Loss of a negative feedback loop between IRF8 and AR promotes prostate cancer growth and enzalutamide resistance

Abstract

In incurable castration-resistant prostate cancer (CRPC), resistance to the novel androgen receptor (AR) antagonist enzalutamide (ENZ) is driven mainly by AR overexpression. Here we report that the expression of interferon regulatory factor 8 (IRF8) is increased in primary prostate cancer (PCa) but decreased in CRPC compared to normal prostate tissue. Decreased expression of IRF8 positively associated with CRPC progression and ENZ resistance. IRF8 interacted with AR and promoted its degradation via activation of the ubiquitin/proteasome systems. Epigenetic knockdown of IRF8 promoted AR-mediated PCa progression and ENZ resistance in vitro and in vivo. Furthermore, IFNα increased expression of IRF8 and improved the efficacy of ENZ in CRPC by targeting the IRF8-AR axis. We also provide preliminary evidence for the efficacy of IFNα with hormonotherapy in a clinical study. Collectively, this study identifies IRF8 both as a tumor suppressor in PCa pathogenesis and a potential alternative therapeutic option to overcome ENZ resistance.

Graphical Abstract

Introduction

Androgen and androgen receptor (AR) signaling pathway play a critical role in the carcinogenesis and progression of prostate cancer (PCa), which is the second leading cause of cancer-related deaths in North America (1). Consequently, androgen depletion therapy (ADT) has been the first-line therapy for primary PCa for decades. Despite the initial efficacy of ADT, regeneration of tumour will occur eventually in almost every patient, leading to alleged castration-resistant prostate cancer (CRPC).

AR-targeted therapy is a gold standard therapy for CRPC (2–4). Enzalutamide (ENZ) is approved for the treatment of CRPC patients, based on its ability to block androgen binding to AR in a competitive manner, inhibiting AR nuclear translocation and DNA fixation (5–7). Despite the success of ENZ in improving the overall survival of CRPC patients, inherent or acquired ENZ resistance (ENZR) remains a major clinical challenge (8–10). AR deregulation, including overexpression of AR full-length (ARfl) and AR variants (ARvs), has been identified as a unique factor consistently associated with the progression of PCa to CRPC and ENZR (9); therefore, studying the mechanisms of AR deregulation is critical to improve the efficacy of current treatments. AR undergoes degradation mainly by the ubiquitin/proteasome system, as well as modification of protein stability triggered by ubiquitin-like signaling pathways, such as ISGylation (Interferon stimulated gene) (11,12). AR is a type I interferon (IFN) regulated protein and disruption of interferon system genes plays a novel function in malignant transformation of PCa (12–15). However, the mechanism for AR maintaining its stability under the influence of interferon system remains unknown.

Activity of the interferon system is mainly regulated by interferon regulatory factor 8 (IRF8), a member of the IRF family (IRF1–9) (16). Loss of IRF8 in immune cells leads to the occurrence of chronic myelogenous leukemia and aberrant methylation of IRF8 gene in nonhemopoietic cells play an increasingly important role in tumorigenesis (17–25). Among all the IRFs, only IRF8 facilitates a protein-protein interactions model by interacts with proteins containing the PEST motif, a region that plays an important role in protein degradation by the proteasome system, including AR degradation (26,27). Emerging evidences suggest that IRF8 may function in the cytosol ubiquitylation system (16,28), but the exact role and the regulation mechanism of IRF8 in cancers, especially in PCa tissues, have not been explored. More importantly, the relationship between IRF8 with AR in PCa and whether IRF8 interacts with AR (containing the PEST motif) and functions in the regulation of AR stability are still unknown.

The present study provides evidences to support an advantaged role for IRF8 in CRPC and ENZR, and this role of IRF8 is likely mediated through regulating AR stability. We then evaluated the potential of IFNα targeting IRF8 to improve the therapeutic efficacy of hormonotherapy in PCa, suggesting that IFNα combined with ENZ is an attractive therapeutic strategy for CRPC and ENZR.

Materials and Methods

Reagents

Fetal bovine serum (FBS) and charcoal-stripped, dextran-treated fetal bovine serum (CSS, depleted androgen and any other steroid) were purchased from Biological Industries (Israel). Enzalutamide (MedChemExpress, USA), R1881, Dihydrotestosterone (DHT) (Meilunbio, China), EGF, IGF-1 were commercially obtained (Peprotech, USA). Serial dilutions of all drugs were made using DMSO.

Cell lines and primary cultures

PC-3 cells were cultured in F12 Medium, HEK293T were cultured in DMEM Medium, 22RV1 cells and LNCaP cells were cultured in RPMI-1640 Medium. PC-3, 22RV1, LNCaP and HEK293T cells were purchased from were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), two stable LNCaP-sh-IRF8-Puro (LNCaP-shIRF8) and LNCaP-sh-negative control (LNCaP-shNC)-Puro cells were purchased from GenePharma Technology Co. Ltd. (GenePharma, Shanghai, China). All cells were authenticated by the Short Tandem Repeat DNA profiling (Cobioer, Nanjing, China) and confirmed Mycoplasma free using GMyc-PCR Mycoplasma Test Kit (YeSen; Shanghai, China, 40601ES10) after last experiment, and used within 15 cell passages after thawing. All cell lines were cultured in medium supplemented with 10% fetal bovine serum (Sigma), 1% Penicillin-Streptomycin (Gibico), 5% CO2, 37 °C.

