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. 2016 Oct 1:117:35-45.
doi: 10.1016/j.bcp.2016.08.009. Epub 2016 Aug 9.

Epigenetic blockade of neoplastic transformation by bromodomain and extra-terminal (BET) domain protein inhibitor JQ-1

Chengyue Zhang  1 , Zheng-Yuan Su  1 , Ling Wang  2 , Limin Shu  1 , Yuqing Yang  1 , Yue Guo  1 , Douglas Pung  1 , Chas Bountra  3 , Ah-Ng Kong  4
Affiliations

Epigenetic blockade of neoplastic transformation by bromodomain and extra-terminal (BET) domain protein inhibitor JQ-1

Chengyue Zhang et al. Biochem Pharmacol. .

Abstract

The neoplastic transformation of cells and inflammation are processes that contribute to tumor initiation. Recently, emerging evidence has suggested that epigenetic alterations are also implicated in the early stages of carcinogenesis. Therefore, potent small molecules targeting epigenetic regulators have been developed as novel cancer therapeutic and preventive strategies. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that play key roles at the interface between chromatin modification and transcriptional regulation. In this study, we investigated the effect of the BET inhibitor JQ-1 on malignant transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 P+ cells. Treatment with JQ-1 effectively impaired TPA-induced colony formation in vitro. At the molecular level, the expression of several key TPA-induced pro-survival and pro-proliferative genes (Bcl2, Cyclin D1, and c-Myc) decreased rapidly after BET inhibition. In addition, JQ-1 treatment attenuated the activation of inflammatory NF-κB signaling triggered by TPA. Luciferase reporter assays using plasmids carrying different elements from the COX2 or IL6 promoters demonstrated that JQ-1 does not directly inhibit interactions between NF-κB and its binding sequence; rather, it affects CRE-element-associated transcriptional enhancement. Through siRNA gene silencing, we found that JQ-1 inhibits the p300-dependent transcriptional activation of COX2, which correlates with the results of the luciferase assay. Chromatin immunoprecipitation assays showed that TPA elevated H3K27Ac enrichment in the COX2 promoter region, which is mediated by p300, and Brd4. JQ-1 treatment did not change H3K27Ac levels but decreased the recruitment of Brd4 and RNA Polymerase II. Collectively, our study reveals that the BET inhibitor JQ-1 exerts potent anti-cancer and anti-inflammatory effects by interfering with the core transcriptional program of neoplastic transformation.

Keywords: Brd4; Bromodomain; JQ-1; NF-κB; Neoplastic transformation.

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Conflict of interest statement

