BRD4 inhibition sensitizes renal cell carcinoma cells to the PI3K/mTOR dual inhibitor VS-5584

VS-5584 inhibits survival, proliferation, cell cycle progression and migration in RCC 786-O cells

The current study tested the potential anti-tumor activity of VS-5584, a novel dual inhibitor of PI3K/mTOR [14, 23], in RCC cells. 786-O RCC cells [12, 24] were treated with different concentrations (0.5-10 μM) of VS-5584. MTT cell viability assay results showed that VS-5584 treatment inhibited 786-O cell survival in a dose- and time-dependent manner (Figure 1A). The IC₅₀ of VS-5584 was between 1-5 μM (at 72/96-h treatment, Figure 1A). A lower dose of VS-5584 (0.5 μM) was unable to significantly inhibit 786-O cell viability (Figure 1A). Results in Figure 1B demonstrated that VS-5584 dose-dependently inhibited PI3K/mTORC1/2 cascade activation in 786-O cells. As shown in Figure 1B, treatment with VS-5584 inhibited the activation of phosphorylated (“p-”) p85, an indicator of PI3K activation, as well as of p-S6K1 (Thr-389) and p-Akt (Ser-473), which are substrates of mTORC1 and mTORC2 [25], respectively. The total protein levels of p85, S6K1, and Akt1/2 remained unchanged (Figure 1B).

Figure 1. VS-5584 inhibits survival, proliferation, cell cycle progression and migration in RCC 786-O cells. RCC 786-O cells were either left untreated (“C”, same for all Figures), or treated with applied concentrations of VS-5584 (0.5-10 μM), cells were further cultured for the indicated time; Cell survival (A, MTT assay), PI3K-mTORC1/2 activation (B, Western blotting), cell proliferation (C–E, BrdU EILSA, soft agar colony formation and EdU incorporation staining assays) and cell cycle progression (F, PI-FACS) were tested, with cell migration examined by “Transwell” assays (G). For “EdU” assays, at least 800 cells in five random views were included to calculate EdU ratio for each treatment (same for all Figures). For “Transwell” assays five random views were included to calculate average number of migrated cells (same for all Figures). Data were presented as mean ± standard deviation (SD, n=5). *p< 0.05 vs. “C” group. The in vitro experiments were repeated four times, and similar results were obtained. Bar = 100 μm (E, G).

To test cell proliferation in vitro, BrdU ELISA and soft agar colony formation assays were performed. VS-5584 treatment (1-10 μM) significantly decreased BrdU ELISA OD (Figure 1C) and the number of 786-O colonies (Figure 1D). These results indicated its anti-proliferative activity. Furthermore, VS-5584 dose-dependently inhibited EdU incorporation in 786-O cells (Figure 1E), further confirming proliferation inhibition.

Analysis of cell cycle distribution by PI-FACS showed that treatment with VS-5584 (2/5 μM) increased the percentage of cells in the G0/G1 phases, while decreasing the percentage of cells in the S and G2/M phases (Figure 1F). Testing cell migration in vitro, using “Transwell” assays, confirmed that VS-5584 (1-10 μM) reduced the number of migrated 786-O cells (Figure 1G). At the lowest concentration (0.5 μM), VS-5584 again failed to inhibit 786-O cell migration in vitro (Figure 1G). Treatment with vehicle control (dimethyl sulfoxide, 0.1-0.5%), as expected, had no significant effect on 786-O cell survival, proliferation and migration (Figure 1C–1G). These results show that VS-5584 inhibited survival, proliferation, cell cycle progression, and migration in RCC 786-O cells.

VS-5584 induces apoptosis activation in RCC 786-O cells

Cell death assay results showed that VS-5584 dose-dependently induced LDH release into the culture medium (Figure 2A), indicating cell death. VS-5584 treatment (1-10 μM) of 786-O cells also increased single strand DNA (ssDNA) production (Figure 2B). Western blotting assay results, Figure 2C, demonstrated that VS-5584 dose-dependently induced cleavages of caspase-3, caspase-9 and PARP (poly ADP-ribose polymerase) in 786-O cells. Additional studies demonstrated that the percentage of TUNEL-positive nuclei was significantly increased with VS-5584 (1-10 μM) treatment (Figure 2D), thereby confirming apoptosis activation. Lower concentrations of VS-5584 (0.5 μM) failed to induce 786-O cell apoptosis (Figure 2A–2D). Collectively, our data suggest that VS-5584 induced apoptosis activation in 786-O RCC cells.

