Brd4 maintains constitutively active NF-κB in cancer cells by binding to acetylated RelA

Abstract

Acetylation of the RelA subunit of NF-κB at lysine-310 regulates the transcriptional activation of NF-κB target genes and contributes to maintaining constitutively active NF-κB in tumors. Bromodomain-containing factor Brd4 has been shown to bind to acetylated lysine-310 (AcLys310) and to regulate the transcriptional activity of NF-κB, but the role of this binding in maintaining constitutively active NF-κB in tumors remains elusive. In this study, we demonstrate the structural basis for the binding of bromodomains (BDs) of bromodomain-containing protein 4 (Brd4) to AcLys310 and identify the BD inhibitor JQ1 as an effective small molecule to block this interaction. JQ1 suppresses TNF-α-mediated NF-κB activation and NF-κB-dependent target gene expression. In addition, JQ1 inhibits the proliferation and transformation potential of A549 lung cancer cells and suppresses the tumorigenicity of A549 cells in severe combined immunodeficiency mice. Furthermore, we demonstrate that depletion of Brd4 or treatment of cells with JQ1 induces the ubiquitination and degradation of the constitutively active nuclear form of RelA. Our results identify a novel function of Brd4 in maintaining the persistently active form of NF-κB found in tumors, and they suggest that interference with the interaction between acetylated RelA and Brd4 could be a potential therapeutic approach for the treatment of NF-κB-driven cancer.

Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest

Figures

Figure 1. Structural basis for the binding of Brd4 to acetylated lysine-310 of RelA

(A) Stereo view of BD1 ligand binding site showing the residues that are in the vicinity of the AcLys310 of RelA. Superimposed is a difference Fourier electron density map (contoured at 2.5σ over background in blue and 8σ over background in red) calculated with coefficients |Fobs| - |Fcalc| and phases from the final refined model with the coordinates of peptide deleted prior to one round of refinement. (B) Stereo view of BD2 ligand binding peptide showing equivalent residues. The superimposed difference Fourier electron density map was calculated as described in (A). (C) Sequence alignment of BD1 and BD2 of Brd4 and the description of various mutations within bromodomains (left panel). Recombinant proteins of GST-Brd4 bromodomains or its various mutants were used to pull down cell lysates containing acetylated RelA (right panel). (D) IL8-luciferase (IL-8-Luc) or E-selectin-luciferase (E-selectin-Luc) reporter plasmids were co-transfected with RelA expression vector, alone or in combination with WT Brd4 or its various mutants, into HEK293T cells. Luciferase activity was measured 30 h after transfection. Results represent the average of three independent experiments +/−SD. (E) HEK293T cells were co-transfected with IL-8-Luc or E-selectin-Luc reporter plasmid alone or in combination with expression vectors for WT Brd4 or its various mutants. Twenty-four hours after transfection, cells were treated with TNF-α (20 ng/ml) for 5 h and luciferase activity was measured as described in (D). (F) HEK293T cells transfected with Brd4 siRNA for 24 h were transfected with 5X-κB-Luc reporter plasmid alone or in combination with expression vectors for mouse WT or mutant Brd4. Twenty-four h after transfection, cells were treated with TNF-α and luciferase activity was measured as described in (D). (G) A549 cells transfected with Brd4 siRNA were reconstituted with mouse WT or mutant Brd4 followed by stimulation for 1 hrs with TNF-α and gene expression was analyzed by quantitative RT-PCR.

Figure 2. JQ1 inhibits the binding of Brd4 to RelA and suppresses NF-κB activation

(A) Purified GST-bromodomains (1 μg) of Brd4 were incubated with a biotin-labeled RelA acetylated K310 peptide (21) bound to Streptavidin agarose beads. Binding of the bromodomains to the peptide in the presence or absence of JQ1 (2 μM) was detected by immunoblotting with anti-GST antibody (upper panel). (B) JQ1 inhibits the interaction between Brd4 and RelA in vivo. HEK293T cells were transfected with the indicated combination of expression vectors for Flag-Brd4, T7-RelA, and HA-p300. Brd4 immunoprecipitation was performed in the presence or absence of JQ1 (0, 10, 20 μM) and immunoprecipitates were immunoblotted with anti-T7 antibodies. (C) A549 cells were treated with 5 μM JQ1 or vehicle for 4 hr. Nuclear extracts were prepared and subject to immunoprecipitation with anti-Brd4 antibodies. Brd4 associated RelA was detected by immunoblotting the Brd4 immunoprecipitates with anti-RelA antibodies. Levels of Brd4 and RelA were shown in the lower panels. (D) JQ1 inhibits TNF-α-mediated activation of NF-κB. HEK293T cells were co-transfected with 5XκB-Luc or IL-8-Luc reporter plasmid. Twenty-four hours after transfection, cells were pretreated with different doses of JQ1 for one hour before treatment with TNF-α for 5 hr, and luciferase activity was measured as in Figure 1D. (E),(F) A549 cells were pretreated with JQ1 (5 μM) for one hr and stimulated with TNF-α for indicated time points. Quantitative RT-PCR was performed to analyze the TNF-α-induced expression of NF-κB (E) or AP1 (F) target genes. Results represent the average of three independent experiments +/−SD.

