QCA570 was synthesized as reported previously (Qin et al., 2018). JQ1, MLN4924 and MG132 were purchased from Med Chem Express (Monmouth Junction, NJ).

The human bladder cancer cell lines T24 and SV-HUC-1 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The 5637, J82, UMUC-3 and EJ-1 cells were obtained from Yuhuangding Hospital (Yantai, China). 5637, EJ-1, UM-UC-3 cells were cultured in RPMI1640 media (Procell, PM150110); J82 cells were cultured in MEM (NEAA) media (Procell, PM150410), and T24 cells were cultured in McCoy’s 5A media (PM150710). A total of 10% FBS (Gibco,1099141C) and 1% antibiotics (penicillin and streptomycin, Procell, PB180120) were added to the basic media. All the cells were cultured at 37°C with 5% CO₂.

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China) with protease inhibitor (Roche, 11697498001) and phosphatase inhibitor (Roche, 4906837001) for 10 min on ice and shaken every 5 min on a vortex mixer for 30 s. Lysates were centrifuged at 12,000 rpm at 4°C for 10 min and the supernatant fraction was retained. Protein concentrations were quantified with BCA Protein Assay Kit (Thermo Fisher Scientific, A53226). Protein samples were separated via 10% SDS-PAGE and then transferred onto PVDF membrane. The membrane was blocked in 5% milk containing TBST (Tris-buffered Saline with Tween 20) at room temperature for 1 h, and this was followed by incubation with indicated primary antibodies overnight at 4°C. The membrane was then washed 3 times with TBST and incubated with secondary antibody for 1 h at room temperature before its development with the ECL kit (Thermo Fisher Scientific, 34,578). Primary antibodies to the following proteins were used: BRD4 (Bethyl Laboratories, A301-985A100), BRD3 (Bethyl Laboratories, A302-368A), BRD2 (Bethyl Laboratories, A302-583A), EZH2 (D2C9) (Cell Signaling Technology Inc, #5246), c-MYC (Abcam, ab32072), caspase-3 (Cell Signaling Technology Inc, #9662) GAPDH (ABclonal, AC033), β-Actin (ABclonal, AC006).

Total RNA was extracted using the RNA-easy Isolation Reagent (Vazyme, R701) according to the manufacturer’s instructions. Complementary DNAs (cDNAs) were synthesized using HiScript III RT Super Mix for qPCR (+gDNA wiper) (Vazyme, R323). qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) and detected with a LightCycler^® 96 Detection System (Roche, Switzerland). The PCR amplification was performed following the program: 95°C for 5 min of initial denaturation, then 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 10 s, followed by a final melting curve step. The relative abundance of transcripts was calculated based on normalization to the GAPDH gene. The primers are shown in Table 1.

5637, T24, EJ-1 at 3,000 cells/well and J82, UMUC-3 at 5,000 cells/well were seeded into 96-well plates. Cell viability and proliferation were measured with CCK-8 (Biosharp, BS350) at designated times after drug treatment. Briefly, 10 µL of CCK-8 solution was added to each well and incubated at 37°C for 2 h before reading the absorbance at 450 nm. The results were analyzed using GraphPad software. All experiments were repeated at least three times.

Collect cells, seed them in 12-well plates, culture until the cells are completely confluent, and use a 200 μL sterilized pipette tip to make scratches perpendicular to the bottom of the culture dish. The original medium was discarded, washed twice with PBS, and the medium containing the compound was added again, and cultured in the incubator for 48 h. They were placed under a microscope and photographed at 8, 12, 24, and 48 h respectively.

Cells were harvested two days after treatment with compounds or DMSO, washed twice with PBS, and fixed with 70% ethanol overnight at 4°C. Then cells were centrifuged at 3,000 rpm for 5 min, washed twice with PBS, and resuspended in PBS. After incubating with 20 μg/mL RNase A (Solarbio Beijing, China), cells were stained with 20 μg/mL propidium iodide (PI) (Solarbio Beijing, China). A Beckman Coulter flow cytometer was used for detection and FlowJo-V10 software was used to analyze the data.

We harvested cells after a 2-day treatment with compounds or DMSO and washed them twice with cold PBS before resuspending them in 1x binding buffer. An Annexin V-FITC/PI Apoptosis Assay Kit (abs50001, Absin) was used to stain cells. After incubation for 15 min in the dark, we analyzed cell apoptosis on a flow cytometer (Beckman Coulter). Data were analyzed with FlowJo-V10 software.

For this experiment, 5 × 10⁵ cells were grown on coverslips. Cell apoptosis was determined using the one-step TUNEL apoptosis assay kit (Roche, 12156792910) according to the manufacturer’s instructions. Following TUNEL staining, the slides were stained with DAPI (Solarbio Beijing, C0060) to highlight the nuclei with fluorescence. Slides were observed under an LSM 510 META Olympus Confocal Laser Scanning Microscope using 488-nm excitation and 530-nm emission, and the images were captured and quantified by image analysis software, ImageJ.

The BRD4 and EZH2 gene expression across tumor and normal samples was analyzed online from TCGA clinical data on the UALCAN database (ualcan.path.uab.edu/home, accessed on may 3, 2022). TPM (transcripts per million) values were employed for the generation of boxplots and to estimate the significance between groups by t-test. Besides, BRD4 and EZH2 gene expression data were also downloaded from the publicly available GEO website (Home - GEO - NCBI (nih.gov), accessed on may 3, 2022) and included GSE48276 and GSE13507 datasets containing the raw RNA microarray data of 208 samples, comprised of 43 control and 165 BC patients. The Kaplan–Meier survival analysis was generated from the OncoLnc database (OncoLnc, accessed on May 3, 2022) which performs in-depth analyses of TCGA gene expression data. A Log-rank test was used for survival comparison.

