p300 acetyltransferase regulates androgen receptor degradation and PTEN-deficient prostate tumorigenesis.

Author Manuscript Published OnlineFirst on January 30, 2014; DOI: 10.1158/0008-5472.CAN-13-2485 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

P300 acetyltransferase regulates androgen receptor degradation and PTEN-deficient prostate tumorigenesis Jian Zhong, Liya Ding, Laura R. Bohrer, et al. Cancer Res Published OnlineFirst January 30, 2014.

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P300 acetyltransferase regulates androgen receptor degradation and PTEN-deficient prostate tumorigenesis

Jian Zhong1,2,3,6, Liya Ding1,2,3,6, Laura R. Bohrer4, Yunqian Pan1, Ping Liu4, Jun Zhang5, Thomas J. Sebo5, R. Jeffrey Karnes2, Donald J. Tindall2,3, Jan van Deursen1,3 and Haojie Huang1,2,3* 1

Department of Biochemistry and Molecular Biology, 2Department of Urology, 3Mayo Clinic

Cancer Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA; 4Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA; 5

Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine,

Rochester, MN 55905, USA. 6These authors contributed equally to this work. Running title: P300 regulates AR degradation and prostate tumorigenesis Keywords: p300, PTEN, AR, protein ubiquitination, proteasome degradation, prostate cancer Financial support: Supported in part by grants from the National Institutes of Health (CA134514 and CA130908 to H.H. and CA091956 to D.J.T.), the Department of Defense (W81XWH-09-1-622 to H.H.) and the T.J. Martell Foundation. Corresponding author: Haojie Huang, Ph.D., 200 First Street SW, Rochester, MN 55905; Phone: (507) 293-1712; Fax: (507) 293-3071; E-mail: [email protected]. Conflicts of interest: The authors declare no conflict of interest in this study. Word count: 4,817 words (excluding references and figure legends).

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Abstract Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer (PCa), but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional co-activator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion-induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human PCa cells where PTEN expression was abolished by RNAi-mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlate positively in human PCa 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 findings, in PTENdeficient PCa in the mouse we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlight p300 as a candidate target to manage PCa, especially in cases marked by PTEN loss.

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Introduction Androgens and the AR are paramount for PCa growth (1, 2). Androgen deprivation therapy (ADT) is the mainstay of treatment for patients with advanced/disseminated PCa. Unfortunately, this treatment is palliative, and the majority of prostate cancers evolve into a disease termed castration-resistant PCa (CRPC). In virtually all cases, castration-resistant progression is accompanied by a rise in the level of prostate-specific antigen (PSA), a well-studied AR transactivated gene (3), indicating that the AR is aberrantly activated under castration conditions. AR protein is expressed in nearly all metastatic CRPC. The importance of persistent AR signaling in CRPC is further demonstrated by the antitumor activity of next-generation androgen-AR axis inhibitors including abiraterone and enzalutamide (4, 5). However, resistance to these drugs has been observed in many patients (6, 7). Thus, there is an unmet need for development of new strategies to eliminate AR protein and functions in PCa.

The tumor suppressor gene PTEN is often mutated, deleted or transcriptionally downregulated in human prostate cancers. Genome-wide integrative genomic profiling analysis showed that PTEN loss and activation of the PI3K pathway occur in up to 70% of metastatic prostate cancers (8). PTEN loss or PI3K activation results in AKT phosphorylation and activation. Importantly, homozygous deletion of Pten invariably induces AKT activation and prostate tumorigenesis in mice (9-11). However, the downstream signaling pathways that contribute to PTEN loss or AKT activation-induced prostate cancer pathogenesis are poorly understood.

P300 was originally identified as a histone acetyltransferase, which acts as a coactivator for many transcription factors including AR, p53 and NFκB (12). Thus, the role of p300 in cancer is

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not entirely clear and may depend on the pathophysiological milieu of the tumors. P300 appears to have an oncogenic potential since its expression is upregulated during human PCa progression (13, 14). Moreover, p300 has been demonstrated to be an essential coactivator of the AR, a key PCa promoter. P300 interacts with and acetylates AR in vitro and in vivo (15-17). Not only is p300 important for androgen-dependent and independent transactivation of the AR (15, 18-20), but also its expression is associated with human PCa proliferation (13). However, the precise roles of p300 in PCa development and progression remain elusive. In the present study we demonstrate that p300 functions as a key regulator of AR protein degradation and PTENdeficient prostate tumorigenesis.

