Resource availabilityLead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Ping Mu (Ping.Mu@UTSouthwestern.edu).

Material availability

All cell lines, plasmids, and other reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement if there is potential for commercial application.

Data and code availability

All the described bulk RNA-seq data has been deposited in the Gene Expression Omnibus under the accession numbers GSE240305, reviewer token is otgrsqsszrydlib. Microscopy data reported in this paper will be shared by the lead contact upon request.

All analysis in this manuscript was performed using open-source software. Bulk RNA-Seq analysis was done using the QBRC Bulk RNA-Seq pipeline and all code could be accessed: https://github.com/QBRC/QBRC_BulkRnaSeqDE. GSEA statistical analysis was carried out with the R package ‘fgsea’ (v1.14.0) in conjunction with the ‘Hallmark’ libraries from MsigDB.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and subject detailsCell lines

The parental LNCaP/AR cell line was obtained from the laboratory of C.L. Sawyers at Memorial Sloan Kettering Cancer Center (MSKCC). DU145 (HTB-81), PC3 (CRL-1435) and HEK293T (CRL-3216) cell lines were purchased from ATCC. LNCaP/AR and PC3 cells were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 1% l-glutamine, 1% penicillin-streptomycin (p/s), 1% HEPES and 1% sodium pyruvate. DU145 and HEK293T cells were cultured in DMEM high-glucose medium supplemented with 10% FBS, 1% p/s and 1% l-glutamine. LNCaP/AR, DU145 and PC3 cells were passaged at a ratio of 1:6 every 3-5 days. HEK293T cells were passaged at a ratio of 1:8 every 3-5 days. The MDA-PCa-2b cell line was purchased from ATCC (CRL-2422) and cultured following the manufacturer’s instruction. More specifically, cells were cultured in Ham’s F 12 K (Kaighn’s) Medium supplemented with 20% FBS, 1% p/s, 25 ng/ml Cholera toxin (Sigma-Aldrich, C8052), 10 ng/ml mouse epidermal growth factor (Fisher Scientific, CB-40010), 0.0005mM O-phosphoethanolamine (Sigma-Aldrich, P0503), 0.1 ng/ml hydrocortisone (Sigma-Aldrich, H0135), 45 nM sodium selenite (Sigma-Aldrich, S9133) and 5 µg/mL human recombinant insulin (Thermo Fisher Scientific, 12-585-014). They were passaged at a ratio of 1:2 or 1:3 (depending on confluency) every 3–4 days and were cultured in Poly-d-Lysine (Gibco™, A3890401) coated plates. When LNCaP/AR cells were treated in charcoal stripped media (CSS), they were cultured in RPMI 1640 medium supplemented with 10% charcoal stripped serum (CSS), as well as all the other supplements mentioned above. All cell cultures were tested monthly for mycoplasma using the MycoAlertTM Plus Mycoplasma Detection kit (Lonza, LT07-710). Cell lines were validated yearly using human short tandem repeat profiling cell authentication and compared to ATCC profiles.

CRISPR-Cas9 and overexpression plasmid

Lentiviral-based constructs were used for CRISPR-based KO and for overexpression of all genes in this study and were modified as described before [24, 29, 30]. Briefly, the All-In-One lentiCRISPRv2 (Addgene, 52961), pLKO5.sgRNA.EFS.RFP (Addgene, 57823) and pLKO5.EFS.GFP (Addgene, 57822) were used to generate the CRISPR and guide RNAs. A non-targeting RNA was used as empty control. The Benchling (https://benchling.com) guide RNA designing tool was used to design the guide RNAs. Sequences of guide RNAs are listed in Table S1. Key Resources. UBE2J1 (NM_016021.2), UBE2J1-HA (NM_016021.2) and AR-FLAG (NM_000044.6) were amplified from cDNA purchased from The McDermott Center for Human Growth and Development at UT Southwestern and cloned into a vector via restriction enzyme cloning. The UBE2J1 overexpression plasmid used for rescue assays was mutated at the PAM sequences as well as in the sequence were the guide RNA targets. All sequences were cloned into pLenti-CMV-P2A-blast vector (Addgene, 17486), or pLVX-IRES-PURO vector (Addgene, 107435).