Plasmids, siRNA and DNA transient transfections

Plasmids including pcDNA3 (Vector), pcDNA3-hIRF8 (IRF8), pcDNA3-hAR (AR), containing the whole CDS domains were constructed by Genscript (Nanjing, China); double luciferase reporter plasmid pEZX-FR03-hIRF8-luc, carrying the IRF8 promoter (−1106-+166bp), was constructed by FulenGen (Guangzhou, China); 3xFlag-Ub plasmid was generously provided by Dr. Guo-qiang Xu (Soochow University, China). For transient transfections, cells were seeded into six-well plates at 150,000 cells per well and transfected with IRF8 or AR expression vectors using empty vector (pvector) as control. The total plasmid DNA was adjusted to the same with empty vector.

siRNA and shRNA targeting IRF8 are as follows: siNC (shNC): TTC TCC GAA CGT GTC ACG TTT C; siRNA1# (shIRF8A): GCA GTT CTA TAA CAG CCA GGG; siRNA2# (shIRF8B): GGG AAG AGT TTC CGG ATA TGG.

Flat clone formation assay

LNCaP-shNC and LNCaP-shIRF8B cells were plated in the 6-well plates in triplicate with 500 cells every well and cultured with complete medium for 14 days. For ENZ sensitivity experiment, once cells were attached, ENZ (10 μM) were added to the media using 0.1%DMSO as control. The complete medium or culture medium containing ENZ or DMSO was replaced every two days, and cells were cultured for 10 days. Crystal violet was used to stain the colonies. Colony number and the inhibition effects of ENZ were calculated according the number of the cloning formation measured by ImagePro plus software.

Western blot analysis

Briefly, whole cell lysates were prepared with ice-cold RIPA lysis buffer or nuclear and cytoplasmic protein extraction lysis buffer with protease inhibitors (Roche). Approx. 30 μg total protein was separated by SDS-polyacrylamide gels and transferred to PVDF transfer membrane and the membranes were incubated with specific antibodies for IRF8 (ab28696, Abcam), AR (ab74272, Abcam), p-STAT1(Tyr701), p-STAT1 (Ser727), STAT1 (Cell Signaling Technology), β-actin (bsm-33036M, Bioss, China). Each experiment was repeated at least twice with similar results. The densitometry data presented below the bands are ‘fold change’ as compared with control normalized to respective β-actin from two independent western blot analyses. Values are expressed as mean.

RNA purification and quantitative real-time PCR (qPCR)

Total RNA was prepared by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using random hexamers, which was used for quantitative real-time PCR using gene-specific primers. Data were normalized by the level of GAPDH expression in each sample. The primer sequences used in this study are as follows: IRF8 forward 5’- TCG GAG TCA GCT CCT TCC AGA CT -3’, reverse 5’- TCG TAG GTG GTG TAC CCC GTC A -3’; AR forward 5’- CTA CTC TTC AGC ATT ATT CCA G -3’, reverse CAT GTG TGA CTT GAT TAG CA- 3’; GAPDH, forward 5’- CAT GAG AAG TAT GAC AAC AGC CT -3’, reverse 5’- AGT CCT TCC ACG ATA CCA AAG T -3’.

Immunoprecipitation

Cells treated with the appropriate stimuli were lysed with Western and IP lysis buffer (Beyotime, China). Aliquots of 600 μg protein from each sample were precleared by incubation with 20 μL of Pierce™ Protein A/G magnetic beads (88803; Thermo Fisher Scientific) for 1 hour at 4°C and then incubated with anti-IRF8 antibody (5628s, Cell Signaling Tech), or anti-AR (ab74272, Abcam) in lysis buffer at 4°C overnight. Protein A/G beads were added and incubated for 2 hours at 4°C. The beads were washed five times with PBS and once with lysis buffer, boiled, separated by 10% SDS-PAGE, and analyzed by Western blotting as described above. For in vitro binding assays, purified recombinant Flag-tagged AR protein was incubated with recombinant His-tagged IRF8, and anti-flag agarose beads (Bimake) in Buffer C. Bound immunocomplexes were washed three times with buffer B for nuclear extracts or buffer C-400mM NaCl for total protein extracts immunoprecipitation and in vitro binding assays.

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was performed using the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore) according to manufacturer’s protocol. Anti-AR antibodies and normal IgG were used to precipitate DNA. The normal IgG was used as a negative control and the PSA enhancer region was used as a positive control. The precipitated DNA was subjected to PCR to amplify the PSA and IRF8 promoter regions with the primers listed as follows, and were quantified with the qPCR using SYBR Green. PSA Enhancer+: TGG GAC AAC TTG CAA ACC TG, PSA Enhancer−: CCA GAG TAG GTC TGT TTT CAA TCC A; IRF8 (−872/−863bp) F: 5’- TGT GTG ATT CTC TAC TGG GCA A -3’, R: 5’- CTG GAA ACG GAA AAA GAA GCG T -3’; IRF8 (−852/−843bp) F: 5’-TTT CTT TTT CCA GTG TCG TTC TCC -3’, R: 5’- GGG CGT TAA GAT GTC CCC T -3’; IRF8 (−896/−783bp) F: 5’- GGA TAG AAC GCG GAA ACG CT -3’, R: 5’- CCT GGG TGT GCA CTG ACA TTT A -3’.

Immunohistochemistry of IRF8 and AR protein expression in PCa cells

Segregation of clinic specimens.

13 benign prostatic hyperplasia (BPH), 20 untreated primary PCa tissues and 13 CRPC paraffin-embedded tissues were collected from the Departments of Urinary surgery at Jiangsu Province Hospital of Traditional Chinese Medicine (TCM), which is approved by the Institutional Review Board of the Jiangsu Province Hospital of TCM for IHC and MSP-PCR analysis.

Tissue microarray.