Materials and Methods Plasmids and reagents HA-p300 and HA-p300∆HAT (lacking amino acids 1430-1504) were provided by Dr. R. Janknecht. AR mutants S81A, S213,791A, K630,632,633A and K630,632,633Q were generated by mutagenesis (Stratagene). V5-tagged CDK1 and cyclin B1 were described previously (21). Antibodies: AR (N20), CDK1, anti-ERG (C20), ERK2, E2F1 (C20), Mme/CD10 (F4), p53 (DO1), p300 (C20), Pb (R15), SP1 (PEP2) (Santa Cruz Biotechnology), AKT-p (Serine 473 phospho-specific), RB-p (Serine 795 phospho-specific), PTEN (Cell Signaling Technology), V5 (Invitrogen), acetyl-lysine, AR-p (Serine 81 phospho-specific) (Millipore), SMA monoclonal (DAKO), Ki-67 (Novocastra) and Nkx3.1 (Novus Biological).

Generation of prostate-specific p300 single and p300 and Pten double deletion mice

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P300 conditional knockout (P300L/L) mice were reported previously (22) and provided by Dr. Jan van Deursen at Mayo Clinic. Pten conditional knockout (PtenL/L) mice were generated originally in the laboratory of Dr. Hong Wu (9) and purchased from Jackson Laboratory (Bar Harbor, Maine). Pb-Cre4 transgenic mice were generated originally in the laboratory of Dr. Pradip Roy-Burman (23) and acquired from the National Cancer Institute (NCI) Mouse Repository. Cohorts of p300pc-/-;Ptenpc-/-, p300L/L;PtenL/L, p300pc-/-, Ptenpc-/- mice were generated from p300pc+/-;Ptenpc+/- males and p300L/+;PtenL/+ 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 Institutional Animal Care and Use Committee and conform to the legal mandates and federal guidelines for the care and maintenance of laboratory animals.

Human PCa specimens and IHC scoring Thirty-two PCa tissues (Gleason score (GS10), 1 case; GS9, 2 cases; GS8, 4 cases; GS7 14 cases and GS6, 11 cases) were selected randomly from patients with biopsy-proven PCa that have been treated at Mayo Clinic by radical retropubic prostatectomy between January 1995 and December 1998 without neoadjuvant therapy. The age of the patients ranged from 47 to 73 (mean 62.9). The study was approved by the Mayo Clinic Institutional Review Board. IHC with antibodies for p300 (C20) and AR (N20) (Santa Cruz) was performed as described above. A staining index (SI) was calculated as follows: Staining intensity and staining percentage of each slide was graded accordingly (intensity: 0 = no staining, 1 = low staining, 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 was obtained by multiplying values obtained from staining percentage and intensity and used for correlation analysis.

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Cell lines, cell culture and transfection Cell lines 22Rv1, PC-3, DU145 and 293T were purchased from ATCC. 22Rv1, PC-3 and DU145 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. 293T cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% FBS. LAPC-4 was kindly provided by Dr. Charles Sawyers and was grown in IMEM supplemented with 10% FBS. These cell lines have been tested and authenticated (karyotyping, AR expression and PTEN mutation status) for fewer than 6 months before the first submission of the manuscript. Transfections were performed by electroporation using an Electro Square Porator ECM 830 (BTX) (24) or by using Lipofectamine 2000 (Invitrogen). Approximately 75–90% transfection efficiencies were routinely achieved.

Immunoprecipitation and western blotting Protein immunoprecipitations were carried out using an immunoprecipitation kit (Roche AppliedScience) as described previously (24), and western blotting was performed as described previously (24). Antibodies used for western blotting were diluted at 1:1,000 to 1:2,000.

Statistical analysis Experiments were carried out with three or more replicates unless otherwise stated. Statistical analyses were performed by Student’s t-test for most studies. For analysis of correlation between p300 and AR protein expression in human PCa specimens, nonparametric Spearman rank correlation was used. Values with P < 0.05 are considered statistically significant.

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Additional methods Additional methods are provided in Supplementary Information.

Results P300 is required for PTEN-deficient prostate tumorigenesis in mice The tumor suppressor PTEN is frequently lost in advanced human PCa (8), and overexpression of p300 is associated with human PCa progression (13, 14). To test whether PTEN-deficient tumors require a functional p300, we investigated whether deletion of p300 impairs Pten knockout-induced tumorigenesis in the mouse prostate. By crossbreeding probasin-Cre transgenic mice (Pb-Cre4) (23) with p300 conditional (p300Loxp/Loxp, p300L/L) (22) and Pten conditional (PtenLoxp/Loxp, PtenL/L) mice (9), we generated four cohorts of animals: prostatespecific Pten knockout (Pb-Cre4;PtenL/L, hereafter termed as Ptenpc-/-), Pten/p300 double knockout (Pb-Cre4;PtenL/L;p300L/L, termed as Ptenpc-/-;p300pc-/-), p300 knockout (PbCre4;p300L/L, termed as p300pc-/-), and Cre-negative PtenL/L;p300L/L control mice (hereafter termed as ‘wild type’). Similar to the knockout of other genes such as Pten (9), the p300 gene was robustly deleted in the different lobes of the prostate in mice (Supplementary Fig. S1). As demonstrated by immunohistochemistry (IHC), Pten protein was readily detected in the prostates of ‘wild-type’ and p300pc-/- mice, but little or no Pten protein was detected in Ptenpc-/- and Ptenpc/-

