PADI2-Mediated Citrullination Promotes Prostate Cancer

Published OnlineFirst August 17, 2017; DOI: 10.1158/0008-5472.CAN-17-0150 Cancer Molecular and Cellular Pathobiology Research

PADI2-Mediated Citrullination Promotes Prostate Cancer Progression Lin Wang1, Guanhua Song2, Xiang Zhang3, Tingting Feng4, Jihong Pan1, Weiwen Chen5, Muyi Yang6, Xinnuo Bai7, Yu Pang8, Jindan Yu9, Jinxiang Han1, and Bo Han4,10

Abstract

Onset of castration-resistance prostate cancer (CRPC) after histone H3 amino acid residue R26. In contrast, mutant PADI2 long-term androgen deprivation therapy remains a major obstacle D180A failed to affect AR stability, nuclear translocation, or in the treatment of prostate cancer. The peptidylarginine deimi- transcriptional activity. PADI2 mediated AR control in a manner nase PADI2 has been implicated in chronic inﬂammatory diseases dependent on its enzymatic activity and nuclear localization, as and cancer. Here we show that PADI2 is an androgen-repressed correlated with increased histone H3 citrullination. Notably, gene and is upregulated in CRPC. PADI2 expression was required coadministration of the PADI inhibitor Cl-Amidine and the AR for survival and cell-cycle progression of prostate cancer cells, and signaling inhibitor enzalutamide synergized in inhibiting CRPC PADI2 promoted proliferation of prostate cancer cells under cell proliferation in vitro and tumor growth in vivo. Overall, our androgen-deprived or castration conditions in vitro and in vivo. results establish PADI2 as a key mediator for AR in prostate Cytoplasmic PADI2 protected the androgen receptor (AR) against cancer progression, especially CRPC, and they suggest PADI as proteasome-mediated degradation and facilitated AR binding to novel therapeutic targets in this disease setting. Cancer Res; 77(21); its target genes after nuclear translocation and citrullination of 1–14. 2017 AACR.

Introduction of response is less than one year (2). Therefore, identification of key molecules to modulate AR and development of novel strat- A major clinical hurdle for the management of patients with egies to target CRPC are urgently needed. advanced prostate cancer is the onset of castration-resistant pros- Dysregulation of posttranslational modification (PTM), tate cancer (CRPC; ref. 1). Androgen receptor (AR) signaling including methylation, acetylation, phosphorylation, ubiquityla- remains active and contributes greatly during CRPC progression tion, and sumoylation, might influence cancer progression (3–6). (2). Despite recent hormonal therapies showing a survival advan- The peptidylarginine deiminases (PADI) is a family of PTMs tage for abiraterone acetate (17a-hydroxylase/C17, 20-lyase enzymes that converts arginine into citrulline, namely citrullina- inhibitor) and enzalutamide (AR inhibitor), the median duration tion or, alternatively, deamination (7). Both PADI2 and PADI4 are key members of the PADI family and each shows a relatively distinct pattern of substrate specificity and tissue distribution (8). 1 Research Center for Medicinal Biotechnology, Key Laboratory for Rare and Histone modifications and the enzymatic machineries are crucial Uncommon Diseases of Shandong Province, Shandong Academy of Medical regulators that control cellular proliferation, differentiation, and Sciences, Jinan, China. 2Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, China. 3Department of Urological Surgery, Shandong malignancy processes (3). Importantly, PADI4, as well as PADI2, University Qilu Hospital, Jinan, China. 4Department of Pathology, Shandong is able to alter gene transcription through citrullinating residues in University Medical School, Jinan, China. 5Department of Biochemistry, Shan- histone tails (8). To date, PADI-mediated citrullination has been dong University Medical School, Jinan, China. 6Department of Oncology and proven to be involved in various inflammatory conditions, such Pathology, Karolinska Institute, Stockholm, Sweden. 7Human Biology Depart- 8 as rheumatoid arthritis (9), multiple sclerosis (10), psoriasis (11), ment, University of Toronto, Toronto, Canada. Department of Pathology, Taian chronic obstructive pulmonary disease (12), as well as various City Central Hospital, Taian, China. 9Division of Hematology/Oncology, Depart- ment of Medicine, Northwestern University Feinberg School of Medicine, Chi- cancers (8). Inhibitors targeting PADIs have been investigated in cago, Illinois. 10Department of Pathology, Shandong University Qilu Hospital, the preclinical setting. Jinan, China. PADI2 is expressed in multiple organs (13) and most recent Note: Supplementary data for this article are available at Cancer Research studies suggested a link with cancer progression. McElwee and Online (http://cancerres.aacrjournals.org/). colleagues demonstrated that PADI2 expression increases during Corresponding Authors: Bo Han, Department of Pathology, Shandong Univer- the transition from benign mammary epithelium to malignant sity Medical School, #44, Wenhua Xi Rd, Jinan 250012, P.R. China. Phone: 531- breast carcinomas (14). In another report, overexpressed PADI2 8216-9225; Fax: 531-8295-1586; E-mail: [email protected]; and Jinxiang Han, in transgenic mice promotes spontaneous skin neoplasia by Shandong Medical Biotechnological Center, Shandong Academy of Medical enhancing inflammation within the tumor microenvironment Sciences, #18877, Jingshi Rd, Jinan 250062, P.R. China. Phone: 531-8262- (15). In addition, Cantarino and colleagues found that down- 9328; Fax:-531-8291-9647; E-mail: [email protected] regulation of PADI2 is an early event in the pathogenesis of doi: 10.1158/0008-5472.CAN-17-0150 colorectal cancer and associated with poor prognosis (16), indi- 2017 American Association for Cancer Research. cating a tissue-specific role of PADI2 in tumor progression. So far,

