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br Results br Discussion ER and GR play
Results
Discussion
ER and GR play opposite phenotypic roles in the normal endometrium, with ER promoting growth and GR inhibiting growth. Here, we show that in endometrial cancer, GR expression is associated with worse outcomes and higher-grade tumors, and this association is observed only in the context of high ER expression. These findings seem to contradict the antigrowth effects of corticosteroids; however, our results show that growth inhibition is no longer observed after estrogen-induced hyperplasia has formed in vivo and that Dex, in combination with E2, promotes endometrial cancer cell growth in culture.
One possible explanation for the difference in GR-induced growth effects is that ER and GR are expressed in different compartments of the endometrium, with GR expressed in stromal Nitrendipine and ER expressed in endometrial glands (Bamberger et al., 2001). GR signaling in stromal cells, through either autocrine or paracrine changes, causes growth inhibition of the normal uterus. However, GR activity in hyperplastic or cancerous endometrial cells, when co-expressed in the same cells as ER, no longer inhibits growth and may lead to more aggressive tumors. In fact, gene expression profiling of cells induced with both Dex and E2 uncovered a downregulation of cell adherence genes, which has the potential to cause a more metastatic phenotype. These findings are consistent with work in breast cancer that showed that GR-expressing tumors differentially regulate cell adhesion genes (Pan et al., 2011). Our findings indicate that the administration of Dex during treatment of endometrioid histology endometrial cancer should be re-evaluated.
Molecular characterization of ER and GR crosstalk in endometrial cancer cells revealed that ER is the dominant steroid hormone receptor in this setting. ER genomic binding is mostly unaffected by the activation of GR. In contrast, more than one-third of GR binding is altered by the activation of ER. It also appears that recruitment of GR to ER-bound sites has a functional consequence because it likely leads to an increase in regulatory activity as measured by H3K27ac and proximity to regulated genes. ER dominance is the opposite pattern that is observed in breast cancer cells, in which GR is dominant in dictating ER binding (Miranda et al., 2013). It is interesting that the difference in dominance between GR and ER is associated with differences in prognosis. GR is dominant over ER in breast cancer cells, resulting in a better prognosis when both are expressed (Pan et al., 2011), whereas ER is dominant over GR in endometrial cancer cells, resulting in a worse prognosis when both are expressed (Figure 1C). This leads to a model in which GR is generally growth inhibitory, ER is generally growth promoting, and the dominant factor dictates the aggressiveness of the tumor. It is unclear why ER is dominant in one setting and GR is dominant in another, but the possibility of differences in co-factor abundance is interesting.
To explore how ER was altering GR genomic binding, we used ATAC-seq to identify changes in chromatin accessibility. We found that the majority of GR-binding sites that are gained upon treatment with both Dex and E2 exhibit increased chromatin accessibility after E2 induction. These results are consistent with ER assisting in loading GR onto genomic loci, which is similar to breast cancer cells, in which GR assists in loading ER onto genomic loci (Miranda et al., 2013). We attempted co-immunoprecipitation of ER and GR and were unable to pull down one factor with an antibody that recognizes the other factor (data not shown), suggesting that ER and GR are not forming heterodimers and further supporting the model that crosstalk between ER and GR is occurring through alteration of each other’s chromatin interactions.
The genomic binding crosstalk between ER and GR has gene expression consequences. When endometrial cancer cells were induced with either Dex or E2 in isolation, there was little overlap in the genes affected. When cells were treated with both Dex and E2, the transcriptional response had greater similarity to an E2 response but with additional genes changing expression, including a downregulation of cell-cell adherens junction genes. A significant fraction of these additional genes are differentially expressed in ER high-GR high endometrial tumors, indicating that many genes are regulated by the combination of ER and GR generally in endometrial cancer. Unlike a recent report in breast cancer (Yang et al., 2017), GR does not appear to repress an E2 transcriptional response in endometrial cancer cells. Overall, approximately one-fifth of genes affected by any treatment exhibited an unexpected gene expression level after double induction as determined by significant interaction terms in a linear model. Taken together, our findings are consistent with ER altering the genomic actions of GR, in which GR switches from regulating a distinct set of genes to promoting and enhancing an E2-driven transcriptional program in endometrial cells.