;p300pc-/- prostates (Fig. 1A-i). In accordance with Pten deletion, Akt phosphorylation robustly

increased in the prostates of both Ptenpc-/- and Ptenpc-/-;p300pc-/- mice compared to the Ptenpositive counterparts (Fig. 1A-ii). Similar to the genomic DNA deletion (Supplementary Fig. S1), p300 protein was effectively depleted in p300pc-/- prostates (Fig. 1A-iii, column 3). As expected, p300 protein was detected in the prostates of Ptenpc-/- and ‘wild-type’, but not in Ptenpc-

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/-

;p300pc-/- mice (Fig. 1A-iii). These data indicate that both Pten and p300 proteins were

effectively deleted in the prostates of mice studied.

As expected, Ptenpc-/- mice at 4 months of age displayed histologic evidence of high-grade prostatic intraepithelial neoplasia (HGPIN) in most acini (Fig. 1B and Supplementary Fig. S2), with occasionally diseased acini suggestive of microinvasive cancer where periglandular smooth muscle actin (SMA)-positive stroma was disrupted or absent (Fig. 1C). In contrast, Ptenpc-/;p300pc-/- mice developed primarily low-grade PIN (LGPIN) but not invasive cancer (Fig. 1B, C and Supplementary Fig. S2). P300 deletion alone had no overt impact on the morphology of prostate epithelium in p300pc-/- mice (Fig. 1B). Quantitative analysis of malignant acini in Ptenpc/-

prostates revealed approximately 90% HGPIN/cancer and approximately 10% LGPIN (Fig.

1D). In contrast, Ptenpc-/-;p300pc-/- prostates exhibited approximately 40% HGPIN, approximately 60% LGPIN, and approximately 1% nonmalignant acini (Fig. 1D). In agreement with the morphology data, the mass of prostate glands in Ptenpc-/- mice was much greater than that in Ptenpc-/-;p300pc-/- mice (Fig. 1E). By following the survival of a cohort of 70 mice for over 12 months, we found that none of 15 Ptenpc-/-;p300pc-/- mice died, whereas 7 out of 15 Ptenpc-/- mice died (Fig. 1F), which was probably due to bladder obstruction and renal failure as reported previously (25). Neither ‘wild type’ (n = 20) nor p300pc-/- (n = 20) mice died during the follow-up period. These data indicate that ablation of p300 impairs Pten deletion-induced prostate tumorigenesis and prolongs survival of mice with Pten-deficient prostate.

P300 deletion decreases cell proliferation and AR protein level in PTEN-null PCa in mice

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To determine the molecular mechanism underlying p300 deletion-imposed inhibition of Ptenknockout tumor progression, we examined cell proliferation in the prostates of Pten and/or p300 knockout mice. Similar to previous reports (9), Ki-67 staining significantly increased in the prostates of Ptenpc-/- mice compared to the normal prostatic epithelium in ‘wild-type’ mice (Fig. 2A and B). Importantly, p300 deletion markedly decreased Ki-67 staining in Pten-deficient prostates (Fig. 2A and B). To our knowledge, this is the first demonstration of the causal role of p300 in PCa cell proliferation in vivo, which is consistent with a previous finding that p300 knockdown significantly inhibits proliferation of PTEN-null human LNCaP PCa cells in culture (26). P300 deletion alone had no overt effect on Ki-67 staining (Fig. 2A and B). Because the background level of Ki-67 staining was quite low in wild-type mice at the age examined (Fig. 2A and B), which is consistent with previous reports (25, 27), our data cannot rule out the possibility that p300 loss alone affects prostatic cell proliferation in mice at different ages or with different genetic backgrounds, and thus further investigation is warranted. Since Pten-deleted murine PCa cells depend on the AR for growth in vitro and in vivo (28), we examined whether p300 deletion affects AR protein levels in both Pten-deficient and wild-type prostates. As evident by IHC and western blot analyses, AR protein was substantially downregulated in prostatic cells in Ptenpc-/mice compared to ‘wild-type’ counterparts (Fig. 2C and D), which is consistent with previous reports (27, 29). Strikingly, p300 deletion further decreased AR protein in the vast majority of prostatic epithelial cells in Ptenpc-/-;p300pc-/- mice compared to Ptenpc-/- counterparts (Fig. 2C), and this result was confirmed by western blot analysis (Fig. 2D). These data suggest that p300 is required for PCa cell proliferation and maintenance of AR protein levels in PTEN-deficient mouse prostate tumors.