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the identified positions of arginine residues include H3R2, H3R8, and authenticated again by short tandem repeat (STR) analysis H3R17, and H3R26 (17). PADI2 has been suggested to be again before and after our study. The cumulative culture length of involved in multiple myeloma and breast cancer by citrullinating the cells between thawing and use in this study was less than 15 histone tails at transcription factor–binding sites, ultimately passages. All of the newly revived cells were tested free of myco- affecting transcription (18–20). Notably, PADI2 expression is plasma contamination by Hoechst 33258 staining (Beyotime). responsive to extracellular stimuli, such as estrogen and EGF For the androgen treatment, cells were hormone-starved for 2 days (14), indicating its dynamic activity in human disease. In this in the media containing 10% charcoal-stripped FBS and treated study, we found that PADI2 expression in prostate cancer cells with R1881for expression assay or ChIP assays. Cl-Amidine (an could be induced by androgen deprivation treatment. More arginine-based PAD inhibitor) was used at a concentration of 100 importantly, PADI2-mediated citrullination activates AR signal- and 200 mmol/L as published previously (14). ing and promotes CRPC progression. Our data supported the potential of PADI as novel therapeutic targets to treat prostate MTS, BrdUrd cell proliferation assay, and flow cytometry cancer patients with CRPC. analysis Cell growth and proliferation were measured using the MTS Patients and Methods and BrdUrd incorporation assay as described previously (22). For Patients cell-cycle analysis, cells were treated with 5 mg/mL of aphidicolin A total of 256 prostate cancer patients participated in our study. (Sigma Aldrich) for additional 24 hours. After fixation and stain- The tumor samples were obtained from Qilu Hospital of Shan- ing with propidium iodide (Sigma Aldrich), the samples were dong University (Jinan, China) and The Affiliated Hospital of analyzed by flow cytometry (FACSCalibur, BD Biosciences). Qingdao University (Qingdao, China) between 2003 and 2007. The first cohort consisted of 229 men with localized prostate Quantitative real time-PCR cancers who have undergone radical prostatectomy. None of the Total RNA was prepared from prostate cancer cells and was patients received preoperative radiation or ADT. The second reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo). cohort included patients with CRPC treated by transurethral qRT-PCR was performed with diluted cDNA using SYBR Green resection of the prostate to relieve symptomatic obstruction due PCR Master Mix (Toyobo) and gene-specific primers (23). The to locally advanced disease (n ¼ 27). The initial treatment for sequences of all primers were listed in Supplementary Table S1. patients was either observation or surgery. Development CRPC fl was treated by utamide or bicalutamide. This study was con- ChIP-qPCR analysis ducted in accordance with the International Ethical Guidelines for Chromatin immunoprecipitation (ChIP) was carried out as Biomedical Research Involving Human Subjects (CIOMS). The described previously (22). ChIP-qPCR enrichment of target study protocol was approved by the Institutional Review Board of loci was normalized to input DNA and all primers were listed Medicine School of Shandong University. The informed written in Supplementary Table S1. Anti-histone H3 (citrulline consent was obtained from the patients. R2þR8þR17), anti-histone H3 (citrulline R26), anti-histone IHC and immunofluorescence H4 (citrulline R3), and anti-histone H3 (trimethyl K4) were IHC and the scoring system were described as reported previ- supplied from Abcam. Anti-histone H3 (acelytated R9), anti- ously (21). Briefly, the slides were incubated overnight with anti- histone H3 (trimethyl K27), and irrelevant antibody (IgG) were PADI2 (Proteintech) or anti-citrulline (Abcam) antibody and from Millipore then evaluated blindly by two independent observers (B. Han and T. Feng) who were blinded to the clinicopathologic data. The Plasmids and siRNAs scoring system of PADI2 and citrullinated protein was as follows: Human PADI2 cDNA encompassing different lengths (1–665 the nuclear and cytoplasmic PADI2 and citrullinated protein aa, 1–653 aa, and 1–437 aa) was amplified by reverse transcrip- staining was scored into four grades, which were classified by its tion PCR from LNCaP and cloned into pCR8/GW/TOPO (Invi- staining intensity: 0, 1þ,2þ, and 3þ. The percentages of PADI2 or trogen). The amino acid residue, Asp180, is located in the N- 2þ citrullinated protein–positive cells were scored into five catego- terminal Ca -binding site of PADI2 and is essential for catalysis ries: 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (24). The D180A mutants of PADI2 (PADI2-665mt, PADI2- (76%–100%). A final score was built by multiplying the scores of 653mt, and PADI2-437mt) were prepared with the QuikChange these two parameters, which were defined as follows: 0, negative; site-directed mutagenesis kit (Stratagene), as described previously 1–3, weak; 4–6, moderate; and 8–12, strong. For analysis, we (24). Lentivirus products were constructed in 293T cells by combined both negative and weakly IHC-positive tumors into cotransfection with psPAX2, pMD2.G, and lentiviral construct one group and compared it with moderately and strongly IHC- (pLentiCMV/TO Puro DEST) expressing PADI2 using Lipofecta- positive prostate cancers. The Ki67 labeling index was defined as mine 2000 (Invitrogen). Stable cells overexpressing PADI2 were described previously (21). For immunofluorescence, cells were selected using puromycin after transfection with lentivirus pro- sequentially probed with primary antibodies and fluorescence- ducts. siRNA targeting PADI2, PADI4, AR, and AllStar negative labeled secondary antibodies (Jackson Immunoresearch). Images control were bought from Qiagen and the sequences were pro- were captured under a confocal microscope (FV3000, Olympus). vided in Supplementary Table S2. The mock group was defined as that supplemented with transfection reagent only. The corre- Cell culture and treatment sponding DNA nucleotides encoding short hairpin RNA showing LNCaP and C4-2B cells were obtained from Dr. Jindan Yu the most silencing efficiency (PADI2 #2) were subcloned into (Northwestern University, Chicago, IL) in 2015. VCaP and pSUPER vector with neomycin-resistant gene. The transfected HEK293T were purchased from ATCC between 2012 and 2015 cells were subjected to resistant selection for one week.

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PADI2-Mediated Citrullination and CRPC

Western blot analysis and immunoprecipitation Results Western blot analysis was performed as described previously PADI2 is androgen-repressed and upregulated in CRPC (25). Band intensity underneath the gel image was measured Androgen–AR signaling is critical for prostate cancer (29). As using ImageJ software, presented as fold change compared PADI2 and PADI4 are involved in various diseases (7, 13–15, with control ones. For immunoprecipitation, total proteins were 18, 30), we first asked whether they are responsive to androgen. As precleared with protein-G sepharose (Invitrogen), which immu- shown in Fig. 1A, PADI2 decreased progressively in LNCaP and noprecipitated with anti-Flag (AR), anti-HA (ubiquitin, Ub), or VCaP cells at both mRNA (top) and protein (bottom) levels as the anti-AR. The nuclear proteins were extracted using Nuclear and dose of synthetic androgen R1881 increased (PSA, positive con- Cytoplasmic Extraction Reagents kits (Thermo Scientific). The trol; Supplementary Fig. S1A and S1B). Furthermore, the decrease antibodies used in this study included anti-AR (Abcam) and of the PADI2 mRNA levels was significant as early as 6 hours after anti-GAPDH (Santa Cruz Biotechnology). Anti-PADI2, anti-Lam- R1881 stimulation (Fig. 1A; Supplementary Fig. S1C and S1D). In inA/C, anti-MDM2, and anti-a-tubulin were from Proteintech. contrast, PADI4 was insensitive to R1881 (Supplementary Fig. S1E). Interestingly, R1881 inhibition of PADI2 was significantly Protein stability and degradation attenuated in LNCaP and VCaP cells after siAR or enzalutamide To assess the protein stability of AR, LNCaP cells were trans- treatment (Fig. 1B), indicating the downregulation of PADI2 by fected with siPADI2, vector expressing PADI2, or their respective R1881 is mediated by AR. Next, motif analysis and ChIP assay controls, and the above cells were then treated with cycloheximide identified a high-ranking androgen response element at PADI2 (Sigma) or MG132 for Western blot analysis using AR and promoter encompassing 395 to 380 bp in LNCaP cells (Fig. GAPDH antibodies. 1C). Further study revealed that R1881 stimulation induces more recruitment of AR at PADI2 promoter (positive control, PSA, Luciferase reporter gene assay FKBP5, and TMPRSS2; negative control, 30 end of PADI2 PSA-luciferase-plasmid, AR expression plasmid, and pRL-TK gene; Fig. 1D). To further support that androgen inhibits PADI2 Renilla plasmid were transfected into 293T cells using Lipofecta- through modulation of active transcription, ChIP-qPCR analysis mine 2000. Luciferase activities were measured using Dual-Lucif- observed a decrease in RNA Pol II occupancy on PADI2 and AR- erase Reporter Assay System (Promega). Luciferase activity was repressed gene (MET), but an increase on AR-induced genes (PSA normalized to the luciferase activity of Renilla of the cells. and TMPRSS2; Supplementary Fig. S1F). Because of its androgen-regulated nature, we hypothesized that Animal treatment PADI2 may be differentially expressed during prostate cancer The animal studies were approved by the Ethical Animal Care progression. Western blot analysis revealed that four prostate and Use Committee of Shandong University and the guidelines cancer cell lines exhibited variably higher PADI2 protein levels were strictly followed. For both prostate cancer progression and compared with that of benign RWPE cell line. PADI2 was dra- CRPC model (25), male athymic nude mice were injected sub- matically upregulated in CRPC cell line C4-2B and LNCaP-AI (Fig. cutaneously with 5 106 stably PADI2-overexpressing LNCaP 1E), the latter of which was established as CRPC model in our or PADI2-knocking down C4-2B (suspended in Matrigel; BD previous study (25). Notably, long-term androgen deprivation Biosciences). For treatment, mice were randomly selected to significantly induced PADI2 expression at both mRNA (Fig. 1F, 10 mg/kg enzalutamide daily plus either 10 mg/kg Cl-Amidine left) and protein levels (Fig. 1F, right) in LNCaP cells. In contrast, or vehicle once daily. Each experimental group consisted of androgen deprivation did not affect PADI4 expression (Supple- 10 mice. Tumor response measurement was conducted as mentary Fig. S1G). described previously (25). Animals were sacrificed after 7 weeks PADI2 expression was then analyzed among clinical speci- of treatment and all mice survived till the end of treatment. Data mens. Both cytoplasmic and nuclear PADI2 were present and points were expressed as mean tumor volume SD. representative IHC images were shown in Fig. 1G. Similar to PADI2, the level of citrullinated protein was higher in CRPC Microarray data acquisition and processing tissues than in the HD tissues and higher levels of PADI2 were We obtained raw Affymetrix Human Exon 1.0 ST Array expres- associated with elevated level of citrullinated protein (Fig. 1H; P ¼ 2 sion data and clinical information for Taylor and colleagues from 0.048, c test) in prostate cancer cases. The relationships between the NCBI Gene Expression Ominibus (GSE21032; ref. 26). Gene PADI2 expression level and clinicopathologic variables in HD expression was calculated as described previously (27). Briefly, cases were analyzed. Notably, PADI2 overexpression was signif- probe set normalization was performed by the Single Channel icantly associated with a high Gleason score (P ¼ 0.018), high T Array Normalization algorithm, which modeled and removed (tumor) stage (P ¼ 0.017), and Ki67 index (P ¼ 0.008). Using a probe- and array-specific background noise using data from each publicly available dataset, patients with high PADI2 mRNA array to individually normalize each sample (28). We used expression display an unfavorable biochemical relapse-free sur- Affymetrix Core level summaries for annotated genes to calculate vival (BRFS) and overall survival (OS) than those with low gene expressions. expression, even though this did not reach a statistical significance (Fig. 1I, P ¼ 0.1734 for BRFS and P ¼ 0.2980 for OS). Remarkably, Statistical analysis among HD cases, 160 (69.1%) showed negative or weak staining All results are expressed as the mean SD. Two-tailed tests, (40 cases: negative; 120 cases: weak), and only 69 (30.1%) had one-way ANOVA, or Wilcoxon matched pairs tests were used moderate or strong staining for PADI2 (30 cases: moderate; 39 for statistical analysis. The differences between tumor volumes cases: strong). However, 14 (51.9%) CRPC cases showed mod- were evaluated by the nonparametric Mann–Whitney–Wilcoxon erate to strong expression (6 cases: moderate; 8 cases: strong), test. P < 0.05 and P < 0.01 were considered significant. whereas 13 (48.1%) were negative or weak (2 cases: negative; 11

