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Tumor and Stem Cell Biology p300 Acetyltransferase Regulates Androgen Receptor Degradation and PTEN-Decient Prostate Tumorigenesis Jian Zhong 1,2,4 , Liya Ding 1,2,4 , Laura R. Bohrer 5 , Yunqian Pan 1 , Ping Liu 5 , Jun Zhang 3 , Thomas J. Sebo 3 , R. Jeffrey Karnes 2 , Donald J. Tindall 2,4 , Jan van Deursen 1,4 , and Haojie Huang 1,2,4 Abstract Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modier of prostate tumorigenesis. In a mouse model of PTEN deletioninduced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This nding was conrmed in human prostate cancer cells in which PTEN expression was abolished by RNA interferencemediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these ndings, in PTEN-decient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss. Cancer Res; 74(6); 187080. Ó2014 AACR. Introduction Androgens and the androgen receptor (AR) are paramount for prostate cancer growth (1, 2). Androgen-deprivation ther- apy (ADT) is the mainstay of treatment for patients with advanced/disseminated prostate cancer. Unfortunately, this treatment is palliative, and the majority of prostate cancers evolve into a disease termed castration-resistant prostate cancer (CRPC). In virtually all cases, castration-resistant pro- gression is accompanied by an increase in the level of prostate- specic antigen (PSA), a well-studied AR transactivated gene (3), indicating that the AR is aberrantly activated under cas- tration conditions. AR protein is expressed in nearly all met- astatic CRPC. The importance of persistent AR signaling in CRPC is further demonstrated by the antitumor activity of next-generation androgenAR axis inhibitors, including abir- aterone and enzalutamide (4, 5). However, resistance to these drugs has been observed in many patients (6, 7). Thus, there is an unmet need for the development of new strategies to eliminate AR protein and functions in prostate cancer. The tumor-suppressor gene PTEN is often mutated, delet- ed, or transcriptionally downregulated in human prostate cancers. Genome-wide integrative genomic proling analy- sis showed that PTEN loss and activation of the phosphoi- nositide 3-kinase (PI3K) pathway occur in up to 70% of metastatic prostate cancers (8). PTEN loss or PI3K activa- tion results in AKT phosphorylation and activation. Impor- tantly, homozygous deletion of Pten invariably induces AKT activation and prostate tumorigenesis in mice (911). How- ever, the downstream signaling pathways that contribute to PTEN loss or AKT activationinduced prostate cancer path- ogenesis are poorly understood. p300 was originally identied as a histone acetyltransfer- ase, which acts as a coactivator for many transcription factors, including AR, p53, and NF-kB (12). Thus, the role of p300 in cancer is not entirely clear and may depend on the pathophysiological milieu of the tumors. P300 seems to have an oncogenic potential because its expression is upregulated during human prostate cancer progression (13, 14). More- over, p300 has been demonstrated to be an essential coacti- vator of the AR, a key prostate cancer promoter. P300 interacts with and acetylates AR in vitro and in vivo (1517). Not only is p300 important for androgen-dependent and independent transactivation of the AR (15, 1820), but also its expression is associated with human prostate cancer prolif- eration (13). However, the precise roles of p300 in prostate cancer development and progression remain elusive. In the present study, we demonstrate that p300 functions as a key regulator of AR protein degradation and PTEN-decient prostate tumorigenesis. Authors' Afliations: 1 Departments of Biochemistry and Molecular Biol- ogy, 2 Urology, and 3 Laboratory Medicine and Pathology; 4 Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester; and 5 Depart- ment of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Zhong and L. Ding contributed equally to this work. Corresponding Author: Haojie Huang, Mayo Clinic, 200 First Street SW, Gugg 1311B, Rochester, MN 55905. Phone: 507-293-1712; Fax: 507-293- 3071; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-2485 Ó2014 American Association for Cancer Research. Cancer Research Cancer Res; 74(6) March 15, 2014 1870 on July 20, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst January 30, 2014; DOI: 10.1158/0008-5472.CAN-13-2485

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Page 1: p300 Acetyltransferase Regulates Androgen Receptor Degradation … · Tumor and Stem Cell Biology p300 Acetyltransferase Regulates Androgen Receptor Degradation and PTEN-Deficient

Tumor and Stem Cell Biology

p300 Acetyltransferase Regulates Androgen ReceptorDegradation and PTEN-Deficient Prostate Tumorigenesis

Jian Zhong1,2,4, Liya Ding1,2,4, Laura R. Bohrer5, Yunqian Pan1, Ping Liu5, Jun Zhang3, Thomas J. Sebo3,R. Jeffrey Karnes2, Donald J. Tindall2,4, Jan van Deursen1,4, and Haojie Huang1,2,4

AbstractOverexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of

prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generictranscriptional coactivator functions as a positive modifier of prostate tumorigenesis. In amousemodel of PTENdeletion–induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR).This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNAinterference–mediated attenuation. These results were consistent with clinical evidence that the expression ofp300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivationincreased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, therebyprecluding its polyubiquitination anddegradation. In support of thesefindings, in PTEN-deficient prostate cancerin the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifiesp300 as amolecular determinant of AR degradation and highlights p300 as a candidate target tomanage prostatecancer, especially in cases marked by PTEN loss. Cancer Res; 74(6); 1870–80. �2014 AACR.

