Loss of a Negative Feedback Loop Between IRF8 and AR Promotes

Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Loss of a negative feedback loop between IRF8 and AR promotes

2 prostate cancer growth and enzalutamide resistance

3 4 Hongxi Wu1, †, Linjun You1, †, Yan Li1, †, Zhili Zhao1, †, Guangjiang Shi1, Zhen 5 Chen1, Zhuo Wang1, Xianjing Li1, Shijia Du1, Wanli Ye1, Xiaofang Gao1, Jingjing 6 Duan1, Yan Cheng1, Weiyan Tao1, Jinsong Bian4, Jin-Rong Zhou3, Qingyi Zhu2, * & 7 Yong Yang1, * 8 9 1State Key Laboratory of Natural Medicines, China Pharmaceutical University, 10 Nanjing 211198, China 11 2Department of Urology, Jiangsu Province Hospital of Traditional Chinese Medicine, 12 Nanjing 210029, China 13 3Nutrition/Metabolism Laboratory, Department of Surgery/General Surgery, Harvard 14 Medical School, Boston, Massachusetts 15 4Department of Pharmacology, Yong Loo Lin School of Medicine, National University 16 of Singapore, 117597, Singapore 17 18 Footnotes: † They contributed equally to this paper. 19 20 Running title: IRF8 inhibits prostate cancer progress. 21 22 Keywords: Prostate cancer; Castration-resistant prostate cancer; Interferon regulatory 23 factor 8; Enzalutamide resistance; Androgen receptor 24 25 Disclosures of Potential Conflicts of Interest: We declare no conflict of interest. 26 27 Corresponding Author: 28 Prof. Yong Yang, State Key Laboratory of Natural Medicines, China Pharmaceutical 29 University, Nanjing 211198, China. Phone and Fax: 86-025-86185622; E-mail: 30 [email protected] 31 Dr. Qingyi Zhu, Department of Urology, Jiangsu Province Hospital of Traditional 32 Chinese Medicine, Nanjing 210029. Phone and Fax: 86-025-86617141-90506; E-mail: 33 [email protected]. 34 35 36 37 38 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

40 Abstract 41 In incurable castration-resistant prostate cancer (CRPC), resistance to the novel

42 androgen receptor (AR) antagonist enzalutamide (ENZ) is driven mainly by AR

43 overexpression. Here we report that the expression of interferon regulatory factor 8

44 (IRF8) is increased in primary prostate cancer (PCa) but decreased in CRPC

45 compared to normal prostate tissue. Decreased expression of IRF8 positively

46 associated with CRPC progression and ENZ resistance. IRF8 interacted with AR and

47 promoted its degradation via activation of the ubiquitin/proteasome systems.

48 Epigenetic knockdown of IRF8 promoted AR-mediated PCa progression and ENZ

49 resistance in vitro and in vivo. Furthermore, IFNα increased expression of IRF8 and

50 improved the efficacy of ENZ in CRPC by targeting the IRF8-AR axis. We also

51 provide preliminary evidence for the efficacy of IFNα with hormonotherapy in a

52 clinical study. Collectively, this study identifies IRF8 both as a tumor suppressor in

53 PCa pathogenesis and a potential alternative therapeutic option to overcome ENZ

54 resistance.

56 The statement of significance

57 Findings identify IRF8-mediated AR degradation as a mechanism of resistance to AR

58 targeted therapy, highlighting the therapeutic potential of IFNα in targeting IRF8-AR

59 axis in CRPC.

61 Introduction 62 Androgen and androgen receptor (AR) signaling pathway play a critical role in the

63 carcinogenesis and progression of prostate cancer (PCa), which is the second leading

64 cause of cancer-related deaths in North America (1). Consequently, androgen

65 depletion therapy (ADT) has been the first-line therapy for primary PCa for decades.

66 Despite the initial efficacy of ADT, regeneration of tumour will occur eventually in

67 almost every patient, leading to alleged castration-resistant prostate cancer (CRPC).

68 AR-targeted therapy is a gold standard therapy for CRPC (2-4). Enzalutamide

69 (ENZ) is approved for the treatment of CRPC patients, based on its ability to block

70 androgen binding to AR in a competitive manner, inhibiting AR nuclear translocation

71 and DNA fixation (5-7). Despite the success of ENZ in improving the overall survival

72 of CRPC patients, inherent or acquired ENZ resistance (ENZR) remains a major

73 clinical challenge (8-10). AR deregulation, including overexpression of AR

74 full-length (ARfl) and AR variants (ARvs), has been identified as a unique factor

75 consistently associated with the progression of PCa to CRPC and ENZR (9); therefore,

76 studying the mechanisms of AR deregulation is critical to improve the efficacy of

77 current treatments. AR undergoes degradation mainly by the ubiquitin/proteasome

78 system, as well as modification of protein stability triggered by ubiquitin-like

79 signaling pathways, such as ISGylation (Interferon stimulated gene) (11,12). AR is a

80 type I interferon (IFN) regulated protein and disruption of interferon system genes

81 plays a novel function in malignant transformation of PCa (12-15). However, the

82 mechanism for AR maintaining its stability under the influence of interferon system

83 remains unknown.

84 Activity of the interferon system is mainly regulated by interferon regulatory

85 factor 8 (IRF8), a member of the IRF family (IRF1-9) (16). Loss of IRF8 in immune

86 cells leads to the occurrence of chronic myelogenous leukemia and aberrant

87 methylation of IRF8 gene in nonhemopoietic cells play an increasingly important role

88 in tumorigenesis (17-25). Among all the IRFs, only IRF8 facilitates a protein-protein

89 interactions model by interacts with proteins containing the PEST motif, a region that

90 plays an important role in protein degradation by the proteasome system, including

91 AR degradation (26,27). Emerging evidences suggest that IRF8 may function in the

92 cytosol ubiquitylation system (16,28), but the exact role and the regulation

93 mechanism of IRF8 in cancers, especially in PCa tissues, have not been explored.

94 More importantly, the relationship between IRF8 with AR in PCa and whether IRF8

95 interacts with AR (containing the PEST motif) and functions in the regulation of AR

96 stability are still unknown.

97 The present study provides evidences to support an advantaged role for IRF8 in

98 CRPC and ENZR, and this role of IRF8 is likely mediated through regulating AR

99 stability. We then evaluated the potential of IFNα targeting IRF8 to improve the

100 therapeutic efficacy of hormonotherapy in PCa, suggesting that IFNα combined with

101 ENZ is an attractive therapeutic strategy for CRPC and ENZR.

102

103 Materials and Methods

104 Reagents

105 Fetal bovine serum (FBS) and charcoal-stripped, dextran-treated fetal bovine serum

106 (CSS, depleted androgen and any other steroid) were purchased from Biological

107 Industries (Israel). Enzalutamide (MedChemExpress, USA), R1881,

108 Dihydrotestosterone (DHT) (Meilunbio, China), EGF, IGF-1 were commercially

109 obtained (Peprotech, USA). Serial dilutions of all drugs were made using DMSO.

