Search

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO , the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR ., (©2017 American Association for Cancer Research.)

This paper reviews work on progesterone and the progesterone receptor (PR) in the mouse mammary gland that has been used extensively as an experimental model. Studies have led to the concept that progesterone controls proliferation and morphogenesis of the luminal epithelium in a tightly orchestrated manner at distinct stages of development by paracrine signaling pathways, including receptor activator of nuclear factor κB ligand (RANKL) as a major paracrine factor. Progesterone also drives expansion of stem cells by paracrine signals to generate progenitors required for alveologenesis. During mid-to-late pregnancy, progesterone has another role to suppress secretory activation until parturition mediated in part by crosstalk between PR and prolactin/Stat5 signaling to inhibit induction of milk protein gene expression, and by inhibiting tight junction closure. In models of hormone-dependent mouse mammary tumors, the progesterone/PR signaling axis enhances pre-neoplastic progression by a switch from a paracrine to an autocrine mode of proliferation and dysregulation of the RANKL signaling pathway. Limited experiments with normal human breast show that progesterone/PR signaling also stimulates epithelial cell proliferation by a paracrine mechanism; however, the signaling pathways and whether RANKL is a major mediator remains unknown. Work with human breast cancer cell lines, patient tumor samples and clinical studies indicates that progesterone is a risk factor for breast cancer and that alteration in progesterone/PR signaling pathways contributes to early stage human breast cancer progression. However, loss of PR expression in primary tumors is associated with a less differentiated more invasive phenotype and worse prognosis, suggesting that PR may limit later stages of tumor progression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Our mechanistic understanding of progesterone's involvement in murine mammary morphogenesis and tumorigenesis is dependent on defining effector pathways responsible for transducing the progesterone signal into a morphogenetic response. Toward this goal, microarray methods were applied to the murine mammary gland to identify novel downstream gene targets of progesterone. Consistent with a tissue undergoing epithelial expansion, mining of the progesterone-responsive transcriptome revealed the up-regulation of functional gene classes involved in epithelial proliferation and survival. Reassuringly, signaling pathways previously reported to be responsive to progesterone were also identified. Mining this informational resource for rapidly induced genes, we identified "inhibitor of differentiation 4" (Id4) as a new molecular target acutely induced by progesterone exposure. Mammary Id4 is transiently induced during early pregnancy and colocalizes with progesterone receptor (PR) expression, suggesting that Id4 mediates the early events of PR-dependent mammary morphogenesis. Chromatin immunoprecipitation assay detecting direct recruitment of ligand occupied PR to the Id4 promoter supports this proposal. Given that Id4 is a member of the Id family of transcriptional regulators that have been linked to the maintenance of proliferative status and tumorigenesis, the establishment of a mechanistic link between PR signaling and Id4 promises to furnish a wider conceptual framework with which to advance our understanding of normal and abnormal mammary epithelial responses to progestins. In sum, the progesterone-responsive transcriptome described herein not only reinforces the importance of progesterone in mammary epithelial expansion but also represents an invaluable information resource with which to identify novel signaling paradigms for mammary PR action.

Human progesterone receptor (PR) contains a motif that interacts with the SH3 domain of Src and mediates rapid activation of Src and downstream MAPK (Erk-1/-2) without relying on the transcriptional activity of the receptor. Here we investigated the role and intracellular location of this nontranscriptional activity of PR. Progestin activation of Src/MAPK occurred outside the nucleus with the B isoform of PR that was distributed between the cytoplasm and nucleus, but not with PR-A that was predominantly nuclear. Breast cancer cells stably expressing wild-type PR-B or PR-B with disrupting point mutations in the SH3 domain binding motif (PR-BDeltaSH3) that do not affect the transcriptional activity of PR, were compared for effects of progestin on endogenous target gene expression and cell proliferation. Progestin induction of the cyclin D1 gene, which lacks a progesterone response element, was dependent on PR activation of the Src/MAPK pathway, whereas induction of the Sgk (serum and glucocorticoid regulated kinase) gene that contains a functional progesterone response element was unaffected by mutations that interfere with PR activation of Src. Progestin induction of cell cycle progression was also abrogated in cells expressing PR-BDeltaSH3, and no effect of progestin on cyclin D1 expression and cell cycle was observed in the presence of PR-A. These results highlight the importance of PR activation of the Src/MAPK signaling pathway for progesterone-induced transcription of select target genes and cell cycle progression.

