Induction Of Progesterone Receptor Research Articles

Progesterone receptor mediates ovulatory transcription through RUNX transcription factor interactions and chromatin remodelling.

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.

Effect of Extremely Low–frequency Electromagnetic Field on Apoptosis Induction and Expression of Estrogen Receptor, Progesterone Receptor, and ERBB2 in BT-474 Cells

Effect of Extremely Low–frequency Electromagnetic Field on Apoptosis Induction and Expression of Estrogen Receptor, Progesterone Receptor, and ERBB2 in BT-474 Cells

Progesterone receptor-Src kinase signaling pathway mediates neuroprogesterone induction of the luteinizing hormone surge in female rats.

Neural circuits in female rats are exposed to sequential estradiol and progesterone to regulate the release of luteinizing hormone (LH) and ultimately ovulation. Estradiol induces progesterone receptors (PGRs) in anteroventral periventricular nucleus (AVPV) kisspeptin neurons, and as estradiol reaches peak concentrations, neuroprogesterone (neuroP) synthesis is induced in hypothalamic astrocytes. This local neuroP signals to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signaling through Src family kinase (Src) underlies the LH surge. As observed in vitro, PGR and Src are co-expressed in AVPV neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Furthermore, estradiol treatment increased the number of AVPV cells that had extranuclear PGR and Src in close proximity (<40nm). Infusion of the Src inhibitor (PP2) into the AVPV region of ovariectomized/adrenalectomized (ovx/adx) rats attenuated the LH surge in trunk blood collected 53h post-estradiol (50µg) injection that induced neuroP synthesis. Although PP2 reduced the LH surge in estradiol benzoate treated ovx/adx rats, activation of either AVPV PGR or Src in 2µg estradiol-primed animals significantly elevated LH concentrations compared to dimethyl sulfoxide infused rats. Finally, antagonism of either AVPV PGR or Src blocked the ability of PGR or Src activation to induce an LH surge in estradiol-primed ovx/adx rats. These results indicate that neuroP, which triggers the LH surge, signals through an extranuclear PGR-Src signaling pathway.

Differential Expression of Estrogen Receptors in the Hypothalamus Underlies the Bimodal Effects of Estradiol on Luteinizing Hormone Release

Low 17β-estradiol (E2) levels suppress luteinizing hormone (LH) release, while high E2 stimulates an LH surge in the positive-feedback required for ovulation. Kisspeptin (Kp) neurons in the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei of hypothalamus have been implicated in E2 positive- and negative-feedback effects, respectively. However, how E2 differentially regulates these two neuronal populations remains unknown. We investigated whether neurons in the AVPV and ARC are differently responsive to changes in E2 levels and the regulatory role of estrogen receptors (ERs). Ovariectomized (OV) rats received oil or E2 at doses of 4 (OVE-4), 40 (OVE-40), or 80 (OVE-80) µg/kg. Rats on diestrus (DI) and proestrus (PRO) were used as physiological controls. OV rats were also treated with 0.02, 0.2 or 2.0 mg/kg of propyl pyrazole triol (PPT), or 6 mg/kg of diarylpropionitrile (DPN). Serial blood samples were collected for hormonal measurements. Brains were processed for immunohistochemistry and qPCR analyses in the AVPV and ARC. The E2 doses gradually increased plasma E2, with PRO levels being attained in OVE-80 rats. OVE-80 rats displayed a PRO-like LH surge, while LH levels were constantly suppressed in OVE-4 rats. Progesterone receptor (PR) was used as an index of E2 responsiveness. PR expression was increased in the AVPV of PRO and OVE-80 rats, associated with c-Fos expression and occurrence of LH surge. In the ARC, both low and high E2 induced PR expression and reduced the number of Kp-immunoreactive (ir) neurons, consistent with the negative feedback effects on LH. E2 at 4 or 80 µg/kg equally induced PR expression in 90% of ARC Kp-ir neurons. Despite the higher sensitivity to E2 in the ARC, the percentage of neurons expressing ERα was lower in the ARC compared with AVPV. However, Esr1 expression was 2-fold higher in the ARC than in AVPV for low E2 levels, whereas both Esr1 and Esr2 were more expressed in the AVPV under high E2 status. Notably, Esr1/Esr2 ratio was twice as high in the ARC as in the AVPV regardless of E2 levels, suggesting a stronger ERβ inhibition over ERα in the AVPV. Accordingly, ERα selective activation with PPT increased PR in the ARC at the doses of 0.2 and 2.0 mg/kg, reducing plasma LH, while only the highest dose was able to stimulate PR expression in the AVPV. ERβ activation with DPN, in turn, had no effect in OV rats but amplified the induction of AVPV PR and the size of the LH surge in OVE-80 rats. Thus, we provide evidence that ARC and AVPV neurons are responsive to low and high E2 levels, respectively. ARC is 10 times more sensitive to ERα activation than AVPV, whereas ERβ positively modulates AVPV responsiveness to high E2. These differential responses to E2 seem to be related to differences in the relative Esr1 and Esr2 expression in the ARC and AVPV. Our findings suggest that hypothalamic differences in the relative expression of ERs play a key role in the bimodal regulation of LH release by E2.

Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma

Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance and loss of efficacy. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease. These results suggest that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC that are dependent on disease stage. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response.

Abstract 627: Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma

Abstract Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease suggesting that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response. Citation Format: Christopher Steven Koivisto, Parrish Melodie, Bonala Santosh, Soo Ngoi, Georges J. Nahhas, Bei Liu, Adrian Torres, James Gallagher, Rebekah Snchez-Hodge, Victor Zeinner, David Cohn, Floor Backes, Paul Goodfellow, Helen Chamberlin, Gustavo Leone. Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 627.

Abstract 4379: Not all selective estrogen receptor degraders are equal - Preclinical comparison of AZD9833, AZD9496 and fulvestrant

Abstract Anti-hormonal therapy has been the mainstay treatment for estrogen receptor (ER) +ve breast cancer for &amp;gt;40 years. The selective ER degrader (SERD) fulvestrant has demonstrated clinical benefit over aromatase inhibitors and the selective ER modulator tamoxifen. However, fulvestrant's efficacy may be limited by the intramuscular route of administration. To overcome this, AstraZeneca has developed orally bioavailable SERDs, the first generation compound AZD9496 and the next generation oral SERD AZD9833. Here we report the preclinical comparison of fulvestrant, AZD9496 and AZD9833. All three compounds induced equal ERα degradation in the ER+ breast cancer cell line MCF7. Importantly, analysis of other ER+ cell lines revealed AZD9833 phenocopied ERα degradation induced by fulvestrant, while AZD9496 only reduced ERα levels to 54% of that achieved by fulvestrant. Activity in the endometrial Ishikawa cell line demonstrated induction of progesterone receptor, an ER target gene, by AZD9496 but not AZD9833. RNAseq analysis of ER+ breast cancer cell lines treated with the three agents with and without estradiol did not reveal differences between the compounds; all estradiol-induced gene expression changes were completely reversed by the SERDs and no ER agonism was detected. By contrast, AZD9833 and fulvestrant produced an equivalent maximal anti-proliferative effect in MCF-7 and CAMA-1 cells, while AZD9496 induced a significantly inferior anti-proliferative effect. The pure anti-estrogen nature of AZD9833 and the partial agonist potential of AZD9496 was confirmed in vivo in a juvenile rat uterine assay. Like fulvestrant, AZD9833 caused a significant reduction in uterine weight and endometrial thickness, while AZD9496 produced a significant increase in both parameters. In MCF7 xenografts, both oral SERDs caused equivalent anti-tumour and pharmacodynamic effects to a supraclinical exposure of fulvestrant. However, in the patient derived xenograft CTC174 harbouring an ESR1 D538G mutation, AZD9833 caused equivalent anti-tumour and ERα degradation effects to supraclinical fulvestrant. By contrast, AZD9496 produced significantly poorer maximal anti-tumour and pharmacodynamic effects in this model compared to fulvestrant. These data demonstrate that AZD9833 phenocopied fulvestrant preclinically, as a SERD and pure anti-estrogen. It is, therefore, a suitable candidate to test the hypothesis that increasing the exposure of a SERD above what is achieved with fulvestrant can provide additional patient benefit. AZD9496, on the other hand, demonstrated inferior ERα degradation in several ER+ breast cancer cell lines and partial agonism. This may explain the result of the clinical window of opportunity study, comparing AZD9496 to fulvestrant, in which AZD9496 failed to demonstrate superior degradation of ERα, downregulation of PR expression and reduction in Ki67 positivity. Citation Format: Mandy Lawson, Natalie Cureton, Michelle DuPont, Oona Delpuech, Dawn Trueman, Pei Zhang, Azadeh Cheraghchi-Bashi-Astaneh, Sladjana Gagrica, Gareth Maglennon, Daniel Sutton, Bairu Zhang, Jonathan Cairns, Jamie Scott, Teresa Klinowska, Christopher J. Morrow. Not all selective estrogen receptor degraders are equal - Preclinical comparison of AZD9833, AZD9496 and fulvestrant [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4379.