Protein expression of IRF8 and AR in clinical prostate cancer tissues were determined using tissue microarray analysis (TMA, Shanghai OUTDO Biotech), containing 64 paired human primary prostate cancer/adjacent noncancerous lesion tissues. Tissue microarray were stained with H&E to verify histology and the immunohistochemistry staining of IRF8 (ab28696; Abcam) and AR (ab74272) were performed. The positive staining rate was estimated in 3 fields with different staining intensity by pathologists. The staining of IRF8 and AR in the TMA was scored independently by two pathologists blinded to the clinical data using the following criterion: the intensity of immunostaining 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%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The final score was calculated using the percentage score × staining intensity score as described previously (29). For tissue samples from clinical PCa patient and animal xenografts, the primary antibodies of the anti-IRF8 antibody (Abcam, 1:200), anti-AR antibody (Abcam, 1:200) or anti-PCNA antibody (Santa Cruz, 1:200), were used for staining at 4°C overnight. Immunostaining pictures were acquired using an inverted fluorescence microscope (Leica) and the Integral optical density (IOD) sum was calculated using ImagePro plus software. The average DAB staining intensity of the selected two cores represents the quantitative protein expression level in these tissues.

DNA affinity binding assay

Nuclear extracts and affinity binding assays were prepared as described previously (30). Briefly, LNCaP cells were treated with 10 nM DHT or 2000 IU/mL IFNα2a for 48 h, using ethanol and medium as control, respectively and lysed with ice-cold nuclear protein extraction lysis buffer. 4.5-μg aliquots DNA formed by 8 pairs of the ISRE and ISRE-like primers (2 WT and 6 mutants, Mut) were conjugated to streptavindin M280 magnetic beads (Dynal) in the presence of 20 μg of salmon sperm DNA (Sigma) for 10 min at room temperature. DNA-coupled magnetic beads were incubated with 1 mg of nuclear extracts for 1 h at 4°C. Beads were washed three times with washing buffer and bound materials were eluted in 1×sodium dodecyl sulfate (SDS) sample buffer. Samples were separated by 8% SDS-PAGE for immunoblot detection with anti-AR or other antibodies as indicated at 4°C overnight, using 30 μg nuclear protein as input and Lamin B as negative control.

Transient transfections for IRF8 promoter activity

HEK293T cells (1×10⁷/100 mm dish) were pre-cultured with 5% CSS overnight and transfected with pcDNA3.1-AR (1 μg). Cells were co-transfected with an artificial promoter luciferase reporter with both the proximal IRF8 5’ flank (flanking regions of IRF8 (−1106/+166bp) and the firefly/renilla luciferase (pEZX-FR03-hIRF8, 3 μg) in the same vector. The empty reporter vector (pEZX-FR03) was used as a control. The transfected cells were cultured with 5% CSS for 24 h and then seed into 96-wells (1×10⁴ cell/well) and treated with DHT (1 nM, 10 nM); R1881(1 nM, 10 nM); IGF-1(10 nM); EGF (10 nM) with or without ENZ (10 μM). DMSO was used as a control. After incubation with these stimuli for 24h, the transfected cells were collected and AR transcriptional activity was detected by dual luciferase assay.

Transient transfections for AR activity

LNCaP cells (1×10⁷/100 mm dish) were pre-cultured with 5% CSS overnight and transfected with various combinations of vectors to express IRF8 or control (0.75, 3 μg), IRF8-specific shRNA or scrambled control (3 μg). Cells were co-transfected with an androgen-dependent firefly luciferase reporter (pMMTV-Luc, 3 μg, for AR activity as previously described) (31) and a renilla luciferase promoter (pRL-SV40, 0.04 μg, for transfection efficiency). The transfected cells were cultured with 5% CSS for 24 h and then seeded into 96-wells (1×10⁴ cell/well) and treated with or without DHT (10 nM).

Luciferase activities were measured 24 h after treatment using the dual luciferase reporter system according to the manufacturer’s instructions (Promega). All samples were tested in triplicate.

Xenografts and animal model

All athymic nu/nu BALB/c (BALB/c nude) and non-obese diabetic severely combined immunodeficient (NOD-SCID) mice, aged 4–6 weeks, were purchased from Lingchang biotech (Shanghai, China) and housed in a specific pathogen free facility and maintained in a standard temperature and light-controlled animal facility for 1 week before used. For transgenic animal experiment, the Hi-myc mice (FVB-Tg (ARR2/Pbsn-MYC) 7Key, strain number: 01XK8) were obtained from the Mouse Repository of the National Cancer Institute. All animal procedures were performed according to the guidelines of the US National Institutes of Health on Animal Care and appropriate institutional certification, with the approval of the Committee on Animal Use and Care of Center for New Drug Evaluation and Research, China Pharmaceutical University (Nanjing, China).

For tumor growth of LNCaP cell xenograft (growing in intact BALB/C nude mice), 1*10⁷ LNCaP-shNC or LNCaP-shIRF8A cells (with higher KD efficiency, indicated as LNCaP-shIRF8) were resuspended in 100 μL medium containing 50% matrigel (BD Biosciences) and 50% growth media and subcutaneously injected in the flank of BALB/c nude mice. For the tumor growth of LNCaP-CRPC xenograft (growing in surgically castrated BALB/C nude mice with low serum androgen), BALB/c nude mice were anesthetized using 10% chloral hydrate. Testes were excised distal to the ligature, and the incision was closed with sterile dissolvable sutures and disinfected with betadine solution and ampicillin. Two weeks later, LNCaP-shNC or LNCaP-shIRF8 cells were inoculated as described above.

