JQ1: A Selective BRD4 Inhibitor Suppressing Retinoblastoma via Cell Cycle Arrest and Apoptosis

Abstract

Retinoblastoma (RB) is the most common intraocular cancer in children, and chemotherapy has been the first-line treatment. However, due to the side effects of chemotherapy drugs, novel treatments must be developed. JQ1, a selective inhibitor of BRD4, suppresses cell growth in several cancers in which BRD4 is overexpressed. In the present study, BRD4 was overexpressed in retinoblastoma, and JQ1 effectively inhibited RB cell proliferation and colony formation by inducing cell cycle arrest and promoting apoptosis. Furthermore, the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways were activated in RB cells treated with JQ1, and an animal experiment suggested that JQ1 significantly inhibited tumour growth in vivo. In conclusion, JQ1 may be a potential drug treatment for retinoblastoma.

Keywords

Retinoblastoma, BRD4, JQ1, Y79, WERI-Rb1

Value of the Data

It provides a potential therapeutic target for retinoblastoma. It may provide a new possibility for chemotherapeutics targeting retinoblastoma. Potential signalling pathways activated by JQ1 in RB cells were explored.

Data Description

Figure 1. BRD4 was highly expressed in retinoblastoma. (A and B) The mRNA and protein levels of BRD4 in retinoblastoma cells and retinal pigment epithelium cells were detected using real-time PCR and western blot assays. (C) Tissue structures were examined with H&E staining, and BRD4 expression in retinoblastoma tissues and adjacent tissues was detected with immunohistochemistry (magnification: 40x was in Supplementary Figure S1). (D) IHC scores for retinoblastoma tissues and adjacent tissues. (*p<0.01)

Figure 2. JQ1 had no adverse effect on the proliferation of ARPE-19 cells, but reduced the growth of RB cells. (A, C, and E) Cell viability of the cells treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 24, 48 and 72 h were determined using the MTT assay. The value of JQ1 concentration on the x-axis was log transformed to -1.301, -0.301, 0.699 and 1. (B, D, and F) The number of colonies formed was calculated and analysed after the cells were treated with DMSO or JQ1 for two weeks. (*p<0.01 and **p<0.001)

Figure 3. JQ1 arrested the RB cell cycle at G0/G1 phase and induced apoptosis. (A) The cell cycle of RB cells treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 48 h was detected using flow cytometry. (B and C) The distribution of cells at G0/G1, S and G2/M phase. (D) The apoptosis of RB cells was detected using FITC-AV/PI staining after treatment with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 72 h. (E and F) The apoptosis ratio of RB cells was calculated and analysed. (G and H) The expression of cleaved caspase-3 and cleaved caspase-9 in RB cells treated with DMSO or JQ1 (0.05, 0.5, 5, 10 μmol/ml) for 72 h was analyzed using western blot. (*p<0.01 and **p<0.001)

Figure 4. JQ1 downregulated the expression of BRD4 at protein levels and activated the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways in RB cells. (A-D) RB cells were treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 48 h, and then the protein expression of BRD4 was measured using western blot assay. (E-J) The Myc, P21, CDK2, CDK6, cyclinD3 expression at RNA and protein levels was respectively detected using real-time PCR and western blot after RB cells were treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 48 h. (*p<0.01 and **p<0.001)

Figure 5. JQ1 inhibited tumour growth in vivo. (A) During the days of treatment, the sizes of the tumours were measured every three days. (B and C) After treatment with DMSO or JQ1 for 21 days, the mice were sacrificed and the tumours were harvested. (D) The average weight of the tumours from each group on the last day of treatment. (E) The body weights of the mice during treatment. (F and G) The BRD4 expression in tumours after treatment for 21 days was detected using western blot. (*p<0.01)

Experimental Design, Materials, and Methods

Introduction

Retinoblastoma is considered the most common primary intraocular cancer in children, although it is rare, and it accounts for approximately 3 percent of childhood cancers. Children with retinoblastoma usually present with features of leukocoria, strabismus, blind eye and loss of vision. Retinoblastoma may even lead to death from systemic metastasis as the disease progresses. Therefore, effective treatment is particularly important for patients with retinoblastoma. Currently, the treatments for retinoblastoma mainly include chemotherapy, radiation therapy, focal therapy, and enucleation, and the choice of these treatments generally depends on the tumour group and stage. Among these management strategies, chemotherapy has been the first-line approach for children with retinoblastoma. Currently, the drugs used for chemotherapy of retinoblastoma mainly include carboplatin, etoposide, vincristine, and melphalan. However, reports of side effects of these drugs have increased in recent years. Therefore, novel therapies for retinoblastoma are urgently needed and should be developed.

