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

Activation of the PI3K/AKT/mTOR pathway promotes the progression of renal cell carcinoma (RCC). This study tested the anti-RCC cell activity of the PI3K/mTOR dual inhibitor, VS-5584. We show that VS-5584 inhibited PI3K/AKT/mTORC1/2 activation in established (786-O and A498 lines) and primary RCC cells, thereby suppressing cell survival, proliferation, migration and cell cycle progression. VS-5584 induced significant apoptosis in RCC cells. A daily single oral dose of VS-5584 (20 mg/kg) significantly inhibited 786-O tumor growth in vivo. VS-5584 treatment of 786-O tumor xenografts and RCC cells resulted in feedback upregulation of bromodomain-containing protein 4 (BRD4). Furthermore, BRD4 inhibition (by JQ1 and CPI203), knockdown or complete knockout potentiated VS-5584-induced RCC cell death and apoptosis. Conversely, forced overexpression of BRD4 attenuated the cytotoxicity of VS-5584 in 786-O cells. Collectively, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.


INTRODUCTION
Renal cell carcinoma (RCC) is the most common renal malignancy globally, causing significant human mortalities each year [1,2]. In clinical practices, nephroureterectomy of the early-stage RCCs is yet the only curable treatment procedure [1]. However, a large proportion of RCC patients are diagnosed at advanced stages. Over 25% of them have local invasion and systematic metastasis [1,3]. These patients often have a poor prognosis [1,3].
VS-5584 is a potent dual inhibitor of PI3K and mTOR [14]. It displays almost equivalent activity against PI3K and mTOR [14]. This dual inhibitor exhibits certain pharmacokinetic properties. It is well-tolerated in animal studies [14]. The current study tested the anti-RCC cell activity of VS-5584.
Bromodomain-containing protein 4 (BRD4), a member of the BET (bromodomain and extraterminal domain) family [15], binds acetylated-histones to participate in epigenetic processes [16][17][18]. It is required for chromatin structure formation in daughter cells in mitosis. BRD4 recruits positive transcription elongation factor b and phosphorylates RNA polymerase II. It is an essential step for transcription elongation and expression of several key oncogenes, including Bcl-2 and c-Myc [17,19].
In cancer cells BRD4 overexpression promotes cell survival, proliferation, and resistance to apoptosis [20]. Recent studies have proposed a pivotal function of BRD4 in chemoresistance. The BRD4 inhibitor JQ1 sensitized highly chemo-resistant pleural mesothelioma cells to cisplatin [21], and pancreatic cancer cells to gemcitabine [22]. The results of this study demonstrated BRD4 is a key resistance factor of VS-5584 in RCC cells.
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-

VS-5584 induces apoptosis activation in RCC 786-O cells
Cell death assay results showed that VS-5584 dosedependently induced LDH release into the culture medium (

AGING
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, antimigration 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).

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).
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,  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  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. AGING 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.

DISCUSSION
There are two mTOR complexes, namely mTORC1 and mTORC2. mTORC1 inhibitors, such as everolimus, have been approved by the FDA for the clinical treatment of certain human RCCs [6,9]. Yet, the clinical application of these inhibitors has several limitations. Rapamycin and its analogs can only partially inhibit mTORC1 activity [29,30]. They fail to directly inhibit mTORC2, which is also important in the progression of RCC [7,31].
VS-5584 is a novel PI3K/mTOR dual inhibitor, showing almost equivalent activity against PI3K and mTOR [14]. We found that VS-5584 blocked both mTORC1 and mTORC2 activation, as well as PI3K-Akt activity in RCC cells. We failed to observe feedback Erk-MAPK activation in VS-5584-treated RCC cells. A single daily oral dose of VS-5584 (20 mg/kg) significantly inhibited 786-O tumor growth in vivo. Hence, our data suggest that inhibition of the entirePI3K/AKT/mTOR cascade by VS-5584 could explain its superior anti-RCC cell activity.
Another important finding of this study was that BRD4, the BET family protein, is a key resistance factor against VS-5584 in RCC cells. VS-5584 treatment induced feedback upregulation of BRD4 in RCC cells, resulting in expression of BRD4 target proteins, Bcl-2 and c-Myc. Co-treatment with BRD4 inhibitors (JQ1/CPI203) potentiated VS-5584-induced RCC cell death and apoptosis. Furthermore, BRD4 knockdown or knockout enhanced VS-5584-induced cytotoxicity in RCC cells. Conversely, forced overexpression of BRD4 attenuated VS-5584-induced 786-O cell apoptosis.
The pharmacological and genetic evidence provided by this study indicate that BRD4 is a VS-5584 drug resistance factor in RCC cells. BRD4 inhibition may be an important strategy to sensitize RCC cells to VS-5584. The observed resistance to a PI3K-Akt inhibitor could be driven by the feedback activation of receptor tyrosine kinases (RTKs) [32]. It has been previously shown that BET inhibitors dissociated BRD4 from chromatin at the regulatory regions of multiple RTKs to reduce their expression level [32], thereby sensitizing a broad range of tumor cell lines to PI3K-Akt inhibitors [32]. Wang et al. demonstrated that BRD4 inhibition suppressed Sonic hedgehog signaling to sensitize pancreatic ductal adenocarcinoma cells to gemcitabine [22]. Moreover, JQ1 in combination with cisplatin induced synergistic inhibitory effects on human malignant pleural mesothelioma cells, possibly via the promotion of cell senescence and apoptosis [21]. Further studies are needed to explore the underlying mechanisms of BRD4 upregulation by VS-5584, and how BRD4 inhibition sensitizes RCC cells to VS-5584.
In summary, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.

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 × 10 3 cells 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.

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 × 10 5 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 × 10 4 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 5 cells/well) were seeded onto the six-well plates. An EdU (5-ethynyl-20deoxyuridine) 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 × 10 5 cells per well). Cell death was examined by measuring the LDH content in the medium, using a 2step 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).
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.

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.

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 mm 3 ). 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.

AUTHOR CONTRIBUTIONS
MX, YW, LX, JZb, YL, JZ conceived, designed, and supervised the study. MX, YW, LX, GD, BX, JZb, YL, JZ collected samples, performed the experiments and analyzed the data. MX, YW, JZb, YL, JZ wrote the paper. All authors reviewed and approved the final manuscript.