Figure 3. JQ1 suppresses the proliferation and tumorigenesis of A549 cells

(A) A549 cells were plated in 96-well plates in DMEM and cultured for three days. Cells were then treated with or without JQ1 as indicated for up to 6 days. Cell proliferation was measured by using the MTS assay. Data represent the average of three independent experiments +/−SD. (B) A total of 5000 A549 cells were suspended in DMEM containing 0.35% SELECT Agar® (Invitrogen) and then plated in 6-well plates coated with an initial underlay of 0.5% SELECT Agar® (Invitrogen) in culture medium. Colony growth was scored after 14 days of cell incubation with or without JQ1 treatment as indicated. Representative photographs were taken at day 14 to show colonies. All the colony formation assays presented in this study were repeated in at least 3 independent experiments. (C) Nude mice bearing A549 xenografts were treated with vehicle or JQ1 daily for the indicated times with the dosage of 50 mg/kg (mpk). Mice (n=3 in each group) were killed when tumor volume reached 2,000 mm3. (D) Summary of the average weight of tumors from (C). Statistical analysis (P value) was performed using a student’s t-test. Data represent mean ± SD (n=3). All experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC).

Figure 4. Brd4 stabilizes nuclear RelA by preventing its ubiquitination

(A) A549 cells were transfected with control or Brd4 siRNA for 48 hr, the nuclear (Nuc.) and cytoplasmic (Cyt.) extracts were immunoblotted for the levels of endogenous RelA and Brd4. HDAC1 and tubulin were used as nuclear or cytoplasmic protein control, respectively. (B) A549 cells were transfected with siRNA as in (A). Forty hr after transfection, cells were treated with MG132 (10 μM) for 8 hr. Nuclear or cytoplasmic extracts were then immunoblotted for levels of RelA, Brd4, HDAC1 and tubulin. (C) A549 cells were transfected with control or Brd4 siRNA for 48 hr before total RNA was extracted. Quantitative RT-PCR was performed to analyze the expression level of RelA. Data represent the average of three independent experiments +/−SD. (D) A549 cells were transfected with control or Brd4 siRNA and nuclear RelA immunoprecipitates were immunoblotted for ubiquitination with anti-ubiquitin antibodies. To prevent the degradation of RelA, cells were treated with MG-132 (10μM) for 2 hr before lysis of cells. (E) HEK293T cells were transfected with indicated combinations of expression vectors for T7-RelA, HA-ubiquitin and Flag-Brd4. T7-RelA immunoprecipitates were immunoblotted for ubiquitination with anti-HA antibodies.

Figure 5. JQ1 induces the ubiquitination and degradation of nuclear RelA

(A) JQ1 reduces the nuclear levels of RelA in a dose- and time-dependent manner. A549 cells were treated with indicated dose of JQ1 for 4 or 8 hr. The nuclear and cytoplasmic extracts were immunoblotted for the levels of endogenous RelA and Brd4 as in Figure 4A. (B) MG-132 reverses the JQ1-induced down-regulation of nuclear RelA. A549 cells were treated with 2.5 μM JQ1 for 8 hr, followed by treatment with or without MG-132 (10 μM) for 4 hr. The nuclear and cytoplasmic extracts were immunoblotted for the indicated proteins. (C). JQ1 does not affect the transcription of RelA. A549 cells were treated with 2.5 μM JQ1 for 8 hr before total RNA was extracted. Complementary DNA was synthesized and quantitative real-time PCR was performed. Levels of RelA mRNA were normalized with the expression of actin. Data represent the average of three independent experiments +/−SD. (D) JQ1 induces the ubiquitination of nuclear RelA. A549 cells were treated with 10 μM JQ1 for 4 hr followed by treatment with MG-132 (10 μM) for 2 hr to prevent the degradation of RelA. Nuclear extracts were isolated and subjected to immunoprecipitation with anti-RelA antibodies. RelA immunoprecipitates were then immunoblotted with anti-ubiquitin antibodies for the ubiquitination of endogenous nuclear RelA. (E) Schematic model for the binding of Brd4 to acetylated lysine-310 of RelA to prevent the ubiquitination and degradation of nuclear RelA and the potential role of this interaction in the maintenance of constitutively active NF-κB and in tumor formation.