Bromodomain-containing protein BRPF1 is a therapeutic target for liver cancer

Epigenetic deregulation plays an essential role in hepatocellular carcinoma (HCC) progression. Bromodomains are epigenetic “readers” of histone acetylation. Recently, bromodomain inhibitors have exhibited promising therapeutic potential for cancer treatment. Using transcriptome sequencing, we identified BRPF1 (bromodomain and PHD finger containing 1) as the most significantly upregulated gene among the 43 bromodomain-containing genes in human HCC. BRPF1 upregulation was significantly associated with poor patient survival. Gene ablation or pharmacological inactivation of BRPF1 significantly attenuated HCC cell growth in vitro and in vivo. BRPF1 was involved in cell cycle progression, senescence and cancer stemness. Transcriptome sequencing revealed that BRPF1 is a master regulator controlling the expression of multiple key oncogenes, including E2F2 and EZH2. We demonstrated that BRPF1 activated E2F2 and EZH2 expression by facilitating promoter H3K14 acetylation through MOZ/MORF complex. In conclusion, BRPF1 is frequently upregulated in human HCCs. Targeting BRPF1 may be an approach for HCC treatment.

. a Expression of BRPF1 in HCC cell lines determined by RNA-Seq. b Knockout efficiency of BRPF1 (sgBRPF1) in MHCC97L validated by PCR and subsequent Sanger sequencing. c Tide analysis demonstrating that the CRISPR knockout successfully induced indels in MHCC97L. d BRPF1 knockout suppressed cell migration in MHCC97L (Scale bar: 0.3 mm). Error bars indicate mean ± SD. Data were compared by independent t-test. Results were repeated at least three times. ***P<0.001 vs. sgNTC as indicated.   Figure S6. a, b The mRNA expression of E2F2 and EZH2 was reduced by GSK5959, OF-1 and NI-57 in a dose-dependent manner after 5-day treatment. c The cell proliferation rate was reduced upon 20 μM OF-1 or 100 μM NI-57 treatment in MHCC97L and Hep3B cells. d 20 μM OF-1 significantly induced cellular senescence after 5-day treatment (Scale bar: 0.3 mm). Serumstarved cells acted as a positive control. Error bars indicate mean ± SD. Data were compared by independent t-test. Results were repeated at least three times. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. mock as indicated.  The RNA-sequencing data on the 16 paired HCC and nontumor samples can be accessed through Bioproject (Accession ID: 294031).

Microarray Data
Publicly released microarray data are available via GEO (accession GSE23450 and GSE23451).
The microarray data showed the BRPF1 expression in CD133+ HCC cell population and CD133counterpart. The BRPF1 expression in CD133+ group was normalized to that in CD133-group. Mithramycin A was purchased from Sigma Aldrich.

Transcriptome Sequencing and Computational Analyses
The global gene expression profiles of mock control cells and HCC cells treated with GSK5959, WM1119 or EPZ6438 were examined by transcriptome sequencing (RNA-seq). The RNA samples were sent for sequencing using Illumina NovaSeq 6000 with the read length of paired-end 150 bp.
The sequencing data were aligned to hg19 using Hisat2, while the expression level in fragments per kilobase of transcript per million mapped reads (FPKM) was called by Stringtie. The expression of bromodomain-containing genes was visualized in a heatmap. The RNA-sequencing raw data can be found in Bioproject (Accession ID: PRJNA701710, PRJNA701712, PRJNA701713 and PRJNA701714). Pathway analysis on the common downregulated genes upon GSK5959 treatment in HCC cells was performed by DAVID functional annotation tool, while the gene set enrichment analysis was performed by R.

Luciferase Reporter Assay
To determine BRPF1 promoter activity, the promoter region from -1000bp to +250bp of BRPF1

Functional Assay
For cell proliferation assay, 1x10 4 cells were seeded into a 24-well plate in triplicate.

Cell Cycle Analysis
To perform cell cycle analysis, 2x10 5 cells were first seeded into a 6-well plate. After 2 days, the cells were trypsinized and washed with 1x PBS. Then the cells were fixed with 70% ethanol for 4 hours. The fixed cells were treated with Rnase A for 5 minutes to degrade RNA and prevent it from interfering with the result. The cells were then stained with a fluorescent DNA-intercalating agent, propidium iodide (Calbiochem) for 20 minutes. The cell cycle profile was examined by flow cytometry (BD Biosciences) and analyzed by FlowJo software.

Apoptosis Assay
Apoptosis assay was performed by using Annexin V-FITC Apoptosis Detection Kit (Vazyme Biotech). Cells together with the culture medium were collected and centrifuged at 250 x g for 5 minutes at room temperature. The cells were then washed with 1x PBS twice and resuspended in 100 μl of binding buffer. 5 μl of Annexin V FITC and 5 μl of PI were added to the resuspended cells for 15-minute incubation in the dark at room temperature. Finally, 400 μl of binding buffer was added to each sample before the samples were analyzed by BD FACS flow cytometer. The cell gating and analysis were done by FlowJo 7.6.

Sphere Formation Assay
The number of viable cells was first counted with trypan blue. 4000 live cells were then suspended in tumorsphere medium (Dulbecco's Modified Eagle Medium/F12 with 20 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor, 5 μg/ml insulin and 0.4% bovine serum albumin). 200 cells in each well were seeded for each treatment group in a 96-well plate. There were 20 replicates for each experimental group. The cells were then incubated for a week without any disturbance. The number of tumorsphere formed was counted under a phase-contrast microscope using 40X magnification lens. After 2 weeks, the mice were divided into 2 groups, treated with mock (DMSO, Sigma Aldrich) or GSK5959 (30 mg/kg) via intraperitoneal injection once per day for 2 weeks. The tumor size (length x width 2 ) and the weight of mice were measured every day. After 2-week treatment, the mice were sacrificed to collect tumors. Tumor weights were measured.

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The mice liver tumors extracted from the orthotopic xenograft experiment were homogenized for RNA extraction. In brief, around 0.2 g of liver tissue was cut into smaller fractions by a blade and then transferred into a 2 ml tube with a screw cap and biomedical beads (MP Biomedicals) inside.
1 ml of Trizol reagent was added into each tube. The liver tissues were homogenized by Minilys personal homogenizer (Bertin) at the highest speed for 30 seconds. The thoroughly homogenized liver tissues were then proceeded to RNA extraction.

RNA Extraction and Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
TRIzol reagent (Invitrogen) was used to extract total RNA. The procedures strictly followed the manufacturer's protocol.

Statistical Analyses
Gene expression levels between HCC samples and nontumorous liver samples were compared by paired t-test. The tumor volumes and tumor weights between the control subcutaneous tumors and the tumors in sgBRPF1 group were also compared by paired t-test. Mann Whitney U test was used to analyze continuous non-parametric data, while independent t-test was used to analyze continuous parametric data. Survival rate analysis was performed by Kaplan-Meier method and Log rank test. Linear regression or Chi-square test was used to test the correlation between two gene expression. Statistical analyses were carried out by Prism 8 software. Data were represented as mean ± standard deviation (error bars). Results were repeated at least three times unless indicated otherwise. P < 0.05 was considered as statistically significant. ns: not significant, * P < 0.05, ** P < 0.01, ***P < 0.001, ****P < 0.0001.