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A E 1.2 1.2

1 1 LNCaP LNCaP PADI2 0.8 VCaP 0.8 VCaP 0.6 0.6 GAPDH

0.4 0.4 0.2 VCaP C4-2B 0.2 RWPE-1LNCaPLNCaP-AI 0 F

0 expression PADI2 Relative Relative PADI2 expression PADI2 Relative Vehicle 0.01 0.1 1 10 nmol/L 061224 48 h 20 18 16 14 Androgen concentration Time after androgen stimulation 12 10 PADI2 PADI2 8 VCaP VCaP PADI2 GAPDH GAPDH 6 4 GAPDH PADI2 PADI2 2 30 6 Months LNCaP expression PADI2 Relative LNCaP 0 GAPDH GAPDH 0 3 6 Months Vehicle 0.01 0.1 1 10 nmol/L 0 6 12 24 48 h G B

1.4 LNCaP 1.4 VCaP G1 G2 1.2 1.2 1 1 0.8 0.8 0.6 0.6

0.4 0.4 G3 G4 0.2 0.2 Relative PADI2 expression PADI2 Relative Relative PADI2 expression PADI2 Relative 0 0 H EtOH + – + – + – + – EtOH + – + – + – + –

R1881 – ++––– ++ R1881 + –– + ––+ +

siCtrl ++ –– – ––– siCtrl ++ –– –––– siAR ––––– ++ – siAR ––++ –– –– H1 H3 Vehicle ––– –––++ Vehicle ––––++ –– Enz ––– ––– ++ Enz –––– –– ++

PADI2 PADI2 LNCaP LNCaP GAPDH GAPDH H2 H4 PADI2 PADI2 VCaP VCaP GAPDH GAPDH EtOH + – + – EtOH + – + – I J R1881 – + – + R1881 – + – +

siCtrl + – + – Vehicle + – + – PADI2_low 1 100 siAR – ––+ Enz – ––+ PADI2_high 0.8 80 0.6 CRPC 60 HD 0.4 free survival C D 0 50 100 150 0.04 Time Month 0.2

0.10 recurrence Biochemical 0 0.03 Negative and weak Moderate and strong AR expression PADI2 of different Percentage 100 0.02 EtOH IgG 0.05

% Input R1881 % Input 0.01 80 PADI2_low 60 PADI2_high 0.00 0

PADI2 PSA FKBP5 TMPRSS2 PD PADI2 PSA TMPRSS2 PD Overall survival J1 J2 0 50 100 150 Time Month

Figure 1. PADI2 is androgen responsive and upregulated in CRPC. A, The levels of PADI2 in LNCaP and VCaP cells were detected by qRT-PCR (top) and Western blot (bottom) analysis after stimulation with the synthetic androgen R1881 (1 nmol/L), with indicated doses of R1881 for 24 hours or for the indicated periods of time. , P < 0.05 versus vehicle or 0 hours. B, mRNA (top) and protein (bottom) levels of PADI2 were analyzed by qRT-PCR and Western blot analysis in LNCaP and VCaP cells after 1 nmol/L R1881 stimulation in the presence of siRNA targeting AR or enzalutamide (Enz; AR inhibitor). , P < 0.05. C, AR and IgG ChIP were performed in LNCaP cells and qPCR was carried out using primers ﬂanking the PADI2 promoter regions and well-established AR target gene enhancers (PSA, FKBP5, and TMPRSS2). The 30 end of the PADI2 gene was used as a negative control (PD). , P < 0.05. D, Recruitments of AR on the promoter of PADI2, or AR target genes (PSA and TMPRSS2), were analyzed by ChIP-qPCR. , P < 0.05. E, PADI2 expression was characterized by Western blot analysis in various prostate cancer cell lines. (Continued on the following page.)

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PADI2-Mediated Citrullination and CRPC

A 0.9 D 1.2 E 1.2 siPADI2#1 0.9 0.8 siPADI2#2 0.8 siPADI2#1 1 1 0.7 siPADI2#2 NC 0.7 0.6 NC 0.8 Mock 0.6 * * 0.8 * 0.5 Mock * siPADI2#1 * * * * Cl-amidine (100 µmol/L) 0.5 0.6 * siPADI2#2 0.6 Cl-amidine (200 µmol/L) 0.4 0.4 * NC 0.3 0.4 Vehicle * 0.3 * Mock 0.4 0.2 0.2

Cell viability (OD) 0.2 0.2 0.1 Cell viability (OD) 0.1 BrdUrd incorporation BrdUrd incorporation 0 0 0 0 LNCaP VCaP LNCaP 24 48 72 VCaP 24 48 72 h LNCaP VCaP * * * * F 100% 100% 90% 90% B 0.7 1.2 Cl-amidine (100 mmol/L) 80% 80% Cl-amidine (100 µmol/L) 70% 0.6 Cl-amidine (200 mmol/L) 70% 1 Cl-amidine (200 µmol/L) 60% Vehicle 60% 0.5 Vehicle 50% 50% 0.8 G0–G1 40% 40% 0.4 30% S

30% Cell cycle 0.6 Cell cycle * 20% 20% G2–M 0.3 * * 10% 10% 0.4 * 0.2 0% 0% VCaP LNCaP NC NC Cell viability (OD) Cell viability (OD) 0.2 NC 0.1 NC Mock Mock Mock Mock

siPADI2#1 siPADI2#1siPADI2#2 siPADI2#1siPADI2#2 0 0 siPADI2#2 siPADI2#1siPADI2#2 Aphidicolin Aphidicolin LNCaP 24 48 72 h VCaP 24 48 72 h Aphidicolin Aphidicolin withdrawal withdrawal * * C G 100% 100% 90% 90% 0.9 0.9 80% 80% * * 70% 70% 0.8 0.8 Vec Ctrl Vec Ctrl 60% 60% 0.7 0.7 PADI2 50% PADI2 * 50% G –G 0.6 0.6 40% 40% 0 1 30% S

Cell cycle 30% Cell cycle 0.5 0.5 20% 20% G2–M 0.4 0.4 10% 10% 0% 0.3 0.3 0% VCaP 0.2 0.2 LNCaP Vehicle Vehicle 0.1 0.1 Vehicle Vehicle CI-Amidine CI-Amidine Aphidicolin Aphidicolin 0 0 CI-AmidineAphidicolin CI-AmidineAphidicolin withdrawal withdrawal LNCaP 24 48 72 h VCaP 24 48 72 h H J 1,400

) ** 3 1,200 Vec Ctrl ** 5 I ** 1,000 PADI2 PADI2 ** 4 800 * 3 600 * 2 Vec Ctrl 400 1 200 Tumor weight (g)