Lentivirus preparation and CRISPR/overexpression cell line construction

The different cell lines utilized in this study were constructed by lentiviral infection as previously described [24, 29, 30], with minor modifications. HEK293T cells were seeded at a concentration of 1.5 × 106 in 2 mL of medium in a 6-well plate 24 h before transfection. Plasmid and virus packaging vectors PspAX2 (Addgene, 12260) and VSV-G (Addgene, 138479), as well as Lipofectamine® 2000 (Thermo Fisher Scientific, 11668500) were diluted with OPTI-MEM I Reduced Serum Medium (Gibco™, 31985062) separately. After the diluted plasmid and the Lipofectamine® 2000 were mixed and incubated for 20 minutes at RT, the mixture was added to HEK293T cells in dropwise manner. The medium was changed 8–16 h after transfection. 24 and 48 h after the transfection, the virus containing medium was filtered with a 0.45 µm syringe filter and saved for transduction. LNCaP/AR, DU145 and PC3 cells were seeded at a concentration of 400,000 cells per well in 2 mL of medium in 6-well plates 24 h before the transduction. MDA-PCa-2b cells were seeded at a concentration of 800,000 cells per well in 2 mL of medium in a six-well plate coated with Poly-d-Lysine 48 h before the transduction. For the transduction the medium was replaced with medium containing 50% virus, 50% growth medium and 5 µg/mL polybrene (Millipore Sigma, TR-1003-G). The virus containing medium was replaced with regular growth medium after 24 h. Cells were selected with 2 µg/mL puromycin (InvivoGen, ANT-PR-1) for 4 days or 10 µg/mL blasticidin (Gibco™, A1113903) for 5 days.

In vivo xenograft experiment

All animal experiments were performed in compliance with the guidelines of the Animal Resource Center at UT Southwestern Medical Center. Animals were housed under humidity and temperature-controlled environment with a 12-h light/12-h night cycle in a pathogen free facility. The in vivo xenograft experiments were performed as previously described [24, 29, 30]. Briefly, 2 × 106 LNCaP/AR cells were suspended in a solution where half the volume was Matrigel (BD Biosciences, 356237) and the other half was growth medium. We subcutaneously injected 100 µL into both flanks of 7-week-old castrated or intact SCID male mice. After 3 days of cell injection mice were gavage with 10 mg/kg Enzalutamide (daily) or vehicle (1% carboxymethyl cellulose, 0.1% Tween 80, 5% DMSO) (daily). All animals were separated into groups at random and tumor size was measured weekly with a digital caliper. The tumor cell injection and tumor treatment were performed by one researcher, while the tumor measurement and data analysis were performed by a different researcher to ensure that the studies were run in a blinded manner. The mice were monitored to minimize discomfort, distress, pain, or injury throughout the course of the xenograft experiments. Animals were removed from the study and euthanized if any signs of pain and distress were detected or if the tumor volume reached 2000 mm3. All procedures were performed in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of UT Southwestern Medical Center (protocol #2018-102461). Male CB17/lcr-Prkdcscid/lcrlcoCrl were purchased from Charles River. No statistical method was used to predetermine samples size, it was decided based on previously established protocols [24, 29, 30].

Cell growth and fluorescence-activated cell sorting (FACS)-based competition assays

For the cell growth assay in Fig. 1K, 9000 LNCaP/AR cells were plated in a 24-well plate in either vehicle (DMSO) or 10 µM ENZ in full serum medium. Cells were manually counted on day five using a hemocytometer. For the cell growth assay in Figs. 7C, 3000 LNCaP/AR cells were plated in a 24-well plate in either vehicle (DMSO), 80 nM AC67 or 16 nM AC176 in full serum medium. Cells were manually counted on day 7 using a hemocytometer. The fluorescence-activated cell sorting (FACS)-based competition assay was performed as described previously in [24, 29, 30]. Briefly, a 50/50 mixture was created with sgNT-RFP and sgUBE2J1-GFP cells and were either treated with vehicle (DMSO), 10 µM ENZ (LNCaP/AR), 16 nM AC176 (LNCaP/AR), 80 nM AC67 (LNCaP/AR) or 1 µM ENZ (MDA-PCa-2b) in full serum medium or treated with charcoal stripped serum (CSS) (LNCaP/AR). The percentages of RFP-positive and GFP-positive cells were measured using Attune NxT (version 4.2.1627.1) at Day 0 and the subsequent different days presented in the figures. Relative cell number fold change was calculated as previously described in [24, 29, 30]. To be certain that the results were not biased, the cell number and percentage were automatically measured by the Attune NxT Acoustic Focusing Cytometer. Bar plots visualizing the relative cell number fold change were created by Prism 10 Sotfware (https://www.graphpad.com/scientific-software/prism/). Three biological triplicates were used, mean ± s.e.m. is reported and experiments were repeated at least two times and achieved similar results. No data points were excluded.

