Onset of castration-resistance prostate cancer (CRPC) after long-term androgen deprivation therapy remains a major obstacle in the treatment of prostate cancer. The peptidylarginine deiminase PADI2 has been implicated in chronic inflammatory diseases and cancer. Here we show that PADI2 is an androgen-repressed gene and is upregulated in CRPC. PADI2 expression was required for survival and cell-cycle progression of prostate cancer cells, and PADI2 promoted proliferation of prostate cancer cells under androgen-deprived or castration conditions in vitro and in vivo. Cytoplasmic PADI2 protected the androgen receptor (AR) against proteasome-mediated degradation and facilitated AR binding to its target genes after nuclear translocation and citrullination of histone H3 amino acid residue R26. In contrast, mutant PADI2 D180A failed to affect AR stability, nuclear translocation, or transcriptional activity. PADI2 mediated AR control in a manner dependent on its enzymatic activity and nuclear localization, as correlated with increased histone H3 citrullination. Notably, coadministration of the PADI inhibitor Cl-Amidine and the AR signaling inhibitor enzalutamide synergized in inhibiting CRPC cell proliferation in vitro and tumor growth in vivo. Overall, our results establish PADI2 as a key mediator for AR in prostate cancer progression, especially CRPC, and they suggest PADI as novel therapeutic targets in this disease setting. Cancer Res; 77(21); 5755–68. ©2017 AACR.

A major clinical hurdle for the management of patients with advanced prostate cancer is the onset of castration-resistant prostate cancer (CRPC; ref. 1). Androgen receptor (AR) signaling remains active and contributes greatly during CRPC progression (2). Despite recent hormonal therapies showing a survival advantage for abiraterone acetate (17α-hydroxylase/C17, 20-lyase inhibitor) and enzalutamide (AR inhibitor), the median duration of response is less than one year (2). Therefore, identification of key molecules to modulate AR and development of novel strategies to target CRPC are urgently needed.

Dysregulation of posttranslational modification (PTM), including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation, might influence cancer progression (3–6). The peptidylarginine deiminases (PADI) is a family of PTMs enzymes that converts arginine into citrulline, namely citrullination 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). Histone modifications and the enzymatic machineries are crucial regulators that control cellular proliferation, differentiation, and malignancy processes (3). Importantly, PADI4, as well as PADI2, is able to alter gene transcription through citrullinating residues in histone tails (8). To date, PADI-mediated citrullination has been proven to be involved in various inflammatory conditions, such as rheumatoid arthritis (9), multiple sclerosis (10), psoriasis (11), chronic obstructive pulmonary disease (12), as well as various cancers (8). Inhibitors targeting PADIs have been investigated in the preclinical setting.

PADI2 is expressed in multiple organs (13) and most recent studies suggested a link with cancer progression. McElwee and colleagues demonstrated that PADI2 expression increases during the transition from benign mammary epithelium to malignant breast carcinomas (14). In another report, overexpressed PADI2 in transgenic mice promotes spontaneous skin neoplasia by enhancing inflammation within the tumor microenvironment (15). In addition, Cantarino and colleagues found that downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer and associated with poor prognosis (16), indicating a tissue-specific role of PADI2 in tumor progression. So far, the identified positions of arginine residues include H3R2, H3R8, H3R17, and H3R26 (17). PADI2 has been suggested to be involved in multiple myeloma and breast cancer by citrullinating histone tails at transcription factor–binding sites, ultimately affecting transcription (18–20). Notably, PADI2 expression is responsive to extracellular stimuli, such as estrogen and EGF (14), indicating its dynamic activity in human disease. In this study, we found that PADI2 expression in prostate cancer cells could be induced by androgen deprivation treatment. More importantly, PADI2-mediated citrullination activates AR signaling and promotes CRPC progression. Our data supported the potential of PADI as novel therapeutic targets to treat prostate cancer patients with CRPC.

Patients

A total of 256 prostate cancer patients participated in our study. The tumor samples were obtained from Qilu Hospital of Shandong University (Jinan, China) and The Affiliated Hospital of Qingdao University (Qingdao, China) between 2003 and 2007. The first cohort consisted of 229 men with localized prostate cancers who have undergone radical prostatectomy. None of the patients received preoperative radiation or ADT. The second cohort included patients with CRPC treated by transurethral resection of the prostate to relieve symptomatic obstruction due to locally advanced disease (n = 27). The initial treatment for patients was either observation or surgery. Development CRPC was treated by flutamide or bicalutamide. This study was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The study protocol was approved by the Institutional Review Board of Medicine School of Shandong University. The informed written consent was obtained from the patients.