Tumor volume (mm 0 0 PADI2 Vec Ctrl 01234567Week

Figure 2. PADI2 acts as an oncogene in prostate cancer progression. Effect of silencing PADI2 (A) or treatment with Cl-Amidine (B) on cell viability of LNCaP and VCaP cells as detected by MTS assay. , P < 0.05 versus negative control (NC) and mock (transfection reagent only) or vehicle. C, The cell viability was evaluated by MTS assay after overexpressing PADI2 in LNCaP cells. , P < 0.05 versus vector control (Vec Ctrl). BrdUrd incorporation assay was performed in LNCaP and VCaP cells after siPADI2 (D) or Cl-Amidine (E) treatment. , P < 0.05 versus negative control and mock, or vehicle. Cell-cycle distribution was detected in LNCaP and VCaP cells after siPADI2 (F) or Cl-Amidine (G) treatment in the presence or absence of aphidicolin. , P < 0.05. H, Male Balb/c athymic nude mice were injected subcutaneously with either 5 106 cells/100 mL of LNCaP-Vec Ctrl or LNCaP-PADI2 cells. Tumor volumes were measured at the indicated weeks. The tumor tissues were weighed at the time point of 7 weeks (I) and are depicted as mean value (J). The number of animal models in each group was 10. , P < 0.05; , P < 0.01 versus vector control in I and J. Two different PADI2 siRNAs, designated as siPADI2 #1 and siPADI2 #2, were used to derive data. cases: weak). Overall, CRPC specimen showed significantly stron- PADI2 functions as an oncogene in prostate cancer progression ger expression of PADI2 than HD samples (Fig. 1J, P ¼ 0.034). We next applied in vitro functional assays to characterize PADI2 staining in HD and CRPC was shown in Fig 1J, 1-2. These PADI2 in prostate cancer. Supplementary Figure S1H illustrates data indicated potential role of PADI2 in prostate cancer progres- two independent siRNA constructs that silence PADI2 expression and CRPC. sion efficiently. Inhibition of PADI2 expression significantly

(Continued.) F, qRT-PCR (left) and Western blot (right) analysis were performed to detect PADI2 expression in LNCaP cells after androgen deprivation treatment (ADT) in charcoal-stripped medium for the indicated time periods. , P < 0.05; , P < 0.01 versus 0 months. G, Representative IHC staining of PADI2 protein in Chinese prostate cancer patients. G1, Negative staining, 200; G2, weak staining, 200; G3, moderate staining, 200; and G4, strong staining, 200. Insets with red boxes show corresponding IHC images with higher magniﬁcation, 400. H, Representative IHC images of both PADI2 and citrullinated protein in prostate cancer. One prostate cancer case with both low expression of PADI2 and citrullinated protein is illustrated in H1 and H3. H1, IHC staining of PADI2; H3, IHC staining of citrullinated protein. One prostate cancer case with both high expression of PADI2 and citrullinated protein is illustrated in H2 and H4. H2, IHC staining of PADI2; H4, IHC staining of citrullinated protein. I, Kaplan–Meier analysis of prostate cancer outcome (BRFS and OS) was compared in a publicly available dataset (GSE21032). PADI2 mRNA expression was represented as fragments per kilobase million. The top and bottom 5% of samples were excluded to minimize the detecting errors. According to the PADI2 expression levels, we arbitrarily deﬁned the top 35% as the PADI2-high group and the bottom 35% as the PADI2-low group. The P values for Kaplan–Meier curves were determined using a log-rank test. J, Percentages of different PADI2 expression levels in hormone dependent (HD; J1) and androgen-independent (AI; J2) tumors. , P < 0.05. Percentage of moderate or strong PADI2 expression in CRPC versus hormone dependent. Results shown in C and D are representatives of at least two independent experiments and A, B, E,and F were duplicated by at least three independent experiments.

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suppressed the viability of LNCaP and VCaP cells either by immunofluorescence analysis in LNCaP (Fig. 3J). In contrast, siRNAs (Fig. 2A) or Cl-Amidine (Fig. 2B). In contrast, over- overexpressed PADI2 in LNCaP and VCaP cells could facilitate expressing PADI2 enhanced the cell viability (Fig. 2C). How- the nuclear translocation of AR with R1881 stimulation, and these ever, no significant alteration of cell viability was identified effects could be antagonized after transfection with its mutant in PADI4-silenced prostate cancer cells (Supplementary type (PADI2-665mt; Supplementary Fig. S2D). Fig. S1I–J). BrdUrd incorporation assay further revealed that DNA synthesis was significantly suppressed in PADI2-silenced PADI2-mediated citrullination in the nucleus activates AR (Fig. 2D) or Cl-Amidine–treated (Fig. 2E) prostate cancer cells. signaling In addition, cell-cycle distribution analysis demonstrated that As shown in Fig. 4A, PADI2 overexpression could recruit more silencing PADI2 (Fig. 2F) or Cl-Amidine treatment (Fig. 2G) AR to the promoter of PSA and TMPRSS2 in LNCaP and VCaP couldleadtoasignificant accumulation in the S-phase of cells. In contrast, siPADI2 (Fig. 4B) or Cl-Amidine treatment (200 LNCaP and VCaP cells after withdrawal of aphidicolin. More mmol/L; Fig. 4C) inhibited its occupancy. These findings were then importantly, in vivo experiments demonstrated that the growth confirmed by measuring the changes of MET gene (Fig. 4A–C). rate and the mean weight of LNCaP-PADI2–derived primary Next, the ChIP-qPCR of RNA pol II revealed a decrease of its tumors is much higher than that of LNCaP-Vec Ctrl–derived occupancy on AR-induced PSA gene (Supplementary Fig. S2E and tumors (133.9 mm3/week vs. 64.1 mm3/week;Fig.2H–J). S2F), but an increase of MET gene (Supplementary Fig. S2G and Collectively, our data suggested that PADI2 is required for the S2H). As illustrated in Fig. 4D and E, the induction of PSA, KLK2, growth of prostate cancer cells. and TMPRSS2 caused by androgen could be significantly attenuated when treated with siPADI2 or Cl-Amidine. Notably, the PADI2 stabilizes AR protein and facilitates its nuclear induction of AR target genes caused by PADI2 overexpression translocation could be completely abolished by siAR or enzalutamide treatment As both PADI2 and AR were upregulated in late-stage (Fig. 4F). prostate cancer (2), we next sought to determine whether Our data revealed that the shuttle of PADI2 from cytoplasm to PADI2 might regulate AR expression. Western blot analysis nucleus could be facilitated with the stimulation of androgen (Fig. showed that siPADI2 (Fig. 3A, top) or Cl-Amidine treatment 4G), suggesting that nuclear translocation may have functional (200 mmol/L; Fig. 3A, bottom) in LNCaP and VCaP could importance for PADI2. Previous study has shown that PADI2- suppress the AR protein level, and the opposite pattern was mediated citrullination of H3R26 facilitated transcriptional acti- observed after overexpressing PADI2 (Fig. 3B). However, no vation induced by estrogen (19). Using confocal immunofluo- visiblechangeofARmRNAwasidentified (Supplementary rescent analysis, we demonstrated a strong colocalization Fig. S2A and S2B). To test whether PADI2 modulated AR between AR and citrullination of H3R26 in LNCaP (Fig. 4H) and expression at posttranscriptional level, we treated LNCaP with VCaP (Fig. 4I) cells with and without R1881 treatment. Next, cycloheximide, a protein biosynthesis inhibitor. Western blot ChIP-qPCR was performed to validate the citrullination of analysis showed that siPADI2 or Cl-Amidine treatment H3R26 of AR target genes, including PSA. Importantly, siPADI2 (200 mmol/L) remarkably accelerated the degradation of AR (Fig. 4J) or Cl-Amidine treatment (Fig. 4K) led to a significant protein (Fig. 3C and D). In contrast, PADI2 overexpression in decrease of citrullinated H3R26, methylated H3K4, and acetylat- LNCaP and VCaP suppressed the degradation of AR protein, ed H3K9, but a concomitant increase of H3K27 methylation. In an which could be blocked by inhibiting its citrullinating activity attempt of validating the specificity of H3R26 citrullination, two after transfection with its mutant type (PADI2-665mt; Sup- other site-specific anti-citrullinated histone antibodies, anti- plementary Fig. S2C). Furthermore, immunoblotting analysis H3Cit2/8/17 and anti-H4Cit3 were also applied. As demonstrat- showed that ectopic expression of PADI2 failed to increase the ed in Supplementary Fig. S3A and S3B, no specific changes protein level of AR in the presence of proteasome inhibitor of H3Cit2/8/17 or H4Cit3 were recognized in the presence of MG132 (Fig. 3E). These findings suggested that PADI2- siPADI2 or Cl-Amidine. facilitated stabilization of AR protein depends on protea- As PADI2 may modulate AR both in cytoplasm and nucleus some-mediated AR degradation. of prostate cancer cells, we next examined which protein Previous studies have shown that AR formed a heterodimer sequences of PADI2 are important for its subcellular localiza- complex with MDM2 (Proto-Oncogene) to protect ligand- tion. NoD software (http://www.compbio.dundee.ac.uk/www- unbound AR from degradation by the ubiquitin–proteasome nod/) identified a 12 aa residing in the C-terminal of PAID2 system (31). In this study, we demonstrated that silencing PADI2 with relatively higher NLS score and truncated PADI2 vector or Cl-Amidine treatment (200 mmol/L) significantly disrupted with deletion of the 12 aa sequence was constructed (PADI2- the association between AR and MDM2 by Western blot analysis 653wt). Besides, the full-length FLAG-tagged PADI2 and a (Fig. 3F). We then performed cotransfection of FLAG-AR, HA- truncated PADI2 vector encoding 437 aa (PADI2-437wt) were ubiquitin, and PADI2 into 293T cells to determine whether PADI2 also prepared (Supplementary Fig. S4A). As demonstrated in affected AR ubiquitination. Our results showed that the presence Supplementary Fig. S4B, PADI2-653wt still could be detected of ubiquitin led to AR protein ubiquitination, which was in the nucleus, even though it could promote AR protein enhanced by suppressing PADI2 expression or activity stability (Supplementary Fig. S4C) and nuclear translocation (Fig. 3G). Concordantly, reverse coimmunoprecipitation assay (Supplementary Fig. S4D) in LNCaP and VCaP. Consistent showed that PADI2 prevents AR from binding to ubiquitin with previous study, PADI2-437wt was sequestered only in the proteins (Fig. 3H). Importantly, Western blot analysis revealed nucleus of LNCaP and VCaP (Supplementary Fig. S4B). that nuclear translocation of AR could be significantly inhibited in Although it failed to regulate the protein stability (Supple- LNCaP and VCaP after transfection with siPADI2 or Cl-Amidine mentary Fig. S4E) and nuclear translocation (Supplementary treatment (Fig. 3I). The above effect was further confirmed by Fig. S4F) of AR, robust of citrullination of H3R26 could be

