Loss of a Negative Feedback Loop Between IRF8

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Loss of a Negative Feedback Loop between IRF8 and AR Promotes Prostate Cancer Growth and Enzalutamide Resistance Hongxi Wu1, Linjun You1, Yan Li1, Zhili Zhao1, Guangjiang Shi1, Zhen Chen1, Zhuo Wang1, Xianjing Li1, Shijia Du1, Wanli Ye1, Xiaofang Gao1, Jingjing Duan1, Yan Cheng1, Weiyan Tao1, Jinsong Bian2, Jin-Rong Zhou3, Qingyi Zhu4, and Yong Yang1

ABSTRACT ◥ In incurable castration-resistant prostate cancer (CRPC), therapy, highlighting the therapeutic potential of IFNa in target- resistance to the novel androgen receptor (AR) antagonist ing IRF8–AR axis in CRPC. enzalutamide is driven mainly by AR overexpression. Here we Graphical Abstract: http://cancerres.aacrjournals.org/content/ report that the expression of interferon regulatory factor 8 canres/80/13/2927/F1.large.jpg. (IRF8) is increased in primary prostate cancer but decreased in CRPC compared with normal prostate tissue. Decreased expression of IRF8 positively associated with CRPC progression and enzalutamide resistance. IRF8 interacted with AR and promoted its degradation via activation of the ubiquitin/ proteasome systems. Epigenetic knockdown of IRF8 promoted AR-mediated prostate cancer progression and enzalutamide resistance in vitro and in vivo. Furthermore, IFNa increased expression of IRF8 and improved the efficacy of enzalutamide in CRPC by targeting the IRF8–AR axis. We also provide preliminary evidence for the efficacy of IFNa with hormonotherapy in a clinical study. Collectively, this study identifies IRF8 both as a tumor suppressor in prostate cancer pathogenesis and a potential alternative therapeutic option to overcome enzalutamide resistance.

Signiﬁcance: These ﬁndings identify IRF8-mediated AR degradation as a mechanism of resistance to AR-targeted

Introduction Despite the initial efficacy of ADT, regeneration of tumor will occur eventually in almost every patient, leading to alleged castration- Androgen and androgen receptor (AR) signaling pathway play a resistant prostate cancer (CRPC). critical role in the carcinogenesis and progression of prostate cancer, AR-targeted therapy is a gold standard therapy for CRPC (2–4). which is the second leading cause of cancer-related deaths in North Enzalutamide is approved for the treatment of patients with CRPC, America (1). Consequently, androgen depletion therapy (ADT) has based on its ability to block androgen binding to AR in a competitive been the first-line therapy for primary prostate cancer for decades. manner, inhibiting AR nuclear translocation and DNA fixation (5–7). Despite the success of enzalutamide in improving the overall survival of patients with CRPC, inherent or acquired enzalutamide resistance 1State Key Laboratory of Natural Medicines, China Pharmaceutical University, – 2 (ENZR) remains a major clinical challenge (8 10). AR deregulation, Nanjing, China. Department of Pharmacology, Yong Loo Lin School of Medicine, fl National University of Singapore, Singapore. 3Nutrition/Metabolism Laboratory, including overexpression (OE) of AR full-length (AR ) and AR fi Department of Surgery/General Surgery, Harvard Medical School, Boston, variants (ARv), has been identi ed as a unique factor consistently Massachusetts. 4Department of Urology, Jiangsu Province Hospital of Tradi- associated with the progression of prostate cancer to CRPC and tional Chinese Medicine, Nanjing, China. ENZR (9); therefore, studying the mechanisms of AR deregulation Note: Supplementary data for this article are available at Cancer Research is critical to improve the efficacy of current treatments. AR undergoes Online (http://cancerres.aacrjournals.org/). degradation mainly by the ubiquitin/proteasome system, as well as fi H. Wu, L. You, Y. Li, and Z. Zhao contributed equally to this article. modi cation of protein stability triggered by ubiquitin-like signaling pathways, such as ISGylation (Interferon stimulated gene; refs. 11, 12). Corresponding Authors: Yong Yang, China Pharmaceutical University, 24 Tongjia Street, Nanjing, Jiangsu 210009, China. Phone/Fax: 8625-8618-5622; AR is a type I IFN-regulated protein and disruption of IFN system E-mail: [email protected]; and Qingyi Zhu, [email protected] genes plays a novel function in malignant transformation of prostate cancer (12–15). However, the mechanism for AR maintaining its Cancer Res 2020;80:2927–39 stability under the influence of IFN system remains unknown. doi: 10.1158/0008-5472.CAN-19-2549 Activity of the IFN system is mainly regulated by IFN regulatory 2020 American Association for Cancer Research. factor 8 (IRF8), a member of the IRF family (IRF1-9; ref. 16). Loss of

