Targeting BRD4 proteins suppresses the growth of NSCLC through downregulation of eIF4E expression

Abstract

Lung cancer is the leading cause of cancer-related death worldwide. Bromodomain and extraterminal domain (BET) proteins act as epigenome readers for gene transcriptional regulation. Among BET family members, BRD4 was well studied, but for its mechanism in non-small cell lung carcinoma has not been elucidated. eIF4E regulates gene translation and has been proved to play an important role in the progression of lung cancer. In this study, we first confirmed that BET inhibitors JQ1 and I-BET151 suppressed the growth of NSCLCs, in parallel with downregulated eIF4E expression. Then we found that knockdown of BRD4 expression using siRNAs inhibited the growth of NSCLCs as well as decreased eIF4E protein levels. Moreover, overexpression of eIF4E partially abrogated the growth inhibitory effect of JQ1, while knockdown of eIF4E enhanced the inhibitory effect of JQ1. Furthermore, JQ1 treatment or knockdown of BRD4 expression decreased eIF4E mRNA levels and inhibited its promoter activity by luciferase reporter assay. JQ1 treatment significantly decreased the binding of eIF4E promoter with BRD4. Finally, JQ1 inhibited the growth of H460 tumors in parallel with downregulated eIF4E mRNA and protein levels in a xenograft mouse model. These findings suggest that inhibition of BET by JQ1, I-BET151, or BRD4 silencing suppresses the growth of non-small cell lung carcinoma through decreasing eIF4E transcription and subsequent mRNA and protein expression. Considering that BET regulates gene transcription epigenetically, our findings not only reveal a new mechanism of BET-regulated eIF4E in lung cancer, but also indicate a novel strategy by co-targeting eIF4E for enhancing BET-targeted cancer therapy.

Keywords: BRD4; eIF4E; epigenetics; molecular targeted therapy; non-small cell lung carcinoma.

Figure 1.

BET inhibitors suppressed the growth of the NSCLCs as well as decreased eIF4E expression. A, NSCLC cells, including Calu-1, H460, H157, A549, and H1299, were treated with different concentrations of JQ1 for 3 days and subjected to SRB assay. B, the NSCLC cells were treated with 0–8 μmol/L JQ1 as indicated for 24h, then the whole-cell lysates were prepared and subjected to western blot assay. C, Calu-1 and H460 cells were treated with I-BET 151 for 3 days and subjected to SRB assay. D, Calu-1 and H460 cells were treated with 0–5μmol/L I-BET 151 as indicated for 24h, then the whole-cell lysates were prepared and subjected to western blot assay. Columns, means of four replicate determinations; bars, SD. *, P < 0.05 vs control. The data are representatives of three independent experiments.

Figure 2.

Knockdown BRD4 expression inhibited the growth of NSCLCs in parallel with downregulated eIF4E expression. A, Calu-1 and H460 cells were transiently transfected with a pool of 3 different sequences of BRD4 siRNAs or the control siRNAs for 48h using lipofectamine 2000. The whole-cell lysates were prepared and subjected to western blot assay. B, the two cell lines were seeded to 6-well plates and transiently transfected with the pool of 3 BRD4 siRNAs and the control siRNAs for 24h. Then the cells were re-seeded to 96-well plates for another 5 days and subjected to SRB assay. Points, means of four replicate determinations; bars, SD. *, P<0.05. The data are representatives of three independent experiments.

Figure 3.

Overexpression or knockdown of eIF4E expression partially abrogated or enhanced the growth inhibitory effect of JQ1 in NSCLCs, respectively. A, Calu-1, H460, and A549 cells were transiently transfected with eIF4E plasmid and the control vector for 48h using lipofectamine 2000, then subjected to western blot assay. B, cells transfected with plasmids as aforementioned for 24 h were re-seeded to 96-well plates, treated with different concentrations of JQ1 as indicated for another 3 days, and subjected to SRB assay. C, Calu-1 and H460 cells were transfected with 2 sequences of eIF4E siRNAs or the control siRNAs for 48h, then subjected to western blot assay. D, cells transfected with the pool of eIF4E siRNAs or the control siRNAs for 24h were re-seeded to 96-well plates and treated with JQ1 as indicated, and then subjected to SRB assay. Points, means of four replicate determinations; bars, SD. *, P < 0.05. The data are representatives of three independent experiments.

Figure 4.

JQ1 decreased eIF4E mRNA expression, the promoter activity, and the binding of eIF4E promoter with BRD4. A and B, cell lines as indicated were treated with 8 μmol/L JQ1 for 6h (A) or 24 h (B), then subjected to qRT-PCR assay. C, Calu-1 and H460 were transiently transfected with the pool of 3 BRD4 siRNAs and the control siRNAs for 24h. Then total RNAs were purified and subjected to qRT-PCR assay. D, Calu-1 and H460 cells were transiently transfected with eIF4E promoter plasmid (pGL3-eIF4E) or the vector (pGL3-basic) using lipofectamine 2000, and treated with 8 μmol/L JQ1 for 24h. Renilla plasmids were co-transfected as loading control. Then the cell lysates were prepared and subjected to luciferase reporter assay. Relative expression of BRD4 and eIF4E mRNA were calculated using the 2^−ΔΔCt method. E, H460 cells were treated with 8 μmol/L JQ1 for 24h, then subjected to ChIP assay using BRD4 antibody. The eIF4E promoter was detected using primers designed from -648 to -489bp upstream of the start site. The PCR products of eIF4E promoter was subjected to quantitative PCR (left), and agarose gel (right). Columns, means of three replicate determinations; bars, SD. *, P < 0.05. The data are representatives of three independent experiments.

Figure 5.

JQ1 suppressed the growth of H460 tumors in parallel with decreased eIF4E expression in a xenograft mouse model. H460 cells were inoculated to the subcutaneous of nude mouse. The mice were treated with 100 mg/kg/d JQ1 or the vehicle for 15 days (n = 7 for each group). A, the size of H460 tumors. Points, means of tumor volume; bars, SD. B, the weight of the tumors. Points, the weight of each tumor; horizontal line, means of tumor weight; bars, SD. C, the photo of the tumors. D and E, the RNAs and the protein lysates of the tumors were prepared and subjected to qRT-PCR assay (D) or western blot assay (E). Relative expression of eIF4E was calculated using the 2^−ΔΔCt method. Columns, means of three replicate determinations; bars, SD. *, P < 0.05.