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Expression of p300 positively correlates with AR protein levels in human PCa specimens To support the clinic relevance of p300 regulation of AR protein observed in p300/Pten knockout mice, we sought to determine if p300 and AR expression correlates in human PCa specimens. To this end, we examined the expression of these proteins by IHC in PCa specimens obtained from a cohort of 32 patients. Examples of both strong and weak staining of p300 and AR and H&E staining are shown in Fig. 3A-C. The p300 and AR protein expression data are summarized in Fig. 3D. Nonparametric Spearman rank correlation analysis indicated that there is a strong correlation between p300 and AR protein expression in the human PCa specimens examined (r = 0.64 , p < 0.001).

P300 precludes complete degradation of AR protein in PTEN-deficient PCa cells Human PCa cell lines were employed to determine mechanistically how p300 regulates AR protein levels. Similar to the results seen in mice (Fig. 2C and D), PTEN knockdown by small interference RNAs (siRNAs) decreased AR protein expression and concomitant knockdown of p300 further reduced AR protein levels in both LAPC4 and 22Rv1 cell lines (Fig. 4A and Supplementary Fig. S3A). Of note, the effect of PTEN and p300 knockdown on AR levels was much greater than that on other cancer-relevant transcription factors examined including p53, E2F1, ERG and SP1 (Supplementary Fig. S3B), suggesting a specific impact of PTEN and p300 on AR protein levels in PCa cells. P300 knockdown alone also decreased AR protein in LAPC4 and 22Rv1 cells (Fig. 4A and Supplementary Fig. S3A). PTEN and/or p300 knockdownmediated decrease in AR protein was largely blocked by the proteasome inhibitor MG132 (Fig. 4A and Supplementary Fig. S3A), suggesting a proteasome-dependent mechanism. Furthermore, PTEN depletion increased AR protein instability, and this effect was further enhanced by p300

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silencing in LAPC4 cells (Supplementary Fig. S3C and D). Neither PTEN nor p300 knockdown had any overt effect on AR mRNA levels in these cell lines (Supplementary Fig. S3E). These data indicate that p300 is a key regulator of proteasome-mediated AR degradation in both PTENproficient and -deficient PCa cells.

P300 inhibits AR polyubiquitination via its acetyltransferase activity Next we sought to determine whether p300 regulates AR protein polyubiquitination. PTEN knockdown increased AR polyubiquitination in LAPC4 cells (Fig. 4B), which is consistent with the finding that AR polyubiquitination is regulated by AKT (30). P300 knockdown alone also increased AR polyubiquitination, and this effect was further enhanced by co-knockdown of PTEN (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 result could be due to the possibility that these lysine residues are ubiquitin-accepting sites or that blockage of acetylation of these sites affects AR ubiquitination through mechanisms other than site competition. To test the second possibility, the lysine residues were converted into glutamine (Q), which structurally mimics the acetylation state of lysine (31). Polyubiquitination on the K630/632/633Q mutant was further reduced compared to the K630/632/633A mutant, although the expression levels of these mutated proteins was comparable (Fig. 4C). Thus, these experiments identified AR acetylation at K630, 632 and 633 residues as an important inhibitory mechanism of AR polyubiquitination in PCa cells. Consistent with these observations, we further showed that ectopic expression of wild-type p300, but not the histone acetyltransferase (HAT)-deficient mutant (p300ΔHAT), diminished AR polyubiquitination (Fig. 4D). In contrast, overexpression

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of p300 had little or no effect on polyubiquitination of the acetylation-resistant K630/632/633A mutant of AR (Fig. 4D). We conclude that p300 inhibits AR polyubiquitination and that this effect is mediated by its acetyltransferase activity.

Serine 81 phosphorylation in AR is required for the p300-AR interaction Overexpression of active AKT triggers AR degradation (30). As expected, PTEN knockdown by siRNAs downregulated AR protein in LAPC4 cells and this was blocked by the AKT inhibitor LY294002 (Fig. 4E left). Similarly, AR degradation was inhibited by LY294002 in PTEN and p300 co-knockdown cells (Fig. 4E right). When two AKT phosphorylation sites in AR (serine 213 and 791) (30) were mutated to alanine, the steady state level of the S213,791A mutant was higher than the wild-type AR (AR-WT) in DU145 cells (Fig. 4F). However, these mutations failed to block AR degradation induced by co-knockdown of PTEN and p300 (Fig. 4F). These data suggest that the effect of p300 on AR degradation is AKT dependent but independent of AKT-mediated phosphorylation of AR at Ser213 and Ser791.