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IRF8 in immune cells leads to the occurrence of chronic myelogenous Flat clone formation assay leukemia and aberrant methylation of IRF8 gene in nonhemopoietic LNCaP-shNC and LNCaP-shIRF8B cells were plated in the 6-well cells play an increasingly important role in tumorigenesis (17–25). plates in triplicate with 500 cells every well and cultured with complete Among all the IRFs, only IRF8 facilitates a protein–protein interaction medium for 14 days. For enzalutamide sensitivity experiment, once model, which interacts with proteins containing the PEST motif, a cells were attached, enzalutamide (10 mmol/L) were added to the media region that plays an important role in protein degradation by the using 0.1%DMSO as control. The complete medium or culture proteasome system, including AR degradation (26, 27). Emerging medium containing enzalutamide or DMSO was replaced every 2 days, evidences suggest that IRF8 may function in the cytosol ubiquitylation and cells were cultured for 10 days. Crystal violet was used to stain the system (16, 28), but the exact role and the regulation mechanism of colonies. Colony number and the inhibition effects of enzalutamide IRF8 in cancers, especially in prostate cancer tissues, have not been were calculated according the number of the cloning formation explored. More importantly, the relationship between IRF8 with AR in measured by ImagePro plus software. prostate cancer and whether IRF8 interacts with AR (containing the PEST motif) and functions in the regulation of AR stability are still Western blot analysis unknown. Briefly, whole cell lysates were prepared with ice-cold RIPA lysis This study provides evidence to support an advantageous role for buffer or nuclear and cytoplasmic protein extraction lysis buffer with IRF8inCRPCandENZR,andthisroleofIRF8islikelymediated protease inhibitors (Roche). Approximately, 30 mg of total protein was through regulating AR stability.Wethenevaluatedthepotential separated by SDS-PAGE and transferred to polyvinylidene difluoride of IFNa targeting IRF8 to improve the therapeutic efficacy of transfer membrane and the membranes were incubated with specific hormonotherapy in prostate cancer, suggesting that IFNa com- antibodies for IRF8 (ab28696, Abcam), AR (ab74272, Abcam), bined with enzalutamide is an attractive therapeutic strategy for p-STAT1(Tyr701), p-STAT1 (Ser727), STAT1 (Cell Signaling Tech- CRPC and ENZR. nology), and b-actin (bsm-33036M, Bioss). Each experiment was repeated at least twice with similar results. The densitometry data presented below the bands are “fold change” as compared with control Materials and Methods normalized to respective b-actin from two independent Western blot Reagents analyses. Values are expressed as mean. FBS and charcoal-stripped, dextran-treated FBS (CSS, depleted androgen, and any other steroid) were purchased from Biological RNA purification and qPCR Industries. Enzalutamide (MedChemExpress), R1881, DHT (Meilun- Total RNA was prepared by using TRIzol reagent (Invitrogen) bio), EGF, and IGF-1 were commercially obtained (Peprotech). Serial according to the manufacturer's instructions. RNA was reverse tran- dilutions of all drugs were made using DMSO. scribed into cDNA using random hexamers, which was used for qPCR using gene-specific primers. Data were normalized by the level of Cell lines and primary cultures GAPDH expression in each sample. The primer sequences used in this PC3 cells were cultured in F12 Medium, HEK293T were cultured in study are as follows: IRF8 forward 50- TCG GAG TCA GCT CCT TCC DMEM, and 22RV1 and LNCaP cells were cultured in RPMI1640 AGA CT -30, reverse 50- TCG TAG GTG GTG TAC CCC GTC A -30; Medium. PC3, 22RV1, LNCaP, and HEK293T cells were purchased AR forward 50- CTA CTC TTC AGC ATT ATT CCA G -30, reverse from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), CAT GTG TGA CTT GAT TAG CA- 30; GAPDH, forward 50- CAT two stable LNCaP-sh-IRF8-Puro (LNCaP-shIRF8) and LNCaP- GAG AAG TAT GAC AAC AGC CT -30, reverse 50- AGT CCT TCC sh-negative control (LNCaP-shNC)-Puro cells were purchased from ACG ATA CCA AAG T -30. GenePharma Technology Co. Ltd. (GenePharma). All cells were authenticated by the short tandem repeat DNA profiling (Cobioer) Immunoprecipitation and confirmed Mycoplasma free using GMyc-PCR Mycoplasma Test Cells treated with the appropriate stimuli were lysed with Western Kit (YeSen, 40601ES10) after last experiment, and used within 15 cell blot analysis and immunoprecipitation (IP) lysis buffer (Beyotime). passages after thawing. All cell lines were cultured in medium sup- Aliquots of 600 mg protein from each sample were precleared by plemented with 10% FBS, 1% penicillin-streptomycin (Gibico), and incubation with 20 mL of Pierce Protein A/G magnetic beads (88803; 5% CO2 at 37 C. Thermo Fisher Scientific) for 1 hour at 4 C and then incubated with anti-IRF8 antibody (5628s, Cell Signaling Technology), or anti-AR Plasmids, siRNA, and DNA transient transfections (ab74272, Abcam) in lysis buffer at 4C overnight. Protein A/G beads Plasmids including pcDNA3.1 (Vector), pcDNA3.1-hIRF8 (IRF8), were added and incubated for 2 hours at 4C. The beads were washed and pcDNA3.1-hAR (AR), containing the whole CDS domains were five times with PBS and once with lysis buffer, boiled, separated by 10% constructed by Genscript; double luciferase reporter plasmid pEZX- SDS-PAGE, and analyzed by Western blotting as described above. For FR03-hIRF8-luc, carrying the IRF8 promoter (-1106-þ166 bp), was in vitro binding assays, purified recombinant Flag-tagged AR protein constructed by FulenGen; 3xFlag-Ub plasmid was generously provid- was incubated with recombinant His-tagged IRF8, and anti-flag ed by Dr. Guo-qiang Xu (Soochow University, Suzhou, China). For agarose beads (Bimake) in buffer C. Bound immunocomplexes were transient transfections, cells were seeded into 6-well plates at 150,000 washed three times with buffer B for nuclear extracts or buffer cells per well and transfected with IRF8 or AR expression vectors using C-400 mmol/L NaCl for total protein extracts IP and in vitro binding empty vector (pvector) as control. The total plasmid DNA was assays. adjusted to the same with empty vector. siRNA and shRNA targeting IRF8 are as follows: siNC (shNC): TTC Chromatin immunoprecipitation assay TCC GAA CGT GTC ACG TTT C; siRNA1# (shIRF8A): GCA GTT The chromatin immunoprecipitation (ChIP) assay was performed CTA TAA CAG CCA GGG; siRNA2# (shIRF8B): GGG AAG AGT using the EZ-Magna ChIP A/G Chromatin Immunoprecipitation TTC CGG ATA TGG. Kit (Millipore) according to the manufacturer's protocol. Anti-AR