110

111 Cell lines and primary cultures

112 PC-3 cells were cultured in F12 Medium, HEK293T were cultured in DMEM

113 Medium, 22RV1 cells and LNCaP cells were cultured in RPMI-1640 Medium. PC-3,

114 22RV1, LNCaP and HEK293T cells were purchased from were purchased from Cell

115 Bank of the Chinese Academy of Sciences (Shanghai, China), two stable

116 LNCaP-sh-IRF8-Puro (LNCaP-shIRF8) and LNCaP-sh-negative control

117 (LNCaP-shNC)-Puro cells were purchased from GenePharma Technology Co. Ltd.

118 (GenePharma, Shanghai, China). All cells were authenticated by the Short Tandem

119 Repeat DNA profiling (Cobioer, Nanjing, China) and confirmed Mycoplasma free

120 using GMyc-PCR Mycoplasma Test Kit (YeSen; Shanghai, China, 40601ES10) after

121 last experiment, and used within 15 cell passages after thawing. All cell lines were

122 cultured in medium supplemented with 10% fetal bovine serum (Sigma), 1%

123 Penicillin-Streptomycin (Gibico), 5% CO2, 37 °C.

124 Plasmids, siRNA and DNA transient transfections

125 Plasmids including pcDNA3 (Vector), pcDNA3-hIRF8 (IRF8), pcDNA3-hAR (AR),

126 containing the whole CDS domains were constructed by Genscript (Nanjing, China);

127 double luciferase reporter plasmid pEZX-FR03-hIRF8-luc, carrying the IRF8

128 promoter (-1106-+166bp), was constructed by FulenGen (Guangzhou, China);

129 3xFlag-Ub plasmid was generously provided by Dr. Guo-qiang Xu (Soochow

130 University, China). For transient transfections, cells were seeded into six-well plates

131 at 150,000 cells per well and transfected with IRF8 or AR expression vectors using

132 empty vector (pvector) as control. The total plasmid DNA was adjusted to the same

133 with empty vector.

134 siRNA and shRNA targeting IRF8 are as follows: siNC (shNC): TTC TCC GAA

135 CGT GTC ACG TTT C; siRNA1# (shIRF8A): GCA GTT CTA TAA CAG CCA GGG;

136 siRNA2# (shIRF8B): GGG AAG AGT TTC CGG ATA TGG.

137

138 Flat clone formation assay

139 LNCaP-shNC and LNCaP-shIRF8B cells were plated in the 6-well plates in triplicate

140 with 500 cells every well and cultured with complete medium for 14 days. For ENZ

141 sensitivity experiment, once cells were attached, ENZ (10 μM) were added to the

142 media using 0.1%DMSO as control. The complete medium or culture medium

143 containing ENZ or DMSO was replaced every two days, and cells were cultured for

144 10 days. Crystal violet was used to stain the colonies. Colony number and the

145 inhibition effects of ENZ were calculated according the number of the cloning

146 formation measured by ImagePro plus software.

147

148 Western blot analysis

149 Briefly, whole cell lysates were prepared with ice-cold RIPA lysis buffer or nuclear

150 and cytoplasmic protein extraction lysis buffer with protease inhibitors (Roche).

151 Approx. 30 μg total protein was separated by SDS-polyacrylamide gels and

152 transferred to PVDF transfer membrane and the membranes were incubated with

153 specific antibodies for IRF8 (ab28696, Abcam), AR (ab74272, Abcam),

154 p-STAT1(Tyr701), p-STAT1 (Ser727), STAT1 (Cell Signaling Technology), β-actin

155 (bsm-33036M, Bioss, China). Each experiment was repeated at least twice with

156 similar results. The densitometry data presented below the bands are ‘fold change’ as

157 compared with control normalized to respective β-actin from two independent western

158 blot analyses. Values are expressed as mean.

159

160 RNA purification and quantitative real-time PCR (qPCR)

161 Total RNA was prepared by using TRIzol reagent (Invitrogen) according to the

162 manufacturer’s instructions. RNA was reverse transcribed into cDNA using random

163 hexamers, which was used for quantitative real-time PCR using gene-specific primers.

164 Data were normalized by the level of GAPDH expression in each sample. The primer

165 sequences used in this study are as follows: IRF8 forward 5’- TCG GAG TCA GCT

166 CCT TCC AGA CT -3’, reverse 5’- TCG TAG GTG GTG TAC CCC GTC A -3’; AR

167 forward 5’- CTA CTC TTC AGC ATT ATT CCA G -3’, reverse CAT GTG TGA CTT

168 GAT TAG CA- 3’; GAPDH, forward 5’- CAT GAG AAG TAT GAC AAC AGC CT

169 -3’, reverse 5’- AGT CCT TCC ACG ATA CCA AAG T -3’.

170

171 Immunoprecipitation

172 Cells treated with the appropriate stimuli were lysed with Western and IP lysis buffer

173 (Beyotime, China). Aliquots of 600 μg protein from each sample were precleared by

174 incubation with 20 μL of PierceTM Protein A/G magnetic beads (88803; Thermo

175 Fisher Scientific) for 1 hour at 4℃ and then incubated with anti-IRF8 antibody (5628s,

176 Cell Signaling Tech), or anti-AR (ab74272, Abcam) in lysis buffer at 4℃ overnight.

177 Protein A/G beads were added and incubated for 2 hours at 4℃. The beads were

178 washed five times with PBS and once with lysis buffer, boiled, separated by 10%

179 SDS-PAGE, and analyzed by Western blotting as described above. For in vitro

180 binding assays, purified recombinant Flag-tagged AR protein was incubated with

181 recombinant His-tagged IRF8, and anti-flag agarose beads (Bimake) in Buffer C.

182 Bound immunocomplexes were washed three times with buffer B for nuclear extracts

183 or buffer C-400mM NaCl for total protein extracts immunoprecipitation and in vitro

184 binding assays.

185

186

187 Chromatin immunoprecipitation (ChIP) assay

188 The ChIP assay was performed using the EZ-Magna ChIPTM A/G Chromatin

189 Immunoprecipitation Kit (Millipore) according to manufacturer’s protocol. Anti-AR

190 antibodies and normal IgG were used to precipitate DNA. The normal IgG was used

191 as a negative control and the PSA enhancer region was used as a positive control.

