RNA-seq and pathway analysis
To examine the direct effect of AR signaling on gene transcription, the cells were perfused with charcoal-dextran stripped fetal bovine serum (CD-FBS) for 3 days, and then treated with 10 nM dihydrotestosterone (DHT) or DMSO for 24 h. Deep sequencing of rRNA-depleted total RNAs was performed in biological duplicates on LNCaP cells. To examine the biological effect of ARHGEF2 in PCa cells, 22RV1 cells were transfected with siARHGEF2 (#1 and #2) and SCRAMBLE siRNAs for 48 h. RNA high throughput sequencing was performed by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA of each sample was extracted using RNeasy Mini Kit (Qiagen). Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1 μg total RNA with RIN value above 6.5 was used for the following library preparation. The poly(A) mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module or rRNA removal Kit. Next generation sequencing library preparations were constructed according to the manufacturer’s protocol. RNA libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Novaseq 6000 instrument with 150 bp paired-end reads. After 3′ adaptor-trimming and low-quality read removing by cutadapt software (v1.9.3), the high-quality clean reads were aligned to the reference genome (UCSC hg19) with hisat2 software (v2.0.4). Then, HTSeq software (v0.9.1) was used to get the raw count, and the edgeR (v3.32.1) Bioconductor package was used to perform normalization, then differentially expressed mRNAs were identified by p-value and fold change. 189 genes were downregulated in 22RV1 siARHGEF2 cells (Fold Change cut-off: 2.0; P-value cut-off: 0.05), which were further analyzed for enriched pathways. Pathway analysis is a functional analysis mapping gene to KEGG pathways. The Fisher p-value denotes the significance of the pathway correlated to the condition that was enriched (P-value cut-off is 0.05.) GSEA analysis was performed in the 22RV1 siARHGEF2 and 22RV1 si-NC groups to explore the biological signaling pathway . The MAPK_Pathway with significant enrichment results was demonstrated on the basis of enrichment score (ES) and p-value.
Human prostate cancer specimens
Tissue microarrays (TMA) for prostate cancer (PCa) specimens were obtained from the second hospital of Tianjin Medical University, acquiring the due consent from the patients and mandatory approval from the Institutional Review Board as described previously .
TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) [C57BL/6] mice were obtained from the Jackson Laboratories repository. Mouse-tail DNA was collected from the litter and subjected to PCR as described previously . Whole murine prostates were micro-dissected from mice at the indicated age, imaged, weighed, and then fixed overnight in 4% PFA. Sections were then transferred to 70% EtOH solution and paraffin embedded, and sectioned.
Immunohistochemistry (IHC) staining
IHC for AR (ab9474, Abcam), FGFR1 (9740, CST), P-ERK (4370, CST), SOX2 (3579, CST), CHGA (ab45179, Abcam), SYP (ab52636, Abcam) and ARHGEF2 (ab155785, Abcam) was performed using PV-6000 system (ZSGB-BIO, China). Briefly, the slides were immersed in 1X Tris-EDTA (pH 9.0) buffer (Solarbio, China) and placed in a microwave oven for 10 min on high heat, and then adjusted to medium-low heat for 10 min to restore the antigen. 3% H2O2 was added to remove endogenous peroxidase in tissue samples. Cover the tissue on the slide with the primary antibody, place it in a humid box, and incubate overnight at 4 °C. After rewarming at room temperature for 30 min, horseradish peroxidase-linked secondary antibody (ZSGB-BIO, PV-6000, China) was added to the specimen and incubate the slides at room temperature for 30 min. After being stained with the DAB solution, the slides were immediately placed in water to stop dyeing, slides were subsequently counterstained with hematoxylin. The tissue is then dehydrated and preserved with neutral balsam (OriGene, ZLI- 9555, China).
Immunostaining was performed as described before . Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).
All the prostate cancer (22RV1, LNCaP, PC3, and DU145) cell lines were obtained from American Type Cell Culture (ATCC) and maintained as per guidelines. Briefly, cells were cultured in the recommended media supplemented with 10% fetal bovine serum (FBS) (Gibco) and 0.5% Penicillin Streptomycin (Thermo Fisher Scientific) in cell culture incubator (Thermo Fisher Scientific) supplied with 5% CO2 at 37 °C. LNCaP-AI was generated as described before and cultured in RPMI 1640 media supplemented with 10% charcoal stripped serum (CD-FBS; Gibco) . For certain experiments, different cell lines were used based on their characteristics and potential significance. Briefly, the LNCaP cell line is an AR positive and androgen sensitive human PCa cell [32, 33]. The LNCaP-AI cell line is an AR positive and androgen independent human PCa cell, generated from LNCaP cells . PC3 and DU145 cell lines are negative for AR expression and show androgen-independent responses [32, 34, 35]. 22Rv1 cells harbor the H874Y mutation in the AR and are resistant to castration [32, 36].