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antibodies and normal IgG were used to precipitate DNA. The normal for immunoblot detection with anti-AR or other antibodies as indi- IgG was used as a negative control and the PSA enhancer region was cated at 4C overnight, using 30 mg nuclear protein as input and Lamin used as a positive control. The precipitated DNA was subjected to PCR B as negative control. to amplify the PSA and IRF8 promoter regions with the primers listed as follows, and were quantified with the qPCR using SYBR Green. PSA Transient transfections for IRF8 promoter activity Enhancerþ: TGG GAC AAC TTG CAA ACC TG, PSA Enhancer: HEK293T cells (1 107/100 mm dish) were precultured with 5% CCA GAG TAG GTC TGT TTT CAA TCC A; IRF8 (-872/-863 bp) F: CSS overnight and transfected with pcDNA3.1-AR (1 mg). Cells were 50- TGT GTG ATT CTC TAC TGG GCA A -30,R:50- CTG GAA ACG cotransfected with an artificial promoter luciferase reporter with both GAA AAA GAA GCG T -30; IRF8 (-852/-843 bp) F: 50-TTT CTT TTT the proximal IRF8 50 flank (flanking regions of IRF8 (1106/þ166 bp) CCA GTG TCG TTC TCC -30,R:50- GGG CGT TAA GAT GTC CCC and the firefly/renilla luciferase (pEZX-FR03-hIRF8, 3 mg) in the same T-30; IRF8 (-896/-783 bp) F: 50- GGA TAG AAC GCG GAA ACG CT vector. The empty reporter vector (pEZX-FR03) was used as a control. -30,R:50- CCT GGG TGT GCA CTG ACA TTT A -30. The transfected cells were cultured with 5% CSS for 24 hours and then seed into 96 wells (1 104 cells/well) and treated with DHT (1 nmol/L, IHC of IRF8 and AR protein expression in prostate cancer cells 10 nmol/L); R1881(1 nmol/L, 10 nmol/L); IGF-1(10 nmol/L); EGF Segregation of clinic specimens. (10 nmol/L) with or without enzalutamide (10 mmol/L). DMSO was A total of 13 benign prostatic hyperplasia (BPH), 20 untreated primary used as a control. After incubation with these stimuli for 24 hours, the prostatic cancer tissues, and 13 CRPC paraffin-embedded tissues were transfected cells were collected and AR transcriptional activity was collected from the Departments of Urinary surgery at Jiangsu Province detected by dual luciferase assay. Hospital of Traditional Chinese Medicine (TCM; Jiangsu, China), which is approved by the Institutional Review Board of the Jiangsu Province Transient transfections for AR activity Hospital of TCM for IHC and MSP-PCR analysis. LNCaP cells (1 107/100 mm dish) were precultured with 5% CSS overnight and transfected with various combinations of vectors to Tissue microarray. express IRF8 or control (0.75, 3 mg), IRF8-specific shRNA or scram- Protein expression of IRF8 and AR in clinical prostate cancer tissues bled control (3 mg). Cells were cotransfected with an androgen- were determined using tissue microarray analysis (TMA, Shanghai dependent firefly luciferase reporter (pMMTV-Luc, 3 mg, for AR OUTDO Biotech), containing 64 paired human primary prostate activity as described previously; ref. 31) and a renilla luciferase cancer/adjacent noncancerous lesion tissues. Tissue microarray were promoter (pRL-SV40, 0.04 mg, for transfection efficiency). The trans- stained with hematoxylin and eosin to verify histology and the IHC fected cells were cultured with 5% CSS for 24 hours and then seeded staining of IRF8 (ab28696; Abcam) and AR (ab74272) were performed. into 96 wells (1 104 cells/well) and treated with or without DHT The positive staining rate was estimated in three fields with different (10 nmol/L). staining intensity by pathologists. The staining of IRF8 and AR in the Luciferase activities were measured 24 hours after treatment using TMA was scored independently by two pathologists blinded to the the dual luciferase reporter system according to the manufacturer's clinical data using the following criterion: the intensity of immunos- instructions (Promega). All samples were tested in triplicate. taining was scored from 0 to 3 (0, negative; 1, weak; 2, moderate; and 3, strong); the percentage of immunoreactive was deemed as 0 (0%–5%), Xenografts and animal model 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The All athymic nu/nu BALB/c (BALB/c nude) and nonobese diabetic final score was calculated using the percentage score staining severely combined immunodeficient (NOD-SCID) mice, ages 4– intensity score as described previously (29). For tissue samples from 6 weeks, were purchased from Lingchang biotech and housed in a patient with clinical prostate cancer and animal xenografts, the specific pathogen-free facility and maintained in a standard temper- primary antibodies of the anti-IRF8 antibody (Abcam, 1:200), anti- ature and light-controlled animal facility for 1 week before used. For AR antibody (Abcam, 1:200) or anti-PCNA antibody (Santa Cruz transgenic animal experiment, the Hi-myc mice [FVB-Tg (ARR2/ Biotechnology, 1:200), were used for staining at 4C overnight. Pbsn-MYC) 7Key, strain number: 01XK8] were obtained from the Immunostaining pictures were acquired using an inverted fluores- Mouse Repository of the NCI. All animal procedures were performed cence microscope (Leica) and the integral optical density (IOD) sum according to the guidelines of the U.S. NIH on Animal Care and was calculated using ImagePro plus software. The average diamino- appropriate institutional certification, with the approval of the Com- benzidine staining intensity of the selected two cores represents the mittee on Animal Use and Care of Center for New Drug Evaluation quantitative protein expression level in these tissues. and Research, China Pharmaceutical University (Nanjing, China). For tumor growth of LNCaP cell xenograft (growing in intact DNA affinity binding assay BALB/C nude mice), 1107 LNCaP-shNC or LNCaP-shIRF8A cells Nuclear extracts and affinity binding assays were prepared as (with higher KD efficiency, indicated as LNCaP-shIRF8) were resus- described previously (30). Briefly, LNCaP cells were treated with 10 pended in 100 mL medium containing 50% matrigel (BD Biosciences) nmol/L DHT or 2,000 IU/mL IFNa2a for 48 hours, using ethanol and and 50% growth media and subcutaneously injected in the flank of medium as control, respectively, and lysed with ice-cold nuclear BALB/c nude mice. For the tumor growth of LNCaP-CRPC xenograft protein extraction lysis buffer. 4.5-mg aliquots DNA formed by 8 pairs (growing in surgically castrated BALB/C nude mice with low serum of the ISRE and ISRE-like primers [2 wild-type (WT) and 6 mutants, androgen), BALB/c nude mice were anesthetized using 10% chloral Mut] were conjugated to streptavindin M280 magnetic beads (Dynal) hydrate. Testes were excised distal to the ligature, and the incision was in the presence of 20 mg of salmon sperm DNA (Sigma) for 10 minutes closed with sterile dissolvable sutures and disinfected with betadine at room temperature. DNA-coupled magnetic beads were incubated solution and ampicillin. Two weeks later, LNCaP-shNC or LNCaP- with 1 mg of nuclear extracts for 1 hour at 4C. Beads were washed shIRF8 cells were inoculated as described above. three times with washing buffer and bound materials were eluted In the enzalutamide sensitivity study, orchiectomized BALB/c or in 1 SDS sample buffer. Samples were separated by 8% SDS-PAGE NOD/SCID male mice were inoculated subcutaneously with 1 107