192 The precipitated DNA was subjected to PCR to amplify the PSA and IRF8 promoter

193 regions with the primers listed as follows, and were quantified with the qPCR using

194 SYBR Green. PSA Enhancer+: TGG GAC AAC TTG CAA ACC TG, PSA Enhancer-:

195 CCA GAG TAG GTC TGT TTT CAA TCC A; IRF8 (-872/-863bp) F: 5’- TGT GTG

196 ATT CTC TAC TGG GCA A -3’, R: 5’- CTG GAA ACG GAA AAA GAA GCG T

197 -3’; IRF8 (-852/-843bp) F: 5’-TTT CTT TTT CCA GTG TCG TTC TCC -3’, R: 5’-

198 GGG CGT TAA GAT GTC CCC T -3’; IRF8 (-896/-783bp) F: 5’- GGA TAG AAC

199 GCG GAA ACG CT -3’, R: 5’- CCT GGG TGT GCA CTG ACA TTT A -3’.

200

201 Immunohistochemistry of IRF8 and AR protein expression in PCa cells

202 Segregation of clinic specimens. 13 benign prostatic hyperplasia (BPH), 20 untreated

203 primary PCa tissues and 13 CRPC paraffin-embedded tissues were collected from the

204 Departments of Urinary surgery at Jiangsu Province Hospital of Traditional Chinese

205 Medicine (TCM), which is approved by the Institutional Review Board of the Jiangsu

206 Province Hospital of TCM for IHC and MSP-PCR analysis.

207 Tissue microarray. Protein expression of IRF8 and AR in clinical prostate cancer

208 tissues were determined using tissue microarray analysis (TMA, Shanghai OUTDO

209 Biotech), containing 64 paired human primary prostate cancer/adjacent noncancerous

210 lesion tissues. Tissue microarray were stained with H&E to verify histology and the

211 immunohistochemistry staining of IRF8 (ab28696; Abcam) and AR (ab74272) were

212 performed. The positive staining rate was estimated in 3 fields with different staining

213 intensity by pathologists. The staining of IRF8 and AR in the TMA was scored

214 independently by two pathologists blinded to the clinical data using the following

215 criterion: the intensity of immunostaining was scored from 0 to 3 (0, negative; 1,

216 weak; 2, moderate; and 3, strong); the percentage of immunoreactive was deemed as 0

217 (0%–5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The final

218 score was calculated using the percentage score × staining intensity score as described

219 previously (29). For tissue samples from clinical PCa patient and animal xenografts,

220 the primary antibodies of the anti-IRF8 antibody (Abcam, 1:200), anti-AR antibody

221 (Abcam, 1:200) or anti-PCNA antibody (Santa Cruz, 1:200), were used for staining at

222 4℃ overnight. Immunostaining pictures were acquired using an inverted fluorescence

223 microscope (Leica) and the Integral optical density (IOD) sum was calculated using

224 ImagePro plus software. The average DAB staining intensity of the selected two cores

225 represents the quantitative protein expression level in these tissues.

226

227

228 DNA affinity binding assay

229 Nuclear extracts and affinity binding assays were prepared as described previously

230 (30). Briefly, LNCaP cells were treated with 10 nM DHT or 2000 IU/mL IFNα2a for

231 48 h, using ethanol and medium as control, respectively and lysed with ice-cold

232 nuclear protein extraction lysis buffer. 4.5-μg aliquots DNA formed by 8 pairs of the

233 ISRE and ISRE-like primers (2 WT and 6 mutants, Mut) were conjugated to

234 streptavindin M280 magnetic beads (Dynal) in the presence of 20 μg of salmon sperm

235 DNA (Sigma) for 10 min at room temperature. DNA-coupled magnetic beads were

236 incubated with 1 mg of nuclear extracts for 1 h at 4°C. Beads were washed three times

237 with washing buffer and bound materials were eluted in 1×sodium dodecyl sulfate

238 (SDS) sample buffer. Samples were separated by 8% SDS-PAGE for immunoblot

239 detection with anti-AR or other antibodies as indicated at 4°C overnight, using 30 μg

240 nuclear protein as input and Lamin B as negative control.

241

242 Transient transfections for IRF8 promoter activity

243 HEK293T cells (1×107/100 mm dish) were pre-cultured with 5% CSS overnight and

244 transfected with pcDNA3.1-AR (1 μg). Cells were co-transfected with an artificial

245 promoter luciferase reporter with both the proximal IRF8 5’ flank (flanking regions of

246 IRF8 (−1106/+166bp) and the firefly/renilla luciferase (pEZX-FR03-hIRF8, 3 μg) in

247 the same vector. The empty reporter vector (pEZX-FR03) was used as a control. The

248 transfected cells were cultured with 5% CSS for 24 h and then seed into 96-wells

249 (1×104 cell/well) and treated with DHT (1 nM, 10 nM); R1881(1 nM, 10 nM);

250 IGF-1(10 nM); EGF (10 nM) with or without ENZ (10 μM). DMSO was used as a

251 control. After incubation with these stimuli for 24h, the transfected cells were

252 collected and AR transcriptional activity was detected by dual luciferase assay.

253

254 Transient transfections for AR activity

255 LNCaP cells (1×107/100 mm dish) were pre-cultured with 5% CSS overnight and

256 transfected with various combinations of vectors to express IRF8 or control (0.75, 3

257 μg), IRF8-specific shRNA or scrambled control (3 μg). Cells were co-transfected with

258 an androgen-dependent firefly luciferase reporter (pMMTV-Luc, 3 μg, for AR activity

259 as previously described) (31) and a renilla luciferase promoter (pRL-SV40, 0.04 μg,

260 for transfection efficiency). The transfected cells were cultured with 5% CSS for 24 h

261 and then seeded into 96-wells (1×104 cell/well) and treated with or without DHT (10

262 nM).

263 Luciferase activities were measured 24 h after treatment using the dual luciferase

264 reporter system according to the manufacturer’s instructions (Promega). All samples

265 were tested in triplicate.

266

267 Xenografts and animal model

268 All athymic nu/nu BALB/c (BALB/c nude) and non-obese diabetic severely

269 combined immunodeficient (NOD-SCID) mice, aged 4-6 weeks, were purchased from

270 Lingchang biotech (Shanghai, China) and housed in a specific pathogen free facility

271 and maintained in a standard temperature and light-controlled animal facility for 1

272 week before used. For transgenic animal experiment, the Hi-myc mice (FVB-Tg

273 (ARR2/Pbsn-MYC) 7Key, strain number: 01XK8) were obtained from the Mouse

274 Repository of the National Cancer Institute. All animal procedures were performed

275 according to the guidelines of the US National Institutes of Health on Animal Care

276 and appropriate institutional certification, with the approval of the Committee on

277 Animal Use and Care of Center for New Drug Evaluation and Research, China

278 Pharmaceutical University (Nanjing, China).

279 For tumor growth of LNCaP cell xenograft (growing in intact BALB/C nude

280 mice), 1*107 LNCaP-shNC or LNCaP-shIRF8A cells (with higher KD efficiency,

281 indicated as LNCaP-shIRF8) were resuspended in 100 μL medium containing 50%

282 matrigel (BD Biosciences) and 50% growth media and subcutaneously injected in the

283 flank of BALB/c nude mice. For the tumor growth of LNCaP-CRPC xenograft

284 (growing in surgically castrated BALB/C nude mice with low serum androgen),

285 BALB/c nude mice were anesthetized using 10% chloral hydrate. Testes were excised

286 distal to the ligature, and the incision was closed with sterile dissolvable sutures and

287 disinfected with betadine solution and ampicillin. Two weeks later, LNCaP-shNC or

288 LNCaP-shIRF8 cells were inoculated as described above.

289 In the ENZ sensitivity study, orchiectomized BALB/c or NOD/SCID male mice

290 were inoculated subcutaneously with 1×107 LNCaP or LNCaP-shNC or

291 LNCaP-shIRF8 cells. Once tumors were established, mice were randomly assigned to

292 vehicle (1% carboxymethyl cellulose, 0.1% Tween-80, 5% DMSO), and ENZ (10

293 mg/kg) treatment groups. In the sensitivity study of ENZ combined IFNα, mice were

294 treated with vehicle alone, enzalutamide (10 mg/kg) alone, IFNα (1.5×107 IU/kg)

295 alone or ENZ (10 mg/kg) combined with IFNα (1.5×107 IU/kg). ENZ and IFNα were

296 administered by oral gavage and subcutaneously daily, respectively. Tumor volume

297 measurements were performed every 2 days and were calculated by the formula:

298 length×width2/2. At experimental endpoint, tumors were harvested and tumor

299 inhibition ratio were calculated and compared to the average tumor weight in the

300 vehicle control.