Androgen stimulation and deprivation
For androgen stimulation, cells were starved for 72 h in RPMI 1640 media (Gibco) supplemented with 10% CD-FBS (Gibco) followed by stimulation with dihydrotestosterone (DHT) (Sigma-Aldrich) at the indicated time points and indicated concentrations. For anti-androgen treatment, LNCaP and 22RV1 cells were hormone-starved for 48 h using RPMI 1640 media supplemented with 10% CD-FBS (Gibco) followed by treatment with enzalutamide (cat. HY-70002, MedChem Express) for 48 h in complete medium.
Transfections with siRNAs (purchased from GenePharma, China) were carried out using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific, USA) with cells at 50% confluence cultured in 6-well plates. The siRNA sequences are listed in Supplementary Table 1. To achieve the maximal inhibition effect, two siRNAs were mixed together and transfected into cells. LNCaP-AI and 22RV1-shARHGEF2 cells were generated using lentiviral shRNA against ARHGEF2 or SCRAMBLE (SWS Biotechnology, Tianjin, China). Overexpression of ARHGEF2 was generated using lentiviral mediated vector (SWS Biotechnology, Tianjin, China), and empty vector was used as control. After infection, cells were selected with puromycin (1 ug/ml) for 3 days to remove uninfected cells, and then maintained in puromycin-containing complete medium.
Total RNA was isolated using TRIzol Reagent (Invitrogen, USA) as the standard RNA isolation procedure. About 5 μg of total RNA with oligo (dT) primers was reverse-transcribed in a 20 μL volume using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) followed by the manufacturer’s protocol. For regular PCR, all reactions were set up as follows: 10 µl 2× Taq PCR MasterMix (with dye), 0.4 µl forward primer (10 nM), 0.4 µl reverse primer (10 nM), 1 µl cDNA and 8.2 µl ddH2O, for a total reaction volume of 20 µl. The reaction system was preheated at 94 °C for 3 min, and then performed using the following thermal cycle program: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and 40 cycles, followed by 72 °C for 5 min. DNA products were analyzed in 1% agarose gel. For quantitative PCR, all reactions were set up as follows: 10 µl FastStart Universal SYBR Green Master (Roche), 0.2 µl forward primer (10 nM), 0.2 µl reverse primer (10 nM), 1 µl cDNA and 8.6 µl ddH2O, for a total reaction volume of 20 µl. The reaction system was preheated at 95 °C for 10 min, and then performed using the following thermal cycle program: 95 °C for 15 s, 72 °C for 20 s, and 40 cycles. Relative expression of target genes was calculated using the 2-ΔΔCt method using GAPDH as an internal control. The primers are listed in Supplementary Table 2.
Cell lysates were prepared in cell lysis buffer (Cat.R0020, Solarbio), supplemented with Protease Inhibitor Cocktail (Roche). Protein concentration was determined by Bradford (Pierce Bradford Assay Kit, Thermo Scientific) at 595 nm. Western blot analysis was performed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 50 µg proteins were separated by 10% SDS-PAGE and then transferred onto an Immobilon-P membrane (Millipore, USA). The membrane was blocked with 5% non−fat dry milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody. Subsequently, blots were washed in 1× tris-buffered saline, 0.1% Tween 20 (TBS-T) buffer and incubated with secondary antibody (Goat anti-mouse IgG (H + L), HRP conjugate, cat. SA00001-1; Goat Anti-Rabbit IgG (H + L), HRP conjugate, cat. SA00001-2, Proteintech) for 1 h, washed, and processed using the Enhanced chemiluminescence (ECL) reagent (Millipore), then visualized by Tanon (model 4500, Tanon) system. The primary antibodies used are as follow: 1:1000 diluted ARHGEF2 (Abcam, cat. ab155785), 1:1000 diluted p44/42 MAPK (ERK) (CST, cat. 9102), 1:1000 diluted phospho-p44/42 MAPK (P-ERK) (Thr202/Tyr204) (CST, cat. 9101), 1:2000 diluted AR (Abcam, cat. Ab133273), 1:1000 diluted KLK3 (CST, cat. 5365), 1:2000 diluted CHGA (Abcam, cat. ab45179), 1:2000 diluted SYP (Abcam, cat. ab52636), 1:1000 diluted SOX2 (CST, cat. 3579), 1:1000 diluted FGFR1 (CST, cat.9740) and 1:5000 diluted GAPDH (Abcam, cat. ab8245).
Chromatin immunoprecipitation (ChIP) assay and ChIP-qPCR
ChIP was performed by EZ-Magna ChIP™ A/G kit (Catalog: 17-10086; Millipore) following the manufacturer’s instructions. Briefly, 1 × 107 cells were cross-linked with 37% formaldehyde (Catalog: F8775; Sigma) at room temperature for 10 min. Nuclei were extracted with Nuclear Lysis Buffer, and cross-linked DNA was sheared to 200–1000 base pairs using SONICS Vibra-Cell™ Ultrasonic Liquid Processors (model VCX130; Sonics & Materials, Inc.). Antibody for ChIP assays was purchased from Millipore (AR, rat, cat. 17-10489). ChIP-qPCR was performed with the following parameters: Initial Denaturation at 94 °C for 10 min, followed by 50 cycles of Denature at 94 °C for 20 s and Anneal and Extension at 60 °C for 1 min. Primers for ChIP-qPCR are listed in Supplementary Table 3.
CUT & Tag (Cleavage under targets and tagmentation)
The CUT and Tag assay (CUT & Tag also referred to as “ChIP-seq”) was performed to determine the AR binding sites in LNCaP cells upon DHT treatment within 24 h. The detailed, step-by-step protocol was followed by Steven Henikoff protocol at https://www.protocols.io/view/bench-top-cut-amp-tag-bcuhiwt6. Briefly, cells were harvested, counted, and centrifuged for 3 min at 600 × g at room temperature, washed twice in 1.5 mL Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail), and incubated with activated concanavalin A coated magnetic beads (Bangs Laboratories) at RT for 15 min. Then, the bead-bound cells were resuspended in 50–100 µL Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin) containing 2 mM EDTA and incubated with 5 µL of the AR primary antibody (Millipore; cat. 17-10489) overnight at 4 °C. Cells were then incubated with pA-Tn5 at RT for 1 h and resuspended in 50–100 µL Tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. 2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/mL Proteinase K was added to stop tagmentation with incubation at 55 °C for 30 min, and then at 70 °C for 20 min to inactivate Proteinase K. Followed by DNA extraction, library amplification, post-PCR clean-up, paired-end NovaSeq 6000 sequencing was performed. Trimmomatic (version 0.40) was used to remove adapters and low-quality reads. Quality distribution plots and base content distribution were generated by FASTQC (version 0.11.9). Before read-mapping, clean reads were obtained from the raw reads by removing the adaptor sequences. The clean reads were then aligned to reference genome (hg38) sequences using the Burrows-Wheeler-Alignment Tool (bwa). The bam file generated by the unique mapped reads as an input file, using macs2 (version 22.214.171.124) for peak calling with cutoff q-value < 0.05. The data have been deposited into the CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0001628.
Plasmids and luciferase assay
The pGL3-ARHGEF2-PP (GEF-H1 PP) construct was obtained by cloning the ARHGEF2 proximal promoter (ARHGEF2-PP) from the pGL3-basic vector (Promega), and pGL3-ARHGEF2-DP (GEF-H1 DP) was generated by cloning the distal promoter of the ARHGEF2 gene in the pGL3-basic vector (Promega). The DNA sequences are listed in Supplementary Table 4. 22RV1 cells were plated at 40–50% confluency in a 24-well plate and were transfected with pGL3-ARHGEF2-PP (500 ng) and pRL-TK vector (5 ng) using Lipofectamine 3000 Transfection Reagent. For androgen stimulation, the 22RV1 cells were serum starved for 48 h and stimulated with DHT at indicated concentrations for 24 h in RPMI1640 media containing 10% CD-FBS. For anti-androgen treatment, 22RV1 cells were treated with enzalutamide (10 µM) for 24 h. After 24 h of transfection with luciferase constructs, cells were harvested using the lysis buffer provided with the Dual-Glo Luciferase assay kit (Promega). Firefly and Renilla luciferase activity were measured according to the manufacturer’s protocol using GloMax Luminometer (Promega). For each sample, firefly luciferase activity was normalized to Renilla luciferase activity.
Cells were harvested, washed, re-suspended, incubated with Propidium Iodide Staining Solution (BD Pharmingen) in the dark for 15 min, and subjected to flow cytometry analysis using ModFit LT software (Verity Software House, Topsham, ME, USA).
MTT and migration assay
Cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide proliferation assays. Migration assay was performed using Transwell chambers of 8μm pore size (Corning) as described before .
Nude mice (6–7 weeks old, n = 10) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The animal studies were approved by Tianjin Institute of Urology, Tianjin, China. Subcutaneous tumor growth assays were performed with 22RV1 shSCR (control) and shARHGEF2 stable cell lines (2 × 106 shSCR cells injected to 5 mice separately, 2 × 106 shARHGEF2 cells injected to another 5 mice separately). At the end point, all mice were sacrificed, and the tumors were harvested under standard, institutionally approved processes.
Statistical analysis was performed using GraphPad Prism 8.0 software (San Diego, CA, USA). Differences were measured using either one-way ANOVA, two-way ANOVA with the post hoc multiple comparisons test, or unpaired two-tailed Student’s t-test or otherwise mentioned. P < 0.05 is considered statistically significant, where *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and NS denotes nonsignificant. The results were shown as mean ± standard deviation (SD).