IHC and immunofluorescence

IHC and the scoring system were described as reported previously (21). Briefly, the slides were incubated overnight with anti-PADI2 (Proteintech) or anti-citrulline (Abcam) antibody and then evaluated blindly by two independent observers (B. Han and T. Feng) who were blinded to the clinicopathologic data. The scoring system of PADI2 and citrullinated protein was as follows: the nuclear and cytoplasmic PADI2 and citrullinated protein staining was scored into four grades, which were classified by its staining intensity: 0, 1+, 2+, and 3+. The percentages of PADI2 or citrullinated protein–positive cells were scored into five categories: 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). A final score was built by multiplying the scores of these two parameters, which were defined as follows: 0, negative; 1–3, weak; 4–6, moderate; and 8–12, strong. For analysis, we combined both negative and weakly IHC-positive tumors into one group and compared it with moderately and strongly IHC-positive prostate cancers. The Ki67 labeling index was defined as described previously (21). For immunofluorescence, cells were sequentially probed with primary antibodies and fluorescence-labeled secondary antibodies (Jackson Immunoresearch). Images were captured under a confocal microscope (FV3000, Olympus).

Cell culture and treatment

LNCaP and C4-2B cells were obtained from Dr. Jindan Yu (Northwestern University, Chicago, IL) in 2015. VCaP and HEK293T were purchased from ATCC between 2012 and 2015 and authenticated again by short tandem repeat (STR) analysis again before and after our study. The cumulative culture length of the cells between thawing and use in this study was less than 15 passages. All of the newly revived cells were tested free of mycoplasma contamination by Hoechst 33258 staining (Beyotime). For the androgen treatment, cells were hormone-starved for 2 days in the media containing 10% charcoal-stripped FBS and treated with R1881for expression assay or ChIP assays. Cl-Amidine (an arginine-based PAD inhibitor) was used at a concentration of 100 and 200 μmol/L as published previously (14).

MTS, BrdUrd cell proliferation assay, and flow cytometry analysis

Cell growth and proliferation were measured using the MTS and BrdUrd incorporation assay as described previously (22). For cell-cycle analysis, cells were treated with 5 μg/mL of aphidicolin (Sigma Aldrich) for additional 24 hours. After fixation and staining with propidium iodide (Sigma Aldrich), the samples were analyzed by flow cytometry (FACSCalibur, BD Biosciences).

Quantitative real time-PCR

Total RNA was prepared from prostate cancer cells and was reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo). qRT-PCR was performed with diluted cDNA using SYBR Green PCR Master Mix (Toyobo) and gene-specific primers (23). The sequences of all primers were listed in Supplementary Table S1.

ChIP-qPCR analysis

Chromatin immunoprecipitation (ChIP) was carried out as described previously (22). ChIP-qPCR enrichment of target loci was normalized to input DNA and all primers were listed in Supplementary Table S1. Anti-histone H3 (citrulline R2+R8+R17), anti-histone H3 (citrulline R26), anti-histone H4 (citrulline R3), and anti-histone H3 (trimethyl K4) were supplied from Abcam. Anti-histone H3 (acelytated R9), anti-histone H3 (trimethyl K27), and irrelevant antibody (IgG) were from Millipore

Plasmids and siRNAs

Human PADI2 cDNA encompassing different lengths (1–665 aa, 1–653 aa, and 1–437 aa) was amplified by reverse transcription PCR from LNCaP and cloned into pCR8/GW/TOPO (Invitrogen). The amino acid residue, Asp180, is located in the N-terminal Ca2+-binding site of PADI2 and is essential for catalysis (24). The D180A mutants of PADI2 (PADI2-665mt, PADI2-653mt, and PADI2-437mt) were prepared with the QuikChange site-directed mutagenesis kit (Stratagene), as described previously (24). Lentivirus products were constructed in 293T cells by cotransfection with psPAX2, pMD2.G, and lentiviral construct (pLentiCMV/TO Puro DEST) expressing PADI2 using Lipofectamine 2000 (Invitrogen). Stable cells overexpressing PADI2 were selected using puromycin after transfection with lentivirus products. siRNA targeting PADI2, PADI4, AR, and AllStar negative control were bought from Qiagen and the sequences were provided in Supplementary Table S2. The mock group was defined as that supplemented with transfection reagent only. The corresponding DNA nucleotides encoding short hairpin RNA showing the most silencing efficiency (PADI2 #2) were subcloned into pSUPER vector with neomycin-resistant gene. The transfected cells were subjected to resistant selection for one week.