According to recent studies, epigenetics will play a central role in cancers. As a member of the bromodomain and extraterminal (BET) family of proteins, BRD4 has emerged as a new epigenetic target for oncology and is recurrently mutated or aberrantly expressed in various malignancies. It contains two N-terminal bromodomains (BDI and BDII), an extraterminal (ET) domain and a C-terminal domain. Among these domains, BDI and BDII bind acetylated chromatin and interact with non-histone proteins to regulate some cellular activities, such as transcription, DNA replication, and cell cycle progression, among others. Thus, BRD4 plays a critical role in the genesis and development of malignant tumours. Some BRD4 inhibitors have been developed, and they have an attractive therapeutic strategy against cancers. Their effects on multiple cancers, including salivary adenoid cystic carcinoma, nasopharyngeal carcinoma, chondrosarcoma, and thyroid cancer, have been described. Excitingly, some bromodomain inhibitors have entered clinical trials for acute leukaemia and lymphoma or multiple myeloma.

JQ1 is a BRD4 inhibitor that has been widely studied in cancer research. It displaces BET bromodomains from chromatin and interferes with BRD4 function, leading to cell cycle arrest and the induction of apoptosis. Recently, JQ1 was reported to suppress the proliferation of various cancer cells. However, the anti-tumour effect of JQ1 on retinoblastoma has not been investigated. Therefore, it is necessary for us to explore whether JQ1 also inhibits the tumor growth in retinoblastoma. In this study, we evaluated the expression of BRD4 in cells and tissues and explored the anti-proliferative effect of JQ1 on RB cells in vitro and in vivo. Furthermore, the expression of Myc and cell cycle-related genes in RB cells was detected to explore potential signalling pathways involved in the effects of JQ1.

Materials and Methods

Chemicals and Cell Culture

The BET bromodomain inhibitor (JQ1) purchased from MedChemExpress of China was dissolved in dimethylsulfoxide (DMSO) at an initial concentration of 10 mmol/ml. Then, the stock solution was diluted to 0.05, 0.5, 5, and 10 μmol/ml for the experiments, and 0.1% DMSO was used as a control. For this study, three cell lines were used: the retinoblastoma cell lines Y79 and WERI-Rb1, and the human retinal pigment epithelium cell line ARPE-19. All cell lines were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). The medium used for culturing cells was RPMI1640 (Gibco, Grand Island, NY) contained 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) and 1% L-glutamine. All cells were placed in a humidified incubator at 37°C with a 5% CO2 for culture.

Real-time Polymerase Chain Reaction

For real-time PCR, the cells were harvested and total RNA was extracted using TRIzol reagent (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer (Thermo Fisher) was used to measure the concentration of total RNA. DNase (Sigma-Aldrich, St. Louis, MO) was used to remove the genomic DNA. Then, 1 μg of RNA was reverse transcribed to cDNA templates using a cDNA Synthesis Kit (Takara, Dalian, China). Real-time PCR was conducted in a 20 μl reaction system containing 10 μl of SYBR Green Reaction Mix (Takara, Dalian, China), 0.4 μl of paired primers, 2 μl of cDNA templates, and 7.2 μl of DEPC. Finally, the relative expression of each RNA was calculated using the 2-ΔΔCt method, as previously described. All primers used in this study are listed in Table 1.