IntroductionAndrogens and the androgen receptor (AR) are paramount

for prostate cancer growth (1, 2). Androgen-deprivation ther-apy (ADT) is the mainstay of treatment for patients withadvanced/disseminated prostate cancer. Unfortunately, thistreatment is palliative, and the majority of prostate cancersevolve into a disease termed castration-resistant prostatecancer (CRPC). In virtually all cases, castration-resistant pro-gression is accompanied by an increase in the level of prostate-specific antigen (PSA), a well-studied AR transactivated gene(3), indicating that the AR is aberrantly activated under cas-tration conditions. AR protein is expressed in nearly all met-astatic CRPC. The importance of persistent AR signaling inCRPC is further demonstrated by the antitumor activity ofnext-generation androgen–AR axis inhibitors, including abir-aterone and enzalutamide (4, 5). However, resistance to thesedrugs has been observed in many patients (6, 7). Thus, there is

an unmet need for the development of new strategies toeliminate AR protein and functions in prostate cancer.

The tumor-suppressor gene PTEN is often mutated, delet-ed, or transcriptionally downregulated in human prostatecancers. Genome-wide integrative genomic profiling analy-sis showed that PTEN loss and activation of the phosphoi-nositide 3-kinase (PI3K) pathway occur in up to 70% ofmetastatic prostate cancers (8). PTEN loss or PI3K activa-tion results in AKT phosphorylation and activation. Impor-tantly, homozygous deletion of Pten invariably induces AKTactivation and prostate tumorigenesis in mice (9–11). How-ever, the downstream signaling pathways that contribute toPTEN loss or AKT activation–induced prostate cancer path-ogenesis are poorly understood.

p300 was originally identified as a histone acetyltransfer-ase, which acts as a coactivator for many transcriptionfactors, including AR, p53, and NF-kB (12). Thus, the roleof p300 in cancer is not entirely clear and may depend on thepathophysiological milieu of the tumors. P300 seems to havean oncogenic potential because its expression is upregulatedduring human prostate cancer progression (13, 14). More-over, p300 has been demonstrated to be an essential coacti-vator of the AR, a key prostate cancer promoter. P300interacts with and acetylates AR in vitro and in vivo (15–17). Not only is p300 important for androgen-dependent andindependent transactivation of the AR (15, 18–20), but also itsexpression is associated with human prostate cancer prolif-eration (13). However, the precise roles of p300 in prostatecancer development and progression remain elusive. In thepresent study, we demonstrate that p300 functions as a keyregulator of AR protein degradation and PTEN-deficientprostate tumorigenesis.

Authors' Affiliations: 1Departments of Biochemistry and Molecular Biol-ogy, 2Urology, and 3Laboratory Medicine and Pathology; 4Mayo ClinicCancer Center, Mayo Clinic College of Medicine, Rochester; and 5Depart-ment of Laboratory Medicine and Pathology, University of Minnesota,Minneapolis, Minnesota

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

J. Zhong and L. Ding contributed equally to this work.

Corresponding Author: Haojie Huang, Mayo Clinic, 200 First Street SW,Gugg 1311B, Rochester, MN 55905. Phone: 507-293-1712; Fax: 507-293-3071; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2485

�2014 American Association for Cancer Research.

CancerResearch

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Materials and MethodsPlasmids and reagentsHA-p300 and HA-p300DHAT (lacking amino acids 1430–

1504) were provided by Dr. R. Janknecht, The University ofOklahoma College of Medicine (Oklahoma City, OK). ARmutants S81A, S213,791A, K630,632,633A, and K630,632,633Qwere generated bymutagenesis (Stratagene). V5-tagged cyclin-dependent kinase 1 (CDK1) and cyclin B1 were describedpreviously (21). Antibodies: AR (N20), CDK1, ERG (C20), ERK2,E2F1 (C20), Mme/CD10 (F4), p53 (DO-1), p300 (C20), Pb (R15),SP1 (PEP2; Santa Cruz Biotechnology), AKT-p (Serine 473phospho-specific), RB-p (Serine 795 phospho-specific), PTEN(Cell Signaling Technology), V5 (Invitrogen), acetyl-lysine, AR-p (Serine 81 phospho-specific; Millipore), smooth muscle actin(SMA) monoclonal (Dako), Ki-67 (Novocastra), and Nkx3.1(Novus Biological).

Generation of prostate-specific p300 single- and p300and Pten double-deletion micep300 conditional knockout (P300L/L) mice were reported

previously (22) and provided by Dr. Jan van Deursen at MayoClinic (Rochester, MN). Pten conditional knockout (PtenL/L)mice were generated originally in the laboratory of Dr. HongWu, University of California Los Angeles (Los Angeles, CA;ref. 9), and purchased from The Jackson Laboratory. Pb-Cre4transgenic mice were generated originally in the laboratoryof Dr. Pradip Roy-Burman, University of Southern California(Los Angeles, CA; ref. 23), and acquired from the NationalCancer Institute (NCI) Mouse Repository. Cohorts ofp300pc�/�;Ptenpc�/�, p300L/L;PtenL/L, p300pc�/�, Ptenpc�/�

mice were generated from p300pcþ/�;Ptenpcþ/� males andp300L/þ;PtenL/þ females, which were obtained by cross-breeding Pb-Cre4 males with p300L/L and PtenL/L females.All procedures were approved by the Mayo Clinic Institu-tional Animal Care and Use Committee and conform to thelegal mandates and federal guidelines for the care andmaintenance of laboratory animals.