In the ENZ sensitivity study, orchiectomized BALB/c or NOD/SCID male mice were inoculated subcutaneously with 1×10⁷ LNCaP or LNCaP-shNC or LNCaP-shIRF8 cells. Once tumors were established, mice were randomly assigned to vehicle (1% carboxymethyl cellulose, 0.1% Tween-80, 5% DMSO), and ENZ (10 mg/kg) treatment groups. In the sensitivity study of ENZ combined IFNα, mice were treated with vehicle alone, enzalutamide (10 mg/kg) alone, IFNα (1.5×10⁷ IU/kg) alone or ENZ (10 mg/kg) combined with IFNα (1.5×10⁷ IU/kg). ENZ and IFNα were administered by oral gavage and subcutaneously daily, respectively. Tumor volume measurements were performed every 2 days and were calculated by the formula: length×width²/2. At experimental endpoint, tumors were harvested and tumor inhibition ratio were calculated and compared to the average tumor weight in the vehicle control.

Clinical case study

Three advanced and untreated PCa patients with bone metastasis were enrolled at the Department of Urinary Surgery at Jiangsu Province Hospital of TCM, and written informed consent was received from participants prior to inclusion in the study, in accordance with the guidelines of the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS), with the approval of the Institutional Ethics Review Boards of Jiangsu Province Hospital of TCM (NO.2017NL-054–02). Inclusion criteria were histologically confirmed prostate adenocarcinoma, untreated, cT3-cT4 M0 and M1, serum prostate-specific antigen (PSA) ≥ 150 ng/mL, Gleason Score ≥ 9 (4+5 or 5+4), age ≤ 80 yr, and normal liver function not suitable for definitive treatment. For CASE 1 clinical study, two patients received Goserelin Acetate Sustained-Release Depot (3.6 mg/28d) plus bicalutamide (50mg/d; Casodex) as maximal androgen blockade (MAB) therapy, one patient received MAB therapy plus recombinant IFNα2a (3*10⁶ IU/3d; Yinterfen). For CASE 2 clinical study, one advanced and untreated PCa patients with continued PSA level rise for 6 months were enrolled and received MAB therapy plus recombinant IFNα2a (3*10⁶ IU/3d; Yinterfen). Patients in both cohorts were discontinued when there was evidence of biochemical progression, defined as an increase of the PSA level ≥ 0.2 ng/mL on two successive occasions at least 1 month apart.

Results

IRF8 expression is increased in primary PCa but decreased in CRPC and ENZR tissues.

To reveal the relationship between IRF8 and AR protein expression in PCa, PCa tissue microarrays were immunohistochemically assessed using optimized anti-IRF8 and anti-AR antibodies. The representative primary PCa specimens showed high IRF8 expression and high AR accumulation compared with the adjacent nontumor tissue (Fig. 1A; Fig. S1A–B). Tumor samples with high expression of AR protein exhibited more IRF8 accumulation and a positive correlation was observed between protein levels of IRF8 versus AR in primary PCa specimens (Fig. S1C–D). Similar findings confirmed that IRF8 and AR was increased in tumour tissues of Hi-myc transgenic (TG) murine PCa models during the PCa progression (≥ 6 months) (Fig. 1B; Fig. S1E–F). Moreover, in LNCaP-CRPC xenografts models, IRF8 expression was significantly decreased in tumours treated with ENZ (Fig. S1G). To clarify this point, we quantified IRF8 and AR protein levels using IHC of paraffin-embedded clinical BPH, primary PCa and CRPC tissues. The results showed that IRF8 and AR were increased in primary PCa tissues compared to BPH, while low IRF8 but high AR were observed in the representative CRPC specimens (Fig. 1C; Fig. S1H). A positive correlation between IRF8 and AR in primary PCa was confirmed. In contrast, statistically significant negative correlation was observed between protein levels of IRF8 versus AR in CRPC specimens (Fig. 1D). Similarity to CRPC, IRF8 was decreased but AR was increased in ENZR xenograft tissues and an inverse correlation between IRF8 with AR protein was observed (Fig. 1E; Fig. S1I–J).

Fig. 1. IRF8 expression is increased in primary PCa but decreased in CRPC and ENZR tissues.

(A) Representative IHC of IRF8 and AR protein expression in primary PCa samples compared with adjacent noncancerous lesion tissues (ADJ) from PCa tissues microarrays (TMA). Scale bar, 100 μm. (B) IHC of IRF8 and AR protein expression in wildtype (WT) and Hi-myc (TG) mice prostate tissues. Scale bar, 100 μm. (C) IHC staining of IRF8 and AR protein expression in BPH(n=11), primary PCa (n=22) and CRPC tissues (n=11). Scale bar, 100 μm. (D) Correlation of IRF8 with AR expression normalized by the average IOD of BPH tissues in primary PCa and CRPC specimens was analyzed, linear regression coefficient and statistical significance are indicated. (E) IHC staining of IRF8 and AR protein expression in ENZ resistant (ENZR) xenograft tissues (n=6). Scale bar, 100 μm. (F) LNCaP cells, pre-cultured with 5% CSS for 3 days, were treated with DHT for 24 h and IFR8 expression was analyzed by qPCR (left panels), western blot (right panels). (G) IRF8 mRNA (left panels) and protein expression (right panels) in LNCaP cells cultured in the presence of the indicated concentration of ENZ for 24 h. (H) HEK293T cells co-transfected with pcDNA3.1-AR and IRF8 promoter luciferase reporter were treated in triplicate with DHT (1, 10 nM); R1881 (1, 10 nM); IGF-1(10 nM); EGF (10 nM) with and without ENZ (10 μM) for 24 h. Luciferase activities were measured 24 h after treatment. (I) Immobilized WT and mutant ISREs were incubated with nuclear extracts from LNCaP cells treated with DHT for 24 h, 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 h. 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.