Besides the AKT pathway, AR proteasome degradation is also regulated by cyclin-dependent kinase 1 (CDK1), and this effect is attributed to CDK1 phosphorylation of AR at multiple serine/threonine sites including serine 81 (Ser81) (32). PTEN loss or AKT activation promotes cell cycle progression by increasing the activity of CDKs (33-36). We therefore examined the role of AR Ser81 phosphorylation in the regulation of p300 and PTEN deficiency-induced AR protein degradation. PTEN knockdown increased RB phosphorylation, an indicator of CDK activation and AR phosphorylation at Ser81 (Fig. 5A). Next, we examined whether AR Ser81 phosphorylation affects the p300-AR interaction. The interaction between these two proteins

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was readily detected, but was largely attenuated by the S81A mutation (Fig. 5B). Accordingly, overexpression of cyclin B1 and CDK1 greatly enhanced p300 interaction with AR-WT but not the S81A mutant (Fig. 5C and Supplementary Fig. S4). Furthermore, p300-AR interaction was completely blocked by the pan CDK inhibitor roscovitine (Fig. 5D). We conclude that loss of PTEN increases 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 ubiquitination We further examined whether AR Ser81 phosphorylation affects the regulation of AR acetylation and polyubiquitination by p300. S81A mutation decreased AR acetylation (Fig. 5E, lane 1 versus 3). Consistent with CDK1/cyclin B1-enhanced interaction between p300 and AR (Fig. 5C), overexpression of CDK1 and cyclin B1 augmented acetylation of AR-WT but not the S81A mutant (Fig. 5E, lane 3 versus 4). Moreover, overexpression of CDK1 and cyclin B1 substantially decreased polyubiquitination of AR-WT, and this effect was partially reversed by p300 knockdown (Fig. 5F). Consistent with the result that acetylation of S81A was lower than AR-WT (Fig. 5E), polyubiquitination of S81A was greater than AR-WT (Fig. 5F). However, in contrast to the effect on AR-WT, ectopic expression of CDK1/cyclin B1 alone or in combination with p300 knockdown had a negligible effect on polyubiquitination of the S81A mutant (Fig. 5F). In agreement with these results (Fig. 5E and F) and the finding that loss of PTEN increases CDK1 phosphorylation of AR at Ser81 (Fig. 5A), we further showed that knockdown of CDK1 accelerated PTEN loss-induced decrease in AR protein level and that this effect was further enhanced by p300 knockdown (Fig. 5G). Thus, besides its role in mediating the interaction

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between p300 and AR, Ser81 phosphorylation is crucial for CDK1-enhanced AR acetylation and p300-mediated inhibition of AR polyubiquitination.

P300 is important for AR transcriptional activity in PTEN-null prostate tumors in mice To gain insight into the biological importance of p300 regulation of AR protein degradation in PTEN-deficient PCa cells, we sought to determine how p300 impacts AR activity by examining AR target gene expression. Consistent with the observation that p300 knockout induced downregulation of AR protein in PTEN-deleted PCa cells in mice (Fig. 2C and D), mRNA levels of AR-regulated genes, including probasin (Pb), Nkx3.1, and Mme, were significantly reduced in Ptenpc-/-;p300pc-/- prostates compared to Ptenpc-/- counterparts (Fig. 6A-C, upper panels). These results were confirmed by IHC (Fig. 6A-C, lower panels). In contrast, there was little 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 deletion does not result in a general downregulation of gene expression in PTEN-deleted PCa cells in vivo. To determine whether p300 exerts a similar effect on AR activity in human PTEN-deficient PCa cells, LAPC4 cells were transfected with PTEN siRNAs in combination with control or p300-specific siRNAs followed by treatment with vehicle or mibolerone, a synthetic androgen. P300 silencing (Supplementary Fig. S5B) markedly decreased the expression of AR target genes PSA, NKX3.1, HK2 and TMPRSS2 in both androgen-untreated and treated PTEN-knockdown cells (Fig. 6D), indicating that p300 is important for AR activation in the presence or absence of androgens in human PTEN-deficient PCa cells. However, no such effects were detected on the expression of p21WAF1, 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

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(Supplementary Fig. S3B), among the transcription factors examined AR activity appears to be mostly affected by p300 depletion. Together, these results suggest that p300 is essential for the transcriptional activity of the AR in PTEN-deficient PCa cells in vitro and in vivo.

Discussion P300 has been implicated in human PCa proliferation and progression (13, 37, 38) although the direct in vivo evidence that p300 is causally involved in prostate oncogenesis is lacking. In the present study we revealed that prostate-specific homozygous deletion of p300 dramatically inhibits Pten-deficient PCa proliferation and progression and prolongs mouse survival. Thus, in the Pten knockout mouse model we demonstrate for the first time that p300 functions as a bona fide oncogenic promoter of PCa. The relevance of this observation is further substantiated by our findings that high expression of p300 correlates with AKT phosphorylation, a surrogate of PTEN inactivation, in a cohort of human prostate tumors examined (Supplementary Fig. S6). It is worth noting that our present study cannot rule out the possibility that p300 is also critical for prostate tumorigenesis induced by other oncogenic factors such as RAS and Myc. It is hence important to address this issue in the future using transgenic mouse models.