Figure 2. VS-5584 induces apoptosis activation in RCC 786-O cells. RCC 786-O cells were treated with applied concentrations of VS-5584 (0.5-10 μM), cells were further cultured for the indicated time; Cell death was tested by LDH medium release assay (A); Cell apoptosis was tested by ssDNA ELISA (B), Western blotting testing apoptosis proteins (C), and nuclei TUNEL staining (D). Data were presented as mean ± standard deviation (SD, n=5). *p< 0.05 vs. “C” group. The in vitro experiments were repeated four times, and similar results were obtained.

VS-5584 exerts anti-survival, anti-proliferative, and pro-apoptotic activity in the established and primary human RCC cells

The anti-tumor effects of VS-5584 were tested on the established human A498 RCC cells and two different primary human RCC cells, RCC1 and RCC2 (see our previous studies [13]). Western blotting results showed that activation of PI3K (“p-p85”), mTORC1 (“p-S6K1”), and mTORC2 (“p-Akt at Ser-473”) was inhibited by VS-5584 treatment (5 μM, 2 h) in A498 and primary human RCC cells (Figure 3A). The basal PI3K/mTORC1/2 activity was low in HK-2 renal epithelial cells (Figure 3B). Treatment with VS-5584 (5 μM) significantly inhibited the viability (MTT OD, Figure 3C) and proliferation (BrdU ELISA OD and nuclei EdU staining, Figure 3D, 3E) of A498 and primary RCC cells. Cell migration, tested by the “Transwell” assay, was largely inhibited in VS-5584-treated RCC cells (Figure 3F).

Figure 3. VS-5584 exerts anti-survival, anti-proliferative, and pro-apoptotic activity in the established and primary human RCC cells. A498 cells, the primary human RCC cells (“RCC1/RCC2”) or HK-2 renal epithelial cells were treated with VS-5584 (5 μM), cells were further cultured for indicated time; PI3K-mTORC1/2 activation (A, B, Western blotting), cell survival (C, MTT), proliferation (D, BrdU EILSA and E, nuclei EdU staining), migration (F, “Transwell” assay) and apoptosis (G, ssDNA ELISA and H, TUNEL staining) were tested. The 786-O xenograft tumor-bearing nude mice were administrated with vehicle control (“Vehicle”, saline), VS-5584 (20 mg/kg, oral administration, daily), the tumor volumes (I) and mice body weights (J) were recorded every five days for a total of 35 days; The estimated daily tumor growth was calculated (K); Data were presented as mean ± standard deviation (SD). *p< 0.05 vs. “C” group (C–H, n=5). *p< 0.05 vs. “Vehicle” (I, J, n=10). The in vitro experiments were repeated four times, and similar results were obtained. Bar = 100 μm (E, F, H).

The ssDNA ELISA OD, an indicator of cell apoptosis, was increased in VS-5584-treated RCC cells (Figure 3G). To further confirm apoptosis activation we show that the ratio of TUNEL-positive nuclei was significantly increased with VS-5584 treatment in the RCC cells (Figure 3H). Whereas in HK-2 renal epithelial cells, the same VS-5584 treatment (5 μM) failed to inhibit cell survival (Figure 3C), proliferation (Figure 3D, 3E) and migration (Figure 3F). Nor did it induce apoptosis activation (Figure 3G, 3H). Thus, VS-5584 induced anti-survival, anti-proliferative, anti-migration and pro-apoptotic activities in established (A498) and primary human RCC cells.

To test the anti-RCC activity of VS-5584 in vivo, nude mice were subcutaneously inoculated with 786-O cells to form xenografts. Tumor growth curve analysis showed that a daily single dose of VS-5584 (20 mg/kg, oral administration) significantly inhibited 786-O tumor growth (Figure 3I). By calculating the estimated daily tumor growth, using the formula (tumor volume at day35− tumor volume at day0) ÷ 35, we show that 786-O xenograft growth in vivo was inhibited following treatment with VS-5584 (Figure 3J). The body weights of the experimental mice were not significantly different between the two groups (Figure 3K). There were no noticeable signs of apparent toxicity, suggesting that the VS-5584 treatment was well tolerated in the xenograft mouse model.