Previous findings revealed that epigenetic mechanisms including mutation and promoter methylation play a crucial role in the IRF8 expression (19,25,32–35). However, IRF8 was not frequently mutated and methylated in clinical PCa tissues and cells (Fig. S1K; Supplementary Table). Lots of AR CHIP-seq data reveal that IRF8 is a putative target of AR upon DHT or R1881 treatment (36–38). Furthermore, we found several AR binding sites at the promoter of IRF8 using JASPAR database. We next tested whether IRF8 expression is regulated upon AR activation and repression. AR activation by dihydrotestosterone (DHT) increased IRF8 mRNA and protein levels in PCa cells (Fig. 1F; Fig. S2A–C). However, AR repression by ENZ treatment decreased IRF8 expression (Fig. 1G; Fig. S2C).

In CRPC, AR can be bypass-activated by growth factors (GFs) such as EGF, IGF-1, at low levels of androgen after ADT (39–42). In the experiments, we treated LNCaP cells with these factors and found that AR activation by GFs reduced IRF8 protein levels (Fig. S2D). We further identified whether these factors regulate IRF8 transcription by AR signaling, and the results showed that DHT and R1881-induced AR activation promoted IRF8 transcription but bypass AR activation by GFs inhibited IRF8 transcription. In contrast, ENZ reduced IRF8 transcription, reversed androgen-mediated IRF8 upregulation and enhanced GFs-mediated IRF8 downregulation (Fig. 1H).

IRF8 transcription is mainly regulated by the binding of p-STAT1 to the interferon-stimulated response element motif (ISRE, TTTC A/G G/C TTTC) (16). Predictably, one canonical ISRE motif and one ISRE-like motif were identified in the IRF8 promoter (Fig. S2E). In our experiments, the amounts of AR bound to the ISRE and ISRE-like motifs were markedly increased after DHT treatment, whereas binding by AR were not increased upon GFs stimulation and only p-AR^Ser81 were activated by DHT (Fig. S2F–G). Furthermore, we found mutations of the GAAA motif greatly diminished binding of AR to the ISRE-like motif while AR binding to ISRE mutant was not reduced (Fig. 1I), indicating the ISRE-like motif has stronger affinity for AR. We further determined whether the IRF8 is a direct AR targeted gene using ChIP-qPCR assays. The results showed that AR was recruited to the ISRE and ISRE-like sites regardless of DHT treatment, whereas occupancy of the ISRE-like site was markedly increased and occupancy of the ISRE site was not changed in LNCaP cells upon DHT stimulation (Fig. 1J; Fig. S2H–I). Together, these results identified AR as a factor is recruited to the ISRE-like motif upon DHT stimulation which promotes IRF8 transcription in an androgen-AR dependent pathway.

IRF8-knockdown promotes the proliferation and tumorigenesis of LNCaP cells through AR activation.

To functionally demonstrate the potential role of IRF8 in tumorigenesis of PCa, we generated two stable IRF8-knockdown (IRF8-KD) LNCaP cell lines (Fig. 2A; Fig. S3A). The proliferation ratios of LNCaP-shIRF8 cells were higher than those in LNCaP-shNC cells (Fig. 2B; Fig. S3B). Furthermore, increased clonogenic ability, number and diameter of tumour cell spheres were also observed in LNCaP-shIRF8 cells (Fig. 2C–D; Fig. S3C–D). In vivo xenograft experiments showed that LNCaP-shIRF8 results in increased tumour growth and proliferating cell nuclear antigen (PCNA) protein expression in intact or castrated BALB/c nude mice (Fig. 2E–G; Fig. S3E–G), whereas 22RV1-IRF8-overexpression (OE) inhibited the proliferation, clone formation and tumour growth (Fig. 2H–I; Fig. S3H). Moreover, IRF8-KD also increased P38/MAPK/ERK phosphorylation and CD133 expression compared to LNCaP-shNC cells (Fig. 2J), indicating that intrinsic IRF8 plays an important role in inhibiting PCa progression.

Fig. 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. (B) Absorbance at 450 nm of LNCaP-shIRF8 cultured with 5% FBS (left panel), 5% CSS (middle panel), or 5% CSS containing 1 nM R1881 (right panel) in 96-well plates for 24–120 h. (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, F) Knockdown of IRF8 in tumorigenesis. Tumour growth of LNCaP-shIRF8 cells in BALB/c nude mice (E, n=9), and in castrated BALB/c nude mice (F, n=9). (G) Representative images of IHC staining of PCNA in LNCaP-shIRF8 BALB/c xenografts. Scale bar, 100 μm. (H, I) OE of IRF8 in tumorigenesis. Proliferation (H) of 22RV1-IRF8 overexpression cells in vitro and tumour growth of 22RV1-IRF8 overexpression cells in vivo (I, n=9). (J) Western blot analysis for p-P38 MAPK, P38 MAPK, p-ERK, ERK, p-AKT(Thr308), p-AKT(Ser473), AKT, CD133, Oct3/4, and β-actin in LNCaP-shNC and LNCaP-shIRF8 cells.

To elucidate the mechanisms responsible for the growth-accelerating effects of IRF8-KD in PCa, we assessed the effects of IRF8 on AR protein expression. IRF8-KD increased and IRF8-OE decreased endogenous AR including AR variants (ARvs) expression, especially in the nucleus (Fig. 3A–B and Fig.S4A–B). Moreover, we demonstrated that IRF8 can reduce ectopic AR protein levels in AR⁻ HEK293T and PC3 cells transfected with AR alone or combined with various amounts of IRF8 expression plasmids (Fig. 3C–D).

