BET Proteins Exhibit Transcriptional and Functional Opposition in the Epithelial-to-Mesenchymal Transition

Abstract

Transcriptional programs in embryogenesis and cancer, such as the epithelial-to-mesenchymal transition (EMT), ensure cellular plasticity, an essential feature of carcinoma progression. As effectors of signal transduction, the bromodomain and extraterminal (BET) proteins are well suited to support plasticity because they function as co-activators or co-repressors of mammalian transcriptomes. Here, using both hormone-sensitive and triple-negative breast cancer (TNBC) model systems, we systematically altered EMT transcriptional profiles by manipulating individual BET proteins and found that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. Knockdown of individual BET proteins revealed independent transcriptional networks that differed from each other and from the small-molecule pan-BET inhibitor JQ1, which previously had been misleadingly asserted to be BRD4-selective. Available small-molecule pan-BET inhibitors, proposed as antiproliferative agents in cancer clinical trials, obscure these biological differences. Transcriptional profiling reveals that individual BET proteins, inhibited separately, engage in and control EMT through unique processes.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/4/580/F1.large.jpg Mol Cancer Res; 16(4); 580-6. ©2018 AACR.

Figure 1

Individual BET proteins control independent EMT transcriptomes. A, Heatmap presenting Z scores of a PCR array of 84 EMT genes expressed in MDA-MB-231 cells upon BET protein depletion by siRNA (50 nM for three days) (n=3). Independent transcriptional signatures relevant to EMT regulation were obtained for each BET protein. Pan-BET inhibition using small molecule JQ1 (400 nM for three days) obscured these individual profiles. A color code is used to illustrate Z score variations. Normalization is set to scramble. B, Systemic analysis of the EMT signatures. BRD2 depletion exhibit a strong association with JQ1 treatment with 25 common genes downregulated. Conversely, BRD3 and BRD4 depletions are mostly associated with a small number of upregulated genes that do not overlap. Most of the BET-regulated genes are EMT transcription factors. Graphs plot Z scores from BET depletion or JQ1 datasets. Signicantly altered genes are indicated (Z score ≥ 2 or ≤ −2, p-value < 0.05). C, Functional analysis of the EMT signatures. Most of the genes modulated by BET depletion are transcription factors, indicating that BET proteins transcriptionally regulate EMT.

Figure 2

BRD2 opposes BRD3 and BRD4 to control key EMT transcription factors. A, Validation of BET depletion by siRNA in MDA-MB-231 and MCF-7 cells (50 nM for three days). B, Protein expression of key EMT transcription factors upon BET protein depletion in MDA-MB-231 or MCF-7 cells. C, Validation of BET overexpression in MDA-MB-231 and MCF-7 cells. D, Protein expression of key EMT transcription factors upon BET protein overexpression in MDA-MB-231 or MCF-7 cells. Blots are representative of three independent experiments. Molecular weights are indicated (kDa).

Figure 3

BET protein manipulation triggers EMT in epithelial breast cancer cells. A, Representative images of MCF-7 depleted for BET proteins (50 nM for three days) and stained for E-cadherin (green), N-cadherin (red) and vimentin (gray). B, Representative images of MCF-7 overexpressing BET proteins and stained for E-cadherin (green), N-cadherin (red) and DNA (DAPI, blue). For measurement of relative fluorescence intensities, lines represent means ± SEM of three independent experiments. Each dot represents a single cell value. For cell area measurement, histograms represent means ± SEM of three independent experiments. Blots depict protein expression of key EMT markers upon BET protein depletion (A) or overexpression (B) in MCF-7 cells. Molecular weights are indicated (kDa). Statistical analyses were conducted by one-way ANOVA. The following symbols were used to indicate significant differences: ns, P > 0.05; **, P < 0.01; ***, P < 0.001. Images and blots are representative of three independent experiments. Bar scale, 10 μm.