Western blot analysis and immunoprecipitation

Western blot analysis was performed as described previously (25). Band intensity underneath the gel image was measured using ImageJ software, presented as fold change compared with control ones. For immunoprecipitation, total proteins were precleared with protein-G sepharose (Invitrogen), which immunoprecipitated with anti-Flag (AR), anti-HA (ubiquitin, Ub), or anti-AR. The nuclear proteins were extracted using Nuclear and Cytoplasmic Extraction Reagents kits (Thermo Scientific). The antibodies used in this study included anti-AR (Abcam) and anti-GAPDH (Santa Cruz Biotechnology). Anti-PADI2, anti-LaminA/C, anti-MDM2, and anti-α-tubulin were from Proteintech.

Protein stability and degradation

To assess the protein stability of AR, LNCaP cells were transfected with siPADI2, vector expressing PADI2, or their respective controls, and the above cells were then treated with cycloheximide (Sigma) or MG132 for Western blot analysis using AR and GAPDH antibodies.

Luciferase reporter gene assay

PSA-luciferase-plasmid, AR expression plasmid, and pRL-TK Renilla plasmid were transfected into 293T cells using Lipofectamine 2000. Luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to the luciferase activity of Renilla of the cells.

Animal treatment

The animal studies were approved by the Ethical Animal Care and Use Committee of Shandong University and the guidelines were strictly followed. For both prostate cancer progression and CRPC model (25), male athymic nude mice were injected subcutaneously with 5 × 106 stably PADI2-overexpressing LNCaP or PADI2-knocking down C4-2B (suspended in Matrigel; BD Biosciences). For treatment, mice were randomly selected to 10 mg/kg enzalutamide daily plus either 10 mg/kg Cl-Amidine or vehicle once daily. Each experimental group consisted of 10 mice. Tumor response measurement was conducted as described previously (25). Animals were sacrificed after 7 weeks of treatment and all mice survived till the end of treatment. Data points were expressed as mean tumor volume ± SD.

Microarray data acquisition and processing

We obtained raw Affymetrix Human Exon 1.0 ST Array expression data and clinical information for Taylor and colleagues from the NCBI Gene Expression Ominibus (GSE21032; ref. 26). Gene expression was calculated as described previously (27). Briefly, probe set normalization was performed by the Single Channel Array Normalization algorithm, which modeled and removed probe- and array-specific background noise using data from each array to individually normalize each sample (28). We used Affymetrix Core level summaries for annotated genes to calculate gene expressions.

Statistical analysis

All results are expressed as the mean ± SD. Two-tailed tests, one-way ANOVA, or Wilcoxon matched pairs tests were used for statistical analysis. The differences between tumor volumes were evaluated by the nonparametric Mann–Whitney–Wilcoxon test. P < 0.05 and P < 0.01 were considered significant.

PADI2 is androgen-repressed and upregulated in CRPC

Androgen–AR signaling is critical for prostate cancer (29). As PADI2 and PADI4 are involved in various diseases (7, 13–15, 18, 30), we first asked whether they are responsive to androgen. As shown in Fig. 1A, PADI2 decreased progressively in LNCaP and VCaP cells at both mRNA (top) and protein (bottom) levels as the dose of synthetic androgen R1881 increased (PSA, positive control; Supplementary Fig. S1A and S1B). Furthermore, the decrease of the PADI2 mRNA levels was significant as early as 6 hours after R1881 stimulation (Fig. 1A; Supplementary Fig. S1C and S1D). In contrast, PADI4 was insensitive to R1881 (Supplementary Fig. S1E). Interestingly, R1881 inhibition of PADI2 was significantly attenuated in LNCaP and VCaP cells after siAR or enzalutamide treatment (Fig. 1B), indicating the downregulation of PADI2 by R1881 is mediated by AR. Next, motif analysis and ChIP assay identified a high-ranking androgen response element at PADI2 promoter encompassing −395 to −380 bp in LNCaP cells (Fig. 1C). Further study revealed that R1881 stimulation induces more recruitment of AR at PADI2 promoter (positive control, PSA, FKBP5, and TMPRSS2; negative control, 3′ end of PADI2 gene; Fig. 1D). To further support that androgen inhibits PADI2 through modulation of active transcription, ChIP-qPCR analysis observed a decrease in RNA Pol II occupancy on PADI2 and AR-repressed gene (MET), but an increase on AR-induced genes (PSA and TMPRSS2; Supplementary Fig. S1F).

