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Prostate cancer

SR9009 inhibits lethal prostate cancer subtype 1 by regulating the LXRα/FOXM1 pathway independently of REV-ERBs

Cell lines

RWPE-1, PC3, 22RV1, DU145, LNCaP, and C4-2B cells were purchased from Shanghai Cell Bank Type Culture Collection Committee (CBTCCC, Shanghai, China). HEK293T cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). PCa cell lines were cultured in RPMI 1640 medium (HyClone, Utah, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% antibiotics (penicillin and streptomycin; HyClone) in a humidified incubator containing 5% CO2 at 37 °C. RWPE-1 cells were cultured in keratinocyte serum-free medium. Cells stably transfected with plasmid were cultured in complete culture medium with additional puromycin (2 μg/mL; KEHBIO, Beijing, China).

Cell transfection

Small interfering RNAs (siRNAs) targeting REV-ERBα, REV-ERBβ, LXRα, and FOXM1 were obtained from RiboBio (Guangzhou, China). The siRNA sequence is shown in Table S1. siRNA (50 nM) was transfected into cells using Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA). FOXM1 overexpression plasmid (pLV.CMV.FOXM1.PGK. Puro) was purchased from PackGene (Guangzhou, China). The lentiviral packaging procedure for the target plasmid has been described previously [17].

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing

Cas9-expressing stable cell lines were constructed by infection with Cas9 lentivirus and further puromycin screening. REV-ERB (NR1D1 and NR1D2)-specific sgRNA oligos were designed and cloned into the pLentiCRISPR V2 plasmid (sequences listed in Table S2). The harvested lentivirus was added to the cell supernatant and centrifuged at 2000 rpm for 1 h, followed by incubation for 1.5 h.

Cell counting kit-8 (CCK-8) assay

SR9009, purchased from MedChemExpress (MCE, NJ, USA), was first dissolved in dimethyl sulfoxide (DMSO) and then diluted to the working concentrations (maximum DMSO concentration < 0.5%). Approximately (2–5) × 103 cells per well were seeded in a 96-well plate. When the cells grew to 70–80% confluence, they were treated with SR9009, DMSO, or siRNAs for 48 h. Thereafter, the culture medium in each well was replaced with 100 μL of fresh complete culture medium containing 10 μL of CCK-8 reagent (Dojindo Molecular Technologies, Rockville, USA). Then, the 96-well plate was placed into an incubator at 37 °C in the dark for 2 h. Finally, the plate was placed in the EonTM Microplate Reader (Bio-Tek, VT, USA) to measure the absorbance at 450 nm. At least 3 duplicate wells were set at the same time.

Colony formation assay

PCa cells were seeded in 6-well plates at 500 cells/well. After 48 h of incubation, the cells were treated with SR9009 (20 μM) or DMSO for another 10–14 days. The cells were washed with phosphate buffered saline (PBS) and fixed with cold methanol for 20 min. After washing with PBS, the cells were stained with crystal violet (Beyotime, Shanghai, China) for 15 min. Subsequently, the cells were washed and imaged using a Celigo Imaging Cytometer.

Cell cycle

Cells were pretreated with SR9009 (20 μM), DMSO or siRNAs for 48 h. Subsequently, they were digested, centrifuged and collected into flow tubes. Then, 500 μL of 70% ice-cold ethanol was added, and the cells were fixed overnight at 4 °C. Cells were washed and filtered before the PI/RNase A (KeyGen Biotech, Jiangsu, China) dye working solution was added. After 30 min of incubation in the dark, we detected and recorded the cell cycle using a CytoFLEX Research Flow Cytometer (Beckman Coulter, CA, USA).

Cell apoptosis

An Annexin V-PE/7-AAD cell apoptosis detection kit was purchased from KeyGen Biotech. Cells were treated with SR9009 (20 μM) or DMSO for 48 h. The cells were then digested and washed twice with cold PBS. Next, 55 μL dye working solution (50 μL binding buffer + 5 μL 7-AAD) was added to the cells and incubated at 37 °C for 5–15 min in the dark. Subsequently, 450 μL binding buffer and 1 μL Annexin V-PE were added and incubated for 5–15 min. Cell apoptosis was assessed by a FACSAria SORP instrument (BD, USA).

Wound healing assay

Cells were digested, suspended and seeded at 3 × 105 cells per well in 6-well plates. When the cells grew to approximately 80–90% confluence, 3 vertical parallel lines were drawn in each well. Cells were washed twice and treated with SR9009 (20 μM) or DMSO for 24 h. Images were immediately taken under an inverted fluorescence Zeiss OBSERVER D1/AX10 CAM HRC microscope (Zeiss), and the sites were recorded. Subsequently, the 6-well plates were placed in the cell incubator for an additional 24 h of incubation and imaged again. ImageJ software (National Institutes of Health, USA; Version 1.48) was applied to calculate the migration distance.

Transwell assay

Transwell migration assays were conducted using a Transwell chamber (Millipore, Massachusetts, USA). Briefly, Transwell chambers were placed on a 24-well plate. Fresh medium containing 10% FBS and 20 μM SR9009 in 600 μL was added to the lower chambers, and (2–5) × 104 cells in 200 μL of medium containing 20 μM SR9009 without FBS were added to the upper chamber. The 24-well plate was incubated at 37 °C for 48 h. Cells that invaded through the chamber were washed, fixed (20 min with 4% paraformaldehyde) and stained (30 min with crystal violet). Then, the upper chambers were washed, photographed and preserved under an inverted fluorescence OBSERVER D1/AX10 cam HRC microscope (Zeiss). Transferred cells were analyzed using ImageJ software.

