Abstract P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer
Authors: G Andrieu, AH Tran, GV Denis
Journal: Cancer Research (2017)

Abstract BACKGROUND. The epithelial-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, but also chemoresistance and cancer stem cell generation. As readers of epigenetic marks, bromodomain and extra-terminal (BET) proteins BRD2, BRD3 and BRD4 participate in the regulation of multiple transcriptional programs implicated in cancer progression. We sought to unravel the roles of BET proteins in EMT in breast cancer. METHODS. Cell line models of the different subtypes of breast cancer were used in this study (luminal A: MCF-7, T47D, basal-like: MDA-MB-231, SUM149PT). BET expression was modulated by using RNA interference or plasmid-mediated overexpression. Small molecule JQ1 was used to inhibit BET proteins. The expression of 84 key EMT genes was monitored by PCR using a Qiagen kit. We also monitored EMT by measuring the expression of epithelial and mesenchymal markers by immunoblot and immunofluorescence staining. RESULTS. Despite their homology, we report that BET proteins differentially regulate EMT. Based on an EMT PCR array, we identified a BRD2-specific transcriptional profile that promotes EMT, whereas BRD3 and BRD4 signatures repress this program. These individual signatures are unidentifiable upon pan-BET inhibition using JQ1, reinforcing the necessity to target each BET member separately to better understand their functions. Upon BRD2 depletion, basal-like breast cancer cells, which present a mesenchymal phenotype, exhibit a reduced expression of mesenchymal markers (N-cadherin, vimentin) and re-express epithelial markers (E-cadherin, cytokeratins). Moreover, a large panel of EMT master transcription factors is downregulated in BRD2-depleted cells, including the Snail and ZEB families or Twist. Interestingly, we found that BRD3 or BRD4 depletion leads to the opposite phenotype: an increase of mesenchymal marker expression and repression of the epithelial markers. In luminal A breast cancer cells which present an epithelial phenotype, BRD2 overexpression leads to the expression of mesenchymal markers. Similar results were obtained by depleting BRD3 or BRD4 in these cells, confirming the differential roles of BET proteins in EMT regulation. CONCLUSION. Taken together, our results establish that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. BET proteins possess separate and opposite biological functions, reinforcing the relevance of an individual targeting instead of a pan-BET inhibition using JQ1. We hypothesize that BET proteins modulate EMT through the regulation of its master transcription factors. We propose that the balance of BET proteins present at the promoters of the EMT genes is a novel mechanism of regulation of this program in breast cancer cells.BACKGROUND. The epithelial-mesenchymal transition (EMT) is a developmental program that cancer cells often activate to acquire a highly plastic phenotype that promotes invasion, metastasis, but also chemoresistance and cancer stem cell generation. As readers of epigenetic marks, bromodomain and extra-terminal (BET) proteins BRD2, BRD3 and BRD4 participate in the regulation of multiple transcriptional programs implicated in cancer progression. We sought to unravel the roles of BET proteins in EMT in breast cancer. METHODS. Cell line models of the different subtypes of breast cancer were used in this study (luminal A: MCF-7, T47D, basal-like: MDA-MB-231, SUM149PT). BET expression was modulated by using RNA interference or plasmid-mediated overexpression. Small molecule JQ1 was used to inhibit BET proteins. The expression of 84 key EMT genes was monitored by PCR using a Qiagen kit. We also monitored EMT by measuring the expression of epithelial and mesenchymal markers by immunoblot and immunofluorescence staining. RESULTS. Despite their homology, we report that BET proteins differentially regulate EMT. Based on an EMT PCR array, we identified a BRD2-specific transcriptional profile that promotes EMT, whereas BRD3 and BRD4 signatures repress this program. These individual signatures are unidentifiable upon pan-BET inhibition using JQ1, reinforcing the necessity to target each BET member separately to better understand their functions. Upon BRD2 depletion, basal-like breast cancer cells, which present a mesenchymal phenotype, exhibit a reduced expression of mesenchymal markers (N-cadherin, vimentin) and re-express epithelial markers (E-cadherin, cytokeratins). Moreover, a large panel of EMT master transcription factors is downregulated in BRD2-depleted cells, including the Snail and ZEB families or Twist. Interestingly, we found that BRD3 or BRD4 depletion leads to the opposite phenotype: an increase of mesenchymal marker expression and repression of the epithelial markers. In luminal A breast cancer cells which present an epithelial phenotype, BRD2 overexpression leads to the expression of mesenchymal markers. Similar results were obtained by depleting BRD3 or BRD4 in these cells, confirming the differential roles of BET proteins in EMT regulation. CONCLUSION. Taken together, our results establish that BRD2 positively regulates EMT, whereas BRD3 and BRD4 repress this program. BET proteins possess separate and opposite biological functions, reinforcing the relevance of an individual targeting instead of a pan-BET inhibition using JQ1. We hypothesize that BET proteins modulate EMT through the regulation of its master transcription factors. We propose that the balance of BET proteins present at the promoters of the EMT genes is a novel mechanism of regulation of this program in breast cancer cells. Citation Format: Andrieu G, Tran AH, Denis GV. Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-04-02.