgen-responsive genes PSA and human kallikrein 2 (hK2) have beenshown to be transcriptionally activated by androgens. To assesswhether p300 activation of the PSA promoter involves androgenresponse elements or is directed through a different mechanism weperformed luciferase reporter experiments using a hK2-ARE-promoter construct with a luciferase reporter (hK2-ARE; ref. 24).The synthetic androgen mibolerone induced hK2-ARE activityf8-fold in LNCaP-IL-6� cells cotransfected with the androgenreceptor (Fig. 4A), whereas p300 had no effect whether in theabsence or presence of androgen receptor. When the sameexperiment was done in LNCaP-IL-6+ cells, p300 increased hK2-ARE activity only marginally in the absence of androgen receptorbut induced a 6-fold activation when cotransfected with theandrogen receptor (Fig. 4B). When we used a reporter encoding a

mutated androgen response element, p300 did not induceactivation (Supplementary Data 1). In contrast, mibolerone hadonly a marginal (1-fold) effect in cells cotransfected with theandrogen receptor (Fig. 4B). Moreover, cells transfected with p300alone and treated with mibolerone showed no induction of thepromoter (data not shown) demonstrating that levels of endoge-nous androgen receptor in these cells are too low to activate geneexpression. These results are similar to those in Fig. 3E , wheremibolerone alone could not induce a strong reporter activity fromthe full-length PSA promoter. This suggests that although part ofthe p300-regulated activation of androgen-responsive genes ismediated through the androgen response elements (thus throughthe androgen receptor), additional activation is mediated throughan androgen receptor–independent mechanism.Many transcriptional coactivators like p300 can act directly on

viral vectors themselves, thereby inducing transcriptional activityof the reporter. Most of the vectors used in our experiments areof viral origin. In order to verify that the p300 effect was due to adirect effect on the PSA promoter rather than an indirect effectvia the viral vector, we studied the effect of p300 on expressionof endogenous PSA. LNCaP-IL-6+ cells were transfected withp300 or empty vector, and RNA was extracted 48 hours later.Cells transfected with p300 showed a marked increase in PSAmRNA, determined by both real-time and semiquantitative PCR(Fig. 5A). Furthermore, we measured the protein levels of PSA in

Figure 5. p300 induces expression ofendogenous PSA, as well as NKX3.1.A, cDNA prepared from RNA ofLNCaP-IL-6+ cells was subjected toreal-time PCR amplification (right) orconventional PCR and electrophoresed ina 1.5% agarose gel (left) using primersspecific to PSA. B, cells (plated in 60 mmplates) transfected with p300 (5 Ag),vector (pcDNA3.1 5 Ag), or PSA-prom(5 Ag) as indicated. DNA content was keptconstant at 10 Ag per plate with pcDNA3.1.Protein extracts from transfected cells wereanalyzed by Western blot using a specificantibody to PSA. Total ERK-2 antibodywas used as loading control. C, cDNAprepared from RNA of LNCaP-IL-6+ cellswas subjected to real-time PCR (left),or conventional PCR and electrophoresedin a 1.5% agarose gel (right) with specificprimers to NKX3.1. Experiments wererepeated at least thrice.

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LNCaP-IL-6+ cells transfected with p300 alone or cotransfectedwith p300 and the PSA promoter (in order to mimic theexperimental conditions in our previous experiments). PSAprotein expression increased 48 hours after transfection of p300(Fig. 5B) confirming that the endogenous PSA gene is regulatedby p300. Because PSA is a well-characterized androgen receptor–dependent gene and is often used to study androgen receptoractivity, we examined the effect of p300 on another androgenreceptor–dependent gene. NKX3.1 is a member of the NKX familyof transcription factors that is highly expressed in prostate cells,and is known to be regulated by androgens (27–29). We measuredRNA levels of NKX3.1 in LNCaP-IL-6+ cells transfected with p300or empty vector. Levels of NKX3.1 RNA increased in cellstransfected with p300 (Fig. 5C), suggesting that the effect ofp300 extends to other androgen-responsive genes.Our results showing increased activation of the androgen-

dependent gene activity in cells with reduced expression of theandrogen receptor suggest that the p300 effects are either bypassingthe androgen receptor, activating extremely low levels of theandrogen receptor, or increasing expression of androgen receptor.Thus, we investigated androgen receptor levels (mRNA and protein)in LNCaP-IL-6+ cells transfected with p300. Forty-eight hours aftertransfection of p300, androgen receptor mRNA levels were higher inthose cells with p300 but not in the vector group (Fig. 6A). However,protein levels of androgen receptor were still undetectable aftertransfection with p300 or cotransfection with p300 and PSA-prom(Fig. 6B), indicating that either the p300 effects are mediatedthrough a different pathway than the androgen receptor or thatWestern blots are not sensitive enough to detect an increase inprotein expression. When p300 was transfected into LNCaP-IL-6�cells, there was no increase in androgen receptor RNA levels (datanot shown). Finally, when LNCaP-IL-6+ cells were transfected witheither p300 or vector alone and subjected to immunocytochemistry,there was no increase in androgen receptor staining (Fig. 6C). Thus,p300 seems to have little, if any, effect on androgen receptor proteinexpression in these cells.