Protein Extraction and Western Blot Analysis

Total proteins of tissue and cells were lysed with RIPA (Solarbio, Beijing, China) containing protease inhibitors (CWBIO, Beijing, China) on ice for 30 min, and then centrifuged at 14000 rpm for 22 minutes at 4°C. After retaining the supernatant, 5x sodium dodecyl sulfate loading buffer was added to the sample and then boiled it at 99°C for 10 minutes. The total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 90 mV for approximately 90 minutes, and then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Bedford, MA) at 350 mA for 90 minutes. Afterwards, the membranes were placed in PBST containing 5% skim milk for approximately 2 hours for blocking before an incubation with primary antibodies at 4°C overnight. The primary antibodies included BRD4 (1:1000; Abcam, Cambridge, UK), Myc (1:1000; Cell Signaling Technology, New York), P21 (1:1000; Cell Signaling Technology, New York), CDK2 (1:1000; Cell Signaling Technology, New York), cyclinD3 (1:1000; Cell Signaling Technology, New York), CDK6 (1:1000; Cell Signaling Technology, New York), cleaved caspase-3 (1:1000; Cell Signaling Technology, New York) and cleaved caspase-9 (1:1000; Cell Signaling Technology, New York). Next, the membranes were washed three times with PBST and incubated with a secondary antibody for 1 hour before three washes with PBST again. Next, the membranes were incubated with ECL (KeyGEN BioTECH, Nanjing, China), and the density of the bands was analysed with ImageJ software.

Haematoxylin and Eosin Staining and Immunohistochemistry

All tissue samples were collected from patients undergoing surgery at the eye hospital of Wenzhou Medical University and The Third Affiliated Hospital of Nanchang University. The study was approved by the Ethics Committee of Institute of Health Sciences. Haematoxylin-eosin staining was performed as described elsewhere, and the prepared sections were stained with H&E to examine the structure. For the immunohistochemistry, paraffin sections were deparaffinized with xylene and then rehydrated with 100, 95, 85 and 70% ethanol. For antigen retrieval and blocking endogenous peroxidase activity, the sections were boiled in Tris-EDTA buffer and treated with a 3% H2O2 solution. Subsequently, the sections were incubated with a rabbit monoclonal BRD4 antibody (1:200; Abcam, Cambridge, UK) at 4°C overnight. On the next day, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG at 37°C for 30 min. Then, the sections were stained with 3,3-diaminobenzidine and counterstained with haematoxylin. Immunohistochemical analysis was performed as previously. Briefly, the results of immunohistochemical staining were evaluated by calculating the immunoreactivity score (immunoreactivity score = intensity score × proportion score). The intensity score was defined as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The proportion score was defined as 0 (negative), 1 (<25%), 2 (26-50%), 3 (51-75%), and 4 (>75%). The Clinical and pathological characteristics of patients with RB were shown in Supplementary Table S1.

MTT Assay

Cell viability was detected using the MTT assay after treatment with DMSO or different concentrations of JQ1. First, 5000 cells were seeded into each well of 96-well plates and cultured in the incubator. On the next day, cells were treated with DMSO or different concentrations of JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 24, 48 or 72 h. Then, 20 μl MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, 5 mg/ml) were added to each well and the cells were incubated for another 4 h in the dark. Finally, formazan crystals were dissolved in DMSO, and cell viability was assessed by evaluating the optical density at 490 nm.

Colony Formation

Soft agarose (A9045-5G, Sigma-Aldrich, St. Louis, MO) was used for colony formation. First, 1.2% and 0.7% agarose gels were prepared and sterilized. The medium was supplemented with 20% FBS and 2% P/S. A mixture of 1.2% agarose gel and medium at a 1:1 ratio was added to six-well plates, with a volume of approximately 2 ml per plate. After the lower gel solidified, the cells were added to the medium containing the same amount of 0.7% agarose gel, and approximately 2 ml were added to the lower gel in each well. The medium containing DMSO and different concentrations of JQ1 (0.05, 0.5, 5, or 10 μmol/ml) was then added for two weeks. Colonies containing more than 50 cells were photographed and counted after an overnight stain with 0.1% crystal violet.

Analysis of Cell Cycle

Cells that had been treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 48 h were collected and washed with PBS. Next, 70% ethanol was added, and the cells were fixed at 4°C for approximately 1 h. The fixative was removed, and the cells were washed with PBS. Then, 500 μl PBS, 5 μl PI (Sigma-Aldrich, St.Louis, MO) and 5 μl RNase (TIANGEN BIOTECH, Beijing, China) were added, and the cells were incubated for approximately 15 min in the dark. A flow cytometry (Becton-Dickinson, San Jose, CA) was used to measure the cells, and the results were analysed using FlowJo software.