Fig. 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), β-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 (AR full-length: ARfl and AR variants: ARvs) in 22RV1 cells 48 h after transfection with IRF8 plasmid (pIRF8) at various amounts (0.125–1 μg). (C) Western blot analysis of IRF8 and AR in HEK293T cells co-transfected with AR plasmid (0.3 μg) and different concentrations of IRF8 plasmid (0.125–1 μg) for 24 h and 48 h. (D) Western blot analysis of exogenous IRF8 and AR in PC3 cells transfected with AR plasmid (0.3 μg) and IRF8 plasmid (1 μg) alone or in combination for 48 h. (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 h. (G) Luciferase reporter assays for AR activity in IRF8-KD (LNCaP-shIRF8; left panels) and IRF8-OE (LNCaP-IRF8; right panels) LNCaP cells treated with DHT. ***p < 0.001 by one-way ANOVA.

Androgen binding to AR results in homo-dimerization and nuclear translocation, and subsequent induction of target gene expression, which is regulated by the ubiquitin/proteasome system (27). Native polyacrylamide gel electrophoresis of lysates from HEK293T cells transfected with AR and IRF8 showed that decreased level of AR protein mediated by IRF8 resulted in decreased expression of AR dimers in a high molecular weight complex to a degree equal to the reduction of ubiquitin-mediated AR dimers (Fig. 3E), indicating that IRF8-mediated AR protein degradation could affect AR homo-dimerization. So we next detected effect of IRF8 on AR transcriptional activity. The AR target prostate-specific antigen (PSA) is the most important marker for prostate cancer assessment. As expected, we found that PSA and AR protein were increased by IRF8 siRNA (Fig. 3F). We next explored AR activity using the pMMTV-luc luciferase reporter in IRF8-KD and IRF8-OE LNCaP cells. IRF8-KD increased and IRF8-OE decreased the DHT-dependent AR activity after androgen stimulation (Fig. 3G, Fig. S4C–D), which was associated with nuclear AR expression mediated by IRF8 in LNCaP cells.

IRF8 physically interacts with AR and enhances AR degradation by ubiquitin-dependent pathways

IRF8 mediated AR reduction could not be reversed by DHT treatment in both the cytosol and nucleus, suggesting that IRF8 has no effect on AR translocation (Fig. S4E–F), while the presence of IRF8 accelerated AR degradation and shortened the half-life of AR (Fig. 4A–B; Fig. S5A–B). IRF8-mediated AR degradation was reversed by proteasome inhibitor MG132, but MG101, calpastatin and lysosomal enzyme inhibitor leupeptin had no effect on IRF8-mediated AR degradation (Fig. 4C–D; Fig. S5C). These results indicated that the ubiquitin/proteasome involved in the IRF8-mediated AR degradation.

Fig. 4. IRF8 enhances AR degradation through ubiquitin-pathways.

(A, B) PC3 cells were co-transfected with AR plus IRF8 or AR plus vector control plasmid for 6h and treated with CHX (100 μg/mL) for the indicated time. Cell lysates were immunoblotted for AR, IRF8 and β-actin. Relative quantification was carried out for three biological replicates using β-actin as loading control. P-values were calculated using Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01. (C) HEK293T cells were co-transfected with pIRF8, pAR or vector control for 48 h and incubated with CHX in the presence of proteasome inhibitor MG132 (10 μM), calpain inhibitors MG101 (10 μM), MG132 (0.1 μM), calpastatin (5 μM) or lysosomal inhibitor leupeptin (Leu, 50 μM) for 5 h, or DMSO as control. Cell lysates were analyzed for IRF-8 and β-actin expression. (D) Cells were cotransfected with the AR and control (pcDNA3.1) or IRF8 plasmids for 24 h, and treated with DMSO or MG132 (10 μM) for 5 h. Cell lysates were immunoblotted with the indicated antibodies. (E) Confocal microscopy for endogenous IRF8 and AR in LNCaP cells treated with DHT (10 nM) for 48 h. (F) Co-IP of IRF8 and AR in HEK293T cells co-transfected with pIRF8, pAR or vector control for 48 h. (G) Western blots 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 h. Cells were treated with MG132(10 μM) for 5 h, followed by nuclear and cytoplasmic protein lysate preparation and cytosolic protein were 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 h 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 h and then transfected again with HA-MDM2 or HA-MDM2 and AR for 48 h. 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.

Cytoplasmic AR undergoes proteasome-mediated degradation in the absence of ligand. Our results showed that ubiquitin or IRF8 mediated AR degradation could be reversed by MG132 and DHT (Fig. S5D–E), indicating that IRF8 and ubiquitin regulate the same cellular machinery responsible for cytoplasmic AR degradation. The subcellular co-localization of IRF8 and AR were both observed by immunofluorescent staining in LNCaP cells and ectopically overexpressed HEK293T cells (Fig. 4E; Fig. S5F). We further found that IRF8 could precipitate with AR and vice versa (Fig. 4F). To confirm this interaction, we proved that IRF8 could be eluted with AR in vitro binding assays (Fig. S5G). To identify region within AR responsible for its binding to IRF8, we co-transfected IRF8 with several N-terminal Flag-tagged AR truncated mutants, including AR full-length (AR-FL), AR-NTD, AR-DBD, AR-Hinge and AR-LBD into HEK293T cells (Fig. S5H). Co-IP assay showed that IRF8 was immunoprecipitated with AR-FL, AR-NTD but not with AR-LBD, AR-Hinge and AR-DBD (Fig. 4G). These results indicated that IRF8 physically interacts with the N terminal domain of AR. We next analyzed the effect of IRF8 on AR ubiquitylation. Results showed that IRF8 enhanced AR polyubiquitination as visualized by staining with Flag-ubiquitin (Fig. 4H). There are two common types of polyubiquitination, which are distinguished by the lysine residue through which formation of K48-chain and K63-chain occurs. IB revealed that both WT and K48 ubiquitin led to poly-ubiquitinated AR, but polyubiquitination was impaired in the presence of K63 ubiquitin (Fig. 4I), suggesting that IRF8-mediated AR polyubiquitination is mainly via the K48 branch. Moreover, the K48-chain of AR polyubiquitination is mainly mediated by mouse double minute 2 homolog (MDM2) (43). Immunoprecipitation experiments confirmed that AR interacted with MDM2 (Fig. S5I). In addition, we detected the interaction between IRF8 and MDM2 (Fig. S5J). Together with previous result that AR interacted with IRF8, we tested whether IRF8 could influence the interaction between AR and MDM2. Immunoprecipitation experiments further supported that the interaction between AR and MDM2 was enhanced upon the expression of IRF8 (Fig. 4J).