To explore the role of BRD4 in BC progression, we analyzed the mRNA expression data from the TCGA database. In bladder tissues from 408 patients, BRD4 was upregulated compared with normal samples (Figure 1A). Data from the GEO website further confirmed that BRD4 was overexpressed in BC tissues (Figure 1B) from these patients. Kaplan-Meier survival analysis of 240 well-defined BC cases derived from the TCGA database showed that high BRD4 expression was associated with poor overall survival (OS) of BC patients (Figure 1C). These results showed the association between BRD4 expression with BC risk and suggest that BRD4 may be involved in a potential therapy target for BC patients.

The PROTAC molecule QCA570 (C₃₉H₃₃N₇O₄S, CAS number 2207569-08–0) developed by Qin et al. showed effective degradation of BET proteins BRD2, BRD3, and BRD4 at low picomolar concentrations and showed more potent proliferation inhibitory activity than other BET degraders such as dBET1, ARV-825, ARV-771, and ZBC260 in human leukemia cell lines. The degradation ability of QCA570 was also validated by Liu et al. in human lung cancer cells. In this study, we evaluated the activity of QCA570 on BC cell lines. As shown in Figures 2A-F, QCA570 induces degradation of BET proteins BRD2, BRD3 and BRD4 in a dose-dependent manner with a treatment of 9 h. BRD4 at 3 nM was largely depleted in each of these five BC cell lines with a DC₅₀ around 1 nM. We next explored the degradation kinetics of QCA570 at 30 nM concentration at various duration times ranging from 1 to 24 h (Figures 2G-K). The BRD4 degradation peaked within 1 h in 5637, T24, UM-UC-3 cells and within 3 h in J82 and EJ-1 cell lines. A time-dependent degradation was also observed with BRD2 and BRD3 proteins.

Based on its PROTAC design, QCA570 forms a ternary complex dependent upon its binding to the target protein and E3 ubiquitin ligase CRBN. Similarly, QCA570 should also be proteasome-dependent. The mechanism used by QCA570 for the degradation of BET proteins was previously validated in RS4; 11 cells. In our study, we also confirmed the degradation mechanism in BC cells (Figure 2L). The Western blotting (WB) result showed that BRD4 degradation was almost completely abolished by pre-treatment with the proteasome inhibitor MG-132, the E1 neddylation inhibitor MLN4924, and the BRD4 ligand, JQ1 in BC cells. These data together provide evidence pertaining to the mechanism for ligands and proteasome-dependent degradation of BET proteins, consistent with its PROTAC character.

Previous studies have reported that BET inhibition blocks expression of certain key oncogenes, such as c-MYC in many cancers (Filippakopoulos et al., 2010; Sahai et al., 2016). The Jiang group recently showed that BRD4 can promote EZH2 gene transcription through upregulation of c-MYC which functions as a transcription factor supporting BC progression (Xie et al., 2020; Wu et al., 2016). The mRNA expression data from TCGA and GEO database shows that EZH2 is upregulated in BC tissues, which suggests that the EZH2 gene is significantly associated with BC cancer risk (Figure 3A, B). To investigate whether BRD4 degradation down-regulates the expression level of EZH2, we determined the mRNA levels of these two genes in BC cell lines J82 and T24. In response to treatment with QCA570, the expression of c-MYC and EZH2 was significantly reduced with BRD4 degradation while the BET inhibitor JQ1 showed less suppression activity (Figures 3C, D). The protein level was also consistent with the qPCR results (Figures 3E-G).

We next examined the antitumor effect of QCA570 on BC cell proliferation. As shown in Figure 4A-E, QCA570 effectively decreased the survival of the five BC cell lines tested, with IC₅₀ values ranging from 2 to 30 nM. Notably, 5637 cells and J82 cells were particularly sensitive to QCA570 treatment, with IC₅₀ values of 2.6 and 10.8 nM. In contrast, JQ1 is much less potent than QCA570 in each BC cell line tested. We also observed that QCA570 induced G2/M phase arrest of the cell cycle in 5637 cells (Figure 4F), further confirming the functional effect of QCA570. We investigated cell migration ability that represents another cancer-related feature which has been associated by wound healing assays with tumor metastasis. The results showed that with a 24 h treatment in 5637, EJ-1, J82 and T24 cells, QCA570 significantly suppressed cell migration ability (Figures 4G-J).

To better understand the antiproliferation mechanism of QCA570, we investigated the effect of QCA570 on cell apoptosis in BC cell lines. Flow cytometry results demonstrated that QCA570 increased cell apoptosis in a dose-dependent manner (Figures 5A-E). In J82 and 5637 cell lines, apoptosis was induced in more than 50% of cells by QCA570 treatment. As a comparison, JQ1 had minimal effect on cell apoptosis. Consistent with the flow cytometry data, WB with cleaved Caspase-3 detection (Figures 5F-H) and a TUNEL assay of DNA break staining (Figures 5I-K) further confirmed that QCA570 induced greater cell apoptosis than in JQ1 in BC cells.