P300 is a well-known acetyltransferase that catalyzes acetylation of both histone and non-histone proteins such as AR and p53 (12, 15, 39). It is generally accepted that p300 is a generic transcription coactivator. Moreover, it has been shown that p300 is targeted by oncogenic viral proteins such as E1A and is often mutated or truncated in endometrial, breast and pancreatic cancer (40), implying that p300 functions as a classical tumor suppressor. However, in contrasting with this concept, different studies invariably demonstrate that p300 is overexpressed

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in human prostate cancers (13, 14). These observations in clinical specimens and our findings in the mouse model clearly support the hypothesis that p300 functions as an oncogene in the prostate. Therefore, our findings as to how p300 functions paradoxically as an oncogene in the prostate are of importance from both basic science and translational perspectives.

Development, differentiation and growth of the prostate rely on androgens and androgen activation of the AR. The same is true for PCa growth and progression. Because of the androgen/AR addiction of prostate cells, ADT is the mainstay of treatment for advanced PCa in the clinic. It is known that p300 promotes AR transactivation by inducing acetylation of AR and histone proteins (15). In the current report we have discovered another important layer of p300 regulation of the AR by demonstrating for the first time that p300 plays an essential role in regulation of AR protein degradation. First, we provide evidence that homozygous deletion of p300 decreases AR protein levels in the mouse prostate. Second, we show that expression of p300 correlates positively with AR protein levels in a cohort of human PCa specimens. Third, using human PCa cell lines as a working model, we demonstrate that p300 inhibits AR protein polyubiquitination and proteasome degradation by inducing AR acetylation at lysine residues 630, 632 and 633. Importantly, the effect of p300 on AR degradation appears to be AR-specific since p300 depletion had no overt effect on other cancer-relevant transcription factors examined such as p53, E2F1, ERG and SP1. Consistent with the essential role of AR in PCa proliferation and growth, we further show that homozygous deletion of p300 significantly decreases PCa cell proliferation in vivo. Together, our findings imply that the oncogenic function of p300 in the prostate could be linked, at least in part, to the role of p300 in regulation of AR protein levels, which is highly relevant to prostate tumorigenesis and progression.

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AR protein stability is known to be regulated by CDK1 in PCa cells, and this effect is likely mediated by CDK1 phosphorylation of multiple serine/threonine residues in the AR protein including the major phosphorylation site Ser81 (32). However, the precise mechanism by which Ser81 phosphorylation influences AR protein stability is unclear. Our data demonstrate that loss of PTEN increases AR phosphorylation at Ser81 and that Ser81 phosphorylation acts as a molecular beacon that is required for the binding of p300, a key event that subsequently leads to AR acetylation, inhibition of AR ubiquitination and AR stabilization (Fig. 6E). Thus, we identify p300 as a key molecular determinant of AR degradation in PTEN-mutated PCa cells. In addition to its role in regulation of AR stability, Ser81 phosphorylation is important for AR transactivation (41, 42). In the present study, we reveal for the first time that Ser81 phosphorylation is essential for p300-AR interaction and AR acetylation. Given that p300 is a key transcriptional coactivator, beside its role in mediating chromatin binding (42), enhanced p300-AR interaction represents a novel mechanism of action of Ser81 phosphorylation in AR transactivation (Fig. 6E). Another important function of AR Ser81 phosphorylation is its role in regulation of PCa cell growth (41). Consistent with these findings, we provide evidence that p300 is important for PCa cell growth in mice. Thus, it can be speculated that Ser81 phosphorylation may promote PCa cell growth via mechanisms such as increased interaction between AR and p300 and subsequent AR activation (Fig. 6E).

The findings that p300 acetylation of AR is important for AR stabilization and transactivation and PCa cell growth and survival (Fig. 6E) (13, 16, 19, 26, 43, 44) imply that p300 could be a potential target for PCa therapy. P300 is a bromodomain protein, and it could potentially be