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LNCaP or LNCaP-shNC or LNCaP-shIRF8 cells. Once tumors were the representative CRPC specimens (Fig. 1C; Supplementary established, mice were randomly assigned to vehicle (1% carboxymethyl Fig. S1H). A positive correlation between IRF8 and AR in primary cellulose, 0.1% Tween-80, 5% DMSO), and enzalutamide (10 mg/kg) prostate cancer was confirmed. In contrast, statistically significant treatment groups. In the sensitivity study of enzalutamide combined negative correlation was observed between protein levels of IRF8 IFNa, mice were treated with vehicle alone, enzalutamide (10 mg/kg) versus AR in CRPC specimens (Fig. 1D). Similarity to CRPC, IRF8 alone, IFNa (1.5 107 IU/kg) alone or enzalutamide (10 mg/kg) was decreased but AR was increased in ENZR xenograft tissues and an combined with IFNa (1.5 107 IU/kg). Enzalutamide and IFNa were inverse correlation between IRF8 with AR protein was observed administered by oral gavage and subcutaneously daily, respectively. (Fig. 1E; Supplementary Fig. S1I and S1J). Tumor volume measurements were performed every 2 days and were Previous findings revealed that epigenetic mechanisms including calculated by the formula: length width2/2. At experimental endpoint, mutation and promoter methylation play a crucial role in the IRF8 tumors were harvested and tumor inhibition ratio were calculated and expression (19, 25, 32–35). However, IRF8 was not frequently mutated compared with the average tumor weight in the vehicle control. and methylated in clinical prostate cancer tissues and cells, using methods of ref.1 in the Supplementary Table (Supplementary Fig. S1K; Clinical case study Supplementary Table). Lots of AR CHIP-seq data reveal that IRF8 is a Three advanced and untreated patients with prostate cancer with putative target of AR upon DHT or R1881 treatment (36–38). Fur- bone metastasis were enrolled at the Department of Urinary Surgery at thermore, we found several AR-binding sites at the promoter of IRF8 Jiangsu Province Hospital of TCM (Jiangsu, China), and written using JASPAR database. We next tested whether IRF8 expression is informed consent was received from participants prior to inclusion regulated upon AR activation and repression. AR activation by DHT in the study, in accordance with the guidelines of the International increased IRF8 mRNA and protein levels in prostate cancer cells Ethical Guidelines for Biomedical Research Involving Human Subjects (Fig. 1F; Supplementary Fig. S2A–S2C). However, AR repression by (CIOMS), with the approval of the Institutional Ethics Review Boards enzalutamide treatment decreased IRF8 expression (Fig. 1G; Supple- of Jiangsu Province Hospital of TCM (number 2017NL-054-02). mentary Fig. S2C). Inclusion criteria were histologically confirmed prostate adenocarci- In CRPC, AR can be bypass-activated by growth factors (GF) such as noma, untreated, cT3-cT4 M0 and M1, serum PSA ≥ 150 ng/mL, EGF, IGF-1, at low levels of androgen after ADT (39–42). In the Gleason score ≥ 9(4þ5or5þ4), age ≤ 80 years, and normal liver experiments, we treated LNCaP cells with these factors and found that function not suitable for definitive treatment. For case 1 clinical study, AR activation by GFs reduced IRF8 protein levels (Supplementary 2 patients received Goserelin Acetate Sustained-Release Depot Fig. S2D). We further identified whether these factors regulate IRF8 (3.6 mg/28 days) plus bicalutamide (50 mg/days; Casodex) as maximal transcription by AR signaling, and the results showed that DHT and androgen blockade (MAB) therapy, one patient received MAB therapy R1881-induced AR activation promoted IRF8 transcription but bypass plus recombinant IFNa2a (3106 IU/3 days; Yintefen). For case 2 AR activation by GFs inhibited IRF8 transcription. In contrast, clinical study, one advanced and untreated patients with prostate enzalutamide reduced IRF8 transcription, reversed androgen- cancer with continued PSA level rise for 6 months were enrolled and mediated IRF8 upregulation and enhanced GFs-mediated IRF8 down- received MAB therapy plus recombinant IFNa2a (3106 IU/3 days; regulation (Fig. 1H). Yintefen). Patients in both cohorts were discontinued when there was IRF8 transcription is mainly regulated by the binding of p-STAT1 to evidence of biochemical progression, defined as an increase of the PSA the IFN-stimulated response element motif (ISRE, TTTC A/G G/C level ≥ 0.2 ng/mL on two successive occasions at least 1 month apart. TTTC; ref. 16). Predictably, one canonical ISRE motif and one ISRE- like motif were identified in the IRF8 promoter (Supplementary Fig. S2E). In our experiments, the amounts of AR bound to the ISRE Results and ISRE-like motifs were markedly increased after DHT treatment, IRF8 expression is increased in primary prostate cancer but whereas binding by AR were not increased upon GFs stimulation and decreased in CRPC and ENZR tissues only p-ARSer81 were activated by DHT (Supplementary Fig. S2F and To reveal the relationship between IRF8 and AR protein expression S2G). Furthermore, we found mutations of the GAAA motif greatly in prostate cancer, prostate cancer tissue microarrays were immuno- diminished binding of AR to the ISRE-like motif while AR binding to histochemically assessed using optimized anti-IRF8 and anti-AR ISRE mutant was not reduced (Fig. 1I), indicating the ISRE-like motif antibodies. The representative primary prostate cancer specimens has stronger affinity for AR. We further determined whether the IRF8 showed high IRF8 expression and high AR accumulation compared is a direct AR-targeted gene using ChIP-qPCR assays. The results with the adjacent nontumor tissue (Fig. 1A; Supplementary Fig. S1A showed that AR was recruited to the ISRE and ISRE-like sites and S1B). Tumor samples with high expression of AR protein exhib- regardless of DHT treatment, whereas occupancy of the ISRE-like ited more IRF8 accumulation and a positive correlation was observed site was markedly increased and occupancy of the ISRE site was not between protein levels of IRF8 versus AR in primary prostate cancer changed in LNCaP cells upon DHT stimulation (Fig. 1J; Supplemen- specimens (Supplementary Fig. S1C and S1D). Similar findings con- tary Fig. S2H and S2I). Together, these results identified AR as a factor firmed that IRF8 and AR were increased in tumor tissues of Hi-myc that is recruited to the ISRE-like motif upon DHT stimulation, which transgenic (TG) murine prostate cancer models during the prostate promotes IRF8 transcription in an androgen–AR dependent pathway. cancer progression (≥6 months; Fig. 1B; Supplementary Fig. S1E and S1F). Moreover, in LNCaP-CRPC xenografts models, IRF8 expression IRF8-knockdown promotes the proliferation and tumorigenesis was significantly decreased in tumors treated with enzalutamide of LNCaP cells through AR activation (Supplementary Fig. S1G). To clarify this point, we quantified IRF8 To functionally demonstrate the potential role of IRF8 in tumor- and AR protein levels using IHC of paraffin-embedded clinical BPH, igenesis of prostate cancer, we generated two stable IRF8-knockdown primary prostate cancer and CRPC tissues. The results showed that (IRF8-KD) LNCaP cell lines (Fig. 2A; Supplementary Fig. S3A). The IRF8 and AR were increased in primary prostate cancer tissues proliferation ratios of LNCaP-shIRF8 cells were higher than those in compared with BPH, while low IRF8 but high AR were observed in LNCaP-shNC cells (Fig. 2B; Supplementary Fig. S3B). Furthermore,