Brain-Selective Estrogen Therapy Prevents Androgen Deprivation-Associated Hot Flushes in a Rat Model.

Hot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17β-estradiol, E2) are produced from testosterone. Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.

Effects of progesterone receptor on proliferation of uterine leiomyoma cells.

In order to study the mechanism of the effect of progesterone receptor on the growth of primary uterine leiomyoma cells, the primary cells were extracted from uterine leiomyoma cells and identified by immunohistochemistry (IHC). Mitochondrial progesterone receptor-positive [PR-M(+)], mitochondrial progesterone receptor-negative [PR-M(-)], progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were screened by Western blotting. Different concentrations of Mifepristone (MIF), a progesterone receptor antagonist, were used to interfere with PR-M(+) and PR-M(-) cell lines, respectively. Proliferation and apoptosis of PR-M(+) and PR-M(-) cell lines were detected by tetramethylazolyl blue method and flow cytometry, respectively. The expression of Caspase-3 and B-cell lymphoma 2 (Bcl-2) protein was detected by Western blotting. The results showed that the growth of PR-M(+) and PR-M(-) uterine leiomyoma cells was inhibited with the increase of MIF concentration. Furthermore, the proliferation inhibition rate and apoptosis rate were gradually increased. However, the expression of Caspase-3 protein on progesterone receptor M increased, while the expression of Bcl-2 decreased. Moreover, progesterone could induce progesterone receptor M to up-regulate apoptotic protein Caspase-3 and down-regulate anti-apoptotic protein Bcl-2, thus it could inhibit the apoptosis of primary cultured uterine leiomyoma cells and promote the proliferation of leiomyoma cells.

Effects of progesterone receptor on proliferation of uterine leiomyoma cells.

In order to study the mechanism of the effect of progesterone receptor on the growth of primary uterine leiomyoma cells, the primary cells were extracted from uterine leiomyoma cells and identified by immunohistochemistry (IHC). Mitochondrial progesterone receptor-positive [PR-M(+)], mitochondrial progesterone receptor-negative [PR-M(-)], progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were screened by Western blotting. Different concentrations of Mifepristone (MIF), a progesterone receptor antagonist, were used to interfere with PR-M(+) and PR-M(-) cell lines, respectively. Proliferation and apoptosis of PR-M(+) and PR-M(-) cell lines were detected by tetramethylazolyl blue method and flow cytometry, respectively. The expression of Caspase-3 and B-cell lymphoma 2 (Bcl-2) protein was detected by Western blotting. The results showed that the growth of PR-M(+) and PR-M(-) uterine leiomyoma cells was inhibited with the increase of MIF concentration. Furthermore, the proliferation inhibition rate and apoptosis rate were gradually increased. However, the expression of Caspase-3 protein on progesterone receptor M increased, while the expression of Bcl-2 decreased. Moreover, progesterone could induce progesterone receptor M to up-regulate apoptotic protein Caspase-3 and down-regulate anti-apoptotic protein Bcl-2, thus it could inhibit the apoptosis of primary cultured uterine leiomyoma cells and promote the proliferation of leiomyoma cells.