301

302 Clinical case study

303 Three advanced and untreated PCa patients with bone metastasis were enrolled at the

304 Department of Urinary Surgery at Jiangsu Province Hospital of TCM, and written

305 informed consent was received from participants prior to inclusion in the study, in

306 accordance with the guidelines of the International Ethical Guidelines for Biomedical

307 Research Involving Human Subjects (CIOMS), with the approval of the Institutional

308 Ethics Review Boards of Jiangsu Province Hospital of TCM (NO.2017NL-054-02).

309 Inclusion criteria were histologically confirmed prostate adenocarcinoma, untreated,

310 cT3-cT4 M0 and M1, serum prostate-specific antigen (PSA) ≥ 150 ng/mL, Gleason

311 Score ≥ 9 (4+5 or 5+4), age ≤ 80 yr, and normal liver function not suitable for

312 definitive treatment. For CASE 1 clinical study, two patients received Goserelin

313 Acetate Sustained-Release Depot (3.6 mg/28d) plus bicalutamide (50mg/d; Casodex)

314 as maximal androgen blockade (MAB) therapy, one patient received MAB therapy

315 plus recombinant IFNα2a (3*106 IU/3d; Yinterfen). For CASE 2 clinical study, one

316 advanced and untreated PCa patients with continued PSA level rise for 6 months were

317 enrolled and received MAB therapy plus recombinant IFNα2a (3*106 IU/3d;

318 Yinterfen). Patients in both cohorts were discontinued when there was evidence of

319 biochemical progression, defined as an increase of the PSA level ≥ 0.2 ng/mL on two

320 successive occasions at least 1 month apart.

321

322 Results

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

324 tissues.

325 To reveal the relationship between IRF8 and AR protein expression in PCa, PCa

326 tissue microarrays were immunohistochemically assessed using optimized anti-IRF8

327 and anti-AR antibodies. The representative primary PCa specimens showed high IRF8

328 expression and high AR accumulation compared with the adjacent nontumor tissue

329 (Fig. 1A; Fig. S1A-B). Tumor samples with high expression of AR protein exhibited

330 more IRF8 accumulation and a positive correlation was observed between protein

331 levels of IRF8 versus AR in primary PCa specimens (Fig. S1C-D). Similar findings

332 confirmed that IRF8 and AR was increased in tumour tissues of Hi-myc transgenic

333 (TG) murine PCa models during the PCa progression (≥ 6 months) (Fig. 1B; Fig.

334 S1E-F). Moreover, in LNCaP-CRPC xenografts models, IRF8 expression was

335 significantly decreased in tumours treated with ENZ (Fig. S1G). To clarify this point,

336 we quantified IRF8 and AR protein levels using IHC of paraffin-embedded clinical

337 BPH, primary PCa and CRPC tissues. The results showed that IRF8 and AR were

338 increased in primary PCa tissues compared to BPH, while low IRF8 but high AR

339 were observed in the representative CRPC specimens (Fig. 1C; Fig. S1H). A positive

340 correlation between IRF8 and AR in primary PCa was confirmed. In contrast,

341 statistically significant negative correlation was observed between protein levels of

342 IRF8 versus AR in CRPC specimens (Fig. 1D). Similarity to CRPC, IRF8 was

343 decreased but AR was increased in ENZR xenograft tissues and an inverse correlation

344 between IRF8 with AR protein was observed (Fig. 1E; Fig. S1I-J).

345 Previous findings revealed that epigenetic mechanisms including mutation and

346 promoter methylation play a crucial role in the IRF8 expression (19,25,32-35).

347 However, IRF8 was not frequently mutated and methylated in clinical PCa tissues and

348 cells (Fig. S1K; Supplementary Table). Lots of AR CHIP-seq data reveal that IRF8 is

349 a putative target of AR upon DHT or R1881 treatment (36-38). Furthermore, we found

350 several AR binding sites at the promoter of IRF8 using JASPAR database. We next

351 tested whether IRF8 expression is regulated upon AR activation and repression. AR

352 activation by dihydrotestosterone (DHT) increased IRF8 mRNA and protein levels in

353 PCa cells (Fig. 1F; Fig. S2A-C). However, AR repression by ENZ treatment

354 decreased IRF8 expression (Fig. 1G; Fig. S2C).

355 In CRPC, AR can be bypass-activated by growth factors (GFs) such as EGF,

356 IGF-1, at low levels of androgen after ADT (39-42). In the experiments, we treated

357 LNCaP cells with these factors and found that AR activation by GFs reduced IRF8

358 protein levels (Fig. S2D). We further identified whether these factors regulate IRF8

359 transcription by AR signaling, and the results showed that DHT and R1881-induced

360 AR activation promoted IRF8 transcription but bypass AR activation by GFs inhibited

361 IRF8 transcription. In contrast, ENZ reduced IRF8 transcription, reversed

362 androgen-mediated IRF8 upregulation and enhanced GFs-mediated IRF8

363 downregulation (Fig. 1H).

364 IRF8 transcription is mainly regulated by the binding of p-STAT1 to the

365 interferon-stimulated response element motif (ISRE, TTTC A/G G/C TTTC) (16).

366 Predictably, one canonical ISRE motif and one ISRE-like motif were identified in the

367 IRF8 promoter (Fig. S2E). In our experiments, the amounts of AR bound to the ISRE

368 and ISRE-like motifs were markedly increased after DHT treatment, whereas binding

369 by AR were not increased upon GFs stimulation and only p-ARSer81 were activated by

370 DHT (Fig. S2F-G). Furthermore, we found mutations of the GAAA motif greatly

371 diminished binding of AR to the ISRE-like motif while AR binding to ISRE mutant

372 was not reduced (Fig. 1I), indicating the ISRE-like motif has stronger affinity for AR.

373 We further determined whether the IRF8 is a direct AR targeted gene using

374 ChIP-qPCR assays. The results showed that AR was recruited to the ISRE and

375 ISRE-like sites regardless of DHT treatment, whereas occupancy of the ISRE-like site

376 was markedly increased and occupancy of the ISRE site was not changed in LNCaP

377 cells upon DHT stimulation (Fig. 1J; Fig. S2H-I). Together, these results identified

378 AR as a factor is recruited to the ISRE-like motif upon DHT stimulation which

379 promotes IRF8 transcription in an androgen-AR dependent pathway.