Luminescent cell viability assay

CellTiterGlo® luminescent cell viability assay kit (Promega, 7570) was used to measure cell growth of LNCaP/AR cells transduced with UBE2J1 overexpression plasmid according to the manufacturer’s instruction. The cells were seeded in a 96-well plate and treated with Veh (DMSO) or AR degrader 16 nM AC176 for 7 days. 100 µL of CellTiterGlo® Reagent was added to each well and the contents were shaken for 12 min in an orbital shaker. The luminescence was then recorded with the Tecan Spark® Cyto plate reader. Treatments were conducted in triplicates and all experiments were repeated at least twice and achieved similar results. No data points were excluded and mean ± s.e.m. were reported.

Relative gene expression via RNA extraction and RT-qPCR

RNA was isolated from cells using TRIzol (Ambion, 15596018), and cDNA was made using SuperScriptTM IV VILOTM Master Mix with ezDNaseTM (Thermo Fisher Scientific, 11766500) and 200 ng/mL RNA template, per manufacturer’s instructions. cDNA was amplified with 2× PowerUPTM SYBRTM Green Master Mix (Thermo Fisher Scientific, A25778). Each reaction was performed in triplicate, data was analyzed by the delta delta Ct method (2−ΔΔCq) and target genes’ expression was normalized to the expression of a house keeping gene. Bars visualizing the relative gene expression were created by Prism 10 Sotfware (https://www.graphpad.com/scientific-software/prism/) with expression fold change normalized to control cell lines. Three biological replicates were used and mean ± s.e.m. is presented. Experiments were repeated at least two times and achieved similar conclusions. No data points were excluded. XBP1 splicing assay was performed after cDNA amplification, the cDNA was run in a 2.5% agarose gel and visualized by ethidium bromide staining. Primers used for qPCR can be found in Table S1. Key Resources.

Protein detection via Western Blot

For western blots, protein was extracted from cell lysates with Radioimmunoprecipitation Assay lysis buffer (RIPA buffer, 150 mM sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) supplemented with protease and phosphatase inhibitors (PierceTM, A32965) and incubated for 15 min on ice. The samples were then centrifuged at 20,000g for 10 min at 4 °C and the supernatant was collected. Protein quantification was performed using a Pierce BCA Protein Assay Kit (PierceTM, 23225). Upon the addition of 5XSDS loading buffer containing 1% 2-mercaptoethanol (BME), protein lysates were boiled at 95 °C for 5 min. The samples were then resolved by SDS-PAGE using 1× NuPAGE MES SDS buffer (InvitrogenTM, NP0002) and then transferred for 2 h at 10 volts using 0.45 µm nitrocellulose membrane in 1× Blot Transfer buffer (InvitrogenTM, BT00061). After the transfer, membranes were blocked with 5% non-fat milk TBST for 1 h at RT before incubation with primary antibody overnight at 4 °C. After incubation with primary antibody, membranes were washed three times with 1× TBST and then incubated with secondary antibody for 1 h at RT. Membranes were washed with TBST three times and incubated with ECL (Thermo Scientific, 32209) or SuperSignal West Pico PLUS (Thermo Scientific, 34580) and developed to X-ray film in a dark room. The following antibodies were used for western blotting (also listed in Table S1. Key Resources): AR (D6F11) Rabbit mAb (Cell Signaling Technology, 5153), UBE2J1 (B-6) Mouse mAb (Santa Cruz Biotechnology, SC-377002), Cyclophilin B Rabbit mAb (Cell Signaling Technology, 43603), b-Actin (8H10D10) Mouse mAb (Cell Signaling Technology, 3700), Dykddddk Tag (D6W5B) Rabbit mAb (Cell Signaling Technology, 14793), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 3724), K48 linkage Specific Polyubiquitin (D9D5) Rabbit mAb (Cell Signaling Technology, 8081), AR-V7 specific Rabbit mAb (Cell Signaling Technology, 68492), peroxidase AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, 115-035-003) and peroxidase AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 111-035-003). Followed manufacturer’s instructions for dilution of all primary antibodies. Dilutions of all secondary antibodies were 1:10,000.