Discussion

The androgen receptor is thought to be functional in late-stageprostate cancer, as evidenced by elevated levels of PSA in serum.PSA is a direct target of the androgen receptor. Serum levels of thecytokine IL-6 are also elevated in late-stage prostate cancer. Inaddition, IL-6 can induce activation of the androgen receptor andmodulate prostate cancer cell proliferation (8, 11, 30). Thisindicates an important role for IL-6 in late-stage prostate cancer.In this study, we show that the PSA promoter can be activated bythe coactivator p300 in cells following long-term treatment withIL-6. Interestingly, androgen receptor protein was nondetectable byWestern blot in these cells, and immunocytochemistry showed verylow levels of the androgen receptor protein. This finding isimportant because it suggests that coactivators may increaseandrogen receptor–dependent genes through mechanisms that areindependent of the androgen receptor, or through much reducedandrogen receptor levels. In this regard, one study has shown theexistence of a region within the PSA promoter that seems to be anandrogen receptor–independent regulatory element of the pro-moter in prostate cancer cells (25). Long-term treatment with IL-6may induce selection of such a region to induce positive regulationof PSA activity independent of androgen receptor. This observationdeserves further investigation.

We have shown previously that p300 is necessary for theandrogen-independent activation of the androgen receptor byIL-6 (21). The observation that cells which underwent long-termtreatment with this cytokine have decreased levels of androgenreceptor mRNA and protein was surprising. However, the fact thatp300 and CBP proteins were also down-regulated reflects acorrelation between the levels of the androgen receptor and someof its coactivators. Whether there is decreased transcription orincreased degradation of the androgen receptor should beexplored. Interestingly, inhibition of methylation with 5-azacytidinefailed to increase androgen receptor mRNA levels (SupplementaryData 2), indicating that methylation is not involved in the down-regulation of the androgen receptor in these cells. We showedpreviously that LNCaP-IL-6+ cells up to passage 70 expressandrogen receptor with higher androgen binding than theirrespective counterpart (13). However, we have noted that LNCaP-IL-6+ passages 80 and higher lack androgen receptor expression.This cannot be attributed simply to high passage number becauseLNCaP-IL-6� of the same passage are androgen receptor–positive.Although androgen receptor is consistently detected in themajority of prostate cancers, it has been also shown that somecell subpopulations lose androgen receptor expression due toepigenetic changes (31, 32). Modification of androgen receptor inLNCaP-IL-6+ cells may reflect some of these changes. IL-6 isthought to induce differentiation in at least some prostate cancers,(33). However, our finding that several androgen-related genes aredown-regulated after long-term treatment with IL-6 suggests thatinduction of differentiation may be lost after prolonged exposure toIL-6. When IL-6 was removed from the medium for over 5 daysthere was no reexpression of androgen receptor (SupplementaryData 3). When LNCaP-IL-6+ cells were treated with the antian-drogen casodex, p300 still activated the PSA promoter, suggestingthat the androgen receptor is not mediating these effects.Furthermore, when androgen receptor was transfected intoLNCaP-IL-6+ cells, the synthetic androgen mibolerone did notinduce a strong activation of the PSA promoter, as did p300. Thesefindings suggest that during long-term exposure to IL-6, prostatecancer cells select mechanisms that are less dependent onandrogen receptor and more dependent on coactivators toactivate androgen receptor–dependent genes. On the other hand,it cannot be ruled out that the very low levels of androgenreceptor are more responsive to coactivators than to androgens.Failure of prostate cancer cells to respond to androgens is acomplex issue that has been reported previously (34). Whetherthe androgen receptor is being degraded at a faster rate or ifthere is a transcriptional repressive complex inhibiting theactivation of the exogenous androgen receptor by androgensshould be considered and deserves further investigation.We have shown previously that LNCaP-IL-6+ cells express a more

aggressive phenotype, as evidenced by a higher proliferation rateand formation of larger tumors in nude mice (13, 14). Whether p300plays a role in this more aggressive phenotype needs to beinvestigated. We found that inhibition of p300 through smallinterfering RNA results in a decreased proliferation in these cells(data not shown), indicating that p300 plays an important role inthe more aggressive behavior of LNCaP-IL-6+ cells.In the normal prostate, as well as in prostate cancer, the

androgen receptor binds to androgen response elements withinandrogen-responsive gene promoters, and induces transcriptionalactivation in the presence of androgens (3, 35). In particular, theandrogen response elements of the PSA and the hK2 promoter





have been extensively studied. In LNCaP-IL-6+ cells, p300induced activation of hK2-ARE to a higher degree than thesynthetic androgen mibolerone when cotransfected with theandrogen receptor. This indicates that p300 induces gene activityto a limited extent through this androgen response element(thus through the androgen receptor). There is a generalassumption that higher levels of PSA or hK2 in patients withadvanced prostate cancer are due to increased androgenreceptor activity (2, 3). Nonetheless, our findings suggest thatthere are additional pathways involved in the induction ofandrogen-responsive genes.The coactivators p300 and CBP are often referred to as if they

were the same protein, and indeed they both play major roles inthe activation of a number of transcription factors (20). However,p300 and CBP are different proteins and have different functions.For example, p300 is important for the initiation of activation ofthe estrogen receptor, whereas CBP is responsible for maintenanceof the estrogen receptor transcription complex (36). Here, we showthe differential effect of these two coactivators where p300 was ableto induce PSA promoter activation in LNCaP-IL-6+ cells, whereas