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Figure 1. IRF8 expression is increased in primary prostate cancer but decreased in CRPC and ENZR tissues. A, Representative IHC of IRF8 and AR protein expression in primary prostate cancer samples compared with adjacent noncancerous lesion tissues from prostate cancer TMAs. Scale bar, 100 mm. B, IHC of IRF8 and AR protein expression in WT and Hi-myc (TG) mice prostate tissues. Scale bar, 100 mm. C, IHC staining of IRF8 and AR protein expression in BPH (n ¼ 11), primary prostate cancer (n ¼ 22), and CRPC tissues (n ¼ 11). Scale bar, 100 mm. D, Correlation of IRF8 with AR expression normalized by the average IOD of BPH tissues in primary prostate cancer and CRPC specimens was analyzed, and linear regression coefficient and statistical significance are indicated. E, IHC staining of IRF8 and AR protein expression in ENZR xenograft tissues (n ¼ 6). Scale bar, 100 mm. F, LNCaP cells, precultured with 5% CSS for 3 days, were treated with DHT for 24 hours and IFR8 expression was analyzed by qPCR (left) and Western blot analysis (right). G, IRF8 mRNA (left) and protein expression (right) in LNCaP cells cultured in the presence of the indicated concentration of enzalutamide (ENZ) for 24 hours. H, HEK293T cells cotransfected with pcDNA3.1-AR and IRF8 promoter luciferase reporter were treated in triplicate with DHT (1, 10 nmol/L), R1881 (1, 10 nmol/L), IGF-1 (10 nmol/L), and EGF (10 nmol/L) with and without enzalutamide (10 mmol/L) for 24 hours. Luciferase activities were measured 24 hours after treatment. I, Immobilized WT and mutant ISREs were incubated with nuclear extracts from LNCaP cells treated with DHT for 24 hours, and bound proteins were detected by IB with anti-AR. J, ChIP analysis to detect AR binding to the IRF8 promoter. LNCaP cells were stimulated by DHT for 48 hours. Equal amounts of chromatin (DNA) were subjected to ChIP assay with AR-specific antibody. Normal IgG and protein A/G beads alone were used as negative controls. AR occupancy of IRF8 promoter is shown relative to background signal with normal IgG control antibody. PCa, prostate cancer. or #, P < 0.05; or ##, P < 0.01; or ###, P < 0.001; calculated using one-way ANOVA; ns, not significant.

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Figure 2. IRF8-KD in LNCaP cells promotes the growth and tumorigenesis in vivo and in vitro. A, Knockdown efﬁciency in LNCaP-shIRF8 cells determined by Western blot analysis. B, Absorbance at 450 nm of LNCaP-shIRF8 cultured with 5% FBS (left), 5% CSS (middle), or 5% CSS containing 1 nmol/L R1881 (right) in 96-well plates for 24– 120 hours. C, Representative images of clone formation of LNCaP-shNC and LNCaP-shIRF8 cells cultured for 10 days. D, Representative sphere formation of LNCaP- shNC and LNCaP-shIRF8 cells cultured for 14 days. E and F, Knockdown of IRF8 in tumorigenesis. Tumor growth of LNCaP-shIRF8 cells in BALB/c nude mice (n ¼ 9; E) and in castrated BALB/c nude mice (n ¼ 9; F). G, Representative images of IHC staining of PCNA in LNCaP-shIRF8 BALB/c xenografts. Scale bar, 100 mm. H and I, OE of IRF8 in tumorigenesis. Proliferation (H) of 22RV1-IRF8-OE cells in vitro and tumor growth of 22RV1-IRF8-OE cells in vivo (n ¼ 9; I). J, Western blot analysis for p-P38 MAPK, P38 MAPK, p-ERK, ERK, p-AKT(Thr308), p-AKT(Ser473), AKT, CD133, Oct3/4, and b-actin in LNCaP-shNC and LNCaP-shIRF8 cells. , P < 0.05; , P < 0.01; , P < 0.001; ns, not signiﬁcant.

increased clonogenic ability, number, and diameter of tumor cell S3G), whereas 22RV1-IRF8- OE inhibited the proliferation, clone spheres were also observed in LNCaP-shIRF8 cells (Fig. 2C and D; formation, and tumor growth (Fig. 2H and I; Supplementary Supplementary Fig. S3C and S3D). In vivo xenograft experiments Fig. S3H). Moreover, IRF8-KD also increased P38/MAPK/ERK phos- showed that LNCaP-shIRF8 results in increased tumor growth and phorylation and CD133 expression compared with LNCaP-shNC cells proliferating cell nuclear antigen (PCNA) protein expression in intact (Fig. 2J), indicating that intrinsic IRF8 plays an important role in or castrated BALB/c nude mice (Fig. 2E–G; Supplementary Fig. S3E– inhibiting prostate cancer progression.

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Figure 3. IRF8 reduces AR protein levels and activity. A, Western blot analysis for IRF8 and AR in the LNCaP-shIRF8 stable cell line for whole cell lysate (WCL), cytoplasm protein (Cyto), and nuclear protein (Nuc), b-actin, GAPDH, and lamin B were used as reference proteins, respectively. B, Western blot analysis for IRF8 and AR in lysates of LNCaP cells or IRF8 and AR (ARﬂ and ARvs) in 22RV1 cells 48 hours after transfection with IRF8 plasmid (pIRF8) at various amounts (0.125–1 mg). C, Western blot analysis of IRF8 and AR in HEK293T cells cotransfected with AR plasmid (0.3 mg) and different concentrations of IRF8 plasmid (0.125–1 mg) for 24 and 48 hours. D, Western blot analysis of exogenous IRF8 and AR in PC3 cells transfected with AR plasmid (0.3 mg) and IRF8 plasmid (1 mg) alone or in combination for 48 hours. E, Western blot analysis of AR dimers under native polyacrylamide gel electrophoresis conditions in HEK293T cells transfected with pAR, pIRF8, and pFlag-Ub vector alone or in combination. F, Western blot analysis of PSA, AR, and IRF8 in LNCaP cells transfected with siRNA targeting IRF8 for 24 hours. G, Luciferase reporter assays for AR activity in IRF8-KD (LNCaP-shIRF8; left) and IRF8-OE (LNCaP-IRF8; right) LNCaP cells treated with DHT. , P < 0.05; , P < 0.01; , P < 0.001 by one-way ANOVA; ns, not signiﬁcant.