Cell Apoptosis Assay

Cells were treated with DMSO or different concentrations of JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 72 h and then harvested and washed with PBS. Next, the cells were resuspended in 500 μl binding buffer containing 5 μl Annexin V-FITC (AV) (KeyGEN BioTECH, Nanjing, China) and 5 μl propidiumiodide (PI) (KeyGEN BioTECH, Nanjing, China), and then incubated the cells at room temperature in the dark for approximately 15 minutes. A flow cytometry and FlowJo software were then used to, respectively, detect and analyse the cells. The cell apoptosis ratio was calculated as previously described. Briefly, region Q1: damaged cells (PI-positive/Annexin V-negative); region Q2: late apoptotic and dead cells (PI-positive/Annexin V-positive); region Q3: early apoptotic cells (PI-negative/Annexin V-positive); and region Q4: vital cells (PI-negative/Annexin V-negative). The sum of the percentages of cells in regions Q2 and Q3 was defined as the cell apoptosis ratio.

Subcutaneous Xenotransplantation Experiments

The Y79 xenograft model has been shown for the aggressive tumour growth characteristics and hence we used Y79 xenograft model than the WERI-Rb1-based xenograft model. In this assay, 3- to 4-week-old male nude mice were chosen. A mixture of 100 μl PBS containing 1×10^7 Y79 cells and 100 μl Matrigel (Invitrogen) was injected into the right submaxillary region of the mice, the animals were observed for the next few weeks. When tumours had formed on each mouse, the mice were divided into two groups: a DMSO-treated group and a JQ1-treated group. The JQ1-treated group was intraperitoneally injected with JQ1 (50 mg/kg) once daily, and the DMSO-treated group was injected with DMSO. The size of the tumour and the weight of the mouse were measured every three days. Animals were sacrificed after 21 days of treatment, and the tumours were harvested to capture images and weigh the samples before being preserved in liquid nitrogen. The animal experiments procedure used with the mice adhered to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Statistical Analysis

All data are reported as the means ± SD, and each experiment was repeated more than three times. The statistical significance of differences was determined using Student’s t-test for the in vitro study. For the in vivo study, two-way ANOVA was used to analyse the differences in tumour volumes and body weights between groups. The difference was considered significant when P < 0.05.

Results

BRD4 is Overexpressed in RB Cells and Tissues

Cells were harvested and qPCR and western blot assays were performed to explore the levels of BRD4 in retinoblastoma. As shown in Figure 1A and 1B, the BRD4 mRNA and protein were expressed at higher levels in retinoblastoma cells (Y79 and WERI-Rb1) than in human retinal pigment epithelium cells (ARPE-19).

In addition, immunohistochemistry was performed to examine BRD4 expression in tissue samples. As shown in Figure 1C and 1D, BRD4 expression was significantly upregulated in retinoblastoma (Mayo Clinic) compared with the adjacent tissues. Based on these results, BRD4 was overexpressed in this pediatric eye cancer, suggesting its role as a potential therapeutic target.

JQ1 Had No Adverse Effects on the Proliferation of the Human Retinal Pigment Epithelium Cells but Reduced RB Cells Proliferation

The effects of JQ1 on the proliferation of ARPE-19 and RB cells were subsequently explored. The cells were treated with DMSO and different concentrations of JQ1 for 24-72 hours, and then analysed using MTT and colony-formation assays. Significant changes in the viability and clonogenic growth of ARPE-19 cells were not observed (Figure 2A and 2B). Conversely, JQ1 effectively reduced the viability of Y79 and WERI-Rb1 cells in a dose- and time-dependent manner (Figure 2C and 2E), and substantially fewer colonies formed from Y79 and WERI-Rb1 cells treated with JQ1 than from cells treated with DMSO (Figure 2D and 2F). Thus, JQ1 had no obvious adverse effect on the growth of the human retinal pigment epithelium cells, but it effectively inhibited RB cell proliferation in vitro.