Human prostate cancer specimens and IHC scoringThirty-two prostate cancer tissues [Gleason score (GS10), 1

case; GS9, 2 cases; GS8, 4 cases; GS7 14 cases; andGS6, 11 cases]were selected randomly from patients with biopsy-provenprostate cancer that have been treated at the Mayo Clinic byradical retropubic prostatectomy between January 1995 andDecember 1998 without neoadjuvant therapy. The age of thepatients ranged from 47 to 73 years (mean 62.9). The study wasapproved by the Mayo Clinic Institutional Review Board.Immunohistochemistry (IHC) with antibodies for p300 (C20)and AR (N20; Santa Cruz Biotechnology) was performed asdescribed above. A staining index (SI) was calculated asfollows: Staining intensity and staining percentage of eachslide were graded accordingly (intensity: 0, no staining; 1, lowstaining; 2, media staining; and 3, strong staining; percentage:1, 0%–33% positive cells; 2, 34%–66% positive cells; and 3, 67%–100% positive cells). A final SI score for each staining wasobtained by multiplying values obtained from staining per-centage and intensity and used for correlation analysis.

Cell lines, cell culture, and transfectionCell lines 22Rv1, PC-3, DU145, and 293T were purchased

from the American Type Culture Collection. 22Rv1, PC-3, andDU145 cells were cultured in RPMI-1640 medium supplemen-ted with 10% FBS. 293T cells were maintained in Dulbecco'sModified Eagle Medium (Invitrogen) supplemented with 10%FBS. LAPC-4 was kindly provided by Dr. Charles Sawyers,Memorial Sloan Kettering Cancer Center (New York, NY), andwas grown in Iscove's Modified Eagle Medium supplementedwith 10% FBS. These cell lines have been tested and authen-ticated (karyotyping, AR expression, and PTEN mutation sta-tus) for fewer than 6 months before the first submission of thearticle. Transfections were performed by electroporation usingan Electro Square Porator ECM 830 (BTX; ref. 24) or by usingLipofectamine 2000 (Invitrogen). Approximately 75% to 90%transfection efficiencies were routinely achieved.

Immunoprecipitation and Western blottingProtein immunoprecipitations were carried out using an

immunoprecipitation kit (Roche Applied Science) as describedpreviously (24), and Western blotting was performed asdescribed previously (24). Antibodies used forWestern blottingwere diluted at 1:1,000 to 1:2,000.

Statistical analysisExperiments were carried out with three or more replicates

unless otherwise stated. Statistical analyses were performed bythe Student t test for most studies. For the analysis of corre-lation between p300 and AR protein expression in humanprostate cancer specimens, nonparametric Spearman rankcorrelation was used. Values with P < 0.05 are consideredstatistically significant.

Additional methodsAdditional methods are provided in Supplementary Materi-

als and Methods.

Resultsp300 is required for PTEN-deficient prostatetumorigenesis in mice

The tumor suppressor PTEN is frequently lost in advancedhuman prostate cancer (8), and overexpression of p300 isassociated with human prostate cancer progression (13, 14).To test whether PTEN-deficient tumors require a functionalp300, we investigated whether deletion of p300 impairs Ptenknockout-induced tumorigenesis in the mouse prostate. Bycross-breeding probasin-Cre transgenic mice (Pb-Cre4; ref. 23)with p300 conditional (p300Loxp/Loxp, p300L/L; ref. 22) and Ptenconditional (PtenLoxp/Loxp, PtenL/L) mice (9), we generated fourcohorts of animals: prostate-specific Pten knockout (Pb-Cre4;PtenL/L, hereafter termed as Ptenpc�/�), Pten/p300 doubleknockout (Pb-Cre4;PtenL/L;p300L/L, termed as Ptenpc�/�;p300pc�/�), p300 knockout (Pb-Cre4;p300L/L, termed asp300pc�/�), and Cre-negative PtenL/L;p300L/L control mice(hereafter termed as "wild-type"). Similar to the knockout ofother genes such as Pten (9), the p300 gene was robustly deletedin thedifferent lobes of theprostate inmice (Supplementary Fig.S1). As demonstrated by IHC, Pten protein was readily detected

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in the prostates of "wild-type" and p300pc�/� mice, but little orno Pten protein was detected in Ptenpc�/� and Ptenpc�/�;p300pc�/� prostates (Fig. 1A, i). In accordance with Pten dele-tion, Akt phosphorylation robustly increased in the prostates ofboth Ptenpc�/� and Ptenpc�/�;p300pc�/� mice compared withthe Pten-positive counterparts (Fig. 1A, ii). Similar to the geno-mic DNA deletion (Supplementary Fig. S1), p300 protein waseffectively depleted in p300pc�/� prostates (Fig. 1A, iii, column3). As expected, p300 protein was detected in the prostates ofPtenpc�/� and "wild-type," but not in Ptenpc�/�;p300pc�/� mice(Fig. 1A, iii). These data indicate that both Pten and p300proteinswere effectively deleted in theprostates ofmice studied.

As expected, Ptenpc�/� mice at 4 months of age displayedhistologic evidence of high-grade prostatic intraepithelial neo-plasia (HGPIN) in most acini (Fig. 1B and Supplementary Fig.S2), with occasionally diseased acini suggestive of microinva-sive cancer in which periglandular SMA-positive stroma wasdisrupted or absent (Fig. 1C). In contrast, Ptenpc�/�;p300pc�/�

mice developed primarily low-grade PIN (LGPIN) but notinvasive cancer (Fig. 1B and C and Supplementary Fig. S2).P300 deletion alone had no overt impact on the morphology ofprostate epithelium in p300pc�/� mice (Fig. 1B). Quantitativeanalysis of malignant acini in Ptenpc�/� prostates revealedapproximately 90% HGPIN/cancer and approximately 10%

Figure 1. p300 deletion inhibitsPTEN-deficient prostatetumorigenesis in mice. A, IHC forPten (i), phosphorylated Akt (Akt-S473-p; ii), and p300 (iii) in prostatesections of 4-month-old "wild-type," Ptenpc�/�, p300pc�/�,p300pc�/�;Ptenpc�/� mice (n ¼ 8/group). The inset shows a high-magnification image of therepresentative (framed) area ineachpanel. Scale bar, 50mm.Scalebar in inset, 10 mm. B, H&E analysisof ventral prostate (VP) of 4-month-old mice with indicated genotypes(n¼ 8/genotype). Scale bar, 50 mm.C, IHC for SMA in the prostateof Ptenpc�/� and p300pc�/�;Ptenpc�/� mice. Arrows, disruptedand absent smooth muscle stromain the Ptenpc�/� mouse. Scale bar,50 mm. D, quantification ofnonmalignant, LGPIN and HGPIN,or cancerous (with areassuggestive of microinvasion) aciniin the prostates (including AP, VP,and dorsolateral prostate) of micewith the indicated genotypes (n ¼8/genotype). E, representatives ofgenitourinary tracts of Ptenpc�/�

and p300pc�/�;Ptenpc�/� mice(n ¼ 6/genotype) that survived to 8months. B, bladder; AP, anteriorprostate. Scale bar, 2 mm. F,Ptenpc�/� mice lacking p300 in theprostates tend to live longer.Survival was monitored for 12months and is displayed usingKaplan–Meier plots.

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LGPIN (Fig. 1D). In contrast, Ptenpc�/�;p300pc�/� prostatesexhibited approximately 40% HGPIN, approximately 60%LGPIN, and approximately 1% nonmalignant acini (Fig. 1D).In agreement with the morphology data, the mass of prostateglands in Ptenpc�/� mice was much greater than that inPtenpc�/�;p300pc�/� mice (Fig. 1E). By following the survivalof a cohort of 70mice for over 12months, we found that none of15 Ptenpc�/�;p300pc�/� mice died, whereas 7 of 15 Ptenpc�/�

mice died (Fig. 1F), which was probably due to bladderobstruction and renal failure as reported previously (25).Neither "wild-type" (n ¼ 20) nor p300pc�/� (n ¼ 20) mice diedduring the follow-up period. These data indicate that ablationof p300 impairs Pten deletion–induced prostate tumorigenesisand prolongs survival of mice with Pten-deficient prostate.

p300deletiondecreases cell proliferationandARproteinlevel in PTEN-null prostate cancer in miceTo determine the molecular mechanism underlying p300

deletion–imposed inhibition of Pten knockout tumor progres-sion, we examined cell proliferation in the prostates of Ptenand/or p300 knockoutmice. Similar to previous reports (9), Ki-67 staining significantly increased in the prostates of Ptenpc�/�

mice compared with the normal prostatic epithelium in "wild-type"mice (Fig. 2A andB). Importantly, p300 deletionmarkedly

decreased Ki-67 staining in Pten-deficient prostates (Fig. 2Aand B). To our knowledge, this is the first demonstration of thecausal role of p300 in prostate cancer cell proliferation in vivo,which is consistent with a previous finding that p300 knock-down significantly inhibits proliferation of PTEN-null humanLNCaPprostate cancer cells in culture (26). P300 deletion alonehad no overt effect on Ki-67 staining (Fig. 2A and B). Becausethe background level of Ki-67 staining was quite low in wild-type mice at the age examined (Fig. 2A and B), which isconsistent with previous reports (25, 27), our data cannot ruleout the possibility that p300 loss alone affects prostatic cellproliferation in mice at different ages or with different geneticbackgrounds, and thus further investigation is warranted.Because Pten-deleted murine prostate cancer cells depend ontheAR for growth in vitro and in vivo (28), we examinedwhetherp300 deletion affects AR protein levels in both Pten-deficientand wild-type prostates. As evident by IHC and Western blotanalyses, AR protein was substantially downregulated in pros-tatic cells in Ptenpc�/� mice compared with "wild-type" coun-terparts (Fig. 2C and D), which is consistent with previousreports (27, 29). Strikingly, p300 deletion further decreased ARprotein in the vast majority of prostatic epithelial cells inPtenpc�/�;p300pc�/� mice compared with Ptenpc�/� counter-parts (Fig. 2C), and this result was confirmed by Western blot

Figure 2. p300 knockout decreasescell proliferation and AR proteinlevels in PTEN-deleted prostatetumors in mice. A, IHC for Ki-67 inthe prostates of 4-month-old micewith indicated genotypes (n ¼ 3/group). The inset in each panelshows a representative(framed) area in high magnification.Scale bar, 50 mm. Scale barin the inset, 10 mm. B, quantitativedata of Ki-67 staining shown in A.Columns, mean values of Ki-67–positive cells among threeindividual mice. Error bars, SD.�, P < 0.01. C, IHC of AR protein inthe prostates of 4-month-oldmice with indicated genotypes(n ¼ 3/group). Scale bar, 50 mm.D, Western blot analysis ofindicated proteins in cell lysatesprepared from the prostates ofthree mice in each group at 4months of age.

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analysis (Fig. 2D). These data suggest that p300 is required forprostate cancer cell proliferation and maintenance of ARprotein levels in PTEN-deficient mouse prostate tumors.