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 3′ 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. 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.

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 3′ 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. 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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Because of its androgen-regulated nature, we hypothesized that PADI2 may be differentially expressed during prostate cancer progression. Western blot analysis revealed that four prostate cancer cell lines exhibited variably higher PADI2 protein levels compared with that of benign RWPE cell line. PADI2 was dramatically upregulated in CRPC cell line C4-2B and LNCaP-AI (Fig. 1E), the latter of which was established as CRPC model in our previous study (25). Notably, long-term androgen deprivation significantly induced PADI2 expression at both mRNA (Fig. 1F, left) and protein levels (Fig. 1F, right) in LNCaP cells. In contrast, androgen deprivation did not affect PADI4 expression (Supplementary Fig. S1G).

PADI2 expression was then analyzed among clinical specimens. Both cytoplasmic and nuclear PADI2 were present and representative IHC images were shown in Fig. 1G. Similar to PADI2, the level of citrullinated protein was higher in CRPC tissues than in the HD tissues and higher levels of PADI2 were associated with elevated level of citrullinated protein (Fig. 1H; P = 0.048, χ2 test) in prostate cancer cases. The relationships between PADI2 expression level and clinicopathologic variables in HD cases were analyzed. Notably, PADI2 overexpression was significantly associated with a high Gleason score (P = 0.018), high T (tumor) stage (P = 0.017), and Ki67 index (P = 0.008). Using a publicly available dataset, patients with high PADI2 mRNA expression display an unfavorable biochemical relapse-free survival (BRFS) and overall survival (OS) than those with low expression, even though this did not reach a statistical significance (Fig. 1I, P = 0.1734 for BRFS and P = 0.2980 for OS). Remarkably, among HD cases, 160 (69.1%) showed negative or weak staining (40 cases: negative; 120 cases: weak), and only 69 (30.1%) had moderate or strong staining for PADI2 (30 cases: moderate; 39 cases: strong). However, 14 (51.9%) CRPC cases showed moderate to strong expression (6 cases: moderate; 8 cases: strong), whereas 13 (48.1%) were negative or weak (2 cases: negative; 11 cases: weak). Overall, CRPC specimen showed significantly stronger expression of PADI2 than HD samples (Fig. 1J, P = 0.034). PADI2 staining in HD and CRPC was shown in Fig 1J, 1-2. These data indicated potential role of PADI2 in prostate cancer progression and CRPC.

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.

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.

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PADI2 functions as an oncogene in prostate cancer progression

We next applied in vitro functional assays to characterize PADI2 in prostate cancer. Supplementary Figure S1H illustrates two independent siRNA constructs that silence PADI2 expression efficiently. Inhibition of PADI2 expression significantly suppressed the viability of LNCaP and VCaP cells either by siRNAs (Fig. 2A) or Cl-Amidine (Fig. 2B). In contrast, overexpressing PADI2 enhanced the cell viability (Fig. 2C). However, no significant alteration of cell viability was identified in PADI4-silenced prostate cancer cells (Supplementary Fig. S1I–J). BrdUrd incorporation assay further revealed that DNA synthesis was significantly suppressed in PADI2-silenced (Fig. 2D) or Cl-Amidine–treated (Fig. 2E) prostate cancer cells. In addition, cell-cycle distribution analysis demonstrated that silencing PADI2 (Fig. 2F) or Cl-Amidine treatment (Fig. 2G) could lead to a significant accumulation in the S-phase of LNCaP and VCaP cells after withdrawal of aphidicolin. More importantly, in vivo experiments demonstrated that the growth rate and the mean weight of LNCaP-PADI2–derived primary tumors is much higher than that of LNCaP-Vec Ctrl–derived tumors (133.9 mm3/week vs. 64.1 mm3/week; Fig. 2H–J). Collectively, our data suggested that PADI2 is required for the growth of prostate cancer cells.