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PADI2-Mediated Citrullination and CRPC

µ A LNCaP VCaP B C CHX (10 g/mL) Timepoints 0 15 30 60 0 15 30 60 min AR LNCaP VCaP AR GAPDH 1 0.68 0.53 0.45 0.92 0.46 0.12 0.08 LNCaP − − AR siCtrl + + GAPDH siPADI2 − + − + GAPDH siCtrl + +++ −−−− Vec Ctrl ++− − −−−− LNCaP VCaP siPADI2 ++++ − − AR PADI2 + + AR GAPDH 1 0.93 0.87 0.81 0.93 0.71 0.43 0.37 VCaP − − Vehicle + + GAPDH Cl-Amidine − + − + siCtrl ++++ − − − − siPADI2 −−−− D CHX (10 µg/mL) ++ ++

Timepoints 0 15 30 60 0 15 30 60 min E Vec Ctrl PADI2 F AR MG132 − + − + IP:AR IP:AR 1 0.91 0.61 0.53 0.95 0.83 0.32 0.17 AR VCaP siPADI2 − + Cl-Amidine − + GAPDH 1 3.31 3.28 3.33 LNCaP AR AR Vehicle ++++ − −−− GAPDH Cl-Amidine −−−−++++ MDM2 MDM2 AR AR Input Input LNCaP 1 3.57 2.79 3.61 VCaP 1 0.92 0.87 0.81 0.98 0.72 0.37 0.11 GAPDH GAPDH

Vehicle ++++ −−−− Cl-Amidine −−− − ++++

G Flag-AR + + + Flag-AR + + + H Flag-AR + + + Flag-AR + + +

HA-Ub − + + HA-Ub − + + HA-Ub − + + HA-Ub − + +

siPADI2 −− + Cl-Amidine −− + siPADI2 −− + Cl-Amidine −− +

IP:HA (Ub) IP:HA (Ub) IP:HA IP:HA

IB:AR IB:AR IB:Ub IB:Ub

I LNCaP VCaP LNCaP VCaP AR AR AR AR

1 0.412 0.331 0.132 1 0.562 0.435 0.226 1 0.731 0.529 0.179 1 0.628 0.445 0.129 Tubulin Tubulin Tubulin Tubulin

Lamin A/C Lamin A/C Lamin A/C Lamin A/C

−− −− −− siCtrl + + siCtrl ++ Vehicle ++ Vehicle ++ −− siPADI2 −− + + siPADI2 − − ++ Cl-Amidine − − ++ Cl-Amidine −− ++

J Si-Ctrl Si-PADI2 Vehicle Cl-amidine

EtOH EtOH

R1881 R1881

Figure 3. PADI2 stabilizes AR and facilitates its nuclear translocation. A and B, Total AR protein levels were detected by Western blot in siPADI2-transfected (top), Cl-Amidine-treated (bottom), or PADI2-overexpressing VCaP and LNCaP (B) cells. C and D, AR protein levels were determined after incubation with cycloheximide (CHX, 10 mg/mL) for the indicated time periods in siPADI2-transfected (C) or Cl-Amidine–treated (D) LNCaP and VCaP cells. E, LNCaP and VCaP cells with vector control (Vec Ctrl) or PADI2 overexpression were treated with control or 10 mmol/L MG132 for 6 hours and subjected to Western blot analysis. F, Effect of siPADI2 or Cl-Amidine treatment on AR/MDM2 association was detected by coimmunoprecipitation in LNCaP cells. G, Western blot analysis of whole-cell lysates conﬁrmed AR protein expression in all experimental conditions. Cell lysates were then subjected to immunoprecipitation (IP) using an anti-HA (Ub) antibody, followed by Western blot analysis using an anti-AR antibody. H, Cell lysates were subjected to immunoprecipitation using an anti-FLAG (AR) antibody in a reverse coimmunoprecipitation, followed by Western blot analysis using an anti-ubiquitin antibody. I, Cytoplasmic and nuclear AR protein levels were analyzed in LNCaP and VCaP cells after siPADI2 or inhibiting the activity of PADI2 by Cl-Amidine. Tubulin and Lamin A/B were used as cytoplasmic and nuclear protein- loading controls, accordingly. J, Immunoﬂuorescent images of AR expression (green) in LNCaP cells after siPADI2 or Cl-Amidine treatment with or without R1881 stimulation. All experiments were performed three times with similar results. Red circle indicates the nuclear compartment.

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Wang et al.

A D * * 0.08 * * * * * 7 * * 0.15 * * 12 * 6 * * * 0.06 * 6 0.06 10 5 * 5 0.1 AR 8 4 4 0.04 IgG 6 0.03 3 3 0.05 4 2 2 % Input 0.02 2 1 1 0 0 0 0 0 LNCaP VCaP LNCaP VCaP 0 PSA TMPRSS2 LNCaP VCaP Relative expression 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 Vec Ctrl PADI2 Vec Ctrl PADI2 Met Vec Ctrl PADI2 Vec Ctrl PADI2 PADI2Vec Ctrl PADI2 Vec Ctrl PSA KLK2 E Negative control Si - PADI2 Negative control Si - PADI2 TMPRSS2 Negative control Si - PADI2 * * B * * 0.05 0.05 * 0.04 6 * 4.5 * 6 * * * * * * * 4 * 0.04 0.04 5 5 0.03 * 3.5 * 4 3 4 0.03 0.03 2.5 3 3 0.02 AR 2 0.02 0.02 IgG 2 1.5 2 % Input 0.01 1 0.01 0.01 1 1 0.5 0 0 0 0 0 Relative expression 0 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 0 0.01 0.1 1 10 LNCaP VCaP LNCaP VCaP LNCaP VCaP PSA TMPRSS2 Met PSA VehicleCl-amidine Vehicle Cl-amidineTMPRSS2 Vehicle Cl-amidine siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siPADI2 siCtrl siPADI2 siCtrl KLK2 C F 0.05 0.06 0.05 * 12 10 * * * * 9 0.05 * 0.04 * 10 0.04 * 8 0.04 7 0.03 8 0.03 * 6 * 0.03 AR 6 5 0.02 0.02 IgG 4 0.02 % Input 4 3 0.01 2 0.01 0.01 2 1 Relative expressiohn 0 0 0 0 Relative expressiohn 0 LNCaP VCaP LNCaP VCaP LNCaP VCaP PSA KLK2 PSA TMPRSS2 Met PSA KLK2 Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Cl-Amidine Vehicle Cl-Amidine Vehicle siCtrl + + + + -- - - + + + + -- - - Vehicle siAR --- -+ + + + -- -+ - + + + Enz - Vtrl-C + + -+ - + - - + + -+- + - - VtrlC PADI2 -+- + -- + + -+- + -+- + PADI2 G EtOH + -- + + -- + + -- + + -- + EtOH LnCaP VCaP R1881 -++ - -++ - -++ - -++ - R1881 PADI2 1 0.365 0.156 0.762 1 0.227 0.127 0.672 Tubulin J K Lamin A/B 0.012 * 0.08 0.02 0.08 * * H3K9ac H3R26 H3K9ac H3R26 R1881 * 0.009 0.06 * 0.015 0.06 IgG IgG IgG IgG * * * H AR PADI2 Merge 0.006 0.04 0.01 0.04 % Input % Input % Input Vehicle 0.003 0.02 0.005 % Input 0.02