Expression of p300 positively correlates with AR proteinlevels in human prostate cancer specimens

To support the clinical relevance of p300 regulation of ARprotein observed in p300/Pten knockout mice, we sought todetermine if p300 and AR expression correlates in humanprostate cancer specimens. To this end, we examined theexpression of these proteins by IHC in prostate cancer speci-mens obtained from a cohort of 32 patients. Examples of bothstrong and weak staining of p300 and AR and hematoxylin andeosin (H&E) staining are shown in Fig. 3A–C. The p300 and ARprotein expression data are summarized in Fig. 3D. Nonpara-metric Spearman rank correlation analysis indicated that thereis a strong correlation between p300 andARprotein expressionin the human prostate cancer specimens examined (r ¼ 0.64;P < 0.001).

p300 precludes complete degradation of AR protein inPTEN-deficient prostate cancer cells

Human prostate cancer cell lines were used to determinemechanistically how p300 regulates AR protein levels. Similarto the results seen inmice (Fig. 2C andD), PTEN knockdown bysiRNAs decreased AR protein expression and concomitantknockdown of p300 further reduced AR protein levels in bothLAPC4 and 22Rv1 cell lines (Fig. 4A and Supplementary Fig.S3A). Of note, the effect of PTEN and p300 knockdown on ARlevels was much greater than that on other cancer-relevant

transcription factors examined, including p53, E2F1, ERG, andSP1 (Supplementary Fig. S3B), suggesting a specific impact ofPTEN and p300 on AR protein levels in prostate cancer cells.P300 knockdown alone also decreased AR protein in LAPC4and 22Rv1 cells (Fig. 4A and Supplementary Fig. S3A). PTENand/or p300 knockdown–mediated decrease in ARproteinwaslargely blocked by the proteasome inhibitor MG132 (Fig. 4Aand Supplementary Fig. S3A), suggesting a proteasome-depen-dent mechanism. Furthermore, PTEN depletion increased ARprotein instability, and this effect was further enhanced byp300 silencing in LAPC4 cells (Supplementary Fig. S3C andS3D). Neither PTEN nor p300 knockdown had any overt effecton ARmRNA levels in these cell lines (Supplementary Fig. S3E).These data indicate that p300 is a key regulator of proteasome-mediated AR degradation in both PTEN-proficient and -defi-cient prostate cancer cells.

p300 inhibits AR polyubiquitination via itsacetyltransferase activity

Next, we sought to determine whether p300 regulates ARprotein polyubiquitination. PTEN knockdown increased ARpolyubiquitination in LAPC4 cells (Fig. 4B), which is consistentwith the finding that AR polyubiquitination is regulated by AKT(30). P300 knockdown alone also increased AR polyubiquitina-tion, and this effect was further enhanced by coknockdown ofPTEN (Fig. 4B). Lysine (K) residues 630, 632, and 633 (K630, 632,and 633) on AR are three known p300 acetylation sites (15).Interestingly, conversion of these residues to alanine (K630/632/633A) diminished AR polyubiquitination (Fig. 4C). This resultcould be due to the possibility that these lysine residues are

Figure 3. IHC of p300 and ARproteins in human prostate cancerspecimens. A–C, representativeimages of H&E staining (A), p300(B), and AR IHC (C) in prostatecancers exhibiting high and lowexpression of p300 and ARproteins. Scale bar, 100 mm. Insets,high-magnification images offramed areas; scale bar, 50 mm.D, scoring data of the stainingindex for expression of p300 andAR proteins in human prostatecancer specimens examined.

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ubiquitin-accepting sites or that blockage of acetylation of thesesites affects AR ubiquitination through mechanisms other thansite competition. To test the second possibility, the lysineresidues were converted into glutamine (Q), which structurallymimics the acetylation state of lysine (31). Polyubiquitination onthe K630/632/633Qmutant was further reduced compared withthe K630/632/633A mutant, although the expression levels ofthese mutated proteins were comparable (Fig. 4C). Thus, theseexperiments identified AR acetylation at K630, 632, and 633residues as an important inhibitory mechanism of AR polyubi-quitination in prostate cancer cells. Consistent with theseobservations, we further showed that ectopic expression of

wild-type p300, but not the histone acetyltransferase (HAT)-deficient mutant (p300DHAT), diminished AR polyubiquitina-tion (Fig. 4D). In contrast, overexpression of p300 had little or noeffect on polyubiquitination of the acetylation-resistant K630/632/633Amutant ofAR (Fig. 4D).Weconclude that p300 inhibitsAR polyubiquitination and that this effect is mediated by itsacetyltransferase activity.

Serine 81 phosphorylation in AR is required for thep300–AR interaction

Overexpression of active AKT triggers ARdegradation (30). Asexpected, PTEN knockdown by siRNAs downregulated AR

Figure 4. p300 regulates ARubiquitination and proteasomedegradation in prostate cancercells. A, p300 knockdown inducesAR proteasome degradation.LAPC4 cells were transfected asindicated. Sixty-four hours later,cells were treated with vehicleor MG132 (20 mmol/L) for 8 hoursfollowed by Western blot analysis.B, LAPC4 cells were transfectedwith indicated siRNAs for72 hours and then treated withMG132 for 8 hours followed byimmunoprecipitation (IP) andWestern blot analyses. C, AR-negative PC-3 cells weretransfected with AR-WT andmutants (K630,632,633A andK630,632,633Q) in combinationwith HA-tagged Ub for 16 hours,followed by treatment with MG132(20 mmol/L) for 8 hours. Celllysates were subjected toimmunoprecipitation and Westernblot as in B. D, PC-3 cells weretransfected with the plasmids asindicated for 16 hours, followed bytreatment with MG132 (20 mmol/L)for 8 hours. Cell lysates weresubjected to immunoprecipitationand Western blot as in B. E, effectof AKT on p300 and PTENregulation of AR degradation.LAPC-4 cells were transfectedwithindicated siRNAs for 72 hours,followed by treatment of LY294002for 12 hours and Western blotanalysis. F, effect of AKTphosphorylation of AR at Ser213and Ser791 on p300 and PTENregulation of AR degradation.DU145 prostate cancer cells weretransfected with plasmids andsiRNAs as indicated for 48 hoursfollowed by Western blot analysis.