380

381 IRF8-knockdown promotes the proliferation and tumorigenesis of LNCaP cells

382 through AR activation.

383 To functionally demonstrate the potential role of IRF8 in tumorigenesis of PCa, we

384 generated two stable IRF8-knockdown (IRF8-KD) LNCaP cell lines (Fig. 2A; Fig.

385 S3A). The proliferation ratios of LNCaP-shIRF8 cells were higher than those in

386 LNCaP-shNC cells (Fig. 2B; Fig. S3B). Furthermore, increased clonogenic ability,

387 number and diameter of tumour cell spheres were also observed in LNCaP-shIRF8

388 cells (Fig. 2C-D; Fig. S3C-D). In vivo xenograft experiments showed that

389 LNCaP-shIRF8 results in increased tumour growth and proliferating cell nuclear

390 antigen (PCNA) protein expression in intact or castrated BALB/c nude mice (Fig.

391 2E-G; Fig. S3E-G), whereas 22RV1-IRF8-overexpression (OE) inhibited the

392 proliferation, clone formation and tumour growth (Fig. 2H-I; Fig. S3H). Moreover,

393 IRF8-KD also increased P38/MAPK/ERK phosphorylation and CD133 expression

394 compared to LNCaP-shNC cells (Fig. 2J), indicating that intrinsic IRF8 plays an

395 important role in inhibiting PCa progression.

396 To elucidate the mechanisms responsible for the growth-accelerating effects of

397 IRF8-KD in PCa, we assessed the effects of IRF8 on AR protein expression.

398 IRF8-KD increased and IRF8-OE decreased endogenous AR including AR variants

399 (ARvs) expression, especially in the nucleus (Fig. 3A-B and Fig.S4A-B). Moreover,

400 we demonstrated that IRF8 can reduce ectopic AR protein levels in AR- HEK293T

401 and PC3 cells transfected with AR alone or combined with various amounts of IRF8

402 expression plasmids (Fig. 3C-D).

403 Androgen binding to AR results in homo-dimerization and nuclear translocation,

404 and subsequent induction of target gene expression, which is regulated by the

405 ubiquitin/proteasome system (27). Native polyacrylamide gel electrophoresis of

406 lysates from HEK293T cells transfected with AR and IRF8 showed that decreased

407 level of AR protein mediated by IRF8 resulted in decreased expression of AR dimers

408 in a high molecular weight complex to a degree equal to the reduction of

409 ubiquitin-mediated AR dimers (Fig. 3E), indicating that IRF8-mediated AR protein

410 degradation could affect AR homo-dimerization. So we next detected effect of IRF8

411 on AR transcriptional activity. The AR target prostate-specific antigen (PSA) is the

412 most important marker for prostate cancer assessment. As expected, we found that

413 PSA and AR protein were increased by IRF8 siRNA (Fig. 3F). We next explored AR

414 activity using the pMMTV-luc luciferase reporter in IRF8-KD and IRF8-OE LNCaP

415 cells. IRF8-KD increased and IRF8-OE decreased the DHT-dependent AR activity

416 after androgen stimulation (Fig. 3G, Fig. S4C-D), which was associated with nuclear

417 AR expression mediated by IRF8 in LNCaP cells.

418

419 IRF8 physically interacts with AR and enhances AR degradation by

420 ubiquitin-dependent pathways

421 IRF8 mediated AR reduction could not be reversed by DHT treatment in both the

422 cytosol and nucleus, suggesting that IRF8 has no effect on AR translocation (Fig.

423 S4E-F), while the presence of IRF8 accelerated AR degradation and shortened the

424 half-life of AR (Fig. 4A-B; Fig. S5A-B). IRF8-mediated AR degradation was

425 reversed by proteasome inhibitor MG132, but MG101, calpastatin and lysosomal

426 enzyme inhibitor leupeptin had no effect on IRF8-mediated AR degradation (Fig.

427 4C-D; Fig. S5C). These results indicated that the ubiquitin/proteasome involved in the

428 IRF8-mediated AR degradation.

429 Cytoplasmic AR undergoes proteasome-mediated degradation in the absence of

430 ligand. Our results showed that ubiquitin or IRF8 mediated AR degradation could be

431 reversed by MG132 and DHT (Fig. S5D-E), indicating that IRF8 and ubiquitin

432 regulate the same cellular machinery responsible for cytoplasmic AR degradation.

433 The subcellular co-localization of IRF8 and AR were both observed by

434 immunofluorescent staining in LNCaP cells and ectopically overexpressed HEK293T

435 cells (Fig. 4E; Fig. S5F). We further found that IRF8 could precipitate with AR and

436 vice versa (Fig. 4F). To confirm this interaction, we proved that IRF8 could be eluted

437 with AR in vitro binding assays (Fig. S5G). To identify region within AR responsible

438 for its binding to IRF8, we co-transfected IRF8 with several N-terminal Flag-tagged

439 AR truncated mutants, including AR full-length (AR-FL), AR-NTD, AR-DBD,

440 AR-Hinge and AR-LBD into HEK293T cells (Fig. S5H). Co-IP assay showed that

441 IRF8 was immunoprecipitated with AR-FL, AR-NTD but not with AR-LBD,

442 AR-Hinge and AR-DBD (Fig. 4G). These results indicated that IRF8 physically

443 interacts with the N terminal domain of AR. We next analyzed the effect of IRF8 on

444 AR ubiquitylation. Results showed that IRF8 enhanced AR polyubiquitination as

445 visualized by staining with Flag-ubiquitin (Fig. 4H). There are two common types of

446 polyubiquitination, which are distinguished by the lysine residue through which

447 formation of K48-chain and K63-chain occurs. IB revealed that both WT and K48

448 ubiquitin led to poly-ubiquitinated AR, but polyubiquitination was impaired in the

449 presence of K63 ubiquitin (Fig. 4I), suggesting that IRF8-mediated AR

450 polyubiquitination is mainly via the K48 branch. Moreover, the K48-chain of AR

451 polyubiquitination is mainly mediated by mouse double minute 2 homolog (MDM2)

452 (43). Immunoprecipitation experiments confirmed that AR interacted with MDM2

453 (Fig. S5I). In addition, we detected the interaction between IRF8 and MDM2 (Fig.

454 S5J). Together with previous result that AR interacted with IRF8, we tested whether

455 IRF8 could influence the interaction between AR and MDM2. Immunoprecipitation

456 experiments further supported that the interaction between AR and MDM2 was

457 enhanced upon the expression of IRF8 (Fig. 4J).

458 Together with the ubiquitination and protein interaction experiments, our results

459 suggested that IRF8 enhanced the interaction between AR and its E3 ligase MDM2

460 and thus promoted the ubiquitination and degradation of AR.