of potential conflicts of interest: No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Inhibitory effects of JQ-1 on TPA-induced transformation of JB6 P+ cells
Cells were seeded in soft agar containing DMSO (vehicle control), 20ng/mL TPA or a combination of TPA and indicated concentration of JQ-1 in 6-well plates and were allowed to grow for 14 days. The colonies exhibiting anchorage-independent growth were imaged under a microscope, and the colony numbers were counted using ImageJ software. A) Representative images of each treatment group. B) Graphical data of colony numbers in each group are presented as the mean ± SD from three independent experiments. JB6 P+ cells in the vehicle control group rarely formed colonies in agar plate unless treated with TPA. JQ-1 significantly inhibited the TPA-induced anchorage independent growth of JB6 P+ cells. *P<0.05 and **P<0.01 indicate significant differences between the JQ-1-treated group and cells treated with TPA alone.
Figure 2
Figure 2. Effect of JQ-1 on the mRNA and protein expression of oncogenic genes induced by TPA
JB6 P+ or HaCaT cells were treated with the indicated concentrations of JQ-1 for 4 h, followed by 20 ng/ml TPA treatment. Cells were harvested 8 h post TPA treatment for RNA extraction and 24 h for protein sample preparation. A) JQ-1 attenuated the TPA-induced phosphorylation of ERK1/2 in JB6 P+ cells; band intensity of the blots were digitized and normalized to the vehicle control as fold change, presented as the mean ± SD in the right panel. B) JQ-1 treatment decreased the TPA-induced transcription of Cyclin D1 and c-Myc in JB6 P+ cells. C) Western blotting images of Bcl2, Cyclin D1, and c-Myc in JB6 P+ cells after JQ-1 treatment; right panel shows the bar graph of relative band intensity as the mean ± SD. D) JQ-1 treatment attenuated c-MYC mRNA levels in HaCaT cells. E) JQ-1 treatment decreased c-MYC and Bcl2 protein levels in HaCaT cells; right panel shows the bar graph of relative band intensity. *P<0.05, **P<0.01, and #P<0.001 indicate significant differences between the JQ-1-treated group and the cells treated with TPA alone.
Figure 3
Figure 3. The effect of JQ-1 on G1 arrest of HCT-116 cells
HCT116 cells were treated with the indicated concentrations of JQ-1 for 24 h. A, B) JQ-1 suppressed cell cycle-related markers such as c-Myc and Cyclins A and D1 at both the mRNA and protein levels; relative mRNA expression or blot band intensity are presented as the mean ± SD; C) JQ-1 significantly induced G1 cell cycle arrest in HCT116 cells. Representative results from three independent experiments are provided in the upper panel whereas the lower panel shows the cell percentage in each phase as the mean ± SD. The statistical analyses were performed using one-way ANOVA with post-hoc Dunnett's test. *P<0.05, **P<0.01, and #P<0.001 indicate significant differences between the JQ-1-treated group and the vehicle control.
Figure 4
Figure 4. JQ-1 altered the protein expression of HDACs in HCT116 cells
The protein expression of HDAC1–4 was determined by western blotting. The relative band intensity (fold change) was calculated by normalizing the intensity of each sample to the vehicle control. Representative bands are shown in the left panel, whereas the bar chart in the right panel presents the mean ± SD of three independent experiments. *P<0.05 indicates significant differences between the JQ-1-treated group and the vehicle control.
Figure 5
Figure 5. Effect of JQ-1 on the activation of NF-κB target genes triggered by TPA
A) JQ-1 treatment decreased the TPA-induced transcription of IL6, uPAR, and COX2 in JB6 P+ cells. B) Western blot images of COX2 expression in JB6 P+ cells after JQ-1 treatment. C) JQ-1 treatment attenuated IL6 and COX2 mRNA levels in HaCaT cells. D) JQ-1 treatment decreased COX2 protein levels in HaCaT cells. *P<0.05, **P<0.01, and #P<0.001 indicate significant differences between the JQ-1-treated group and cells treated with TPA alone.
Figure 6
Figure 6. JQ-1 is not a direct inhibitor of NF-κB
A) Different constructs of luciferase reporter genes. B) HaCaT cells were co-transfected with 400 ng of the designated pGL4.15 firefly luciferase reporters and 100 ng of pGL4.75 [hRluc/CMV] Renilla luciferase plasmids as an internal control. The transfected cells were treated with DMSO, 20 ng/ml TPA, 500 nM JQ-1, or the combination for 20 h. The firefly luciferase readings were normalized to the Renilla luciferase reading first; then, the fold changes were calculated by comparing each treatment group to the vehicle control. JQ-1 inhibits the luciferase activity in the cells transfected with the plasmids carrying CRE elements (#P<0.001). No significant changes were observed after JQ-1 treatment in the cells transfected with the plasmids carrying the NF-κB binding sequence alone.
Figure 7
Figure 7. Effect of JQ-1 on the recruitment of the transcriptional machinery to COX2 under TPA stimulation
ChIP assays were performed to analyze the enrichment of H3K27Ac, Brd4, and Pol II on the promoter or transcription start site of COX2 in JB6 P+ cells. The immunoprecipitated DNA was used as a template for qPCR and the enrichment was quantified as the proportion ratio of its self- input. Relative fold change was then calculated by normalizing the ratio to that of the vehicle control. *P<0.05 and **P<0.01 indicate significant differences between the TPA-treated group and the indicated groups.
Figure 8
Figure 8. JQ-1 inhibits the p300-dependent activation of COX2
JB6 P+ cells were transfected with siRNA against p300 or non-specific negative control, and the cells were then further treated with DMSO, 20 ng/ml TPA alone or 20 ng/ml TPA in combination with 500 nM JQ-1 for 24 h. ChIP assays were performed to examine the enrichment of H3K27Ac and Brd4 after TPA stimulation in the p300 or negative control siRNA treated cells. The mRNA and protein levels of COX2 were assessed respectively. A) TPA increased the Ac-H3K27 enrichment on COX2 gene promoter and Brd4 enrichment on TSS. Knock down of p300 attenuated these TPA induced alterations. B) The mRNA levels of p300 and COX2 were determined by quantitative PCR. C) Representative western blot images of p300 and COX2 protein levels. Transfection of p300 siRNA attenuated TPA-induced COX2 expression at both the mRNA and protein levels. JQ-1 treatment did not show further inhibitory effects after p300 knockdown.
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