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protein in LAPC4 cells and thiswas blockedby theAKT inhibitorLY294002 (Fig. 4E, left). Similarly, AR degradation was inhibitedby LY294002 in PTEN and p300 coknockdown cells (Fig. 4E,right). When two AKT phosphorylation sites in AR (serine 213and791; ref. 30)weremutated toalanine, the steady-state level ofthe S213,791A mutant was higher than the wild-type AR (AR-WT) in DU145 cells (Fig. 4F). However, these mutations failed toblock AR degradation induced by coknockdown of PTEN andp300 (Fig. 4F). These data suggest that the effect of p300 on ARdegradation is AKT dependent but independent of AKT-medi-ated phosphorylation of AR at Ser213 and Ser791.

Besides the AKT pathway, AR proteasome degradation isalso regulated by CDK1, and this effect is attributed to CDK1

phosphorylation of AR at multiple serine/threonine sites,including serine 81 (Ser81; ref. 32). PTEN loss or AKT activationpromotes cell-cycle progression by increasing the activity ofCDKs (33–36). We therefore examined the role of AR Ser81phosphorylation in the regulation of p300 and PTEN deficien-cy–induced AR protein degradation. PTEN knockdownincreased RB phosphorylation, an indicator of CDK activationand AR phosphorylation at Ser81 (Fig. 5A). Next, we examinedwhether AR Ser81 phosphorylation affects the p300–AR inter-action. The interaction between these two proteins was readilydetected, butwas largely attenuated by the S81Amutation (Fig.5B). Accordingly, overexpression of cyclin B1 and CDK1 greatlyenhanced p300 interaction with AR-WT but not the S81A

Figure 5. ARSer81 phosphorylationby CDK1 is crucial for p300–ARinteraction, AR acetylation, andp300 regulation of ARubiquitination. A, LAPC4 cells weretransfected with indicated siRNAsfor 72 hours followed byWestern blot analysis. B, 293T cellswere transfected with wild-typeand S81A-mutated AR for 24hours followed by treatmentwith 10 nmol/L mibolerone (Mib)for additional 24 hours. Celllysates were subjected toimmunoprecipitation and Westernblot. C, 293T cells were transfectedwith AR-WT and S81A mutant incombination with or withoutCDK1 and cyclin B1 for 24 hoursfollowed by treatment with 10nmol/L Mib for additional 24 hours.Cell lysates were subjected toimmunoprecipitation and Westernblot. D, 293T cells were transfectedwith the indicated plasmids for 24hours followed by treatment with10 nmol/L Mib in combination withorwithout 10mmol/L roscovitine for24 hours. Cell lysates weresubjected to immunoprecipitationand Western blot. E, 293T cellswere transfectedwith the indicatedplasmids for 24 hours and treatedwith 10 nmol/L Mib for 24 hours.Cell lysates were subjected toimmunoprecipitation and Westernblot analysis. F, 293T cells weretransfected with the plasmids andsiRNAs as indicated for 24 hoursand treated with 10 nmol/L Mib for24 hours. Cell lysates weresubjected to immunoprecipitationand Western blot. G, LAPC4 cellswere transfected with indicatedsiRNAs for 72 hours followed byWestern blot analysis.

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mutant (Fig. 5C and Supplementary Fig. S4). Furthermore,p300–AR interaction was completely blocked by the pan CDKinhibitor roscovitine (Fig. 5D). We conclude that loss of PTENincreases CDK1-mediated phosphorylation of AR at Ser81,which enhances the interaction between p300 and AR.

Ser81 phosphorylation in AR is crucial for p300-mediated inhibition of AR ubiquitinationWe further examined whether AR Ser81 phosphorylation

affects the regulation of AR acetylation and polyubiquitinationby p300. S81Amutation decreased AR acetylation (Fig. 5E, lane1 vs. lane 3). Consistent with CDK1/cyclin B1–enhanced inter-action between p300 and AR (Fig. 5C), overexpression of CDK1and cyclin B1 augmented acetylation of AR-WT but not theS81A mutant (Fig. 5E, lane 3 vs. lane 4). Moreover, over-expression of CDK1 and cyclin B1 substantially decreasedpolyubiquitination of AR-WT, and this effect was partiallyreversed by p300 knockdown (Fig. 5F). Consistent with theresult that acetylation of S81A was lower than AR-WT (Fig.5E), polyubiquitination of S81A was greater than AR-WT(Fig. 5F). However, in contrast with the effect on AR-WT,ectopic expression of CDK1/cyclin B1 alone or in combina-tion with p300 knockdown had a negligible effect on poly-ubiquitination of the S81A mutant (Fig. 5F). In agreementwith these results (Fig. 5E and F) and the finding that loss ofPTEN increases CDK1 phosphorylation of AR at Ser81 (Fig.5A), we further showed that knockdown of CDK1 acceleratedPTEN loss–induced decrease in AR protein level and thatthis effect was further enhanced by p300 knockdown (Fig.5G). Thus, besides its role in mediating the interactionbetween p300 and AR, Ser81 phosphorylation is crucial forCDK1-enhanced AR acetylation and p300-mediated inhibi-tion of AR polyubiquitination.