BRD4 inhibition potentiates VS-5584-induced RCC cell death and apoptosis

Although VS-5584 exerts anti-tumor effects against human RCC cells, its efficacy appears to be relatively low with an IC50 of 1-5 μM (Figures 1, 2), suggesting that RCC cells show resistance to VS-558. The BET family protein BRD4 is required for transcription elongation [17]. The BRD4-dependent proteins, Bcl-2 [26] and c-Myc [27, 28], are key oncogenic proteins. To examine the potential activity of BRD4 in chemoresistance, Western blotting was used to analyze BRD4 protein levels in tumor tissue lysates (Figure 3I). Results showed that BRD4 protein levels were significantly increased in VS-5584-treated 786-O tumor tissues compared with those in vehicle control-treated tumor tissues (Figure 4A). Therefore, VS-5584 administration in vivo induced BRD4 expression. Similarly, the protein levels of BRD4, Bcl-2, and c-Myc were increased in VS-5584 (2/5 μM)-treated 786-O cells in vitro (Figure 4B).

Figure 4. BRD4 inhibition potentiates VS-5584-induced RCC cell death and apoptosis. The 786-O xenograft tumor-bearing nude mice were administrated with vehicle control or VS-5584 (20 mg/kg, oral administration, daily), at treatment Day-2 and Day-4, 4 h after the VS-5584 or vehicle administration, two tumors (“Set-1/Set-2”) of each group were isolated, expression of BRD4 and Tubulin in tumor lysates was shown (A). 786-O cells (B) and primary human RCC cells (“RCC1”, E) were treated VS-5584 (or plus BRD4 inhibitors, B) for 24 h, listed proteins in total cell lysates were tested by Western blotting. 786-O cells (C, D), RCC1 primary cancer cells (F, G) or HK-2 cells (H, I) were pretreated with JQ1 (500 nM) or CPI203 (500 nM) for 30 min, followed by VS-5584 (2/5 μM) treatment for 48/72 h, cell survival and apoptosis were tested by MTT (C, F, H) and ssDNA ELISA (D, G, I), respectively. The listed proteins were quantified (B, E). Data were presented as mean ± standard deviation (SD, n=5). *p< 0.05 vs. “C” group. ^#p< 0.05.

To confirm BRD4-induced RCC resistance to VS-5584, two known BRD4 inhibitors, JQ1 and CPI203, were utilized. Both BRD4 inhibitors blocked VS-5584 (5 μM)-induced Bcl-2 and c-Myc upregulation (Figure 4B). Furthermore, treatment with JQ1 and CPI203 significantly enhanced the ability of VS-5584 (2/5 μM) to decrease 786-O cell viability(Figure 4C) and to enhance apoptosis (Figure 4D). Treatment with JQ1 or CPI203 alone induced minor but significant 786-O cell death and apoptosis (Figure 4C, 4D).

In primary RCC cells (“RCC1”), VS-5584 treatment (5 μM, 24 h) induced feedback upregulation of BRD4, Bcl-2, and c-Myc (Figure 4E). Furthermore, treatment with JQ1 or CPI203 potently enhanced the cytotoxicity of VS-5584 in primary cancer cells (Figure 4F, 4G). Co-treatment with VS-5584 and the BRD4 inhibitors (JQ1/CPI203) failed to induce significant reduction in cell viability (Figure 4H) and apoptosis (Figure 4I) in HK-2 epithelial cells.

BRD4 is the primary resistance factor of VS-5584 in RCC 786-O cells

Because the pharmacological BRD4 inhibitors (JQ1 and CPI203) might have off-target toxicities, genetic strategies were employed to alter BRD4 expression in 786-O cells. Two lentiviral BRD4 shRNAs, with non-overlapping sequences (“sh-BRD4-S1/S2”), were transfected into 786-O cells. Western blotting results showed that the protein expression of BRD4, as well as the BRD4-regulated c-Myc gene were significantly downregulated by BRD4 shRNA treatment (Figure 5A). Importantly, 786-O cells transduced with BRD4 shRNA were more vulnerable to VS-5584 treatment, showing an increased viability reduction (Figure 5B) and apoptosis (Figure 5C).