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targeted for cancer treatment because inhibition of other bromodomain proteins can effectively block tumor growth (45, 46). A virtual screen for inhibitors of p300 and CBP, a homolog protein of p300, has been conducted, and a selective small molecule inhibitor C646 has been identified. The anti-tumor effect of C646 was demonstrated in human PCa cells cultured in vitro (26). However, the in vivo application of C646 is unlikely since it can be inhibited by serum (26). Interestingly, curcumin (diferuloylmethane), a phenolic compound derived from the root of the plant Curcuma longa, and procyanidin B3, a compound extracted from grape seeds, have been demonstrated as natural anti-p300 substances (47, 48). Indeed, treatment of the liposome form of curcumin not only inhibits murine Pten knockout PCa progression, but also induces downregulation of AR protein (49), which is highly consistent with our findings in p300-Pten double knockout mice. Procyanidin B3 treatment also inhibits PCa cell proliferation by mitigating p300-dependent acetylation of AR (48). Hence, natural p300 inhibitors are strong candidates for pharmacological intervention of p300 function in cancers such as PCa, although the specificity of these compounds in inhibiting p300 is an open question since numerous targets of dietary compounds have been reported. Thus, while p300 is an attractive anti-PCa target, novel strategies are needed to specifically abolish the oncogenic function of p300 in PCa. In this report we demonstrate that p300 favors PTEN-deficient PCa progression by protecting AR protein from degradation. Because this role of p300 depends on the interaction between p300 and AR, its blockage of AR degradation can be overcome by disrupting their physical interaction without disturbing p300 regulation of other signaling pathways. Our finding therefore opens a unique avenue for development of novel therapeutics for PCa via specific elimination of p300mediated protection of AR proteins in PCa, especially those without a functional PTEN.

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Figure Legends Figure 1. P300 deletion inhibits PTEN-deficient prostate tumorigenesis in mice. A, IHC for Pten (i), phosphorylated Akt (Akt-S473-p) (ii) and p300 (iii) in prostate sections of 4-month-old ‘wild-type’, Ptenpc-/-, p300pc-/-, p300pc-/-;Ptenpc-/- mice (n = 8/group). The inset shows a high magnification image of the representative (framed) area in each panel. Scar bar, 50 µm. Scare bar in inset, 10 µm. B, H&E analysis of ventral prostate (VP) of 4-month-old mice with indicated genotypes (n = 8/genotype). Scar bar, 50 µm. C, IHC for smooth muscle actin (SMA) in the prostate of Ptenpc-/- and p300pc-/-;Ptenpc-/- mice. Arrows: disrupted and absent smooth muscle stroma in the Ptenpc-/- mouse. Scar bar, 50 µm. D, quantification of nonmalignant, lowgrade PIN (LGPIN) and high-grade PIN (HGPIN) or cancerous (with areas suggestive of microinvasion) acini in the prostates (including AP, VP and DLP) of mice with the indicated genotypes (n = 8/genotype). E, representatives of genitourinary tracts of Ptenpc-/- and p300pc-/;Ptenpc-/- mice (n = 6/genotype) that survived to 8 months. B, bladder; AP, anterior prostate; VP, ventral prostate. Scar bar, 2 mm. F, Ptenpc-/- mice lacking p300 in the prostates tend to live longer. Survival was monitored for 12 months and is displayed using Kaplan-Meyer plots.

Figure 2. P300 knockout decreases cell proliferation and AR protein levels in PTEN-deleted prostate tumors in mice. A, IHC for Ki-67 in the prostates of 4-month-old mice with indicated genotypes (n = 3/group). The inset in each panel shows a representative (framed) area in high magnification. Scar bar, 50 µm. Scare bar in inset, 10 µm. B, quantitative data of Ki-67 staining shown in (A). Columns, mean values of Ki-67 positive cells among three individual mice. Error bars, SD. *, p < 0.01. C, IHC of AR protein in the prostates of 4-month-old mice with indicated

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genotypes (n = 3/group). Scar bar, 50 µm. D, western blot analysis of indicated proteins in cell lysates prepared from the prostates of three mice in each group at 4 months of age.

Figure 3. IHC of p300 and AR proteins in human PCa specimens. A-C, representative images of H&E staining (A), p300 (B) and AR IHC (C) in prostate cancers exhibiting high and low expression of p300 and AR proteins. Scale bar, 100 µm. Insets show high magnification images of framed areas, scale bar, 50 µm. D, scoring data of the staining index for expression of p300 and AR proteins in human PCa specimens examined.

Figure 4. P300 regulates AR ubiquitination and proteasome degradation in PCa cells. A, p300 knockdown induces AR proteasome degradation. LAPC4 cells were transfected as indicated. 64 h later, cells were treated with vehicle or MG132 (20 uM) for 8 h followed by western blot (WB) analysis. B, LAPC4 cells were transfected with indicated siRNAs for 72 h and then treated with MG132 for 8 h followed by IP and WB analyses. C, AR-negative PC-3 cells were transfected with wild-type AR (AR-WT) and mutants (K630,632,633A and K630,632,633Q) in combination with HA-tagged Ub for 16 h, followed by treatment with MG132 (20 uM ) for 8 h. Cell lysates were subjected to IP and WB as in (B). D, PC-3 cells were transfected with the plasmids as indicated for 16 h, followed by treatment with MG132 (20 uM ) for 8 h. Cell lysates were subjected to IP and WB as in (B). E, effect of AKT on p300 and PTEN regulation of AR degradation. LAPC-4 cells were transfected with indicated siRNAs for 72 h, followed by treatment of LY294002 for 12 h and WB analysis. F, effect of AKT phosphorylation of AR at Ser213 and Ser791 on p300 and PTEN regulation of AR degradation. DU145 PCa cells were transfected with plasmids and siRNAs as indicated for 48 h followed by WB analysis.