PADI2 stabilizes AR protein and facilitates its nuclear translocation

As both PADI2 and AR were upregulated in late-stage prostate cancer (2), we next sought to determine whether PADI2 might regulate AR expression. Western blot analysis showed that siPADI2 (Fig. 3A, top) or Cl-Amidine treatment (200 μmol/L; Fig. 3A, bottom) in LNCaP and VCaP could suppress the AR protein level, and the opposite pattern was observed after overexpressing PADI2 (Fig. 3B). However, no visible change of AR mRNA was identified (Supplementary Fig. S2A and S2B). To test whether PADI2 modulated AR expression at posttranscriptional level, we treated LNCaP with cycloheximide, a protein biosynthesis inhibitor. Western blot analysis showed that siPADI2 or Cl-Amidine treatment (200 μmol/L) remarkably accelerated the degradation of AR protein (Fig. 3C and D). In contrast, PADI2 overexpression in LNCaP and VCaP suppressed the degradation of AR protein, which could be blocked by inhibiting its citrullinating activity after transfection with its mutant type (PADI2-665mt; Supplementary Fig. S2C). Furthermore, immunoblotting analysis showed that ectopic expression of PADI2 failed to increase the protein level of AR in the presence of proteasome inhibitor MG132 (Fig. 3E). These findings suggested that PADI2-facilitated stabilization of AR protein depends on proteasome-mediated AR degradation.

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 μg/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.

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 μg/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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Previous studies have shown that AR formed a heterodimer complex with MDM2 (Proto-Oncogene) to protect ligand-unbound AR from degradation by the ubiquitin–proteasome system (31). In this study, we demonstrated that silencing PADI2 or Cl-Amidine treatment (200 μmol/L) significantly disrupted the association between AR and MDM2 by Western blot analysis (Fig. 3F). We then performed cotransfection of FLAG-AR, HA-ubiquitin, and PADI2 into 293T cells to determine whether PADI2 affected AR ubiquitination. Our results showed that the presence of ubiquitin led to AR protein ubiquitination, which was enhanced by suppressing PADI2 expression or activity (Fig. 3G). Concordantly, reverse coimmunoprecipitation assay showed that PADI2 prevents AR from binding to ubiquitin proteins (Fig. 3H). Importantly, Western blot analysis revealed that nuclear translocation of AR could be significantly inhibited in LNCaP and VCaP after transfection with siPADI2 or Cl-Amidine treatment (Fig. 3I). The above effect was further confirmed by immunofluorescence analysis in LNCaP (Fig. 3J). In contrast, overexpressed PADI2 in LNCaP and VCaP cells could facilitate the nuclear translocation of AR with R1881 stimulation, and these effects could be antagonized after transfection with its mutant type (PADI2-665mt; Supplementary Fig. S2D).

PADI2-mediated citrullination in the nucleus activates AR signaling

As shown in Fig. 4A, PADI2 overexpression could recruit more AR to the promoter of PSA and TMPRSS2 in LNCaP and VCaP cells. In contrast, siPADI2 (Fig. 4B) or Cl-Amidine treatment (200 μmol/L; Fig. 4C) inhibited its occupancy. These findings were then confirmed by measuring the changes of MET gene (Fig. 4A–C). Next, the ChIP-qPCR of RNA pol II revealed a decrease of its occupancy on AR-induced PSA gene (Supplementary Fig. S2E and S2F), but an increase of MET gene (Supplementary Fig. S2G and S2H). As illustrated in Fig. 4D and E, the induction of PSA, KLK2, and TMPRSS2 caused by androgen could be significantly attenuated when treated with siPADI2 or Cl-Amidine. Notably, the induction of AR target genes caused by PADI2 overexpression could be completely abolished by siAR or enzalutamide treatment (Fig. 4F).

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 μmol/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, and KLK2) 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.

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 μmol/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, and KLK2) 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.