JQ1 Arrested the RB Cell Cycle at G0/G1 Phase and Induced Apoptosis

The cell cycle distribution of RB cells was detected using flow cytometry. The results were shown in Figure 3A-C. After the cells had been treated with DMSO or JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 48 h, the percentage of cells in G0/G1 phase gradually increased as the concentration of JQ1 increased, and the number of cells in S phase gradually decreased. Based on these results, the cell cycle of Y79 and WERI-Rb1 cells treated with JQ1 was arrested at G0/G1 phase. Then, apoptosis assays were performed, and more RB cells in region Q2 and Q3 were observed after treatment with JQ1 (0.05, 0.5, 5, or 10 μmol/ml) for 72 h than after treatment with DMSO (Figure 3D-F). Thus, JQ1 significantly increased the cell apoptosis ratio compared with the control. To further confirm the pro-apoptotic effects of JQ1, the expression of key enzyme involved in apoptosis was evaluated using western blot assay. As shown in Figure 3G and 3H, the protein expression of cleaved caspase-3 and cleaved caspase-9 was significantly upregulated after 72 h treatment with JQ1, consistent with the results of flow cytometry. Thus, JQ1 effectively suppressed the cell cycle and induced apoptosis in RB cells, thereby inhibiting cell proliferation.

JQ1 Regulated BRD4 Expression in RB Cells and Activated the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 Pathways

Next, the effects of JQ1 on the expression of the BRD4 protein in RB cells were explored, and the BRD4-protein expression descended gradually in cells treated with increasing concentrations of JQ1 for 48 h (Figure 4A-D). Considering above results demonstrated that JQ1 may inhibit RB cell proliferation by arresting the cell cycle at G0/G1 phase, the expression of Myc and its downstream cell cycle-related factors that play a crucial role in the progression from G1 phase to S phase were then evaluated to identify potential signalling pathways involved in the effects of JQ1. The mRNA and protein levels in the RB cells were detected after DMSO and JQ1 treatment for 48 h, and the results were shown in Figure 4E-J, the expression of Myc was downregulated after JQ1 treatment. Accompanied with it, the expression of P21 was upregulated and the expression levels of CDK2, cyclinD3 and CDK6 were reduced. These results showed that JQ1 suppressed BRD4 expression on protein levels and inhibit cell proliferation by activating the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways in RB cells.

JQ1 Inhibited Tumour Growth in Vivo

We next probed whether JQ1 exerted an anti-tumour effect in vivo. Thus, a subcutaneous xenograft model was established in nude mice. After several days of treatment, the tumour volumes in the nude mice injected with JQ1 were smaller than in mice injected with DMSO (Figure 5A-C). On day 21 of treatment, the tumours were harvested and weighed. A lower tumour weight was observed in the JQ1-treated group than in the DMSO-treated group (Figure 5D). Throughout treatment, there was no significant difference in body weight of mice between the JQ1-treated group and the DMSO-treated group (Figure 5E). It means that JQ1 had no significant toxicity to the mice. Moreover, total proteins were extracted from the tumour to analyse BRD4 expression. The western blot results showed that the BRD4 expression was significantly downregulated in the JQ1-treated group compared with the DMSO-treated group (Figure 5F and 5G). All of these results confirmed that JQ1 effectively suppressed tumour growth in vivo.

Discussion

The anti-tumour effect of JQ1 on several types of cancer has been described. However, its role in retinoblastoma remains unclear. In the present study, BRD4 was overexpressed in RB cells and retinoblastoma tissues compared to retinal pigment epithelium cells and adjacent tissues. JQ1, a selective inhibitor of BRD4, had no obvious adverse effects on human retinal pigment epithelium cells, but effectively inhibited retinoblastoma cell proliferation by arresting the cell cycle and inducing apoptosis. In addition, JQ1 activated the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways in RB cells. An animal experiment further confirmed the anti-tumour effect of JQ1 in vivo.

BRD4 has been a novel therapeutic target in a number of cancers, and it is overexpressed in several cancer cells. For example, it has been reported that BRD4 protein was unregulated in differentiated thyroid cancer cells, exerting a pro-oncogenic function in cancer progression. Overexpression of BRD4 protein was also found in squamous cell carcinoma, where it affected the cell proliferation and invasion. Therefore, the expression of BRD4 in retinoblastoma was detected here, and the results showed that BRD4 was overexpressed in RB cells and tissues, suggesting that BRD4 may also be a potential therapeutic target for retinoblastoma.