461

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

463 We further explored the effects of IRF8 on sensitivity to ENZ therapy in vitro and in

464 vivo. LNCaP viability treated with ENZ was decreased, whereas LNCaP-shIRF8 cells

465 were resistant to ENZ treatment with increased AR expression (Fig. 5A-B; Fig.

466 S6A-B). Moreover, the IRF8-OE 22RV1 cell was more sensitive to ENZ treatment

467 (Fig. S6C). Furthermore, the colony number with ENZ treatment was significantly

468 decreased in LNCaP-shIRF8 cells compared to controls (P<0.05) (Fig. 5C; Fig.

469 S6D-E). Notably, IRF8-KD decreased and IRF8-OE promoted caspase-3 activity

470 induced by ENZ treatment (Fig. 5D-E). We further confirmed that IRF8-KD could

471 induce ENZR in castrated BALB/c nude and NOD-SCID mice. The ENZ treatment

472 inhibited tumour growth of LNCaP-shNC (P<0.05), while it did not significantly alter

473 the growth of LNCaP-shIRF8 tumours (Fig. 5F; Fig. S6F-G).

474

475 IFNα enhances ENZ sensitivity by targeting the IRF8-AR axis

476 Since decreased IRF8 induces ENZR during ADT, we next explored a novel strategy

477 to attenuate the resistance. The IRF8 expression could be induced by IFNs through

478 phosphorylation and dimerization of STAT1 (p-STAT1Tyr701 and p-STAT1Ser727)

479 (19,44,45). IFNγ receptor is frequently inactivation but expression of IFNA receptors

480 is much higher than IFNγ receptors in human PCa cells (1,3). Fundamental studies

481 have shown IFNα phosphorylates STAT1 (p-STAT1Tyr701 and p-STAT1Ser727) and

482 activates IRF8 gene expression by binding to the GAS element in human NK and T

483 cells (4-6). We explored whether IFNα could improve ENZ efficiency in PCa by

484 targeting the IRF8-AR axis. We found that IFNα increased IRF8 and decreased AR

485 including ARvs expression in PCa cells (Fig. 6A; Fig. S7A). DNA pull-down assays

486 showed that IFNα treatment promotes AR binding to both WT and mutant ISRE-like

487 motif of the IRF8 promoter rather than affecting only the canonical STAT1 pathway

488 (Fig. 6B-C; Fig. S7B). Furthermore, AR degradation by the ubiquitin pathway was

489 accelerated upon IFNα treatment, and the IFNα-induced AR reduction was inhibited

490 by MG132, however, this was greatly diminished when IRF8 was KD in 22RV1 cells

491 (Fig. 6D-F). These results demonstrated that IFNα mediated IRF8 expression

492 promotes AR degradation by the ubiquitin pathway. We next examined the synergy

493 between IFNα and ENZ in inhibiting the cancer cells growth. While IFNα alone had

494 limited effect on cancer cell death (Fig. S7C), its combination with ENZ markedly

495 dropped the IC50 value of ENZ in PCa cells (Fig. 6G).

496 We further assessed the therapeutic activity of IFNα and ENZ combination in

497 CRPC mice. IFNα combined with ENZ showed the better curative effects compared

498 to IFNα or ENZ alone, whereas there were no significant changes among all the

499 treatments in IRF8-KD groups (Fig. 6H; Fig. S7D-F), indicating that IRF8 may have

500 an important role in the therapeutic effect of IFNα-ENZ combination therapy.

501 We retrospectively designed a clinical case study to evaluate the effects of IFNα

502 combined with maximal androgen blockade (MAB) therapy on advanced metastatic

503 PCa patients. In the CASE one clinical study, after three months of MAB alone therapy,

504 both patients had reduced serum PSA levels, and after six months, serum PSA levels

505 were increased. In the MAB and IFNα combination treatment group, the patient serum

506 PSA level continued to decrease with treatment and remained below the biological

507 progression line during follow-up treatment for eleven months (Fig. S7G, CASE 1). In

508 the CASE two clinical study, the PSA level of the one untreated PCa patient was

509 continuously increased over the course of six months. Once the patient received MAB

510 combined with IFNα therapy, the PSA level declined continuously for three months.

511 The combination therapy was stopped when the patient developed a fever, and the PSA

512 level subsequently increased slightly (Fig. S7G, CASE 2). These clinical studies

513 indicated that the combination of IFNα and hormonal therapies may enhance the

514 treatment efficacy.

515

516 Discussion 517 In this work, we demonstrate the precise mechanisms of how IRF8 was involved in

518 PCa progression and ENZR. We report that IRF8 could reduce AR protein levels and

519 block AR activation via the ubiquitin/proteasome systems (Fig.6I). IRF8 is induced by

520 IFNs as a transcription factor in the IFN system, it is thought to mainly function in the

521 nucleus. We found IRF8 protein levels are upregulated in primary PCa and its

522 expression is elevated upon DHT stimulation; in contrast, its levels are downregulated

523 in CRPC and ENZR tissues upon bypassing AR activation and AR repression by ENZ.

524 In CRPC after ADT, IGF-1 and EGF can also enhance the bypass of AR activation

525 via AR phosphorylation at sites different than that phosphorylated by DHT treatment

526 (p-ARSer81) (46-48) and we found that p-ARSer81 is responsible for the different effects

527 on IRF8 expression caused by androgen-AR activation and bypass of AR activation

528 (Fig. S2F-G). Moreover, we found that decreased IRF8 expression in CRPC, resulted

529 in tumour proliferation even under ADT and ENZR, accompanied by a significant

530 upregulation of AR. Accordingly IRF8 overexpression decreased AR expression. We

531 found that IRF8 promoted AR degradation dependent on enhanced AR ubiquitylation

532 in the cytosol. The function of IRF8 in the cytosol and ubiquitylation is less

533 understood (49,50). We report here that AR interacts with IRF8 in the cytosol through

534 its AR-NTD and regulates its ubiquitylation. IRF8-mediated AR polyubiquitination is

535 mainly branched through K48, and we verified MDM2 as the E3 ligase responsible

536 for IRF8-mediated cellular AR polyubiquitination and degradation. Although we

537 found that IRF8 promoted the interaction of AR and its E3 ligase MDM2 in the

538 cytosol, the detailed mechanisms underlying the involvement of IRF8 in

539 MDM2-mediated AR degradation need to be further investigated. We suggest that

540 IRF8 is part of the signaling complex that includes AR and an E2

541 ubiquitin-conjugating enzyme, such as the MDM2-Ubc5 complex, altering the

542 activity of the MDM2-Ubc5 complex (43). There may be additional E3 ligases that

543 modulate IRF8-mediated AR responses, and further work is required to fully reveal

544 the roles of IRF8 in the regulation of AR signaling pathway.

545 For translational medicine, we found that IFNα increased IRF8 expression and

546 promoted AR degradation through both STAT1 and AR pathways activation (Fig. 6).