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Our data revealed that the shuttle of PADI2 from cytoplasm to nucleus could be facilitated with the stimulation of androgen (Fig. 4G), suggesting that nuclear translocation may have functional importance for PADI2. Previous study has shown that PADI2-mediated citrullination of H3R26 facilitated transcriptional activation induced by estrogen (19). Using confocal immunofluorescent analysis, we demonstrated a strong colocalization between AR and citrullination of H3R26 in LNCaP (Fig. 4H) and VCaP (Fig. 4I) cells with and without R1881 treatment. Next, ChIP-qPCR was performed to validate the citrullination of H3R26 of AR target genes, including PSA. Importantly, siPADI2 (Fig. 4J) or Cl-Amidine treatment (Fig. 4K) led to a significant decrease of citrullinated H3R26, methylated H3K4, and acetylated H3K9, but a concomitant increase of H3K27 methylation. In an attempt of validating the specificity of H3R26 citrullination, two other site-specific anti-citrullinated histone antibodies, anti-H3Cit2/8/17 and anti-H4Cit3 were also applied. As demonstrated in Supplementary Fig. S3A and S3B, no specific changes of H3Cit2/8/17 or H4Cit3 were recognized in the presence of siPADI2 or Cl-Amidine.

As PADI2 may modulate AR both in cytoplasm and nucleus of prostate cancer cells, we next examined which protein sequences of PADI2 are important for its subcellular localization. NoD software (http://www.compbio.dundee.ac.uk/www-nod/) identified a 12 aa residing in the C-terminal of PAID2 with relatively higher NLS score and truncated PADI2 vector with deletion of the 12 aa sequence was constructed (PADI2-653wt). Besides, the full-length FLAG-tagged PADI2 and a truncated PADI2 vector encoding 437 aa (PADI2-437wt) were also prepared (Supplementary Fig. S4A). As demonstrated in Supplementary Fig. S4B, PADI2-653wt still could be detected in the nucleus, even though it could promote AR protein stability (Supplementary Fig. S4C) and nuclear translocation (Supplementary Fig. S4D) in LNCaP and VCaP. Consistent with previous study, PADI2-437wt was sequestered only in the nucleus of LNCaP and VCaP (Supplementary Fig. S4B). Although it failed to regulate the protein stability (Supplementary Fig. S4E) and nuclear translocation (Supplementary Fig. S4F) of AR, robust of citrullination of H3R26 could be identified in PADI2-437wt—transfected LNCaP or VCaP cells (Supplementary Fig. S4G). In addition, the induction of AR-targeted genes (PSA, TMPRSS2, and KLK2) caused by R1881 stimulation was significantly enhanced after overexpressing PADI2-437wt in LNCaP and VCaP cells, even though to a less extent than those of PADI2-665wt–transfected cells (Supplementary Fig. S4H and S4I). Importantly, these effects could be completely blocked by inhibiting citrullinating activity of PADI2 through site mutation of D180A (Supplementary Fig. S4H and S4I).

Overexpression of PADI2 results in a CRPC-like phenotype through AR signaling

Next, we sought to determine whether PADI2 is functional in the absence of androgen. As shown in Fig. 5, silencing PADI2 or Cl-Amidine treatment showed similar effects on cell viability (Fig. 5A), BrdUrd incorporation (Fig. 5B), and cell cycle (Fig. 5C) in LNCaP-AI and C4-2B without androgen as those of LNCaP and VCaP cells with androgen. Importantly, LNCaP-PADI2–derived tumors grew more rapidly than controls under the castration condition (21.2 mm3/week vs. 61.1 mm3/week; Fig. 5D), which was also confirmed by the size and weight in isolated tumors (Fig. 5E and F). Furthermore, ectopic expression of PADI2 could promote the cell growth of LNCaP under hormone-free conditions in vitro (Fig. 5G and H). In contrast, shPADI2 in C4-2B suppressed the tumor growth under the castration condition (20.3 mm3/week vs. 33.9 mm3/week; Fig. 5I). Images of isolated tumors further showed differences in tumor size and shape (Fig. 5J).

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 = 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 + siAR or PADI2 + 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 = 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.