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Figure 5. AR Ser81 phosphorylation by CDK1 is crucial for p300-AR interaction, AR acetylation and p300 regulation of AR ubiquitination. A, LAPC4 cells were transfected with indicated siRNAs for 72 h followed by WB analysis. B, 293T cells were transfected with wildtype and S81A mutated AR for 24 h followed by treatment with 10 nM mibolerone (Mib) for additional 24 h. Cell lysates were subjected to IP and WB. C, 293T cells were transfected with AR-WT and S81A mutant in combination with or without CDK1 and cyclin B1 for 24 h followed by treatment with 10 nM Mib for additional 24 h. Cell lysates were subjected to IP and WB. D, 293T cells were transfected with the indicated plasmids for 24 h followed by treatment with 10 nM Mib in combination with or without 10 µM roscovitine for 24 h. Cell lysates were subjected to IP and WB. E, 293T cells were transfected with the indicated plasmids for 24 h and treated with 10 nM Mib for 24 h. Cell lysates were subjected to IP and WB analysis. F, 293T cells were transfected with the plasmids and siRNAs as indicated for 24 h and treated 10 nM Mib for 24 h. Cell lysates were subjected to IP and WB. G, LAPC4 cells were transfected with indicated siRNAs for 72 h followed by WB analysis.

Figure 6. P300 depletion impairs AR target gene expression in PTEN-deficiency PCa cells. AC, RT-qPCR (upper panels) and IHC (lower panels) analyses of expression of AR target genes Pb (A), Nkx3.1 (B) and Mme (C) in prostate tissues from mice (n = 3) with the indicated genotypes at 4 months of age. Columns, mean values among three replicates; error bars, SD. *, p < 0.01. Lower panels: representative IHC staining of Pb (cytoplasmic and secreted out of cells), Nkx3.1 (cytoplasmic) and Mme (plasma membrane). D, LAPC4 cells were transfected as indicated for 48 h and treated with 10 nM Mib for 24 h. Expression of indicated genes was

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analyzed by RT-qPCR. Columns, mean values among three replicates; error bars, SD. *, p < 0.01. E, A diagram depicts the role of p300 and CDK phosphorylation of AR at Ser81 in regulation of AR stabilization and function by inhibiting PTEN loss or AKT activation-induced AR ubiquitination. Ac with circle, acetylation. P with circle, phosphorylation. Ub, ubiquitin.

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mTOR pathways in prostate cancer.

Androgen receptor DNA methylation regulates the timing and androgen sensitivity of mouse prostate ductal development.

Acetylation of androgen receptor by ARD1 promotes dissociation from HSP90 complex and prostate tumorigenesis.

Hdac3 Interaction with p300 Histone Acetyltransferase Regulates the Oligodendrocyte and Astrocyte Lineage Fate Switch.

Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor.

b Enhances Proteasomal Degradation of Androgen Receptor Liganded by Antiandrogens in Prostate Cancer.

FOXA1 regulates androgen receptor variant activity in models of castrate-resistant prostate cancer.

Androgen receptor antagonists for prostate cancer therapy.

fibronectin.

Chrebp regulates the transcriptional activity of androgen receptor in prostate cancer.

Erratum: Stromal androgen receptor regulates the composition of the microenvironment to influence prostate cancer outcome.

The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells.

Androgen receptor signaling in prostate cancer.

PARP-1 regulates epithelial-mesenchymal transition (EMT) in prostate tumorigenesis.

Targeting the androgen receptor in prostate cancer.

Vitamin D3 regulates the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

Metformin represses androgen-dependent and androgen-independent prostate cancers by targeting androgen receptor.

G protein-coupled estrogen receptor regulates mammary tumorigenesis and metastasis.

Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation.

NQO1 suppresses NF-κB-p300 interaction to regulate inflammatory mediators associated with prostate tumorigenesis.

Androgen receptor mutations and polymorphisms in African American prostate cancer.

The histone acetyltransferase p300 regulates the expression of pluripotency factors and odontogenic differentiation of human dental pulp cells.

Stromal androgen receptor in prostate development and cancer.

BAP18 coactivates androgen receptor action and promotes prostate cancer progression.