To elucidate the mechanisms responsible for the growth-accelerating IRF8-mediated AR protein degradation could affect AR homodimer- effects of IRF8-KD in prostate cancer, we assessed the effects of IRF8 on ization. So we next detected effect of IRF8 on AR transcriptional AR protein expression. IRF8-KD increased and IRF8-OE decreased activity. The AR-targeted PSA is the most important marker for endogenous AR including ARvs expression, especially in the nucleus prostate cancer assessment. As expected, we found that PSA and AR (Fig. 3A and B; Supplementary Fig. S4A and S4B). Moreover, we protein were increased by IRF8 siRNA (Fig. 3F). We next explored AR demonstrated that IRF8 can reduce ectopic AR protein levels in AR activity using the pMMTV-luc luciferase reporter in IRF8-KD and HEK293T and PC3 cells transfected with AR alone or combined with IRF8-OE LNCaP cells. IRF8-KD increased and IRF8-OE decreased the various amounts of IRF8 expression plasmids (Fig. 3C and D). DHT-dependent AR activity after androgen stimulation (Fig. 3G; Androgen binding to AR results in homodimerization and nuclear Supplementary Fig. S4C and S4D), which was associated with nuclear translocation, and subsequent induction of target gene expression, AR expression mediated by IRF8 in LNCaP cells. which is regulated by the ubiquitin/proteasome system (27). Native polyacrylamide gel electrophoresis of lysates from HEK293T cells IRF8 physically interacts with AR and enhances AR degradation transfected with AR and IRF8 showed that decreased level of AR by ubiquitin-dependent pathways protein mediated by IRF8 resulted in decreased expression of AR IRF8-mediated AR reduction could not be reversed by DHT dimers in a high molecular weight complex to a degree equal to the treatment in both the cytosol and nucleus, suggesting that IRF8 has reduction of ubiquitin-mediated AR dimers (Fig. 3E), indicating that no effect on AR translocation (Supplementary Fig. S4E and S4F),

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2934 Cancer Res; 80(13) July 1, 2020 CANCER RESEARCH IRF8 Inhibits Prostate Cancer Progress

while the presence of IRF8 accelerated AR degradation and short- between AR and its E3 ligase MDM2 and thus promoted the ubiqui- ened the half-life of AR (Fig. 4A and B; Supplementary Fig. S5A and tination and degradation of AR. S5B). IRF8-mediated AR degradation was reversed by proteasome inhibitor MG132, but MG101, calpastatin, and lysosomal enzyme Low expression of IRF8 in LNCaP cells induces resistance to inhibitor leupeptin had no effect on IRF8-mediated AR degradation enzalutamide treatment (Fig. 4C and D; Supplementary Fig. S5C). These results indicated We further explored the effects of IRF8 on sensitivity to enzaluta- that the ubiquitin/proteasome involved in the IRF8-mediated AR mide therapy in vitro and in vivo. LNCaP viability treated with degradation. enzalutamide was decreased, whereas LNCaP-shIRF8 cells were resis- Cytoplasmic AR undergoes proteasome-mediated degradation in the tant to enzalutamide treatment with increased AR expression (Fig. 5A absence of ligand. Our results showed that ubiquitin or IRF8-mediated and B; Supplementary Fig. S6A and S6B). Moreover, the IRF8-OE AR degradation could be reversed by MG132 and DHT (Supplementary 22RV1 cell was more sensitive to enzalutamide treatment (Supple- Fig. S5D and S5E), indicating that IRF8 and ubiquitin regulate the same mentary Fig. S6C). Furthermore, the colony number with enzaluta- cellular machinery responsible for cytoplasmic AR degradation. The mide treatment was significantly decreased in LNCaP-shIRF8 cells subcellular colocalization of IRF8 and AR were both observed by compared with controls (P < 0.05; Fig. 5C; Supplementary Fig. S6D immunofluorescent staining in LNCaP cells and ectopically overex- and S6E). Notably, IRF8-KD decreased and IRF8-OE promoted pressed HEK293T cells (Fig. 4E; Supplementary Fig. S5F). We further caspase-3 activity induced by enzalutamide treatment (Fig. 5D found that IRF8 could precipitate with AR and vice versa (Fig. 4F). To and E). We further confirmed that IRF8-KD could induce ENZR in confirm this interaction, we proved that IRF8 could be eluted with AR castrated BALB/c nude and NOD-SCID mice. The enzalutamide in vitro binding assays (Supplementary Fig. S5G). To identify region treatment inhibited tumor growth of LNCaP-shNC (P < 0.05), while within AR responsible for its binding to IRF8, we cotransfected IRF8 it did not significantly alter the growth of LNCaP-shIRF8 tumors with several N-terminal Flag-tagged AR-truncated mutants, including (Fig. 5F; Supplementary Fig. S6F and S6G). ARfl, AR-NTD, AR-DBD, AR-Hinge, and AR-LBD into HEK293T cells (Supplementary Fig. S5H). Co-IP assay showed that IRF8 was immu- IFNa enhances enzalutamide sensitivity by targeting the IRF8– noprecipitated with ARfl, AR-NTD but not with AR-LBD, AR-Hinge, AR axis and AR-DBD (Fig. 4G). These results indicated that IRF8 physically Because decreased IRF8 induces ENZR during ADT, we next interacts with the N-terminal domain of AR. We next analyzed the effect explored a novel strategy to attenuate the resistance. The IRF8 of IRF8 on AR ubiquitylation. Results showed that IRF8 enhanced AR expression could be induced by IFNs through phosphorylation and polyubiquitination as visualized by staining with Flag-ubiquitin dimerization of STAT1 (p-STAT1Tyr701 and p-STAT1Ser727; (Fig. 4H). There are two common types of polyubiquitination, which refs. 19, 44, 45). IFNg receptor is frequently inactivation but expression are distinguished by the lysine residue through which formation of K48- of IFNA receptors is much higher than IFNg receptors in human chain and K63-chain occurs. Immunoblotting (IB) revealed that both prostate cancer cells (1, 3). Fundamental studies have shown IFNa WT and K48 ubiquitin led to polyubiquitinated AR, but polyubiqui- phosphorylates STAT1 (p-STAT1Tyr701 and p-STAT1Ser727) and tination was impaired in the presence of K63 ubiquitin (Fig. 4I), activates IRF8 gene expression by binding to the GAS element in suggesting that IRF8-mediated AR polyubiquitination is mainly via the human NK and T cells (4–6). We explored whether IFNa could K48 branch. Moreover, the K48-chain of AR polyubiquitination is improve enzalutamide efficiency in prostate cancer by targeting mainly mediated by mouse double minute 2 homolog (MDM2; ref. 43). the IRF8–AR axis. We found that IFNa increased IRF8 and decreased IP experiments confirmed that AR interacted with MDM2 (Supple- AR including ARvs expression in prostate cancer cells (Fig. 6A; mentary Fig. S5I). In addition, we detected the interaction between IRF8 Supplementary Fig. S7A). DNA pull-down assays showed that IFNa and MDM2 (Supplementary Fig. S5J). Together with previous result treatment promotes AR binding to both WT and mutant ISRE-like that AR interacted with IRF8, we tested whether IRF8 could influence motif of the IRF8 promoter rather than affecting only the canonical the interaction between AR and MDM2. IP experiments further sup- STAT1 pathway (Fig. 6B and C; Supplementary Fig. S7B). Further- ported that the interaction between AR and MDM2 was enhanced upon more, AR degradation by the ubiquitin pathway was accelerated the expression of IRF8 (Fig. 4J). upon IFNa treatment, and the IFNa-induced AR reduction was Together with the ubiquitination and protein interaction experi- inhibited by MG132; however, this was greatly diminished when IRF8 ments, our results suggested that IRF8 enhanced the interaction was KD in 22RV1 cells (Fig. 6D–F). These results demonstrated that