547 Without androgen, treatment with IFNα alone promoted AR binding to both the WT

548 and mutant ISRE-like domains in the IRF8 promoter, while the canonical

549 phosphorylation and dimerization of the STAT1 pathway (p-STAT1Tyr701 and

550 p-STAT1Ser727) did not compete with binding to the same site. In vivo and in vitro

551 studies, although AR could be activated by IFNα, treatment with IFNα alone had

552 limited effect on PCa cell proliferation. It is possible that activation of the AR

553 pathway by IFNα only promotes AR bound to ISRE-like motifs in the IRF8 promoter

554 rather than enhancing AR bound to the androgen response element in the

555 proliferation-related target gene promoter. Treatment of IFNα combined with ENZ

556 significantly improved efficiency in suppressing tumour growth of LNCaP-CRPC,

557 while the enhanced anti-tumour activity was attenuated in LNCaP-shIRF8 CRPC.

558 Most importantly, in our clinical case study, IFNα combined with hormonotherapy

559 MAB was more efficacious than MAB alone. Unfortunately, although the potential

560 for combination treatment is obvious, the side effects of IFNα therapy, such as fever

561 and excessive perspiration, may limit its clinical use as an anti-tumour therapy.

562 Drug-resistant CRPC is current common and a major challenge in the management of

563 PCa, and targeting the AR axis by disrupting androgen-AR interactions remains the

564 primary treatment for CRPC. We suggest that alternative drugs can induce IRF8

565 expression could be combined with ENZ therapy to target the IRF8-AR axis. In

566 conclusion, we showed that decreased IRF8 in PCa cells impair its interaction with

567 AR and promotion on AR degradation via the ubiquitin/proteasome pathway,

568 accompanied AR activation, AR-mediated CRPC progression and ENZR, suggesting

569 that IRF8 could be a promising target to overcome ENZ resistance in CRPC.

570

571 Acknowledgments

572 We thank Dr. Lutz Birnbaumer (NIEH) for critical comments and assistance with

573 preparation of the manuscript. We thank pathologists Mrs. Ning Su and Mr. Hongbao

574 Yang for H&E to verify histology and the immunohistochemistry staining of IRF8

575 studies. These studies were supported by the National Natural Science Foundation of

576 China (Nos. 81903656 to HW, 81772732 to QZ, 81673468 to YY, 81672752 to ZC),

577 “Double First-Class” University project (No. CPU2018GF10 and No. CPU2018GY46

578 to YY), Natural Science Foundation of Jiangsu Province (No. BK20180560 to HW),

579 and China Postdoctoral Science Foundation (No. 2018M632430 to HW).

580

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745

746 Figure legends

747

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

749 ENZR tissues. (A) Representative IHC of IRF8 and AR protein expression in primary

750 PCa samples compared with adjacent noncancerous lesion tissues (ADJ) from PCa

751 tissues microarrays (TMA). Scale bar, 100 μm. (B) IHC of IRF8 and AR protein

752 expression in wildtype (WT) and Hi-myc (TG) mice prostate tissues. Scale bar, 100

753 μm. (C) IHC staining of IRF8 and AR protein expression in BPH(n=11), primary PCa

754 (n=22) and CRPC tissues (n=11). Scale bar, 100 μm. (D) Correlation of IRF8 with AR

755 expression normalized by the average IOD of BPH tissues in primary PCa and CRPC

756 specimens was analyzed, linear regression coefficient and statistical significance are

757 indicated. (E) IHC staining of IRF8 and AR protein expression in ENZ resistant

758 (ENZR) xenograft tissues (n=6). Scale bar, 100 μm. (F) LNCaP cells, pre-cultured with

759 5% CSS for 3 days, were treated with DHT for 24 h and IFR8 expression was analyzed

760 by qPCR (left panels), western blot (right panels). (G) IRF8 mRNA (left panels) and

761 protein expression (right panels) in LNCaP cells cultured in the presence of the

762 indicated concentration of ENZ for 24 h. (H) HEK293T cells co-transfected with

763 pcDNA3.1-AR and IRF8 promoter luciferase reporter were treated in triplicate with

764 DHT (1, 10 nM); R1881 (1, 10 nM); IGF-1(10 nM); EGF (10 nM) with and without

765 ENZ (10 μM) for 24 h. Luciferase activities were measured 24 h after treatment. (I)

766 Immobilized WT and mutant ISREs were incubated with nuclear extracts from LNCaP

767 cells treated with DHT for 24 h, and bound proteins were detected by IB with anti-AR.

768 (J) ChIP analysis to detect AR binding to the IRF8 promoter. LNCaP cells were

769 stimulated by DHT for 48 h. Equal amounts of chromatin (DNA) were subjected to

770 ChIP assay with AR-specific antibody. Normal IgG and protein A/G beads alone were

771 used as negative controls. AR occupancy of IRF8 promoter is shown relative to

772 background signal with normal IgG control antibody.

773

774 Fig. 2. IRF8-KD in LNCaP cells promotes the growth and tumorigenesis in vivo

775 and in vitro. (A) Knockdown efficiency in LNCaP-shIRF8 cells determined by

776 western blot. (B) Absorbance at 450 nm of LNCaP-shIRF8 cultured with 5% FBS

777 (left panel), 5% CSS (middle panel), or 5% CSS containing 1 nM R1881 (right panel)

778 in 96-well plates for 24-120 h. (C) Representative images of clone formation of

779 LNCaP-shNC and LNCaP-shIRF8 cells cultured for 10 days. (D) Representative

780 sphere formation of LNCaP-shNC and LNCaP-shIRF8 cells cultured for 14 days. (E,

781 F) Knockdown of IRF8 in tumorigenesis. Tumour growth of LNCaP-shIRF8 cells in

782 BALB/c nude mice (E, n=9), and in castrated BALB/c nude mice (F, n=9). (G)

783 Representative images of IHC staining of PCNA in LNCaP-shIRF8 BALB/c

784 xenografts. Scale bar, 100 μm. (H, I) OE of IRF8 in tumorigenesis. Proliferation (H)

785 of 22RV1-IRF8 overexpression cells in vitro and tumour growth of 22RV1-IRF8

786 overexpression cells in vivo (I, n=9). (J) Western blot analysis for p-P38 MAPK, P38

787 MAPK, p-ERK, ERK, p-AKT(Thr308), p-AKT(Ser473), AKT, CD133, Oct3/4, and

788 β-actin in LNCaP-shNC and LNCaP-shIRF8 cells.

789

790 Fig. 3. IRF8 reduces AR protein levels and activity. (A) Western blot analysis for

791 IRF8 and AR in the LNCaP-shIRF8 stable cell line for whole cell lysate (WCL),

792 cytoplasm protein(Cyto), and nuclear protein(Nuc), β-actin, GAPDH, and lamin B

793 were used as reference proteins, respectively. (B) Western blot analysis for IRF8 and

794 AR in lysates of LNCaP cells or IRF8 and AR (AR full-length: ARfl and AR variants:

795 ARvs) in 22RV1 cells 48 h after transfection with IRF8 plasmid (pIRF8) at various

796 amounts (0.125–1 μg). (C) Western blot analysis of IRF8 and AR in HEK293T cells

797 co-transfected with AR plasmid (0.3 μg) and different concentrations of IRF8 plasmid

798 (0.125–1 μg) for 24 h and 48 h. (D) Western blot analysis of exogenous IRF8 and AR

799 in PC3 cells transfected with AR plasmid (0.3 μg) and IRF8 plasmid (1 μg) alone or

800 in combination for 48 h. (E) Western blot analysis of AR dimers under

801 native polyacrylamide gel electrophoresis conditions in HEK293T cells transfected

802 with pAR, pIRF8 and pFlag-Ub vector alone or in combination. (F) Western blot

803 analysis of PSA, AR and IRF8 in LNCaP cells transfected with siRNA targeting IRF8

804 for 24 h. (G) Luciferase reporter assays for AR activity in IRF8-KD (LNCaP-shIRF8;

805 left panels) and IRF8-OE (LNCaP-IRF8; right panels) LNCaP cells treated with DHT.

806 ***p < 0.001 by one-way ANOVA.

807

808 Fig. 4. IRF8 enhances AR degradation through ubiquitin-pathways. (A, B) PC3

809 cells were co-transfected with AR plus IRF8 or AR plus vector control plasmid for 6h

810 and treated with CHX (100 μg/mL) for the indicated time. Cell lysates were

811 immunoblotted for AR, IRF8 and β-actin. Relative quantification was carried out for

812 three biological replicates using β-actin as loading control. P-values were calculated

813 using Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01. (C) HEK293T cells were

814 co-transfected with pIRF8, pAR or vector control for 48 h and incubated with CHX in

815 the presence of proteasome inhibitor MG132 (10 μM), calpain inhibitors MG101 (10

816 μM), MG132 (0.1 μM), calpastatin (5 μM) or lysosomal inhibitor leupeptin (Leu, 50

817 μM) for 5 h, or DMSO as control. Cell lysates were analyzed for IRF-8 and β-actin

818 expression. (D) Cells were cotransfected with the AR and control (pcDNA3.1) or

819 IRF8 plasmids for 24 h, and treated with DMSO or MG132 (10 μM) for 5 h. Cell

820 lysates were immunoblotted with the indicated antibodies. (E) Confocal microscopy

821 for endogenous IRF8 and AR in LNCaP cells treated with DHT (10 nM) for 48 h. (F)

822 Co-IP of IRF8 and AR in HEK293T cells co-transfected with pIRF8, pAR or vector

823 control for 48 h. (G) Western blots were performed using indicated antibodies after IP

824 of the full-length and truncated forms of AR from HEK293T WCL using the Flag

825 antibody. (H) HEK293T cells were transfected with various combinations of pAR,

826 pIRF8, and pFlag-Ub plasmids for 48 h. Cells were treated with MG132(10 μM) for 5

827 h, followed by nuclear and cytoplasmic protein lysate preparation and cytosolic

828 protein were used for IP with an anti-AR antibody and IB with the indicated

829 antibodies. (I) HEK293 cells were transfected with AR, IRF8, and WT, K48, or K63

830 ubiquitin (Ub) plasmids. Cells lysates were subjected to IP 48 h later with an anti-AR

831 antibody followed by IB with an anti-HA antibody. (J) PC3 cells were first

832 transfected with pcDNA3.1 or IRF8 for 12 h and then transfected again with

833 HA-MDM2 or HA-MDM2 and AR for 48 h. The amount of plasmids was slightly

834 adjusted to make the proteins expressed in similar level in different samples. Cell

835 lysates were subjected to IP and followed by IB with the indicated antibodies.

836

837 Fig. 5. LNCaP-shIRF8 cells are less sensitive to ENZ treatment. (A) LNCaP-shNC

838 and LNCaP-shIRF8 cells were treated with different doses of ENZ for 48h (left panel)

839 and 72 h (right panel), and cell viability compared to DMSO control was tested using

840 a mitochondrial activity-based cell counting kit–8 (CCK-8) assay. (B, C)

841 LNCaP-shNC and LNCaP-shIRF8 cells were treated with ENZ (10 μM), and the

842 growth curve during 4 d was determined by CCK-8 assay (OD450, B), the inhibition

843 ratio of ENZ in LNCaP-shNC and LNCaP-shIRF8 cells was detected by plate colony

844 formation assay (C). (D, E) Caspase 3 activity in LNCaP-shIRF8 and LNCaP-IRF8

845 cells treated with ENZ (5 μM) for 72 h and 48 h, respectively. (F) Tumour growth and

846 final tumour weight of LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated

847 BALB/c nude mice treated with ENZ (10 mg/kg/d) i.g. for 28 days.

848

849 Fig. 6. IFNα enhances ENZ sensitivity by targeting the AR-IRF8 axis. (A)

850 Western blot analysis for IRF8 and AR (ARfl and ARvs) in 22RV1 cells treated with

851 IFNα at the indicated concentrations for 24 h. (B, C) DNA affinity binding assay for

852 AR, IRF8 (B), p-STAT (Tyr 701), p-STAT1 (Ser 727), total STAT1 and lamin B (C)

853 bound to ISRE-wt2 and ISRE-mut2 in LNCaP cells treated with IFNα (1000 IU/mL,

854 48 h). (D, E) 22RV1 cells were treated with IFNα (2000IU/mL, 48 h) and then treated

855 with CHX (200 μg/mL) for the indicated times (D), or with degradation inhibitor

856 MG132(10 μM, 5 h, E). WCLs were subjected to IB for AR. (F) 22RV1 cells were

857 treated with IFNα (2000 IU/mL, 24 h) in presence of two IRF8-specific siRNA or

858 negative control, prior to treatment with MG132 (10 μM, 5 h), the cytoplasmic lysates

859 were subjected to IP with anti-AR antibody and subsequent IB with the indicated

860 antibodies. (G) Cell inhibition of LNCaP and 22RV1 cells treated with various

861 concentrations of ENZ in the presence of IFNα (1000 IU/mL). (H) Tumour growth of

862 LNCaP-shNC and LNCaP-shIRF8 xenografts in castrated NOD-SCID mice treated as

863 described in the Methods section. (I) The correlation of gene expression levels

864 between IRF8 and AR in the development and progression of prostate cancer as well

865 as their regulatory mechanisms in tumor growth and ENZ resistance.

866

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AR Author ManuscriptPublishedOnlineFirstonApril27,2020;DOI:10.1158/0008-5472.CAN-19-2549 AR AR cancerres.aacrjournals.org ENZ X IFN Tumor growthandENZresistance IRF8 Proliferation-related gene expression α improvesenzalutamideefcacybytargetingtheIRF8-ARaxis. IRF8 Stabilized ARprotein on September 30, 2021. © 2020American Association for Cancer Research. Feedback degradationofARmediatedbyIRF8 IRF8 IRF8 AR Ub

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AR Author Manuscript Published OnlineFirst on April 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2549 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

Hongxi Wu, Linjun You, Yan Li, et al.

Cancer Res Published OnlineFirst April 27, 2020.

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