Figure 5. BRD4 is the primary resistance factor of VS-5584 in RCC 786-O cells. In VS-5584-treated stable 786-O cells with BRD4 shRNA (“sh-BRD4-S1/S2”, A–C), CRISPR-Cas9-BRD4-KO plasmid (D–F) or BRD4-expression vector (“BRD4-GFP”, G–I), BRD4, c-Myc and tubulin expression was shown (A, D, G). Cell survival and apoptosis were tested by MTT (after 72 h, B, E, H) and ssDNA ELISA (after 48 h, C, F, I), respectively. The listed proteins were quantified (A, D, G). Data were presented as mean ± standard deviation (SD, n=5). *p< 0.05 vs. “C” group. ^#p< 0.05.

To confirm BRD4 knockdown results, a CRISPR-Cas9-BRD4-KO plasmid was transfected into RCC 786-O cells to completely knockout BRD4 protein in the stable cells. In BRD4-KO cells, no BRD4 protein expression was observed even with VS-5584 treatment (5 μM, 24 h; Figure 4D). c-Myc expression was significantly decreased (Figure 5D). Compared with control cells, BRD4-KO 786-O cells were significantly more sensitive to VS-5584 (Figure 5E, 5F).

Based on the above results, we predicted that forced BRD4 overexpression shall inhibit VS-5584 activity. To test this hypothesis, a lentiviral BRD4-expression vector was transfected into 786-O cells. After puromycin selection, the stable cells showed exogenous BRD4 expression (tagged with GFP, Figure 5G). c-Myc expression was increased in BRD4-overexpressing cells (Figure 5G). Compared with the vector control cells, BRD4-overexpressing cells showed significant reduction in cell death (Figure 5H) and apoptosis activation (Figure 5I) following VS-5584 treatment. Collectively, these results confirm that BRD4 is the primary factor of VS-5584 resistance in RCC cells.

Chemicals and reagents

VS-5584, JQ1, and CPI203 were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture reagents were purchased from Gibco (Grand Island, NY). The antibodies were purchased from Cell Signaling Technology (Danvers, MA). Puromycin was obtained from Sigma-Aldrich.

Cell culture

Established human RCC cell lines (786-O and A498) as well as HK-2 human renal epithelial cells were obtained as described previously [13, 33]. The primary human RCC cells, derived from two different primary RCC patients (“RCC1” and “RCC2”, PTEN-null), were reported early [13]. The primary human cells were cultured in an appropriate medium as described previously [34].

Methylthiazol tetrazolium (MTT) assay

Cells were seeded onto a 96-well tissue culture plate (3 × 10³cells per well). MTT assay was performed to test cell viability, according to the manufacturer’s instructions (Sigma-Aldrich). The MTT optical density (OD) at 590 nm was recorded.

Soft agar colony formation assay

A total of 10,000 RCC 786-O cells per treatment were seeded on the top layer of 0.35% solidified agar in complete medium in 10-cm culture dishes, with the bottom layer containing 0.8% agar. VS-5584 was added to the complete medium and replaced every two days for a total of 10 days. Following this, colonies were stained with crystal violet (Sigma) and counted.

BrdU (5-bromo-2-deoxyuridine) enzyme-linked immunosorbent assay (ELISA)

Cells were seeded onto 96-well tissue culture plates (3 × 10³ cells per well). The BrdU ELISA kit (Roche Diagnostics, Basel, Switzerland) was utilized to test cell proliferation in vitro. The BrdU ELISA absorbance at 405 nm was recorded.

Cell cycle assay

The propidium iodide (PI; Invitrogen, Carlsbad, CA) flow cytometry assay was applied to test cell cycle distribution. Cells were seeded onto 6-well tissue culture plates (2 × 10⁵ cells per well). Following the applied treatment, cells were washed, fixed, and incubated with DNase-free RNase and PI. Cells were tested using a FACSCalibur instrument (BD Biosciences, Shanghai, China).