Immunohistochemistry (IHC) and immunofluorescence (IF) staining

Tumors were collected from mice, washed with PBS and immediately fixed with 10% Neutral Buffered Formalin (StatLab Medical Products, 28600-1) at 4 °C overnight. Then the tumors embedded in paraffin by the UT Southwestern Tissue Management Shared Resource Core. After embedding, tumors were sectioned at 5 µm using a standard rotary microtome (Leica, Germany) and IHC staining was performed using standard protocol as previously described in [24, 29]. Briefly, slides were deparaffinized in xylene. Xylene was then removed with 100% ethanol and slides were hydrated in a series of ethanol dilutions until finally water was used. Sodium citrate buffer was utilized for antigen retrieval. Then endogenous peroxidase activity was blocked using 3% H2O2 in methanol. Slides were blocked with 3% BSA in PBST for 30 min at RT and incubated with primary antibody (AR (D6F11) Rabbit mAb (Cell Signaling Technology, 5153), Nkx3.1 (D6D2Z) XP® Rabbit mAb (Cell Signaling Technology, 92998), Ki 67 (D3B5) Rabbit mAb (Cell Signaling Technology, 9129), UBE2J1 Rabbit pAb (Sigma-Aldrich, HPA003509)), overnight at 4 °C. After incubating with primary antibodies, VECTASTAIN ABC HRP Kit (Peroxidase, Rabbit IgG) or Biotin-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, 711-065-152) and peroxidase Streptavidin (Fisher Scientific, NC9705430) were used, followed by ImmPACT DAB Peroxidase (HRP) Substrate. Images were taken with the Leica DMi8 microscope and were quantified using Fiji ImageJ. Dot plots visualizing the quantification were created using Prism 10 Sotfware (https://www.graphpad.com/scientific-software/prism/). Immunofluorescence (IF) staining was performed as previously described in [24, 29]. Briefly, LNCaP/AR cells were plated on glass coverslips with Veh (DMSO) or 10 µM enzalutamide (pretreated for 5 days) and after 24 h they were fixed using 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, blocked with 3% BSA in PBST and incubated in a humidified chamber with primary antibody (AR (D6F11) Rabbit mAb (Cell Signaling Technology, 5153), Nkx3.1 (D6D2Z) XP® Rabbit mAb (Cell Signaling Technology, 92998), Ndrg1 (D8G9) XP® Rabbit mAb (Cell Signaling Technology, 9485), UBE2J1 (B-6) Mouse mAb (Santa Cruz Biotechnology, SC-377002)) overnight at 4 °C. After washing with PBS, cells were incubated in the dark with Alexa Fluor 647 or Alexa Fluor 488 conjugated mouse or rabbit secondary antibody for 1 h at RT and then stained with DAPI. Images were acquired using a Zeiss LSM 700 confocal laser-scanning microscope. The following secondary antibodies were used for IF staining: Alexa Fluor 647-conjugated AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, 115-605-003), Alexa Fluor 647-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 111-605-144), Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, 115-545-003) and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 111-545-003). Used the following dilutions for primary antibodies for IHC: AR 1:400, UBE2J1 1:400, Ki67 1:400 and NKX3.1 1:200. Used the following dilutions for primary antibodies for IF: AR 1:600, NKX3.1 1:250, NRDG1 1:200 and UBE2J1 1:50. Dilutions of all secondary antibodies for IF were 1:500. The pictures were coded to blind researchers to treatment or genotype groups prior to data analysis to avoid bias.

AR ChIP-qPCR

ChIP-qPCR experiments were performed as previously described in [30]. Control and UBE2J1 KO LNCaP/AR cells were grown in RPMI-1640 media supplemented with 10% Charcoal Stripped FBS (CSS) or regular FBS for 3 days. Cells were plated in a 15 cm dish a day prior and cells grown in CSS were treated with 10 nM Dihydrotestosterone (DHT) or Veh for 2 h. Cells were then fixed with 1% paraformaldehyde for 15 min and quenched with 0.125 M glycine for 5 min. Then the cells were rinsed with cold 1× PBS twice and lysed in 300 µL ChIP lysis buffer with protease and phosphatase inhibitors (PierceTM, A32965). Chromatin was sonicated by Bioruptor® Pico to obtain an average length of 200–300 base pairs. 1% of the sample was saved as input, and the rest was incubated with 5 µg Anti-AR Antibody (Abcam, ab108341) or 5 µg Normal Rabbit IgG Polyclonal Antibody (Millipore Sigma, 12-370) overnight at 4 °C. After overnight incubation, the samples were incubated for 4 h at 4 °C with Dynabeads Protein G (Fisher Scientific, 10-003-D). The beads were washed with standard wash buffers (low-salt, high-salt and LiCl wash buffer) and finally with TE buffer. The chromatin was eluted from the beads, followed by decrosslinking using 0.2 M NaCl at 65 °C for 4 h. Input and ChIPed DNA were purified with a MiniElute PCR purification kit (Qiagen, 28004) and the concentration was determined by Qubit. For ChIP-qPCR, DNA was amplified with 2× PowerUPTM SYBRTM Green Master Mix (Thermo Fisher Scientific, A25778). Triplicates were conducted for each reaction and the enrichment percentage was calculated using the input. Three biological replicates were used and mean ± s.e.m. is presented. Experiments were repeated at least 2 times and similar conclusions were achieved. No data points were excluded. Primer sequences are listed in Table S1. Key Resources.

Bulk RNA-Seq preparation and analysis

RNA was extracted from LNCaP/AR cells using TRIzol (Ambion, 15596018). The extracted total RNAs were sent to BGI Genomics Global to perform bulk RNA-Seq. RNA-Seq libraries were then prepared using the stranded Illumina TruSeq mRNA kit. The preparation began with 500 ng of total RNA and included 10 cycles of PCR amplification. These barcoded RNA-Seq libraries were run as paired-end, 50-nucleotide reads on an Illumina HiSeq 2500 and were selectively filtered by poly(A). The alignment of the reads to the human reference genome (GRCh38) was executed using STAR (v2.7.2b)72 [49], while FeatureCounts (v1.6.4)73 [50] was employed for gene counts, biotype counts, and rRNA estimation. The differential expression analysis was conducted using the R package DEseq2 (v1.26)74 [51], with selected cutoff values of an absolute fold change greater than 2 and a false discovery rate of less than 0.1. The differentially expressed genes were then identified, and GSEA was performed using the R package fgsea (v1.14.0) in conjunction with the ‘Hallmark’ libraries from MsigDB [52].

Patient derived explants (PDE), patient derived organoids (PDO) and FFPE samples

PDE models were established from prostate cancers (male) in the Raj laboratory, around 1 mm3 samples were embedded in a sponge and cultured in RPMI-1640 media with 10% FBS, 1% penicillin-streptomycin, 0.01 mg/mL hydrocortisone and 0.01 mg/mL insulin [29]. PDEs were treated with 10 µM enzalutamide or DMSO for 24 h and then RNA was extracted. RT-qPCR was performed and analyzed using the protocol mentioned in Relative gene expression via RNA extraction and RT-qPCR. PDO models were established in the Chen laboratory [53]. PDOs were cultured in 3D Matrigel with human organoid medium and were split at a 1:3 ratio every 7 days using trypsin or a sterile glass pipette. When treated with Veh (DMSO), 1 µM ENZ or 16 nM AC176, these organoids were cultured in typical human organoid medium supplemented with drugs. 1 µM ENZ was used as the concentration because patients-derived organoids in 3D culture are more sensitive to enzalutamide. Images were acquired with Leica DMi8 microscope. Diameter was quantified using Fiji ImageJ. Bar plots visualizing the measurements were created using Prism 10 Sotfware (https://www.graphpad.com/scientific-software/prism/). Benign prostate tissue and PCa frozen samples were established in Strand and Raj laboratories. De-identified human PCa Formalin-Fixed Paraffin Embedded (FFPE) slides were purchased from UT Southwestern Tissue Management Shared Resource Core and IHC was performed as mentioned above in Immunohistochemistry (IHC) and Immunofluorescence (IF) staining. The pictures were coded to blind researchers from treatment or genotype groups prior to data analysis to avoid bias.