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 = 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 + siAR or PADI2 + 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 = 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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To further elucidate the critical role of AR signaling on the function of PADI2, we demonstrated that the increase of cell viability of LNCaP caused by PADI2 overexpression could be blocked after siAR (Fig. 5G) and enzalutamide treatment (Fig. 5H) without androgen. Thus, AR signaling potentiates the oncogenic function of PADI2 to exacerbate CRPC. Importantly, PADI2 enhances AR responsiveness in the presence of diverse ligands, such as enzalutamide, flutamide, and estrogen (Fig. 5K), indicating a potential role of treatment resistance in prostate cancer progression.

PADI2 is required for activation of AR signaling in the absence of androgen

Next, we asked whether PADI2 is required for activation of AR signaling under androgen-deprived condition. Western blot analysis showed that siPADI2 (Fig. 6A) or Cl-Amidine treatment (Fig. 6B) could suppress AR protein in the absence of androgen. In addition, siPADI2 (left) or Cl-Amidine treatment (right) could sequester more AR protein in cytoplasm (Fig. 6C and D). Western blot analysis further revealed the presence of PADI2 protein in the nuclear compartment in CRPC cell lines (Fig. 6E) and androgen deprivation could also induce nucleus–cytoplasm translocation (Fig. 6F). More importantly, colocalization of citrullination of H3R26 and AR was identified in LNCaP-AI cells after R1881 stimulation even at extremely low concentration (0.01 nmol/L; Fig. 6F). PADI2 knockdown (Fig. 6G) or Cl-Amidine treatment (Fig. 6H) could then partially abolish the AR-binding events in LNCaP-AI and C4-2B cells. Furthermore, the above treatments suppressed the citrullination of H3R26, methylation of H3K4 and acetylation of H3K9, but induced methylation of H3K27 (Fig. 6I and J). In addition, the occupancy of RNA Pol II decreased after siPADI2 or Cl-Amidine treatment (Supplementary Fig. S5). Taken together, our results demonstrated that PADI2 is required to activate AR signaling in the presence or absence of androgen.

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 μmol/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, and MET. *, 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.

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 μmol/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, and MET. *, 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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Combined inhibition of AR and PADI2 synergistically delays CRPC growth both in vitro and in vivo

Although enzalutamide has been approved for therapy of prostate cancer patients with CRPC, clinical applications revealed that approximately 30% to 40% of patients acquire resistance after a short period of treatment (2). Considering of the important role of PADI2 in CRPC, we then evaluated whether PADI2 inhibition potentiates the effect of enzalutamide treatment. LNCaP-AI cells were then given siPADI2 or Cl-Amidine, followed by enzalutamide treatment. As shown in Fig. 7A and B, siPADI2 or Cl-Amidine treatment significantly enhanced the effect of enzalutamide on reducing cell viability of LNCaP cells. We next evaluated the effects of cotargeting the AR and PADI2 using in vivo models under castration condition. As shown in Fig. 7C and D, Cl-Amidine plus enzalutamide treatment reduced the tumor volume (537.8 mm3) more effectively as compared with that of enzalutamide treatment alone (685.7 mm3) by 7 weeks (Fig. 7C and D). The weight in isolated tumors confirmed the above effects as well (Fig. 7E). These data suggested that combined inhibition of AR and PADI2 synergistically delays CRPC growth both in vitro and in vivo.

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.

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.

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Protein citrullination is emerging as one of the critical PTMs in developmental biology, inflammation, and cancer progression (8, 32, 33). The PADIs are a family of PTM enzymes that mediate citrullination (8). Five PAD family members (PADs 1–4 and 6) have been described (34) and are closely related with tumor progression. Among them, PADI4 is most extensively studied (35). PADI4 mainly acts as a transcriptional coregulator with p53, ELK1, or TAL1, possibly via histone citrullination after translocation into the nucleus (36–38). Recently, PADI2 had been identified as a potential target for breast cancer via citrullinating arginine residues 2/8/17 on histone H3 tails of PTN and MAGEA12 (17). Furthermore, PADI1 and PADI2 expression were significantly increased in mutant HRAS-driven skin carcinogenesis (39), further supporting the dynamic involvement of PADs in skin tumor. In addition, PADI2 overexpression in transgenic mice can promote spontaneous skin neoplasia by disturbing tumor microenvironment and facilitating epithelial-to-mesenchymal transition of tumor cells (15). More importantly, Zhang and colleagues reported that estrogen stimulation induced the recruitment of PADI2 to target promoters by ERα, whereby PADI2 then citrullinated H3R26, which led to local chromatin become less condensed for transcriptional activation (19). In this study, we further demonstrated that PADI2, but not PADI4, could promote protein stability, nuclear translocation, and transcriptional activation of AR in prostate cancer cells. Notably, citrullination of H3R26 contributes significantly to activate AR signaling. In addition, inhibition of citrullinating activity in its nuclear form of 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 to improve our understanding of how AR regulates gene transcription via altering chromatin structure.