The female sex steroid hormones 17beta-estradiol and progesterone mediate their biological effects on development, differentiation, and maintenance of reproductive tract and other target tissues through gene regulation by nuclear steroid receptors that function as ligand-dependent transcription factors. However, not all effects of 17beta-estradiol and progesterone are mediated by direct control of gene expression. These hormones also have rapid stimulatory effects on the activities of a variety of signal transduction molecules and pathways and, in many cases, these effects appear to be initiated from the plasma cell membrane. There is growing evidence that a subpopulation of the conventional nuclear steroid receptor localized at the cell membrane mediates many of the rapid signaling actions of steroid hormones; however, novel membrane receptors unrelated to conventional steroid receptors have also been implicated. This chapter reviews the nature of the receptors that mediate rapid signaling actions of estrogen and progesterone and describes the signaling molecules and pathways involved, the mechanisms by which receptors couple with components of signaling complexes and trigger responses, and the target tissues and cell functions regulated by this mode of steroid hormone action.

The effects of progesterone on target tissues are mediated by progesterone receptors (PRs), which belong to a family of nuclear receptors and function as ligand-activated transcription factors to regulate the expression of specific sets of target genes. Progesterone antagonists repress the biological actions of progesterone by "actively" inhibiting PR activation. This work discusses the first clinically used progesterone antagonist RU486 and closely related compounds in terms of how these compounds inhibit progesterone action through heterodimerization and competition for DNA binding and by the recruitment of corepressors to promoters of target genes to repress transcription. We discuss cellular factors that may influence the activity of these compounds, such as the availability of coactivators and corepressors and the context of specific target promoters in any given cell type. We also discuss steroidal and nonsteroidal antagonist selectivity for PR versus other steroid hormone receptors and suggest that it may be possible to develop tissue/cell specific modulators of PR.

The progesterone receptor (PR), as a member of the nuclear receptor superfamily of ligand-dependent transcription factors, activates gene transcription through binding to specific palindromic progesterone response elements (PRE) in the promoter region of progestin-responsive genes. The progesterone antagonists ZK98299 (Onapristone) and RU 486 (Mifepristone) inhibit the transcriptional activity of PR by complex mechanisms at concentrations much lower than the progestins. Altered conformation is central to antagonist inhibition of the transcriptional activity of PR. Antagonists also promote inappropriate association of PR with corepressors. We speculate that the different PR conformations induced by agonist and antagonists results in an asymmetric agonist/antagonist heterodimer that binds inefficiently to palindromic PREs. PR, under the same cellular conditions but with different promotor contexts, can repress (beta-casein) or enhance (3 beta-HSD) signal transducer and activator of transcription (Stat5)-mediated gene activation. The beta-casein promoter appears to contain a composite DNA-binding element for PR and Stat5 and that occupancy by PR in response to progestins or antagonists suppresses Stat5 transactivation function.

Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.

The use of steroid hormones in postmenopausal replacement therapy has been associated with prevention of cardiovascular disease. Although the contribution of estradiol to endothelial cell function has been addressed, little information is available on the effect of progestins on this cell type. Here, we provide direct evidence for the presence of functional nuclear progesterone receptor in endothelial cells and demonstrate that physiological levels of progesterone inhibit proliferation through a nuclear receptor-mediated mechanism. The effects of progesterone were blocked by pretreatment with a progesterone receptor antagonist, and progesterone receptor-deficient endothelial cells failed to respond to the hormone. We evaluated the effect of progesterone by analysis of aorta re-endothelialization experiments in wild-type and progesterone receptor knockout mice. The rate of re-endothelialization was significantly decreased in wild-type mice when in the presence of progesterone, whereas there was no difference between control and progesterone-treated progesterone receptor knockout mice. FACS analysis showed that progestins arrest endothelial cell cycle in G1. The lag in cell cycle progression involved reduction in cyclin-dependent kinase activity, as shown by down-regulation in retinoblastoma protein phosphorylation. In addition, treatment of endothelial cells with progestins altered the expression of cyclin E and A in accordance with G1 arrest. These results have important implications to our current knowledge of the effect of steroids on endothelial cell function and to the overall contribution of progesterone to vascular repair.

This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

The rat is frequently used as a model to study the role of progesterone (P) in regulating FSH secretion and synthesis. The ability of P to modulate rat FSH-beta mRNA levels suggests the presence of a functional hormone response element. We have found three PRE-like sequences upstream of the transcription start site in the rat FSH-beta gene. These sequences are herein referred to as PRE-like sequence #1, #2 and #3 with #1 being most distal from the start site. The current studies determined whether these PRE-like sequences bound P receptor (PR) and were functional in regulating the induction of expression by P. Electrophoretic mobility shift assays (EMSA) demonstrated that a single 289 base pair (bp) DNA fragment encompassing all three PRE-like sequences specifically bound PR. Further, PR bound with high affinity to double-stranded oligonucleotides representing individual PRE-like sequences #1, #2 and, with lower affinity to a double-stranded oligonucleotide representing PRE-like sequence, #3. We have cloned a 361 bp sequence from the promoter region of the rat FSH-beta gene encompassing all three PRE-like sequences into a luciferase reporter vector (pGL3-promoter) yielding pFSHbeta361-luc+ which when transiently transfected into primary rat pituitary cell cultures, conferred P-responsiveness to a heterologous promoter. P-responsiveness was dependent upon the presence of PR and was blocked by the PR antagonist RU-486. These data strongly suggest the presence of functional PRE's in the rat FSH-beta gene promoter.