0 0 0 0 LNCaP VCaP LNCaP VCaP LNCaP VCaP LNCaP VCaP siCtrl siPADI2 siCtrl siPADI2 R1881 siCtrl siPADI2 siCtrl siPADI2 Vehicle Cl-Amidine VehicleCl-Amidine VehicleCl-Amidine VehicleCl-Amidine

0.04 * * * 0.05 * 0.03 H3K27me AR PADI2 Merge 0.06 H3K4me3 * I H3K4me3 H3K27me 0.03 0.04 IgG IgG * IgG * IgG 0.02 * 0.04 0.03 Vehicle 0.02

% Input 0.02 % Input % input

% Input 0.01 0.01 0.02 0.01

R1881 0 0 0 0 LNCaPCV aP LNCaPCaP V LNCaP VCaP LNCaP VCaP siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-AmidineVehicle Cl-Amidine

Figure 4. PADI2-mediated citrlullination regulates the activity of AR in the nucleus. ChIP assays were conducted in LNCaP and VCaP cells treated with vector control (Vec Ctrl) and PADI2 (A), negative control, and siPADI2 (B), or vehicle and 200 mmol/L Cl-Amidine (C) for 24 hours using anti-AR or control IgG antibodies at androgen response elements in the promoter/enhancer regions of AR target genes (PSA, TMPRSS2, and MET). qRT-PCR analysis of mRNA levels of AR target genes (PSA, TMPRSS2,andKLK2) in LNCaP cells after siPADI2 (D) or Cl-Amidine (E) treatment with vehicle or 10 nmol/L R1881 for 24 hours. F, The mRNA levels of PSA and KLK2 in PADI2-overexpressing LNCaP were detected by qRT-PCR in the presence of negative control or siAR (left), and vehicle or enzalutamide (Enz; right). Values represent mean SD of technical duplicates from a representative experiment. G, Change in levels of nuclear/ cytoplasmic expression of PADI2 was determined by Western blot analysis in hormone-deprived LNCaP and VCaP cells treated with vehicle or R1881. Localization of PADI2 and AR (H) or citrullinated H3R26 and AR (I) in LNCaP and VCaP cells was veriﬁed by immunoﬂuorescent staining with or without R1881 stimulation. LNCaP and VCaP cells treated with siCtrl or siPADI2 (J) and vehicle or Cl-Amidine (K) were subjected to ChIP-qPCR using anti-H3R26, H3K27me3, H3K4me3, H4K9ac, or IgG antibodies at PSA. All experiments were performed three times with similar results. A mixture of siPADI2#1 and siPADI2#2 at equal ratio was further utilized and designated as siPADI2. , P < 0.05; , P < 0.01.

identiﬁed in PADI2-437wt—transfected LNCaP or VCaP cells Overexpression of PADI2 results in a CRPC-like phenotype (Supplementary Fig. S4G). In addition, the induction of AR- through AR signaling targeted genes (PSA, TMPRSS2,andKLK2) caused by R1881 Next, we sought to determine whether PADI2 is functional in stimulation was signiﬁcantly enhanced after overexpressing the absence of androgen. As shown in Fig. 5, silencing PADI2 or PADI2-437wt in LNCaP and VCaP cells, even though to a less Cl-Amidine treatment showed similar effects on cell viability (Fig. extent than those of PADI2-665wt–transfected cells (Supple- 5A), BrdUrd incorporation (Fig. 5B), and cell cycle (Fig. 5C) in mentary Fig. S4H and S4I). Importantly, these effects could LNCaP-AI and C4-2B without androgen as those of LNCaP and be completely blocked by inhibiting citrullinating activity of VCaP cells with androgen. Importantly, LNCaP-PADI2–derived PADI2 through site mutation of D180A (Supplementary tumors grew more rapidly than controls under the castration Fig. S4H and S4I). condition (21.2 mm3/week vs. 61.1 mm3/week; Fig. 5D), which

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PADI2-Mediated Citrullination and CRPC

A 1 0.8 1 siPADI2#1 Cl-amidine (100 nmol/L) 1 siPADI2#1 0.9 0.7 Cl-amidine (100 nmol/L) siPADI2#2 Cl-amidine (200 nmol/L) siPADI2#2 0.8 Cl-amidine (200 nmol/L) 0.6 Vehicle 0.8 NC 0.8 0.7 NC Vehicle 0.5 Mock 0.6 Mock 0.6 0.6 0.5 0.4 0.4 * 0.3 0.4 * 0.4 * * * * * 0.3 0.2 0.2 0.2 0.2 Cell viability (OD) viability Cell 0.1 (OD) viability Cell 0.1 (OD) viability Cell (OD) viability Cell 0 0 0 0 LNCaP-AI 24 48 72 h LNCaP-AI 24 48 72 h C42B 24 48 72 h C42B 24 48 72 h B 1.2 1.2 C * * * * 1 1 100% 100% 90% 90% 80% 0.8 * 0.8 80% * * 70% * siPADI2#1 * * 70% 0.6 * Cl-amidine (100 nmol/L) 60% 60% siPADI2#2 0.6 * 50% Cl-amidine (200 nmol/L) 50% NC 40% G0–G1 0.4 0.4 Vehicle 40% Mock Cell cycle 30% 30% S

20% Cell cycle 0.2 0.2 20% G –M 10% 10% 2

BrdUrd incorporation BrdUrd incorporation 0% 0 0 0% LNCaP-AI NC NC LNCaP-AIC42B LNCaP-AI C42B C42B NC NC Mock Mock Mock Mock

siPADI2#1siPADI2#2 siPADI2#1siPADI2#2 siPADI2#1siPADI2#2 siPADI2#1siPADI2#2 Aphidicolin Aphidicolin Aphidicolin Aphidicolin withdrawal withdrawal D E * * 1,000 100% 100% 3 900 Vec Ctrl * 90% * 90% 800 80% 80% PADI2 70% 700 * PADI2 70% 60% 60% 600 * 50% 50% 500 40% 40% G0–G1 400 30% 30% S Cell cycle Vec Ctrl Cell cycle 20% 300 20% 10% 10% G2–M 200 0% 0% 100

Tumor volume (mm ) LNCaP-AI C42B 0 Vehicle Vehicle Vehicle Vehicle CI-Amidine CI-Amidine 76543210 Week CI-Amidine CI-Amidine Aphidicolin Aphidicolin Aphidicolin Aphidicolin withdrawal withdrawal

F G Vec Ctrl 1 H 2.5 PADI2 Vec Ctrl 0.9 0.9 * PADI2+siCon 0.8 PADI2 2 0.8 PADI2+Vehicle 0.7 PADI2+siAR 0.7 0.6 PADI2+Enz 1.5 0.6 0.5 0.5 * 1 0.4 * 0.4 * 0.3 0.3 * 0.2 0.2 Tumor weight (g) Tumor 0.5 Cell viability (OD) Cell 0.1 viability (OD) Cell 0.1 0 0 0 PADI2 Vec Ctrl 24 48 72 h 24 48 72 h

I J K 40 600 35

3 550 30 Vec Ctrl 500 shPADI2 25 PADI2 450 20 shCtrl 15 400

PSA-Luc (F/R) PSA-Luc * * 10 * 350 * * * * * * shPADI2 5 300 * * shCtrl 0

Tumor volume) (mm 250 1 10 1 10 10 100 10 50 6543210 DHT E2 Flu Enz (nmol/L) (nmol/L) (µmol/L) (µmol/L)

Figure 5. PADI2 induces a CRPC-like phenotype of prostate cancer through AR signaling. A, The cell viability of LNCaP-AI and C4-2B was detected by MTS assay after siPADI2 or Cl-Amidine treatment. , P < 0.05 versus negative control (NC) and mock, or vehicle. The mock was deﬁned as the one supplemented with the transfection reagent only. B, BrdUrd incorporation assay was performed in LNCaP-AI and C4-2B cells after siPADI2 or Cl-Amidine treatment. , P < 0.05 versus NC and mock, or vehicle. C, Cell-cycle analysis was performed in LNCaP-AI and C4-2B cells after siPADI2 or Cl-Amidine treatment. , P < 0.05. D, Nude mice were injected subcutaneously with either 6 106 LNCaP-Vec Ctrl or LNCaP-PADI2 cells and were castrated once the tumors reached approximately 350 mm3. Tumor volumes formed by LNCaP-PADI2 or its control in castrated mice were measured at the indicated weeks. , P < 0.05 versus vector control (Vec Ctrl). The tumors were weighed at the timepoint of 7 weeks (E) and depicted as mean value (F). n ¼ 10 in each group. , P < 0.05 versus vector control. The cell viability was determined by MTS assay in LNCaP after overexpressing PADI2 under androgen-deprived conditions in the presence of negative control or siAR (G), and vehicle or enzalutamide (H). , P < 0.05, PADI2 versus PADI2 þ siAR or PADI2 þ enzalutamide. I and J, C4-2B-shPADI2 and its control cells were injected into nude mice and were castrated once the tumors reached approximately 300 mm3. The tumor volumes were monitored as the indicated timepoint. n ¼ 10 in each group. , P < 0.05 versus shCtrl. K, The luciferase activity of PSA promoter was measured in PADI2- or vector control– transfected 293T cells in the presence of indicated stimulation. , P < 0.05.