p300 is important for AR transcriptional activity inPTEN-null prostate tumors in miceTo gain insight into the biologic importance of p300 regu-

lation of AR protein degradation in PTEN-deficient prostatecancer cells, we sought to determine how p300 affects ARactivity by examining AR target gene expression. Consistentwith the observation that p300 knockout induced downregula-tion of AR protein in PTEN-deleted prostate cancer cells inmice (Fig. 2C and D), mRNA levels of AR-regulated genes,including probasin (Pb), Nkx3.1, and Mme, were significantlyreduced in Ptenpc�/�;p300pc�/� prostates compared withPtenpc�/� counterparts (Fig. 6A–C, top). These results wereconfirmed by IHC (Fig. 6A–C, bottom). In contrast, there waslittle or no difference in expression of Pai-1, a non–androgen-regulated gene, between Ptenpc�/� and Ptenpc�/�;p300pc�/�

prostates (Supplementary Fig. S5A), arguing that p300 deletiondoes not result in a general downregulation of gene expressionin PTEN-deleted prostate cancer cells in vivo. To determinewhether p300 exerts a similar effect on AR activity in humanPTEN-deficient prostate cancer cells, LAPC4 cells were trans-fected with PTEN siRNAs in combination with control or p300-specific siRNAs followed by treatment with vehicle or mibo-lerone, a synthetic androgen. P300 silencing (SupplementaryFig. S5B)markedly decreased the expression of AR target genes

PSA, NKX3.1, HK2, and TMPRSS2 in both androgen-untreatedand -treated PTEN knockdown cells (Fig. 6D), indicating thatp300 is important for AR activation in the presence or absenceof androgens in human PTEN-deficient prostate cancer cells.However, no such effects were detected on the expression ofp21WAF1, E2F1, PLAT, and PAI-1, target genes of p53, E2F1, ERG,and SP1 transcription factors, respectively (Supplementary Fig.S5C). Consistent with the protein expression results (Supple-mentary Fig. S3B), among the transcription factors examinedAR activity seems to be mostly affected by p300 depletion.Together, these results suggest that p300 is essential for thetranscriptional activity of the AR in PTEN-deficient prostatecancer cells in vitro and in vivo.

Discussionp300 has been implicated in human prostate cancer prolif-

eration and progression (13, 37, 38), although the direct in vivoevidence that p300 is causally involved in prostate oncogenesisis lacking. In the present study, we revealed that prostate-specific homozygous deletion of p300 dramatically inhibitsPten-deficient prostate cancer proliferation and progressionand prolongs mouse survival. Thus, in the Pten knockoutmouse model, we demonstrate for the first time that p300functions as a bona fide oncogenic promoter of prostate cancer.The relevance of this observation is further substantiated byour findings that high expression of p300 correlates with AKTphosphorylation, a surrogate of PTEN inactivation, in a cohortof human prostate tumors examined (Supplementary Fig. S6).It is worth noting that our present study cannot rule out thepossibility that p300 is also critical for prostate tumorigenesisinduced by other oncogenic factors such as RAS and Myc. It ishence important to address this issue in the future usingtransgenic mouse models.

p300 is a well-known acetyltransferase that catalyzes acety-lation of both histone and nonhistone proteins such as AR andp53 (12, 15, 39). It is generally accepted that p300 is a generictranscriptioncoactivator.Moreover, it hasbeenshown thatp300is targeted by oncogenic viral proteins such as E1A and is oftenmutated or truncated in endometrial, breast, and pancreaticcancers (40), implying that p300 functions as a classic tumorsuppressor. However, in contrast with this concept, differentstudies invariably demonstrate that p300 is overexpressed inhuman prostate cancers (13, 14). These observations in clinicalspecimens and our findings in the mouse model clearly supportthe hypothesis that p300 functions as an oncogene in theprostate. Therefore, our findings about how p300 functionsparadoxically as an oncogene in the prostate are of importancefrom both basic science and translational perspectives.

Development, differentiation, and growth of the prostaterely on androgens and androgen activation of the AR. The sameis true for prostate cancer growth and progression. Because ofthe androgen/AR addiction of prostate cells, ADT is themainstay of treatment for advanced prostate cancer in theclinic. It is known that p300 promotes AR transactivation byinducing acetylation of AR and histone proteins (15). In thecurrent report, we have discovered another important layer ofp300 regulation of the AR by demonstrating for the first time

p300 Regulates AR Degradation and Prostate Tumorigenesis

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that p300 plays an essential role in regulation of AR proteindegradation. First, we provide evidence that homozygousdeletion of p300 decreases AR protein levels in the mouseprostate. Second, we show that expression of p300 correlatespositively with AR protein levels in a cohort of human prostatecancer specimens. Third, using human prostate cancer celllines as aworkingmodel, we demonstrate that p300 inhibits ARprotein polyubiquitination and proteasome degradation byinducing AR acetylation at lysine residues 630, 632, and 633.Importantly, the effect of p300 on AR degradation seems to beAR-specific because p300 depletion had no overt effect onother cancer-relevant transcription factors examined such asp53, E2F1, ERG, and SP1. Consistent with the essential role ofAR in prostate cancer proliferation and growth, we furthershow that homozygous deletion of p300 significantly decreases

prostate cancer cell proliferation in vivo. Together, our findingsimply that the oncogenic function of p300 in the prostate couldbe linked, at least in part, to the role of p300 in regulation of ARprotein levels, which is highly relevant to prostate tumorigen-esis and progression.