Together with the ubiquitination and protein interaction experiments, our results suggested that IRF8 enhanced the interaction between AR and its E3 ligase MDM2 and thus promoted the ubiquitination and degradation of AR.

Low expression of IRF8 in LNCaP cells induces resistance to ENZ treatment.

We further explored the effects of IRF8 on sensitivity to ENZ therapy in vitro and in vivo. LNCaP viability treated with ENZ was decreased, whereas LNCaP-shIRF8 cells were resistant to ENZ treatment with increased AR expression (Fig. 5A–B; Fig. S6A–B). Moreover, the IRF8-OE 22RV1 cell was more sensitive to ENZ treatment (Fig. S6C). Furthermore, the colony number with ENZ treatment was significantly decreased in LNCaP-shIRF8 cells compared to controls (P<0.05) (Fig. 5C; Fig. S6D–E). Notably, IRF8-KD decreased and IRF8-OE promoted caspase-3 activity induced by ENZ treatment (Fig. 5D–E). We further confirmed that IRF8-KD could induce ENZR in castrated BALB/c nude and NOD-SCID mice. The ENZ treatment inhibited tumour growth of LNCaP-shNC (P<0.05), while it did not significantly alter the growth of LNCaP-shIRF8 tumours (Fig. 5F; Fig. S6F–G).

Fig. 5. LNCaP-shIRF8 cells are less sensitive to ENZ treatment.

(A) LNCaP-shNC and LNCaP-shIRF8 cells were treated with different doses of ENZ for 48h (left panel) and 72 h (right panel), and cell viability compared to DMSO control was tested using a mitochondrial activity-based cell counting kit–8 (CCK-8) assay. (B, C) LNCaP-shNC and LNCaP-shIRF8 cells were treated with ENZ (10 μM), and the growth curve during 4 d was determined by CCK-8 assay (OD450, B), the inhibition ratio of ENZ in LNCaP-shNC and LNCaP-shIRF8 cells was detected by plate colony formation assay (C). (D, E) Caspase 3 activity in LNCaP-shIRF8 and LNCaP-IRF8 cells treated with ENZ (5 μM) for 72 h and 48 h, respectively. (F) Tumour growth and final tumour weight of LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated BALB/c nude mice treated with ENZ (10 mg/kg/d) i.g. for 28 days.

IFNα enhances ENZ sensitivity by targeting the IRF8-AR axis

Since decreased IRF8 induces ENZR during ADT, we next explored a novel strategy to attenuate the resistance. The IRF8 expression could be induced by IFNs through phosphorylation and dimerization of STAT1 (p-STAT1^Tyr701 and p-STAT1^Ser727) (19,44,45). IFNγ receptor is frequently inactivation but expression of IFNA receptors is much higher than IFNγ receptors in human PCa cells (1,3). Fundamental studies have shown IFNα phosphorylates STAT1 (p-STAT1^Tyr701 and p-STAT1Ser⁷²⁷) and activates IRF8 gene expression by binding to the GAS element in human NK and T cells (4–6). We explored whether IFNα could improve ENZ efficiency in PCa by targeting the IRF8-AR axis. We found that IFNα increased IRF8 and decreased AR including ARvs expression in PCa cells (Fig. 6A; Fig. S7A). DNA pull-down assays showed that IFNα treatment promotes AR binding to both WT and mutant ISRE-like motif of the IRF8 promoter rather than affecting only the canonical STAT1 pathway (Fig. 6B–C; Fig. S7B). Furthermore, AR degradation by the ubiquitin pathway was accelerated upon IFNα treatment, and the IFNα-induced AR reduction was inhibited by MG132, however, this was greatly diminished when IRF8 was KD in 22RV1 cells (Fig. 6D–F). These results demonstrated that IFNα mediated IRF8 expression promotes AR degradation by the ubiquitin pathway. We next examined the synergy between IFNα and ENZ in inhibiting the cancer cells growth. While IFNα alone had limited effect on cancer cell death (Fig. S7C), its combination with ENZ markedly dropped the IC50 value of ENZ in PCa cells (Fig. 6G).

Fig. 6. IFNα enhances ENZ sensitivity by targeting the AR-IRF8 axis.

(A) Western blot analysis for IRF8 and AR (ARfl and ARvs) in 22RV1 cells treated with IFNα at the indicated concentrations for 24 h. (B, C) DNA affinity binding 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 IFNα (1000 IU/mL, 48 h). (D, E) 22RV1 cells were treated with IFNα (2000IU/mL, 48 h) and then treated with CHX (200 μg/mL) for the indicated times (D), or with degradation inhibitor MG132(10 μM, 5 h, E). WCLs were subjected to IB for AR. (F) 22RV1 cells were treated with IFNα (2000 IU/mL, 24 h) in presence of two IRF8-specific siRNA or negative control, prior to treatment with MG132 (10 μM, 5 h), 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 ENZ in the presence of IFNα (1000 IU/mL). (H) Tumour growth of LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated NOD-SCID mice treated as described in the Methods section. (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 ENZ resistance.