Figure 4. IRF8 enhances AR degradation through ubiquitin pathways. A and B, PC3 cells were cotransfected with AR plus IRF8 or AR plus vector control plasmid for 6 hours and treated with cycloheximide (CHX; 100 mg/mL) for the indicated time. Cell lysates were immunoblotted for AR, IRF8, and b-actin. Relative quantiﬁcation was carried out for three biological replicates using b-actin as loading control. P values were calculated using Student t test. , P < 0.05; , P < 0.01. C, HEK293T cells were cotransfected with pIRF8, pAR, or vector control for 48 hours and incubated with cycloheximide in the presence of proteasome inhibitor MG132 (10 mmol/L), calpain inhibitors MG101 (10 mmol/L), MG132 (0.1 mmol/L), calpastatin (5 mmol/L) or lysosomal inhibitor leupeptin (Leu, 50 mmol/L) for 5 hours, or DMSO as control. Cell lysates were analyzed for IRF8 and b-actin expression. D, Cells were cotransfected with the AR and control (pcDNA3.1) or IRF8 plasmids for 24 hours and treated with DMSO or MG132 (10 mmol/L) for 5 hours. Cell lysates were immunoblotted with the indicated antibodies. E, Confocal microscopy for endogenous IRF8 and AR in LNCaP cells treated with DHT (10 nmol/L) for 48 hours. F, Coimmunoprecipitation of IRF8 and AR in HEK293T cells cotransfected with pIRF8, pAR, or vector control for 48 hours. G, Western blot analyses were performed using indicated antibodies after IP of the full-length and truncated forms of AR from HEK293T WCL using the Flag antibody. H, HEK293T cells were transfected with various combinations of pAR, pIRF8, and pFlag-Ub plasmids for 48 hours. Cells were treated with MG132 (10 mmol/L) for 5 hours, followed by nuclear and cytoplasmic protein lysate preparation, and cytosolic protein was used for IP with an anti-AR antibody, and IB with the indicated antibodies. I, HEK293 cells were transfected with AR, IRF8, and WT, K48, or K63 ubiquitin (Ub) plasmids. Cells lysates were subjected to IP 48 hours later with an anti-AR antibody, followed by IB with an anti-HA antibody. J, PC3 cells were ﬁrst transfected with pcDNA3.1 or IRF8 for 12 hours and then transfected again with HA-MDM2 or HA-MDM2 and AR for 48 hours. The amount of plasmids was slightly adjusted to make the proteins expressed in similar level in different samples. Cell lysates were subjected to IP and followed by IB with the indicated antibodies.

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Figure 5. LNCaP-shIRF8 cells are less sensitive to enzalutamide treatment. A, LNCaP-shNC and LNCaP-shIRF8 cells were treated with different doses of enzalutamide (ENZ) for 48 hours (left) and 72 hours (right), and cell viability compared with DMSO control was tested using a mitochondrial activity–based cell counting kit–8 (CCK-8) assay. B and C, LNCaP-shNC and LNCaP-shIRF8 cells were treated with enzalutamide (10 mmol/L), and the growth curve during 4 days was determined by CCK-8 assay (OD450; B); the inhibition ratio of enzalutamide in LNCaP-shNC and LNCaP-shIRF8 cells was detected by plate colony formation assay (C). D and E, Caspase-3 activity in LNCaP-shIRF8 and LNCaP-IRF8 cells treated with enzalutamide (5 mmol/L) for 72 and 48 hours, respectively. F, Tumor growth and ﬁnal tumor weight of LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated BALB/c nude mice treated with enzalutamide (10 mg/kg/day) i.g. for 28 days. , P < 0.05; , P < 0.01; , P < 0.001; ns, not signiﬁcant.

IFNa-mediated IRF8 expression promotes AR degradation by the all the treatments in IRF8-KD groups (Fig. 6H; Supplementary ubiquitin pathway. We next examined the synergy between IFNa and Fig. S7D–S7F), indicating that IRF8 may have an important role in enzalutamide in inhibiting the cancer cells growth. While IFNa alone the therapeutic effect of IFNa- enzalutamide combination therapy. had limited effect on cancer cell death (Supplementary Fig. S7C), its We retrospectively designed a clinical case study to evaluate the combination with enzalutamide markedly dropped the IC50 value of effects of IFNa combined with MAB therapy on advanced metastatic enzalutamide in prostate cancer cells (Fig. 6G). patients with prostate cancer. In the case 1 clinical study, after 3 months We further assessed the therapeutic activity of IFNa and enzalu- of MAB alone therapy, both patients had reduced serum PSA levels, tamide combination in CRPC mice. IFNa combined with enzaluta- and after 6 months, serum PSA levels were increased. In the MAB and mide showed the better curative effects compared with IFNa or IFNa combination treatment group, the patient serum PSA level enzalutamide alone, whereas there were no signiﬁcant changes among continued to decrease with treatment and remained below the