Tissue interactions and estrogenic response during human female fetal reproductive tract development

The role of tissue interactions was explored to determine whether epithelial differentiation within the developing human reproductive tract is induced and specified by mesenchyme in tissue recombinants composed of mouse vaginal mesenchyme + human uterine tubal epithelium (mVgM+hTubE). The tissue recombinants were grown in DES-treated ovariectomized athymic mice. After 2–4 weeks of in vivo growth, several vaginal specific features were expressed in the human tubal epithelium. The mesenchyme-induced effects included morphological change as well as expression of several immunohistochemical markers. Although the mesenchyme-induced shift in vaginal differentiation in the human tubal epithelium was not complete, the partial induction of vaginal markers in human tubal epithelium verifies the importance of mesenchymal-epithelial interactions in development of the human female reproductive tract.In a separate experiment, DES-induction of uterine epithelial progesterone receptor (PGR) and estrogen receptor 1 (ESR1) was explored in tissue recombinants composed of wild-type or Esr1KO mouse uterine mesenchyme + human fetal uterine epithelium (wt UtM+hUtE and Esr1KO UtM+hUtE). The rationale of this experiment was to determine whether DES-induction of PGR and ESR1 is mediated directly via epithelial ESR1 or indirectly (paracrine mechanism) via mesenchymal ESR1. DES-induction of uterine epithelial ESR1 and PGR in Esr1KO UtM+hUtE tissue recombinants (devoid of mesenchymal ESR1) formally eliminates the paracrine mechanism and demonstrates that DES induction of human uterine epithelial ESR1 and PGR is directly mediated via epithelial ESR1.

Cadmium induces progesterone receptor gene expression via activation of estrogen receptor in human ovarian cancer cells.

Cadmium (Cd) as a metalloesterogen may have a role in development of ovarian cancer. One of the critical target genes of estrogens is progesterone receptors (PRs). There are controversial studies on association between Cd, PRs, and cell proliferation. This study investigates the effect of Cd on proliferation of ovarian cancer cell lines, PRA and PRB expression and their relationship. OVCAR3 and SKOV3 cells were treated with CdCl2 (1-100 nM) and cell proliferation was assayed using bromodeoxyuridine (BrdU) method. The mechanism underlying the proliferative effect of Cd mediated by PRs was examined using cell transfection with PR- small interfering RNA (siRNA) and western blot analysis. Our results showed the involvement of PRs in Cd induced proliferation of ovarian cancer cells. Progesterone receptors are involved in proliferative effect of Cd. Moreover, Cd modified the expression of PRA and PRB and induced ovarian cancer cell proliferation through the change of PRA/PRB ratio. In conclusion, there is a mechanistic association between Cd effects on ovarian cancer cell proliferation, estrogen receptors and PRs expression.

Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

Context:In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)–like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans.Objective:To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries.Design and Participants:Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients.Main Outcome Measures:The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured.Results:PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes.Conclusions:This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.

Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM–10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).

Genome-wide transcriptional regulation of estrogen receptor targets in fallopian tube cells and the role of selective estrogen receptor modulators.

BackgroundThe fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer (OVCA) risk. Despite estrogen’s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20 % response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors arising from this cell type, such as HGSC.ResultsUsing normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation. Further, using RNAseq, the oviduct specific transcriptional genes targets of ER when stimulated by estradiol and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated including Kuramochi, OVSAHO, OVKATE, OVCAR3, and OVCAR4. OVSAHO demonstrated receptor expression and response, which highlights the need for additional models of ovarian cancer that are estrogen responsive.ConclusionsOverall, the fallopian tube has specific gene targets of estrogen receptor and demonstrates a tissue specific response to SERMs consistent with antagonistic action.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-016-0213-3) contains supplementary material, which is available to authorized users.

Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells.

Induction and subcellular redistribution of progesterone receptor A and B by tamoxifen in the hypothalamic ventromedial neurons of young adult female Wistar rats

The ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is a brain center for estrogen-dependent triggering of female sexual behavior upon progesterone receptor (PR) activation. We examined the agonistic and antagonistic actions of tamoxifen in this nucleus by analyzing its effects on the total number of PR-immunoreactive neurons, PR mRNA and protein levels, and subcellular location of PRs in ovariectomized Wistar rats. The results show that tamoxifen has no agonistic action in the number of PR-immunoreactive neurons, but increases PR expression and labeling in the nucleus and cytoplasm of VMNvl neurons that constitutively express PRs. As an antagonist, tamoxifen partially inhibited the estradiol-dependent increase in the number of PR-immunoreactive neurons and in PR mRNA and protein levels, without interfering with the subcellular location of the protein. We suggest that tamoxifen influence on PR expression in the VMNvl critically depends on the presence or absence of estradiol.

Uterine Leiomyoma Stem Cells: Linking Progesterone to Growth.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/β-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

Abstract P3-04-15: Ethinylestradiol treatment downregulates ER and upregulates PgR; Immunohistochemical analysis in postmenopausal breast cancer tissues after prior long-term estrogen-deprivation therapy

Abstract Background: Estrogen receptor (ER) positive breast cancer can often be treated by hormone therapy; however a certain population of ER-positive patients becomes resistant to hormone therapy after long-term hormone treatment. Ethinylestradiol (EE2) is a derivative of estrogen which has shown promising effects on in these patients. Methods: We successfully obtained tissue samples from 6 patients undergoing EE2 treatment and examined 13 well-known breast cancer-related factors by analyzing their gene expression and by immunohistochemistry. Of the 6 patients, 5 responded but one patient did not. Results: Before EE2 treatment, staining for both ER and androgen receptor (AR) was strong in the nucleus, with weak staining of the progesterone receptor (PgR). EE2 treatment significantly down-regulated ER and up-regulated PgR while nuclear and cytosolic AR were oppositely down- and up-regulated, respectively, by EE2. Cytosolic staining of BRCA1 was significantly up-regulated by EE2 whereas nuclear staining tended to decrease. Individual comparisons suggested less induction of PgR and decreasing AKT but increasing pAKT in the non-responder following EE2 treatment. Conclusion: Our observations revealed that EE2 activated ER downstream genes, although it did not stimulate cell growth. This suggests that hormone resistant cells might receive growth signals from a non-genomic pathway and this may be reflected in their sensitivity to EE2 treatment. Citation Format: Hirotaka Iwase, Yoko Omoto, Takashi Takeshita, Mutsuko Yamamoto-Ibusuki, Mitsuhiro Hayashi, Aiko Sueta, Saori Fujiwara, Yutaka Yamamoto. Ethinylestradiol treatment downregulates ER and upregulates PgR; Immunohistochemical analysis in postmenopausal breast cancer tissues after prior long-term estrogen-deprivation therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-15.

Immunohistochemical analysis in ethinylestradiol-treated breast cancers after prior long-term estrogen-deprivation therapy.

BackgroundEstrogen receptor (ER) positive breast cancer can often be treated by hormone therapy; however a certain population of ER-positive patients become resistant to hormone therapy after long-term hormone treatment. Ethinylestradiol (EE2) is a derivative of estrogen, which has shown promising effects in these patients.MethodsWe successfully obtained tissue samples from 6 patients undergoing EE2 treatment and examined 13 well-known breast cancer-related factors by immunohistochemistry. Of the 6 patients, 5 responded but one patient did not.ResultsBefore EE2 treatment, staining for both ER and androgen receptor (AR) was strong in the nucleus, and the progesterone receptor (PgR) was almost no staining. EE2 treatment significantly down-regulated ER and up-regulated PgR while nuclear and cytosolic AR were oppositely down- and up-regulated, respectively. Cytosolic staining of BRCA1 was significantly up-regulated by EE2 whereas nuclear staining tended to decrease. Individual comparisons suggested less induction of PgR and decreasing AKT but increasing pAKT in the non-responder following EE2 treatment.ConclusionsOur observations revealed that EE2 activated ER downstream genes; however it did not stimulate cell growth. This suggests that hormone resistant cells might receive growth signals from a non-genomic pathway and this may be reflected in their sensitivity to EE2 treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-0851-8) contains supplementary material, which is available to authorized users.