Choosing the right partner in hormone-dependent gene regulation: Glucocorticoid and progesterone receptors crosstalk in breast cancer cells.

Steroid hormone receptors (SHRs) belong to a large family of ligand-activated nuclear receptors that share certain characteristics and possess others that make them unique. It was thought for many years that the specificity of hormone response lay in the ligand. Although this may be true for pure agonists, the natural ligands as progesterone, corticosterone and cortisol present a broader effect by simultaneous activation of several SHRs. Moreover, SHRs share structural and functional characteristics that range from similarities between ligand-binding pockets to recognition of specific DNA sequences. These properties are clearly evident in progesterone (PR) and glucocorticoid receptors (GR); however, the biological responses triggered by each receptor in the presence of its ligand are different, and in some cases, even opposite. Thus, what confers the specificity of response to a given receptor is a long-standing topic of discussion that has not yet been unveiled. The levels of expression of each receptor, the differential interaction with coregulators, the chromatin accessibility as well as the DNA sequence of the target regions in the genome, are reliable sources of variability in hormone action that could explain the results obtained so far. Yet, to add further complexity to this scenario, it has been described that receptors can form heterocomplexes which can either compromise or potentiate the respective hormone-activated pathways with its possible impact on the pathological condition. In the present review, we summarized the state of the art of the functional cross-talk between PR and GR in breast cancer cells and we also discussed new paradigms of specificity in hormone action.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

The glucocorticoid receptor interferes with progesterone receptor-dependent genomic regulation in breast cancer cells.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation.

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3'-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes.

Glucocorticoids repress bcl-X expression in lymphoid cells by recruiting STAT5B to the P4 promoter.

The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-X(L) (antiapoptotic)/bcl-X(S) (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X(L)/bcl-X(S) favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4.

Steroid hormones induce bcl-X gene expression through direct activation of distal promoter P4.

Bcl-X exists in at least five different isoforms with complex effects on programmed cell death. Glucocorticoids and progestins control bcl-X expression and influence the ratio between bcl-X(L) (antiapoptotic isoform) and bcl-X(S) (proapoptotic isoform) in different tissues. The 5'-UTR region of the mouse bcl-X gene contains at least five different promoters, which exhibit a tissue-specific pattern of promoter usage. Several mRNAs with different 5'-leading exons can be generated upon promoter activation. Here we explore the potential of the various bcl-X gene promoters to be regulated by glucocorticoids or progestins. We found that the region located immediately upstream of promoter 4 (P4) contains two hormone response element (HRE)-like sequences at positions -3040 (HRE I) and -3001 (HRE II) relative to the translation initiation codon. These HRE-like sequences confer hormone responsiveness to a core promoter and bind glucocorticoid or progesterone receptors in vitro. Point mutations of both HREs that prevent steroid receptor binding also eliminate hormonal inducibility. In cells treated with glucocorticoids, the hormone receptor is recruited to the P4 region containing the HREs. Analysis of the products of the endogenous bcl-X in epithelial mammary cells showed that only transcripts originating from P4 increased upon hormone treatment. This observation correlates with the induction of the bcl-X(L) mRNA, suggesting that P4 is one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform.

Differences in nuclear retention characteristics of agonist-activated glucocorticoid receptor may determine specific responses.

We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.