AR protein stability is known to be regulated by CDK1 inprostate cancer cells, and this effect is likely mediated byCDK1 phosphorylation of multiple serine/threonine residuesin the AR protein, including the major phosphorylation siteSer81 (32). However, the precise mechanism by which Ser81phosphorylation influences AR protein stability is unclear.Our data demonstrate that loss of PTEN increases ARphosphorylation at Ser81 and that Ser81 phosphorylationacts as a molecular beacon that is required for the binding ofp300, a key event that subsequently leads to AR acetylation,

Figure 6. p300 depletion impairs ARtarget gene expression in PTEN-deficiency prostate cancer (PCa)cells. A–C, reverse transcriptionquantitative PCR (RT-qPCR; top)and IHC (bottom) analyses ofexpression of AR target genes Pb(A), Nkx3.1 (B), and Mme (C) inprostate tissues from mice (n ¼ 3)with the indicated genotypes at 4months of age. Columns, meanvalues among three replicates;error bars, SD. �, P < 0.01. Bottom,representative IHC staining of Pb(cytoplasmic and secreted out ofcells), Nkx3.1 (cytoplasmic), andMme (plasma membrane). D,LAPC4 cells were transfected asindicated for 48 hours and treatedwith 10 nmol/L Mib for 24 hours.Expression of indicated genes wasanalyzed by RT-qPCR. Columns,mean values among threereplicates; error bars, SD.�,P < 0.01. E, a diagram depicts therole of p300 and CDKphosphorylation of AR at Ser81 inregulation of AR stabilization andfunction by inhibiting PTEN loss orAKT activation–induced ARubiquitination. Ac with circle,acetylation; P with circle,phosphorylation; Ub, ubiquitin.

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inhibition of AR ubiquitination, and AR stabilization (Fig.6E). Thus, we identify p300 as a key molecular determinantof AR degradation in PTEN-mutated prostate cancer cells. Inaddition to its role in regulation of AR stability, Ser81phosphorylation is important for AR transactivation (41,42). In the present study, we reveal for the first time thatSer81 phosphorylation is essential for p300–AR interactionand AR acetylation. Given that p300 is a key transcriptionalcoactivator, beside its role in mediating chromatin binding(42), enhanced p300–AR interaction represents a novelmechanism of action of Ser81 phosphorylation in AR trans-activation (Fig. 6E). Another important function of AR Ser81phosphorylation is its role in regulation of prostate cancercell growth (41). Consistent with these findings, we provideevidence that p300 is important for prostate cancer cellgrowth in mice. Thus, it can be speculated that Ser81phosphorylation may promote prostate cancer cell growthvia mechanisms such as increased interaction between ARand p300 and subsequent AR activation (Fig. 6E).The findings that p300 acetylation of AR is important for AR

stabilization and transactivation and prostate cancer cellgrowth and survival (Fig. 6E; refs. 13, 16, 19, 26, 43, 44) implythat p300 could be a potential target for prostate cancertherapy. P300 is a bromodomain protein, and it could poten-tially be targeted for cancer treatment because inhibition ofother bromodomain proteins can effectively block tumorgrowth (45, 46). A virtual screen for inhibitors of p300 andCBP, a homolog protein of p300, has been conducted, and aselective small molecule inhibitor C646 has been identified.The antitumor effect of C646 was demonstrated in humanprostate cancer cells cultured in vitro (26). However, the in vivoapplication of C646 is unlikely because it can be inhibited byserum (26). Interestingly, curcumin (diferuloylmethane), aphenolic compound derived from the root of the plant Cur-cuma longa, and procyanidin B3, a compound extracted fromgrape seeds, have been demonstrated as natural anti-p300substances (47, 48). Indeed, treatment of the liposome formof curcumin not only inhibits murine Pten knockout prostatecancer progression, but also induces downregulation of ARprotein (49), which is highly consistent with our findings inp300-Pten double knockout mice. Procyanidin B3 treatmentalso inhibits prostate cancer cell proliferation by mitigatingp300-dependent acetylation of AR (48). Hence, natural p300

inhibitors are strong candidates for pharmacologic interven-tion of p300 function in cancers such as prostate cancer,although the specificity of these compounds in inhibitingp300 is an open question because numerous targets of dietarycompounds have been reported. Thus, although p300 is anattractive antiprostate cancer target, novel strategies areneeded to specifically abolish the oncogenic function ofp300 in prostate cancer. In this report, we demonstratethat p300 favors PTEN-deficient prostate cancer progressionby protecting ARprotein fromdegradation. Because this role ofp300 depends on the interaction between p300 and AR, itsblockage of AR degradation can be overcome by disruptingtheir physical interactionwithout disturbing p300 regulation ofother signaling pathways. Ourfinding therefore opens a uniqueavenue for development of novel therapeutics for prostatecancer via specific elimination of p300-mediated protectionof AR proteins in prostate cancer, especially those without afunctional PTEN.

Disclosure of Potential Conflicts of InterestD.J. Tindall is a consultant/advisory board member of Medivation. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Zhong, L. Ding, T.J. Sebo, D.J. Tindall, J. van Deursen,H. HuangDevelopment of methodology: L. Ding, J. Zhang, T.J. SeboAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Zhong, L.R. Bohrer, J. Zhang, T.J. Sebo, R.J. Karnes,J. van DeursenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Zhong, L. Ding, T.J. SeboWriting, review, and/or revision of themanuscript: L. Ding, P. Liu, J. Zhang,T.J. Sebo, R.J. Karnes, D.J. Tindall, J. van Deursen, H. HuangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Ding, P. LiuStudy supervision: J. Zhang, H. Huang

Grant SupportThis study was supported in part by grants from the NIH (CA134514 and

CA130908 to H. Huang; CA091956 to D.J. Tindall), the Department of Defense(W81XWH-09-1-622 to H. Huang), and the T.J. Martell Foundation.

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

Received August 28, 2013; revised December 12, 2013; accepted January 2, 2014;published OnlineFirst January 30, 2014.

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2014;74:1870-1880. Published OnlineFirst January 30, 2014.Cancer Res   Jian Zhong, Liya Ding, Laura R. Bohrer, et al.   and PTEN-Deficient Prostate Tumorigenesisp300 Acetyltransferase Regulates Androgen Receptor Degradation

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Published OnlineFirst January 30, 2014; DOI: 10.1158/0008-5472.CAN-13-2485