We further assessed the therapeutic activity of IFNα and ENZ combination in CRPC mice. IFNα combined with ENZ showed the better curative effects compared to IFNα or ENZ alone, whereas there were no significant changes among all the treatments in IRF8-KD groups (Fig. 6H; Fig. S7D–F), indicating that IRF8 may have an important role in the therapeutic effect of IFNα-ENZ combination therapy.

We retrospectively designed a clinical case study to evaluate the effects of IFNα combined with maximal androgen blockade (MAB) therapy on advanced metastatic PCa patients. In the CASE one clinical study, after three months of MAB alone therapy, both patients had reduced serum PSA levels, and after six months, serum PSA levels were increased. In the MAB and IFNα combination treatment group, the patient serum PSA level continued to decrease with treatment and remained below the biological progression line during follow-up treatment for eleven months (Fig. S7G, CASE 1). In the CASE two clinical study, the PSA level of the one untreated PCa patient was continuously increased over the course of six months. Once the patient received MAB combined with IFNα therapy, the PSA level declined continuously for three months. The combination therapy was stopped when the patient developed a fever, and the PSA level subsequently increased slightly (Fig. S7G, CASE 2). These clinical studies indicated that the combination of IFNα and hormonal therapies may enhance the treatment efficacy.

Discussion

In this work, we demonstrate the precise mechanisms of how IRF8 was involved in PCa progression and ENZR. We report that IRF8 could reduce AR protein levels and block AR activation via the ubiquitin/proteasome systems (Fig. 6I). IRF8 is induced by IFNs as a transcription factor in the IFN system, it is thought to mainly function in the nucleus. We found IRF8 protein levels are upregulated in primary PCa and its expression is elevated upon DHT stimulation; in contrast, its levels are downregulated in CRPC and ENZR tissues upon bypassing AR activation and AR repression by ENZ. In CRPC after ADT, IGF-1 and EGF can also enhance the bypass of AR activation via AR phosphorylation at sites different than that phosphorylated by DHT treatment (p-AR^Ser81) (46–48) and we found that p-AR^Ser81 is responsible for the different effects on IRF8 expression caused by androgen-AR activation and bypass of AR activation (Fig. S2F–G). Moreover, we found that decreased IRF8 expression in CRPC, resulted in tumour proliferation even under ADT and ENZR, accompanied by a significant upregulation of AR. Accordingly IRF8 overexpression decreased AR expression. We found that IRF8 promoted AR degradation dependent on enhanced AR ubiquitylation in the cytosol. The function of IRF8 in the cytosol and ubiquitylation is less understood (49,50). We report here that AR interacts with IRF8 in the cytosol through its AR-NTD and regulates its ubiquitylation. IRF8-mediated AR polyubiquitination is mainly branched through K48, and we verified MDM2 as the E3 ligase responsible for IRF8-mediated cellular AR polyubiquitination and degradation. Although we found that IRF8 promoted the interaction of AR and its E3 ligase MDM2 in the cytosol, the detailed mechanisms underlying the involvement of IRF8 in MDM2-mediated AR degradation need to be further investigated. We suggest that IRF8 is part of the signaling complex that includes AR and an E2 ubiquitin-conjugating enzyme, such as the MDM2-Ubc5 complex, altering the activity of the MDM2-Ubc5 complex (43). There may be additional E3 ligases that modulate IRF8-mediated AR responses, and further work is required to fully reveal the roles of IRF8 in the regulation of AR signaling pathway.

For translational medicine, we found that IFNα increased IRF8 expression and promoted AR degradation through both STAT1 and AR pathways activation (Fig. 6). Without androgen, treatment with IFNα alone promoted AR binding to both the WT and mutant ISRE-like domains in the IRF8 promoter, while the canonical phosphorylation and dimerization of the STAT1 pathway (p-STAT1^Tyr701 and p-STAT1^Ser727) did not compete with binding to the same site. In vivo and in vitro studies, although AR could be activated by IFNα, treatment with IFNα alone had limited effect on PCa cell proliferation. It is possible that activation of the AR pathway by IFNα only promotes AR bound to ISRE-like motifs in the IRF8 promoter rather than enhancing AR bound to the androgen response element in the proliferation-related target gene promoter. Treatment of IFNα combined with ENZ significantly improved efficiency in suppressing tumour growth of LNCaP-CRPC, while the enhanced anti-tumour activity was attenuated in LNCaP-shIRF8 CRPC. Most importantly, in our clinical case study, IFNα combined with hormonotherapy MAB was more efficacious than MAB alone. Unfortunately, although the potential for combination treatment is obvious, the side effects of IFNα therapy, such as fever and excessive perspiration, may limit its clinical use as an anti-tumour therapy. Drug-resistant CRPC is current common and a major challenge in the management of PCa, and targeting the AR axis by disrupting androgen-AR interactions remains the primary treatment for CRPC. We suggest that alternative drugs can induce IRF8 expression could be combined with ENZ therapy to target the IRF8-AR axis. In conclusion, we showed that decreased IRF8 in PCa cells impair its interaction with AR and promotion on AR degradation via the ubiquitin/proteasome pathway, accompanied AR activation, AR-mediated CRPC progression and ENZR, suggesting that IRF8 could be a promising target to overcome ENZ resistance in CRPC.

The statement of significance.

Findings identify IRF8-mediated AR degradation as a mechanism of resistance to AR targeted therapy, highlighting the therapeutic potential of IFNα in targeting IRF8-AR axis in CRPC.