CBP induced little or no activity. In contrast, CBP alone inducedPSA expression in IL-6� cells, whereas p300 alone had little or noeffect. Whether this lack of effect of CBP applies to othertranscriptional factors remains to be investigated.p300 has been shown to have various roles in the

transcription process depending on the model or system beingexamined. p300 could serve as a bridge between the generaltranscription factors and the basal machinery, or it could be ascaffold to bring specific transcription factors together withgeneral transcription factors. Also, p300 induces histoneacetylation through its histone acetyltransferase activity, thusmaking the DNA template more susceptible to binding bydifferent transcription factors (20). In our experiments, we foundthat the histone acetyltransferase activity of p300 is critical foractivation of the PSA promoter.Acetylation of androgen receptor by p300 increases the ability of

the androgen receptor to induce proliferation in prostate cells (37).However, the ability of p300 to modulate transcription of theandrogen receptor has not been shown until now. One mechanismby which p300 activates the PSA promoter in the presence of very

Figure 6. p300 differentially regulates androgenreceptor RNA and protein in LNCaP-IL-6+ cells.A, cDNA prepared from LNCaP-IL-6+ cells(60 mm plates) transfected with p300 (5 Ag) orempty vector pcDNA3.1 (5 Ag) was subjected toamplification by real-time PCR (left) orconventional PCR and electrophoresed in anagarose gel (right) using specific primers to theandrogen receptor. B, LNCaP-IL-6+ cells weretransfected with p300 or empty vector(as described in Fig. 4B). Forty-eight hours aftertransfection, protein was extracted and analyzedby Western blot using a specific antibody toandrogen receptor. Protein extract fromLNCaP-IL-6� was used as a positive control forandrogen receptor. Total ERK-2 antibody wasused as a loading control. C, LNCaP-IL-6+ cells(six-well plate) were transfected with p300 (2 Ag) orempty vector pcDNA3.1 (2 Ag) as indicated.After 48 hours, cells were subjected toimmunofluorescence using specific antibodies top300 and androgen receptor. Experiments in (A)and (B) were repeated at least thrice, theexperiment in (C ) was repeated twice.

Cancer Research




low levels of androgen receptor protein might be throughtranscription of androgen receptor mRNA. Nonetheless, we foundthat although there were minimum changes in androgen receptormRNA levels after p300 transfection, protein levels of the androgenreceptor were unaffected. This finding is controversial because itsuggests that p300 can elevate androgen receptor RNA levels but notprotein. In this regard, it should be noted that androgens have beenpreviously shown to differentially regulate RNA and protein levels inLNCaP cells (35). Also, it is possible that there is an increaseddegradation of the androgen receptor protein in these cells.Our experiments show that p300 induces mRNA and protein

expression of the endogenous PSA in IL-6+ cells. We also show thatp300 induces increased levels of endogenous NKX3.1 RNA. In-terestingly, the finding that p300 exerts a differential effect at thetranscriptional and translational levels, suggests that p300 regulatesdifferent transcriptional factors that ultimately will affect proteinfunction or degradation of androgen receptor–dependent genes.

Taken together, these data provide new insights into themechanism by which androgen receptor and androgen receptor–dependent genes are regulated in late-stage prostate cancer. Theysuggest that cytokines and coactivators may have important rolesin bypassing androgen receptor function. The mechanism by whichp300 can induce PSA activity with low to nondetectable androgenreceptor protein levels warrants further investigation.

Acknowledgments

Received 8/8/2004; revised 2/2/2005; accepted 3/2/2005.Grant support: NIH grants DK60920, CA91956, the T.J. Martell Foundation, the

Prostate Cancer Foundation, and the Austrian National Bank grant 9581. S.M. Dehm isa Research Fellow of the Terry Fox Foundation through a grant from the NationalCancer Institute of Canada.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. J. Boyes, C. Young, and O. Janne for providing plasmids; E. Poeschlalab for sharing equipment; and G. Sierek for expert technical assistance.

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2005;65:5965-5973. Cancer Res Jose D. Debes, Barbara Comuzzi, Lucy J. Schmidt, et al. Chronically with Interleukin-6

Treatedof Prostate-Specific Antigen in Prostate Cancer Cells Independent Expression−p300 Regulates Androgen Receptor

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