In vitro cell migration assay

As described human RCC cells or the HK-1 cells (4 × 10⁴ cells of each condition in 200 μL serum-free medium) were seeded on the upper surfaces of “Transwell” chambers, coated with Matrigel (Sigma) [35, 36]. The lower compartments were filled with FBS-containing complete medium. Following incubation, the migrated cells to the lower chambers were fixed, stained and counted.

EdU assay of cell proliferation

RCC cells or the HK-1 cells (1 × 10 ⁵cells/well) were seeded onto the six-well plates. An EdU (5-ethynyl-20-deoxyuridine) Apollo-488 In Vitro Imaging Kit (Ribo-Bio, Guangzhou, China) [37] was applied to examine and quantify cell proliferation. In brief, EdU (2.5 μM) dye was added to RCC cells or the HK-1 cells for 6-8h. Cell nuclei were co-stained with DAPI for 15 min, and visualized via a fluorescent microscope (Leica).

Lactate dehydrogenase (LDH) assay for cell death

Cells were seeded onto 6-well tissue culture plates (2 × 10⁵cells per well). Cell death was examined by measuring the LDH content in the medium, using a 2-step enzymatic reaction LDH assay kit (Takara, Tokyo, Japan). Percentage of LDH release = LDH released in conditional medium ÷ (LDH released in conditional medium + LDH in cell lysates).

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

As described previously [33], cells were seeded onto 6-well tissue culture plates (2 × 10⁵cells per well). TUNEL In Situ Cell Death Detection Kit (Roche Diagnostics, Shanghai, China) was utilized to quantify the number of TUNEL-labeled apoptotic nuclei.

Western blotting

After the applied treatment, cells were treated with lysis buffer [38]. The total cell protein lysates (30 μg per treatment) were analyzed. Western blotting was performed following a previously described protocol [33]. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Pierce, Suzhou, China), and quantified using the ImageJ software (National Institutes of Health).

Single stranded DNA (ssDNA) ELISA

ssDNA accumulation is a characteristic marker of cell apoptosis. For each treatment, 30 μg of cell lysate (using the lysis buffer for western blotting) was analyzed. A ssDNA ELISA kit (Roche Diagnostics) was utilized to quantify DNA fragmentation. The ssDNA ELISA absorbance was recorded at 450 nm.

BRD4 shRNA

Two different lentiviral BRD4 shRNAs, with unique and non-overlapping sequences (“S1/S2”), were provided by Dr. Zhao [39]. 786-O cells were seeded onto 6-well tissue culture plates (2 × 10⁵cells per well). Cells were transfected with BRD4 shRNA lentivirus for 24 h. Puromycin (2 μg/mL) was then used to select stable cells (4-5 passages). BRD4 knockdown in the stable cells was confirmed by Western blotting. Control cells were transfected with lentiviral scramble control shRNA (Santa Cruz Biotechnology).

Exogenous BRD4 overexpression

The pSUPER-puro-BRD4-GFP expression vector was provided by Dr. Zhao [39], and was transfected into HEK-293T cells together with the viral packaging proteins VSVG and Hit-60 (Promega, Shanghai, China). After 48 h, the medium containing the virus particles was filtered, and 786-O cells were incubated in this medium for additional 48 h. Puromycin was used to select the stable cells (4-5 passages). Exogenous BRD4 overexpression in stable cells was confirmed by western blotting.

BRD4 knockout (KO)

The CRISPR/Cas9 BRD4 KO plasmid (sc-400519-KO-2; Santa Cruz Biotechnology) was transfected to 786-O cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China), and selected with puromycin after 4-5 passages. Control cells were treated with an empty vector with control small guide RNA (sgRNA; Santa Cruz Biotechnology). BRD4 expression in stable cells was tested by western blotting.

Xenograft assay

The female nude mice were provided by the Animal Center of Chinese Academy of Science (Shanghai, China). 786-O cells were injected subcutaneously (s.c.) to the flanks of the nude mice. Within 20 days subcutaneous xenografts were established (around 100 mm³). Mice (n=10 each group) were treated with VS-5584. Mice body weight and bi-dimensional tumor measurements were taken every five days for a total of 35 days [40]. The animal protocol was approved by the Ethics Committee of Wenzhou Medical University.

Statistical analysis

Quantitative results were presented as mean ± standard deviation (SD). Results were compared by one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS version 21.0, Chicago, IL). Values of p< 0.05 were considered as statistically significant.