RNA-sequencing (RNA-seq)

PC3 cells were treated with 20 μM SR9009 or DMSO for 48 h. Then, the cells were harvested, and RNA was stored using TRIzol (Invitrogen, CA, USA). Novogene (Beijing, China) was entrusted to perform RNA-seq. Briefly, RNA samples were extracted, and RNA sample quantification and qualification were performed. Then, the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) was selected to generate sequencing libraries following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. After clustering and sequencing (Novogene Experimental Department), data analysis was performed through the following steps: quality control, read mapping to the reference, and quantification of the gene expression level (fragments per kilobase million was calculated). Differential expression analysis was performed using the DESeq2 R package (1.16.1). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis were implemented by the clusterProfiler R package.

RNA extraction and real-time quantitative polymerase chain reaction (qPCR)

Total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Texas, USA) according to manual protocol. A total of 1 μg of RNA was added to synthesize first-strand complementary DNA (cDNA) using the Thermo Scientific RevertAid RT kit (Vilnius, Lithuania) with Oligo (dT)18. Quantitative PCR (qPCR) was performed using the QuantiNova SYBR Green PCR kit (Qiagen), and reactions were performed on the CFX96 Touch Real-Time PCR System (Bio-Rad, California, USA). The PCR amplification settings were as follows: 50 °C for 2 min and 95 °C for 10 min; 40 cycles of 98 °C for 5 s; and 59 °C for 10 s. β-actin was used for normalization, and each sample was repeated at least three times. The data were analyzed using the 2−ΔΔCt method. Primer sequences were acquired from the PrimerBank website (https://pga.mgh.harvard.edu/primerbank/) and synthesized by Sangon Biotech (Shanghai, China). The primers used in this study are shown in Table S2.

Western blot analysis

Proteins were extracted, and the concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo). Proteins were denatured at 100 °C for 10 min. After the proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE, Epizyme, Shanghai, China), the gels were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) and run at 250 mA for 90 min. Subsequently, the membranes were cut, blocked (5% skim milk powder), and incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies were as follows: anti-GAPDH (ZEN-BIO 200306-7E4), anti-β-actin (ZEN-BIO 250132), anti-vinculin (ZEN-BIO R26085), anti-NR1D1 (ab174309), anti-NR1D2 (ab251948 and Protein Tech 13906-1-AP), anti-FOXM1 (CST20459), anti-LXRα (ab41902), anti-CCNB1 (CST12231), anti-CCNB2 (ab185622), anti-CENPA (CST2186), anti-CENPF (CST58982), anti-CDK1 (ZEN-BIO 200544), anti-survivin (CST2808), and anti-ARNTL (Protein Tech 14268-1-AP). The membranes were washed and incubated with secondary antibodies for 1 h according to the primary antibody sources. Immunoreactivity was visualized using enhanced chemiluminescent (ECL) chromogenic substrate (Millipore). The membranes were finally detected by using a ChemiDoc MP Imager System (Bio-Rad).

Immunohistochemistry (IHC)

Specimens were fixed in 4% paraformaldehyde at room temperature and embedded in paraffin. Then, tissues were cut into 4 μm thick sections. Subsequently, we dewaxed, hydrated and incubated the tissues with antibodies overnight at 4 °C. After incubation with the corresponding secondary antibodies, the sections were stained with diaminobenzidine and reverse stained with hematoxylin.

Mouse model

Male nude BALB/c mice (18–20 g each) at 6 weeks of age were purchased from Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, China). Mice were castrated with goserelin (MCE, daily for 19 days) subcutaneously. At the same time, cultured 22RV1 cells were collected and suspended in PBS. A 100 μL cell suspension with 5 × 106 cells was subcutaneously injected into the right flank of the mouse to establish a subcutaneous xenograft model. The weight and tumor volume of the mice were measured every 3 days, and the formula for calculating volume was (length × width2)/2 [18]. When the tumor volume increased up to 100–200 cm3, the mice were randomly divided into two groups (with 4 mice in each group). The investigator was not blinded to the group allocation during the whole experiment. SR9009 was dissolved in 15% Cremophor and administered twice daily (100 mg/kg) [10] in the experimental group through intraperitoneal injection, and the control group was given the same volume of Cremophor. When there was a significant difference between the two groups or the tumor volume exceeded 1000 cm3, mice were sacrificed, and subcutaneous tumors were harvested for hematoxylin-eosin staining and IHC.

Bioinformatic analysis

GEPIA2 (http://gepia2.cancer-pku.cn/#index) was used to analyze the gene expression differences between normal and tumor samples in prostate adenocarcinoma in The Cancer Genome Atlas (TCGA-PRAD). GEPIA2 was also applied to analyze the associations of gene expression (median cutoff value) and disease-free survival (DFS). mRNA expression Z scores relative to diploid samples were analyzed using cBioPortal (http://www.cbioportal.org). Protein levels of LXRα and FOXM1 were obtained from The Human Protein Atlas. The level 3 HTSeq-FPKM data in TCGA-PRAD were downloaded from https://portal.gdc.cancer.gov/. RNA-seq data in FPKM format were transformed into transcripts per million reads (TPM) format with log2 transformation. Correlation analyses were performed using the R package ggplot2.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Data were statistically analyzed using GraphPad Prism software (Version 6.02; CA, USA). Student’s t test, ANOVA, or Wilcoxon rank sum test were applied as appropriate. Homogeneity of variance was tested using the Shapiro‒Wilk normality test for equality of variances. A two-tailed P value lower than 0.05 indicated statistical significance, which was labeled as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.