JQ1 is a new potent and selective inhibitor of BRD4, and many studies have confirmed its ability to significantly inhibit cell viability in vitro and in vivo in some tumour cells overexpressed BRD4. Thus, the effect of JQ1 on RB cell growth was then explored after BRD4 overexpression in retinoblastoma was observed. In the present study, we observed that JQ1 reduced Y79 and WERI-RB1 cell viability and colony formation, as expected. According to a previous study, BRD4 plays a critical role in proper G1 progression, leading to transcriptional elongation following its recruitment to G1 genes and stimulation of the binding of P-TEFb and pol II to the promoters. In addition, it has been demonstrated that treatment with JQ1 in chondrosarcoma cells significantly induced G0/G1 cell cycle arrest and retarded S phase entering. In thyroid cancer, it was found that JQ1 arrested TC cells at G0/G1 phase. Moreover, many other studies have reported effects of JQ1 on the cell cycle of cancer cells. Similar to previous studies, our results showed that the number of cells treated with JQ1 arrested at G0/G1 phase was obviously greater than cells treated with DMSO, and the number of cells at S phase was reduced after JQ1 treatment. Apparently, JQ1 regulated the RB cell cycle and arrested it at G0/G1 phase. In addition, JQ1 not only arrested the cell cycle but also induced apoptosis. That JQ1 induced apoptosis in some cancers has been suggested in recent years. Similarly, the results of apoptosis and western blot assays showed that the number of apoptotic cells increased and the expression of cleaved caspase-3 and cleaved caspase-9 was upregulated after JQ1 treatment for 72 h, indicating that JQ1 induced RB cell apoptosis. In conclusion, JQ1 effectively inhibited RB cell growth in vitro. Additionally, our subcutaneous xenograft experiment further confirmed the growth-inhibitory function of JQ1 in vivo.

The effect of JQ1 on BRD4 expression in RB cells was explored to obtain a better understanding of the anti-tumour mechanisms of JQ1. As previously reported, JQ1 competitively binds to the acetyl-lysine recognition hydrophobic pocket of BRD4 with the highest affinity and interferes with BRD4 function, leading to cell cycle arrest and the induction of apoptosis. However, the BRD4 expression is differentially altered by JQ1 in various cancers. Although Fiskus W et al. and Gao et al. both showed no effects of JQ1 on the expression of BRD4, other researchers have found that JQ1 downregulated the expression of BRD4 in salivary adenoid cystic carcinoma and oral squamous cell carcinoma. In our study, JQ1 suppressed the expression of BRD4 at the protein level, indicating that JQ1 inhibited retinoblastoma cell proliferation with affecting BRD4 expression.

CDK6 and CDK2, the members of the cyclin-dependent kinase (CDK) family, both play a critical role in the promotion of the cell cycle progression, especially in the promotion of G1/S transition. Myc, an oncogene, stimulates cell-cycle progression through the regulation of many genes related to cell-cycle control. In our research, following Myc inhibition, P21 expression was upregulated and the levels of CDK2, cyclinD3 and CDK6 were reduced in RB cells after JQ1 treatment for 48 h. This result was supported by previous studies that Myc, a cell-cycle brake releaser, targets CDK6 associated with D-type cyclins and activates CDK2 by suppressing P21 which is a cyclin-dependent kinase inhibitor (CDKIs) to promote G1/S cell cycle progression. Once Myc was suppressed, CDK6 binded with cyclinD3 and CDK2 regulated by P21 were inhibited and the cell cycle was then arrested at G0/G1 phase.

In summary, we reported the aberrant expression of BRD4 in retinoblastoma, and JQ1 effectively inhibited retinoblastoma growth in vitro and in vivo. Our results provided the possibility of JQ1 treatment for retinoblastoma. However, additional studies of biochemical mechanisms and clinical translational research are still necessary.

Highlights

BRD4 may be a new therapeutic target for retinoblastoma. JQ1, a BRD4 inhibitor, can effectively inhibit retinoblastoma cells growth in vitro and vivo. JQ1 arrested the cell cycle in Y79 cells by activating Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways.