Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression

The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL₂-L₁ and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL ₂ -L ₁ gene. UPA decreased cell proliferation and repressed BCL_2-L₁ expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.     

Progesterone receptors in the human uterus and their possible role in parturition

An overview is given on the role of progesterone in parturition in the human. Progesterone withdrawal is considered to be a major event for the beginning of parturition. However, in the human, no evidence exists in favour of a decline in placental progesterone production prior to labour. Progesterone actions are mediated by two functionally different but structurally highly related intranuclear proteins, progesterone receptor (PR) A and PRB. In the human, functional progesterone withdrawal is thought to play a role. This may be mediated by a change in the expression of the two isoforms of the PR, with an increase in the PRA:PRB ratio, and this is accompanied by an increase in the expression of the estrogen receptor. These mechanisms are considered to be critical for the endocrine control of parturition.     

p38 and p42/44 MAPKs differentially regulate progesterone receptor A and B isoform stabilization

Progesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis.     

Progesterone Acts via the Nuclear Glucocorticoid Receptor to Suppress IL-1β-Induced COX-2 Expression in Human Term Myometrial Cells

Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via the inhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsible for this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1β) inhibits progesterone driven PRE activation via p65 activation and that IL-1β reduced progesterone driven gene expression (FKBP5). Conversely, we found that the activity of a p65-driven NFκB reporter construct was reduced by overexpression of progesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA and progesterone suppressed IL-1β-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-tagged PRB, but not PRA, bound to p65 and that in IL-1β-treated cells, with no overexpression of either PR or p65, activated p65 bound to PR. However, we found that the ability of MPA to repress IL-1β-driven COX-2 expression was not enhanced by overexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects of progesterone and MPA, the specific PR antagonist, {"type":"entrez-protein","attrs":{"text":"Org31710","term_id":"1179178813","term_text":"ORG31710"}}Org31710, did not, suggesting that progesterone and MPA act via GR and not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repress IL-1β-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1β-driven COX-2 activation and that although the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression it does not seem to be responsible for progesterone repression of IL-1β-induced COX-2 expression.     

Effect of overexpression of progesterone receptor A on endogenous progestin-sensitive endpoints in breast cancer cells.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, which differ in the N-terminal region and exhibit different activities in vitro, with PRA demonstrating dominant negative inhibitory effects on the activity of PRB and other nuclear receptors. PRA and PRB are expressed in target tissues at comparable levels although cells expressing a predominance of one isoform can be identified. In breast cancers, PRA is expressed at high levels in some tumors, and this may be associated with features of poorer prognosis. To investigate the role of PRA overexpression in PR-positive target cells, the effect of PRA induction on cell proliferation and expression of endogenous progestin-sensitive genes, SOX4 and fatty acid synthetase (FAS), was examined using PR-positive T-47D cell lines, which express a predominance of PRB, in which PRA could be increased 2- to 20-fold over basal levels. No effect of PRA induction was noted on cell proliferation, but marked changes in morphology, consistent with loss of adherent properties, were observed. Increases up to 4-fold in the relative PRA levels augmented progestin induction of SOX4 mRNA expression, and RU486 treatment revealed a progestin agonist effect. There was no consistent effect of PRA induction on progestin-mediated increases in FAS mRNA levels under these conditions. Clones with PRA:PRB ratios greater than 15 were associated with diminished progestin responses on both SOX4 and FAS mRNA expression. These data show that PRA overexpression is associated with alteration in adhesive properties in breast cancer cells and effects on endogenous progestin targets that were dependent on the cellular ratio of PRA:PRB. The results of this study are consistent with the view that PRA expression can fluctuate within a broad range in target cells without influencing the nature of progestin action on downstream targets, but that overexpression of PRA, such as is seen in a proportion of breast cancers, may be associated with inhibition of progestin action and features of poor prognosis.     

A Computer Simulation of Progesterone and Cox2 Inhibitor Treatment for Preterm Labor

Background

Sufficient information from in vitro and in vivo studies has become available to permit computer modeling of the processes that occur in the myometrium during labor. This development allows the in silico investigation of pathological mechanisms and the trialing of potential treatments.

Methods/Results

Based on the human literature, we developed a computer model of the immune-endocrine environment of the myometrial cell. The interactions between molecules are represented by differential equations. The model is designed to simulate the estrogen and progesterone receptor changes during pregnancy and particularly the changes in the progesterone receptor (PR) isoforms A and B that are thought to mediate functional progesterone withdrawal in the human at labor. Parturition is represented by an increase in the PRA to PRB ratio to levels seen in women in labor. Infection is shown by inducing inflammation in the system by increasing phospho-IkB kinase concentration (IKK) levels; which lead to increased NF-κB activation, causing an increase in the PRA/PRB ratio. We examined the effects of progesterone or cyclo-oxygenase 2 (Cox2) inhibitor treatments on the PRA/PRB ratio in silico. The model predicted that high doses of progesterone and Cox2 inhibition would be effective in preventing an NF-κB-induced PRA/PRB ratio increase to the levels found during labor.

Conclusions

Our data illustrate the use of dynamic biological computer simulations to test the effectiveness of therapeutic interventions. This may allow the early rejection of ineffective therapies prior to expensive field trials.     

Effect of long-term treatment with steroid hormones or tamoxifen on the progesterone receptor and androgen receptor in the endometrium of ovariectomized cynomolgus macaques

The progesterone receptor (PR) and androgen receptor (AR) belong to the nuclear receptor superfamily. Two isoforms of PR (A and B) have been identified with different functions. The expression of AR, each isoform of PR and their involvement in long-term effects on the endometrium after hormonal replacement therapy (HRT) or tamoxifen (TAM) treatment is not known. The aims of this study were to determine PR(A+B), PRB and AR distribution by immunohistochemistry in the macaque (Macaca fascicularis) endometrium. Ovariectomized (OVX) animals were orally treated continuously for 35 months with either conjugated equine estrogens (CEE); medroxyprogesterone acetate (MPA); the combination of CEE/MPA; or TAM. Treatment with CEE/MPA tended to down-regulate PR in the superficial glands, but increased it in the stroma. TAM treatment increased both the PR and PRB levels in the stroma. Overall, less than 20% of the cells were positive for the PRB isoform and less variation was observed after steroid treatment. AR was found in the stroma, mainly distributed in the basal layer of the endometrium in the OVX and steroid treated groups, but was absent in the TAM treated group. No AR was found in the glandular epithelium. The present data show that long-term hormone treatment affects the PR level, and also the ratio between PRA and PRB in the endometrium.     

Heregulin induces transcriptional activation of the progesterone receptor by a mechanism that requires functional ErbB-2 and mitogen-activated protein kinase activation in breast cancer cells

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestin-dependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Paracrine regulation of epithelial progesterone receptor and lactoferrin by progesterone in the mouse uterus

The objective of this study was to determine whether uterine stromal and/or epithelial progesterone receptor (PR) is required for the antagonism by progesterone (P(4)) of estradiol-17beta (E(2)) action on expression of PR and lactoferrin in uterine epithelium. Uterine tissue recombinants were prepared with epithelium (E) and stroma (S) from wild-type (wt) and PR knockout (PRKO) mice: wt-S+wt-E and PRKO-S+wt-E. P(4) action on epithelial PR expression was studied in wt-S+wt-E and PRKO-S+wt-E tissue recombinants. E(2) down-regulated epithelial PR in both types of tissue recombinants, but P(4) blocked E(2)-induced down-regulation of epithelial PR only in wt-S+wt-E tissue recombinants. Thus, P(4) requires stromal PR to inhibit E(2)-induced down-regulation of epithelial PR. Epithelial PR is not sufficient in itself. The inhibitory effect of P(4) on lactoferrin expression was studied in 4 types of tissue recombinants (wt-S+wt-E, PRKO-S+wt-E, wt-S+PRKO-E, and PRKO-S+PRKO-E). E(2) induced lactoferrin in all 4 types of tissue recombinants. P(4) blocked E(2)-induced lactoferrin expression only in wt-S+wt-E tissue recombinants. In wt-S+PRKO-E tissue recombinants, P(4) inhibited lactoferrin expression only partially. P(4) failed to block E(2)-induced lactoferrin expression in PRKO-S+wt-E and PRKO-S+PRKO-E tissue recombinants. Thus, both epithelial and stromal PR are essential for full P(4) inhibition of E(2)-induced lactoferrin expression.