Co-immunoprecipitation (Co-IP)

Co-immunoprecipitation was performed as previously described in [24] with some modifications. For western blotting in Fig. 5D 1.0 × 106 HEK293T cells were seeded into each well of a six-well plate, 2 µg plasmids (pLVX-IRES-PURO-FLAG-AR, pLenti-CMV-P2A-blast UBE2J1, pLenti-CMV-P2A-blast EV) were mixed with 12 µL Lipofectamine® 2000 in 200 µL OPTI-MEM I Reduced Serum Medium for 20 min at RT. The mixture was added in a dropwise manner to the cells and swirled to mix. After 24 h the cells were lysed in 200 µL ice-cold mammalian IP lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% NP40, 10 mM N-ethylmaleimide) supplemented with 1× Pierce protease and phosphatase inhibitors (PierceTM, A32965) for 30 min with constant rotation at 4 °C. The samples were then centrifuged at 20,000g for 10 min at 4 °C, 40 µL of the supernatant were collected for input (whole cell lysate). The rest of the supernatant was incubated with 5 µL of pre-washed and pre-blocked Anti-FLAG® M2 Magnetic Beads (Sigma-Aldrich, M8823) (blocked with 3% BSA in PBS for 30 min at 4 °C with constant rotation) overnight at 4 °C with constant rotation. The next day the magnetic beads in combination with any of the targets were isolated by magnet (InvitrogenTM, 12321D). Beads were washed with cold IP lysis buffer supplemented with 1 mM PMSF AND 0.5 mM DTT 3 times. After the final wash, IP and input samples were boiled with 1×SDS loading buffer + 1% BME at 95 °C for 5 min. For Fig. 5A LNCaP/AR cells transduced with pLenti-CMV-P2A-blast or pLenti-CMV-P2A-blast UBE2J1 HA were seeded in a 10 cm plate and then followed the same steps as previously mentioned with some changes. Cells were lysed with 1 mL of ice-cold lysis buffer (same as previously mentioned). The supernatant was incubated with 15 µL pre-washed PierceTM Anti-HA Magnetic Beads (Thermo Fisher Scientific, 88836) pre-blocked with 3% BSA in PBS for 30 min at 4 °C with constant rotation, then followed the same steps previously mentioned. For Fig. 5C LNCaP/AR cells transduced with Cas9 and annotated guides were seeded in a 10 cm plate and lysed with 1 mL ice-cold lysis buffer (same as before). Supernatant was incubated with 0.4 µL rabbit IgG (Millipore Sigma, 12-370) or 8 µL AR (D6F11) Rabbit mAb (Cell Signaling Technology, 5153). After overnight incubation at 4 °C with constant rotation, samples were incubated with 15 µL Pierce Protein A/G Magnetic Beads (PierceTM, 88802) for 3 h at 4 °C with constant rotation. After that, followed the same steps previously mentioned. All experiments were repeated at least twice and achieved similar conclusions.

Statistical methods

Statistical details of each experiment were shown in figure legends. Two-tailed t-test with Welch’s correction for unequal variances was used to compare two groups of independent datasets that fit normality and homoscedasticity. When normality and homoscedasticity were not satisfied, Mann Whitney U Test (nonparametric Wilcoxon Rank Sum Test) was used when comparing gene expressions between two patients’ groups. For comparisons involving more than two groups, one-way or two-way ANOVA and Kruskal-Wallis nonparametric ANOVA were used as appropriate. mean ± s.e.m were reported, and p values were calculated and adjusted for multiple comparisons (Bonferroni or Benjamini correction) when applicable. For survival studies, the Kaplan-Meier method was used to estimate and plot the survival curve, and the log-rank test analysis was used to evaluate differences in survival data among different groups. For all in vitro experiments, three biological replicates were performed except when noted differently in figure legends.

Analysis of human prostate cancer dataset

Processed 444 SU2C metastatic prostate cancer patient cohort data, including RNA-Seq data and enzalutamide/abiraterone treatment data were downloaded from cBioPortal (RRID: SCR_014555, http://www.cbioportal.org/) [22, 23]. The cohort of 69 patients with clinical response data were examined for progression free survival. The probability of treatment duration figure was generated by prism 10 using Mantel-Cox test. The TCGA PRAD dataset, including gene expression FPKM values of 498 primary tumors and 52 normal tissue controls, was downloaded from the Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/). The Akumal 2020 cohort was obtained from the clinical trial Genetic and Molecular Mechanisms in Assessing Response in Patients with Prostate Cancer Receiving Enzalutamide Treatment [39]. The cohort included 25 patients with mCRPC who had not previously received enzalutamide that had data from RNA-Sequencing (RNA-Seq). The probability of treatment duration figure was generated by prism 10 using Mantel-Cox test.