AR signaling plays a central role for the survival and the growth of most CRPCs (2). Previously, others and we have reported that AR reactivation can occur through deregulation of coactivators in the absence of androgen, such as the P160 SRC (steroid receptor coactivator) family genes (40), the fork head protein FoxA1 (41) and transcription factor, GATA2 (42), and cochaperones, including HSP27 (43), HSP90 (44) and TXNDC5 (25), finally leading to CRPC progression. In this study, we provided several lines of evidence to suggest an important role of PADI2 in CRPC. First, clinical data suggested that PADI2 is significantly overexpressed in patients with CRPC. Second, PADI2 can activate AR signaling, under androgen-deprived conditions. Third, overexpressing PADI2 makes prostate cancer cells more adaptive to survive and proliferate in the absence of androgen and eventually led to CRPC-like phenotype. In contrast, suppression of PADI2 expression and activity inhibits the cell viability of CRPC cells more significantly than those of androgen-dependent prostate cancer cells. Together, these findings indicate that PADI2 is required for prostate cancer progression and CRPC.

Recently, a broad range of therapeutic options have become available for patients with CRPC in a variety of settings, including chemotherapeutic agents (cabazitaxel; ref. 45), abiraterone acetate (46), enzalutamide (47), and immunotherapy (sipuleucel-T; ref. 48). Among them, enzalutamide is a next-generation anti-androgen and a potent AR inhibitor. Although enzalutamide 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 or PAD inhibitors can delay prostate cancer progression in vitro and in vivo, suggesting that the inhibition of PADI2-mediated citrullination may have therapeutic effect in prostate cancer. Remarkably, our study demonstrated that Cl-Amidine treatment could suppress the activity of AR signaling, supporting the cell-specific effects of Cl-Amidine on prostate cancer cells. As Cl-Amidine has been identified to be held the potential to treat certain inflammatory diseases and cancers through targeting dendritic cells, T cells, and endothelial cells (49, 50), we cannot exclude the possibility that the antitumor effects of Cl-Amidine in vivo may be partially done through targeting some other signaling pathways.

Intriguingly, our study demonstrated that the combination of Cl-Amidine with enzalutamide results in synergistic inhibition of cell proliferation and tumor growth in vitro and in vivo under androgen-deprived or castration conditions. This combined treatment strategy may be potentially beneficial to a subset of CRPC patients with PADI2 overexpression. Further investigation will also be needed to evaluate whether PADIs inhibition is effective as a preventive strategy for prostate cancer progression, especially CRPC.

In conclusion, our study defines a novel mechanism that PADI2-mediated citrullination can promote prostate cancer progression and CRPC (Fig. 7F). Treatment with PADIs inhibitor can suppress prostate cancer progression. Most importantly, we suggested a novel potential therapeutic approach with combination of PADIs inhibitor with enzalutamide to treat prostate cancer patients with CRPC.

No potential conflicts of interest were disclosed.

Conception and design: L. Wang, J. Han, B. Han

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Feng

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Wang, G. Song, J. Pan, X Zhang

Writing, review, and/or revision of the manuscript: L. Wang, W. Chen, J. Yu, M. Yang, X. Bai, J. Han, B. Han

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Song, T. Feng, J. Pan, W. Chen, M. Yang, X. Bai, Y. Pang, J. Han

Study supervision: J. Han, B. Han

We thank Drs. Hao Dou, Yongxin Zou, and Yaoqin Gong (Department of Genetics, Shandong University Medical School) for valuable discussion of the study.

This work was supported by National Natural Science Foundation of China (grant nos. 81572544, 81672554, 81472417, 81528015 and 81572254), The Key Research and Development Project of Shandong (2016GSF201166), The Shandong Taishan Scholarship (tsqn20161076), and The Innovation Project of Shandong Academy of Medical Sciences.

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

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