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Wang et al.

A C LNCaP-AI LNCaP-AI E LNCaP-AI LNCaP-AI C4-2B AR AR PADI2 AR 1 0.779 0.573 0.268 1 0.539 0.657 0.068 1 0.427 0.512 0.813 GAPDH Tubulin Tubulin Tubulin

siCtrl Lamin A/B Lamin A/B Lamin A/B

siPADI2 EtOH siCtrl Vehicle

siPADI2 Cl-Amidine R1881

C4-2B C4-2B C4-2B B LNCaP-AI C4-2B D AR AR PADI2 AR 1 0.475 0.268 0.117 1 0.839 0.487 0.109 1 0.113 0.317 0.587 GAPDH Tubulin Tubulin Tubulin

Vehicle Lamin A/B Lamin A/B Lamin A/B Cl-Amidine siCtrl Vehicle EtOH

siPADI2 Cl-Amidine R1881 G 0.06 0.05 0.05

0.04 0.04 * F 0.04 * 0.03 0.03 AR AR AR LNCaP-AI AR H3R26 Merge IgG 0.02 * IgG 0.02 IgG

% Input 0.02 * * * 0.01 0.01 Vehicle

0 0 0 PSA LNCaP-AIC42B TMPRSS2 LNCaP-AI C42B MET LNCaP-AI C42B siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siPADI2 siCtrl siPADI2 siCtrl R1881 H 0.05 0.06 0.05

0.04 0.05 0.04 * * 0.04 0.03 0.03 AR 0.03 AR AR 0.02 * IgG IgG 0.02 IgG % Input 0.02 * * * 0.01 0.01 0.01

0 0 0 PSA LNCaP-AI C42B TMPRSS2 LNCaP-AI C42B MET LNCaP-AI C42B Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Cl-Amidine Vehicle Cl-Amidine Vehicle I 0.015 0.05 0.05 * H3R26 0.06 H3K4me3 H3K27me H3K9ac 0.04 0.04 IgG IgG 0.01 IgG IgG 0.03 0.03 *

* 0.03 0.02 0.02 * * * % Input 0.005 * * 0.01 0.01

0 0 0 0 LNCaP-AI C42B LNCaP-AI C42B LNCaP-AI C42B LNCaP-AI C42B siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 siCtrl siPADI2 J 0.02 0.08 0.08 0.04

0.015 H3R26 H3K4me3 H3K27me 0.06 H3K9ac 0.06 0.03 IgG IgG IgG * IgG 0.01 * 0.04 0.02 nput 0.04 I

% * * 0.005 0.02 * 0.02 * 0.01 * * 0 0 0 0 C42B LNCaP-AI LNCaP-AI C42B LNCaP-AI C42B LNCaP-AI C42B Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine Vehicle Cl-Amidine

Figure 6. PADI2 activates AR signaling under androgen-deprived conditions. The protein levels of total AR were detected by Western blot analysis in LNCaP-AI and C4-2B cells after siPADI2 (A), Cl-Amidine treatment (B). Changes in levels of cytoplasmic and nuclear AR protein levels were analyzed after siPADI2 (left) or Cl-Amidine treatment (right) in LNCaP-AI (C) and C4-2B (D) cells, accordingly. E, Under androgen-deprived conditions, cytoplasmic and nuclear PADI2 protein levels were analyzed by Western blot in LNCAP-AI and C4-2B with or without R1881 stimulation. F, Immunoﬂuorescent images of AR and citrullination of H3R26 in LNCAP-AI or its parental control. Nucleus is outlined by blue circle. ChIP-qPCR assays were performed in LNCaP-AI or C4-2B cells treated with either siPADI2 (G) or 200 mmol/L Cl-Amidine (H) for 24 hours using anti-AR or control IgG antibodies at androgen response elements in the promoter/enhancer regions of AR target genes, PSA, TMPRSS2,andMET. , P < 0.05 versus siCtrl or vehicle. LNCaP and VCaP cells treated with siCtrl or siPADI2 (I) and vehicle or Cl-Amidine (J) were subjected to ChIP-qPCR using anti- H3R26, H3K27me3, H3K4me3, H4K9ac, or IgG antibodies at PSA. , P < 0.05.

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PADI2-Mediated Citrullination and CRPC

A B F 0.85 Vehicle 0.85 Vehicle+siCtrl 0.75 Enz 0.75 Enz Enz+Cl-Amidine Enz+siPADI2 0.65 0.65 0.55 0.55

0.45 0.45 * 0.35 0.35 Cell viability (OD) * * Cell viability (OD) 0.25 0.25 * 24 48 72 h 24 48 72 h C D

860 Vehicle )

3 Vehicle Enz 760 Cl-Amidine Enz+Cl-Amidine 660 Cl-Amidine

560 Enz 460 * * *

Tumor volume (mm 360 Enz+Cl-Amidine 76543210 Week E 6

3 * 2 * Tumor weight (g) 1 ** 0 Vehicle Cl-Amidine Enz Enz+Cl-Amidine

Figure 7. Inhibition of PAD activity is effective in suppression of prostate cancer progression. Cell viability of LNCaP was detected by MTS assay after enzalutamide (Enz) treatment in combination with siPADI2 (A) or Cl-Amidine (B). C and D, The tumor volume was measured in nude mice, with castration as the indicated treatment. E, To recapitulate CRPC model, male nude mice bearing LNCaP xenografts were castrated when the level of serum PSA reached 75 ng/mL and followed until serum PSA and tumor growth rates increased back to precastrated levels, indicating progression to castration resistance. The tumor weight was measured at the timepoint of 7 weeks. The number of animal models in each group was 10. , P < 0.05 versus enzalutamide. F, Schematic of a functional map between PADI2/H3R26 citrullination and AR signaling, which exacerbates prostate cancer progression, especially CRPC. In the cytoplasm, PADI2 could protect AR against proteasome-mediated degradation of AR. After translocation of PADI2 into the nucleus, the binding of AR to its target genes could be facilitated in the presence of citrullination of H3R26, which was mediated by PADI2. Furthermore, the repression of androgen on PADI2 expression could be recovered in the absence of androgen, making it function efﬁciently under androgen-deprived condition. ARE, androgen response element.

was also confirmed by the size and weight in isolated tumors androgen. In addition, siPADI2 (left) or Cl-Amidine treatment (Fig. 5E and F). Furthermore, ectopic expression of PADI2 could (right) could sequester more AR protein in cytoplasm (Fig. 6C promote the cell growth of LNCaP under hormone-free condi- and D). Western blot analysis further revealed the presence of tions in vitro (Fig. 5G and H). In contrast, shPADI2 in C4-2B PADI2 protein in the nuclear compartment in CRPC cell lines suppressed the tumor growth under the castration condition (Fig. 6E) and androgen deprivation could also induce nucleus– (20.3 mm3/week vs. 33.9 mm3/week; Fig. 5I). Images of isolated cytoplasm translocation (Fig. 6F). More importantly, colocali- tumors further showed differences in tumor size and shape zation of citrullination of H3R26 and AR was identified in (Fig. 5J). LNCaP-AI cells after R1881 stimulation even at extremely low To further elucidate the critical role of AR signaling on the concentration (0.01 nmol/L; Fig. 6F). PADI2 knockdown function of PADI2, we demonstrated that the increase of cell (Fig. 6G) or Cl-Amidine treatment (Fig. 6H) could then par- viability of LNCaP caused by PADI2 overexpression could be tially abolish the AR-binding events in LNCaP-AI and C4-2B blocked after siAR (Fig. 5G) and enzalutamide treatment (Fig. 5H) cells. Furthermore, the above treatments suppressed the citrul- without androgen. Thus, AR signaling potentiates the oncogenic lination of H3R26, methylation of H3K4 and acetylation of function of PADI2 to exacerbate CRPC. Importantly, PADI2 H3K9, but induced methylation of H3K27 (Fig. 6I and J). In enhances AR responsiveness in the presence of diverse ligands, addition, the occupancy of RNA Pol II decreased after siPADI2 such as enzalutamide, flutamide, and estrogen (Fig. 5K), indicat- or Cl-Amidine treatment (Supplementary Fig. S5). Taken ing a potential role of treatment resistance in prostate cancer together, our results demonstrated that PADI2 is required to progression. activate AR signaling in the presence or absence of androgen.

