congresos y reuniones científicas
Functional crosstalk between progesterone and glucocorticoid receptors in breast cancer cells
Simposio; EMBO Conference on Nuclear Receptors: From Molecules to Humans; 2015
Institución organizadora:
The glucocorticoid and progesterone receptors (GR and PR, respectively) share many similar structural and functional characteristics, including their DNA sequence recognition motif; however the cognate hormones display very distinct physiological responses. In the mammary gland, the activation of PR is associated with cell proliferation and tumor progression while activated GR was demonstrated to favor cell differentiation. Thus, the relative abundance of both receptors may modulate the proliferative and differentiation response of the mammary epithelium. The aim of this project was to identify -at genome-wide level- PR and GR binding regions in the human epithelial breast cancer cell line T47D/1-2, which expresses comparable levels of both receptors. Cells were treated for 1 h with the progestin analog R5020 or the synthetic glucocorticoid dexamethasone (Dex). By using ChIP-sequencing we found many more de novo binding events for the PR than for the GR (28,563 and 18,538, respectively) upon treatment with the respective hormone. Interestingly, most of the binding sites for both receptors were located in intergenic regions and introns, and only a small fraction was found in promoter regions. Of note, an important fraction of the GR binding regions upon Dex treatment (74%) mapped to regions that were bound by PR upon R5020 stimulation. Consistently, almost half of the PR binding regions (48%) was also bound by the GR upon Dex treatment. PR and GR effect on the surrounded chromatin accessibility was also monitored by DNAse-seq. We found that both receptors trigger similar chromatin remodeling once bound to their target genomic loci. To evaluate whether GR affects PR function, we analyzed the expression of several progestin-target genes involved in epithelial-mesenchymal transition, cell differentiation, cell cycling and proliferation, which presented binding sites for both receptors. We found that some of these genes exhibited a better response to progestins in T47D-WT compared to T47D/A1-2 cells, suggesting that GR would interfere with the function of PR during gene expression induction and repression. In fact, depletion of GR by siRNA knockdown restores the progestin-dependent expression levels in T47D/A1-2 cells. To assess if PR and GR have the potential to form complexes in vivo, T47D-WT cells were transfected with expression vectors encoding for both receptors fused to fluorescent proteins (eGFP-PR and mCherry-GR) and incubated with Dex and/or R5020. Then, to evaluate mCherry-GR and eGFP-PR associations, cross correlation analysis of the intensity fluctuations was performed by confocal microscopy. In the absence of hormone, a symmetric cross correlation (Brightness cross correlation (Bcc) centered on zero) in eGFP-PR and mCherry-GR particles was observed, indicating an absence of interaction. However, in the presence of R5020 or Dex mCherry-GR and eGFP-PR showed an asymmetric, positive Bcc value, suggesting that hormonal treatment induces PR and GR complex formation. Moreover, when cells were treated simultaneously with both hormones, the positive Bcc value significantly increased, indicating an increase in the number of complexes containing both activated receptors. Taken together, our results support the notion that PR and GR could be recruited to common regions in the genome as heterodimers, suggesting the idea that PR and GR crosstalk could play a role during mammary gland development, function and disease.