The human progesterone receptor (hPR) exists as two isoforms, hPR-A and hPR-B, which differ only in that hPR-A lacks 164 amino acids present at the amino-terminus of hPR-B. In this study we have separately expressed hPR-A and hPR-B and asked whether the progesterone-binding mechanisms are the same or different for the two forms of hPR and for their mixture. We investigated 1) the cooperativity of binding [3H]progesterone to the receptor, as measured by the Hill coefficient (nH); and 2) the dissociation rate of [3H]progesterone from the receptor. To compare the effects of dimerization, these ligand-binding properties were measured over a range of receptor concentrations. Binding of [3H]progesterone to hPR-A was positively cooperative at all concentrations used; the limiting value for the Hill coefficient was 1.47 +/- 0.11 at high receptor concentrations (5-19 nM) and 1.31 +/- 0.06 at low receptor concentrations (1-4 nM). Similarly, little change was observed in the dissociation rate constant over the same concentration range; the values at high and low concentrations were 4.59 +/- 0.15 and 3.03 +/- 0.25 x 10(-3) min-1, respectively. By contrast, the hPR-B concentration had a marked effect on positive cooperative binding and the dissociation rate of progesterone. At high hPR-B concentrations (3-5 nM), the limiting Hill coefficient was 1.49 +/- 0.11, which is indicative of moderately strong positive cooperativity, whereas at lower hPR-B concentrations (1-3 nM), the Hill coefficient was reduced to 1.1, which is essentially noncooperative. The [3H]progesterone dissociation rate was 4.52 +/- 0.44 x 10(-3) min-1 at the higher concentrations of hPR-B and was increased to 1.6 +/- 0.11 x 10(-3) min-1 at the lower concentrations. Thus, over the same concentration range where hPR-A exhibited no significant change in positive cooperativity or the dissociation rate, these progesterone-binding properties were highly dependent on the concentration of hPR-B. When hPR-A and hPR-B were mixed, positive cooperative binding and the dissociation rate were more similar to hPR-B than to hPR-A, in that both binding parameters were dependent on the concentration of receptor. However, the hPR-AB mixture differed from hPR-B alone in that the mixture required a greater receptor concentration (7-10 vs. 3-5 nM) to exhibit positive cooperativity and the increased dissociation rate. These results show, first, that each hPR isoform displays different [3H]progesterone-binding properties, which are most prominent at low concentrations of receptor, and second, that one isoform can influence the other. As the two receptor forms differ only at the N-terminus, yet positive cooperativity and changes in the dissociation rate constant are indicative of conformational changes affecting hormone binding, these results also strongly suggest that the N-terminus may directly or indirectly interact with the C-terminal ligand-binding domain.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

Progesterone (P4) can alter the synthesis and secretion of FSH from pituitary gonadotropes of sheep. In this study, the 5'-flanking region (4.7 kilobases) of the ovine FSH beta gene was tested for binding by human progesterone receptors (hPR), using an immunoprecipitation technique. Three fragments were bound by hPR. Competition experiments using homologous and heterologous DNA fragments revealed this binding to be specific and of high affinity (Kd = 1.2-47 nM). The fragment sequences were screened for potential P4 response elements (PREs). Six PRE-like elements were found among the three immunoprecipitated fragments. Band shift experiments discerned that each of these PRE-like sequences could be bound by hPR. In functional studies, each of the PRE-like elements could enhance the expression of a reporter gene driven by a heterologous promoter in a hormone-dependent manner. The 5'-flanking region of the ovine FSH beta gene was tested for P4 responsiveness using a luciferase reporter. In the presence of P4, there was a 2- to 3-fold increase in luciferase activity when the entire 4.7 kilobases of the 5'-flanking sequence were present, whereas no increase was seen in a construct that contained only 84 basepairs 5' to the transcription start site. This effect on transcription was dose dependent for P4. Deletion studies revealed that the three PRE-like elements closest to the transcription start site (-250 to -137) were sufficient to create the hormone-dependent enhancement. These results indicate that the 5'-flanking sequence of the ovine FSH beta gene contains sequences capable of being bound by hPR and may be responsible for the effects of P4 on FSH beta synthesis and secretion. This study is the first to show binding and function of PR for a gonadotropin gene.

Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.