PADI2 is required for activation of AR signaling in the absence Combined inhibition of AR and PADI2 synergistically delays of androgen CRPC growth both in vitro and in vivo Next, we asked whether PADI2 is required for activation of Although enzalutamide has been approved for therapy of AR signaling under androgen-deprived condition. Western blot prostate cancer patients with CRPC, clinical applications revealed analysis showed that siPADI2 (Fig. 6A) or Cl-Amidine treat- that approximately 30% to 40% of patients acquire resistance ment (Fig. 6B) could suppress AR protein in the absence of after a short period of treatment (2). Considering of the important

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role of PADI2 in CRPC, we then evaluated whether PADI2 under androgen-deprived conditions. Third, overexpressing inhibition potentiates the effect of enzalutamide treatment. PADI2 makes prostate cancer cells more adaptive to survive and LNCaP-AI cells were then given siPADI2 or Cl-Amidine, followed proliferate in the absence of androgen and eventually led to by enzalutamide treatment. As shown in Fig. 7A and B, siPADI2 or CRPC-like phenotype. In contrast, suppression of PADI2 expres- Cl-Amidine treatment significantly enhanced the effect of enza- sion and activity inhibits the cell viability of CRPC cells more lutamide on reducing cell viability of LNCaP cells. We next significantly than those of androgen-dependent prostate cancer evaluated the effects of cotargeting the AR and PADI2 using cells. Together, these findings indicate that PADI2 is required for in vivo models under castration condition. As shown in Fig. 7C prostate cancer progression and CRPC. and D, Cl-Amidine plus enzalutamide treatment reduced the Recently, a broad range of therapeutic options have become tumor volume (537.8 mm3) more effectively as compared with available for patients with CRPC in a variety of settings, including that of enzalutamide treatment alone (685.7 mm3) by 7 weeks chemotherapeutic agents (cabazitaxel; ref. 45), abiraterone ace- (Fig. 7C and D). The weight in isolated tumors confirmed the tate (46), enzalutamide (47), and immunotherapy (sipuleucel-T; above effects as well (Fig. 7E). These data suggested that combined ref. 48). Among them, enzalutamide is a next-generation anti- inhibition of AR and PADI2 synergistically delays CRPC growth androgen and a potent AR inhibitor. Although enzalutamide both in vitro and in vivo. improves outcomes for patients with CRPC, it is not universally effective and the responses are not durable (47). Here, we found that the treatment of prostate cancer cells with PADI2 knockdown Discussion or PAD inhibitors can delay prostate cancer progression in vitro Protein citrullination is emerging as one of the critical PTMs in and in vivo, suggesting that the inhibition of PADI2-mediated developmental biology, inflammation, and cancer progression citrullination may have therapeutic effect in prostate cancer. (8, 32, 33). The PADIs are a family of PTM enzymes that mediate Remarkably, our study demonstrated that Cl-Amidine treatment citrullination (8). Five PAD family members (PADs 1–4 and 6) could suppress the activity of AR signaling, supporting the cell- have been described (34) and are closely related with tumor specific effects of Cl-Amidine on prostate cancer cells. As Cl- progression. Among them, PADI4 is most extensively studied Amidine has been identified to be held the potential to treat (35). PADI4 mainly acts as a transcriptional coregulator with certain inflammatory diseases and cancers through targeting p53, ELK1, or TAL1, possibly via histone citrullination after dendritic cells, T cells, and endothelial cells (49, 50), we cannot translocation into the nucleus (36–38). Recently, PADI2 had exclude the possibility that the antitumor effects of Cl-Amidine in been identified as a potential target for breast cancer via citrulli- vivo may be partially done through targeting some other signaling nating arginine residues 2/8/17 on histone H3 tails of PTN and pathways. MAGEA12 (17). Furthermore, PADI1 and PADI2 expression were Intriguingly, our study demonstrated that the combination significantly increased in mutant HRAS-driven skin carcinogene- of Cl-Amidine with enzalutamide results in synergistic inhibi- sis (39), further supporting the dynamic involvement of PADs in tion of cell proliferation and tumor growth in vitro and in vivo skin tumor. In addition, PADI2 overexpression in transgenic mice under androgen-deprived or castration conditions. This com- can promote spontaneous skin neoplasia by disturbing tumor bined treatment strategy may be potentially beneficial to a microenvironment and facilitating epithelial-to-mesenchymal subset of CRPC patients with PADI2 overexpression. Further transition of tumor cells (15). More importantly, Zhang and investigation will also be needed to evaluate whether PADIs colleagues reported that estrogen stimulation induced the recruit- inhibition is effective as a preventive strategy for prostate cancer ment of PADI2 to target promoters by ERa, whereby PADI2 then progression, especially CRPC. citrullinated H3R26, which led to local chromatin become less In conclusion, our study defines a novel mechanism that condensed for transcriptional activation (19). In this study, we PADI2-mediated citrullination can promote prostate cancer pro- further demonstrated that PADI2, but not PADI4, could promote gression and CRPC (Fig. 7F). Treatment with PADIs inhibitor can protein stability, nuclear translocation, and transcriptional acti- suppress prostate cancer progression. Most importantly, we sug- vation of AR in prostate cancer cells. Notably, citrullination of gested a novel potential therapeutic approach with combination H3R26 contributes significantly to activate AR signaling. In addi- of PADIs inhibitor with enzalutamide to treat prostate cancer tion, inhibition of citrullinating activity in its nuclear form of patients with CRPC. PADI2 suppresses the transcriptional activity of AR most efficiently. In all, this is the first study to establish a link between protein citrullination and progression of CRPC. These findings may help Disclosure of Potential Conflicts of Interest fl to improve our understanding of how AR regulates gene tran- No potential con icts of interest were disclosed. scription via altering chromatin structure. AR signaling plays a central role for the survival and the growth Authors' Contributions of most CRPCs (2). Previously, others and we have reported that Conception and design: L. Wang, J. Han, B. Han AR reactivation can occur through deregulation of coactivators in Acquisition of data (provided animals, acquired and managed patients, the absence of androgen, such as the P160 SRC (steroid receptor provided facilities, etc.): T. Feng coactivator) family genes (40), the fork head protein FoxA1 (41) Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and transcription factor, GATA2 (42), and cochaperones, includ- computational analysis): L. Wang, G. Song, J. Pan, X Zhang ing HSP27 (43), HSP90 (44) and TXNDC5 (25), finally leading to Writing, review, and/or revision of the manuscript: L. Wang, W. Chen, J. Yu, CRPC progression. In this study, we provided several lines of M. Yang, X. Bai, J. Han, B. Han Administrative, technical, or material support (i.e., reporting or organizing evidence to suggest an important role of PADI2 in CRPC. First, data, constructing databases): G. Song, T. Feng, J. Pan, W. Chen, M. Yang, X. Bai, clinical data suggested that PADI2 is significantly overexpressed in Y. Pang, J. Han patients with CRPC. Second, PADI2 can activate AR signaling, Study supervision: J. Han, B. Han

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Acknowledgments Shandong Taishan Scholarship (tsqn20161076), and The Innovation Project of We thank Drs. Hao Dou, Yongxin Zou, and Yaoqin Gong (Department of Shandong Academy of Medical Sciences. Genetics, Shandong University Medical School) for valuable discussion of The costs of publication of this article were defrayed in part by the the study. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Grant Support this fact. This work was supported by National Natural Science Foundation of China (grant nos. 81572544, 81672554, 81472417, 81528015 and 81572254), The Received January 17, 2017; revised May 31, 2017; accepted August 11, 2017; Key Research and Development Project of Shandong (2016GSF201166), The published OnlineFirst August 17, 2017.

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PADI2-Mediated Citrullination Promotes Prostate Cancer Progression

Lin Wang, Guanhua Song, Xiang Zhang, et al.

Cancer Res Published OnlineFirst August 17, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0150

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