2936 Cancer Res; 80(13) July 1, 2020 CANCER RESEARCH IRF8 Inhibits Prostate Cancer Progress

Figure 6. IFNa enhances enzalutamide (ENZ) sensitivity by targeting the AR–IRF8 axis. A, Western blot analysis for IRF8 and AR (ARfl and ARvs) in 22RV1 cells treated with IFNa at theindicated concentrations for 24 hours. B and C, DNA affinitybinding assay for AR, IRF8 (B), p-STAT (Tyr 701), p-STAT1 (Ser 727), total STAT1 and lamin B (C) bound to ISRE-wt2 and ISRE-mut2 in LNCaP cells treated with IFNa (1,000 IU/mL, 48 hours). D and E, 22RV1 cells were treated with IFNa (2,000 IU/mL, 48 hours) and then treated with cycloheximide (CHX; 200 mg/mL) for the indicated times (D), or with degradation inhibitor MG132(10 mmol/L, 5 hours, E). WCLswere subjected to IB for AR. F, 22RV1 cells were treated with IFNa (2,000 IU/mL, 24 hours) in presence of two IRF8-specific siRNA or negative control; prior to treatment with MG132 (10 mmol/L, 5 hours), the cytoplasmic lysates were subjected to IP with anti-AR antibody and subsequent IB with the indicated antibodies. G, Cell inhibition of LNCaP and 22RV1 cells treated with various concentrations of enzalutamide in the presence of IFNa (1,000 IU/mL). H, Tumor growth of LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated NOD-SCID mice treated as described in Materials and Methods. I, The correlation of gene expression levels between IRF8 and AR in the development and progression of prostate cancer as well as their regulatory mechanisms in tumor growth and enzalutamide resistance. PCa, prostate cancer; ns, not significant. biological progression line during follow-up treatment for 11 months ubiquitin/proteasome systems (Fig. 6I). IRF8 is induced by IFNs as a (Supplementary Fig. S7G, case 1). In the case 2 clinical study, the PSA transcription factor in the IFN system, it is thought to mainly function in level of the one untreated patient with prostate cancer was continu- the nucleus. We found IRF8 protein levels are upregulated in primary ously increased over the course of 6 months. Once the patient received prostate cancer and its expression is elevated upon DHT stimulation; in MAB combined with IFNa therapy, the PSA level declined contin- contrast, its levels are downregulated in CRPC and ENZR tissues upon uously for 3 months. The combination therapy was stopped when the bypassing AR activation and AR repression by ENZ. In CRPC after patient developed a fever, and the PSA level subsequently increased ADT, IGF-1 and EGF can also enhance the bypass of AR activation via slightly (Supplementary Fig. S7G, case 2). These clinical studies AR phosphorylation at sites different than that phosphorylated by DHT indicated that the combination of IFNa and hormonal therapies may treatment (p-ARSer81;refs.46–48) and we found that p-ARSer81 is enhance the treatment efficacy. responsible for the different effects on IRF8 expression caused by androgen–AR activation and bypass of AR activation (Supplementary Fig. S2F and S2G). Moreover, we found that decreased IRF8 expression Discussion in CRPC, resulted in tumor proliferation even under ADT and ENZR, In this work, we demonstrate the precise mechanisms of how IRF8 accompanied by a significant upregulation of AR. Accordingly, IRF8- was involved in prostate cancer progression and ENZR. We report that OE decreased AR expression. We found that IRF8 promoted AR IRF8 could reduce AR protein levels and block AR activation via the degradation dependent on enhanced AR ubiquitylation in the cytosol.

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The function of IRF8 in the cytosol and ubiquitylation is less under- ment for CRPC. We suggest that alternative drugs can induce IRF8 stood (49, 50). We report here that AR interacts with IRF8 in the cytosol expression could be combined with enzalutamide therapy to target through its AR-NTD and regulates its ubiquitylation. IRF8-mediated the IRF8–AR axis. In conclusion, we showed that decreased IRF8 in AR polyubiquitination is mainly branched through K48, and we verified prostate cancer cells impair its interaction with AR and promotion MDM2 as the E3 ligase responsible for IRF8-mediated cellular AR on AR degradation via the ubiquitin/proteasome pathway, accom- polyubiquitination and degradation. Although we found that IRF8 panied AR activation, AR-mediated CRPC progression, and ENZR, promoted the interaction of AR and its E3 ligase MDM2 in the cytosol, suggesting that IRF8 could be a promising target to overcome the detailed mechanisms underlying the involvement of IRF8 in enzalutamide resistance in CRPC. MDM2-mediated AR degradation need to be further investigated. We suggest that IRF8 is part of the signaling complex that includes AR and Disclosure of Potential Conflicts of Interest an E2 ubiquitin-conjugating enzyme, such as the MDM2–Ubc5 com- No potential conflicts of interest were disclosed. plex, altering the activity of the MDM2–Ubc5 complex (43). There may be additional E3 ligases that modulate IRF8-mediated AR responses, Authors’ Contributions and further work is required to fully reveal the roles of IRF8 in the Conception and design: H. Wu, J.-R. Zhou, Y. Yang regulation of AR signaling pathway. Development of methodology: H. Wu, L. You, Y. Li, Z. Zhao, G. Shi, Z. Chen, Z. Wang, X. Li, S. Du, W. Ye, X. Gao, J. Duan, W. Tao, Q. Zhu, Y. Yang For translational medicine, we found that IFNa increased IRF8 Acquisition of data (provided animals, acquired and managed patients, provided expression and promoted AR degradation through both STAT1 and facilities, etc.): H. Wu, L. You, Y. Li, S. Du, W. Tao, Y. Yang AR pathways activation (Fig. 6). Without androgen, treatment with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, IFNa alone promoted AR binding to both the WT and mutant ISRE- computational analysis): H. Wu, J.-R. Zhou, Y. Yang like domains in the IRF8 promoter, while the canonical phosphory- Writing, review, and/or revision of the manuscript: H. Wu, Y. Cheng, J. Bian, lation and dimerization of the STAT1 pathway (p-STAT1Tyr701 and J.-R. Zhou, Y. Yang Administrative, technical, or material support (i.e., reporting or organizing data, p-STAT1Ser727) did not compete with binding to the same site. In vivo in vitro constructing databases): Z. Chen, Q. Zhu, Y. Yang and studies, although AR could be activated by IFNa, Study supervision: Y. Yang treatment with IFNa alone had limited effect on prostate cancer cell proliferation. It is possible that activation of the AR pathway by IFNa Acknowledgments only promotes AR bound to ISRE-like motifs in the IRF8 promoter We thank Dr. Lutz Birnbaumer (NIEH) for critical comments and assistance with rather than enhancing AR bound to the androgen response element in preparation of the manuscript. We thank pathologists Mrs. Ning Su and Mr. Hongbao the proliferation-related target gene promoter. Treatment of IFNa Yang for H&E to verify histology and the IHC staining of IRF8 studies. These studies combined with enzalutamide significantly improved efficiency in were supported by the National Natural Science Foundation of China (grant numbers 81903656 to H. Wu; 81772732 to Q. Zhu; 81673468 to Y. Yang; 81672752 to Z. Chen), suppressing tumor growth of LNCaP-CRPC, while the enhanced “Double First-Class” University project (numbers CPU2018GF10 and antitumor activity was attenuated in LNCaP-shIRF8 CRPC. Most CPU2018GY46 to Y. Yang), Natural Science Foundation of Jiangsu Province (number importantly, in our clinical case study, IFNa combined with hormo- BK20180560 to H. Wu), and China Postdoctoral Science Foundation (number notherapy MAB was more efficacious than MAB alone. Unfortunately, 2018M632430 to H. Wu). although the potential for combination treatment is obvious, the side effects of IFNa therapy, such as fever and excessive perspira- 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 tion, may limit its clinical use as an antitumor therapy. Drug- with 18 U.S.C. Section 1734 solely to indicate this fact. resistant CRPC is current common and a major challenge in the management of prostate cancer, and targeting the AR axis by Received August 19, 2019; revised January 2, 2020; accepted April 16, 2020; disrupting androgen–AR interactions remains the primary treat- published first April 27, 2020.

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