Progesterone Receptor Activity Research Articles

A deletion mutation within the goat AKAP13 gene is significantly associated with litter size

A-kinase anchoring protein 13 (AKAP13) is one of the AKAP protein family members, which is correlated with estrogen receptors (ERs) and progesterone receptor (PR) activity. Consequently, the AKAP13 gene is considered to be one of the candidate genes for regulating female fertility. Hence, the objectives of this study were to discover the potential insertion/deletion (indel) variants within the AKAP13 gene and evaluate their associations with litter size of Shaanbei white cashmere goats (SBWC) to screen candidate genes for the molecular marker-assisted selection (MAS). Ultimately, we found the 16-bp deletion of AKAP13 gene which displayed three genotypes (II, ID and DD). However, it was not confirmed to Hardy-Weinberg equilibrium (HWE) in the tested population. Statistical analysis demonstrated that this 16-bp indel locus was significantly associated with litter size in goats (p < 0.05), in which the ID genotype was a key genotype for increasing litter size in goats. Besides, independent χ 2 tests between different genotypes and litter size showed that high-prolific groups had higher frequency of the ‘D’ allele (p < 0.05). Briefly, AKAP13 gene is a candidate gene for improving fertility, and its 16-bp indel locus can be used as a valid DNA molecular marker for the MAS in goat breeding.

Secoiridoids from Dogwood (Cornus officinalis) Potentiate Progesterone Signaling.

The use of botanical dietary supplements for the alleviation of conditions such as hot flashes, premenstrual syndrome, and fertility is prolific worldwide. Estrogen and progesterone receptors (ER and PR) and their corresponding steroid hormones are critical for the relief of hot flashes and the treatment of patients who develop endometriosis, and these pathways can influence the development of endometrial, ovarian, and breast cancers. However, few studies have investigated or identified the natural product components in herbal supplements that act on the PR. In the current study, a new secoiridoid, demethoxy-cornuside (1), along with six known secoiridoids (2-7) were isolated from the twigs of dogwood (Cornus officinalis) by bioassay-guided isolation with a progesterone response element (PRE)/luciferase (Luc) reporter assay in Ishikawa cells. Four phytoprogestins (1, 2, 6, 7) potentiated the effect of progesterone in the PRE/Luc assay. This study demonstrates that C. officinalis components might potentiate progesterone signaling in the presence of progesterone, which could modify progesterone receptor action in hormone-responsive tissues such as the uterus and mammary gland.

Progesterone Receptors in AVPV Kisspeptin Neurons Are Sufficient for Positive Feedback Induction of the LH Surge.

Kisspeptin, encoded by Kiss1, stimulates gonadotropin-releasing hormone neurons to govern reproduction. In female rodents, estrogen-sensitive kisspeptin neurons in the rostral anteroventral periventricular (AVPV) hypothalamus are thought to mediate estradiol (E2)-induced positive feedback induction of the preovulatory luteinizing hormone (LH) surge. AVPV kisspeptin neurons coexpress estrogen and progesterone receptors (PGRs) and are activated during the LH surge. While E2 effects on kisspeptin neurons have been well studied, progesterone's regulation of kisspeptin neurons is less understood. Using transgenic mice lacking PGR exclusively in kisspeptin cells (termed KissPRKOs), we previously demonstrated that progesterone action specifically in kisspeptin cells is essential for ovulation and normal fertility. Unlike control females, KissPRKO females did not generate proper LH surges, indicating that PGR signaling in kisspeptin cells is required for positive feedback. However, because PGR was knocked out from all kisspeptin neurons in the brain, that study was unable to determine the specific kisspeptin population mediating PGR action on the LH surge. Here, we used targeted Cre-mediated adeno-associated virus (AAV) technology to reintroduce PGR selectively into AVPV kisspeptin neurons of adult KissPRKO females, and tested whether this rescues occurrence of the LH surge. We found that targeted upregulation of PGR in kisspeptin neurons exclusively in the AVPV is sufficient to restore proper E2-induced LH surges in KissPRKO females, suggesting that this specific kisspeptin population is a key target of the necessary progesterone action for the surge. These findings further highlight the critical importance of progesterone signaling, along with E2 signaling, in the positive feedback induction of LH surges and ovulation.

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract 2513: Convergence of cancer associated fibroblasts (CAFs) and progesterone receptor (PR) signaling in ER+ luminal breast cancer

Abstract Background: Cancer associated fibroblasts (CAFs) in the tumor microenvironment and the circulation secrete a complex array of paracrine factors that contribute to the development of metastasis, stemness and chemotherapy resistance. CAF-derived factors are primary candidates for initiating signaling pathways in ER+/PR+ breast cancer cells even in low/no hormone conditions and particularly at genes that specify stemness and survival in the circulation. Herein, we investigate the convergence of CAFs and PR signaling in ER(+) luminal breast cancer. Methods: Tumorsphere assay: BC cells were grown in B27-supplemented tumorsphere media in ULA plates for 5-10 days. Soft agar colony formation assay: BC cells were grown in soft agar under serum starvation for 3 weeks. Clustering assay: Cell tracker-labeled CAF or BC cells were cultured together in ULA plates for 24 hrs. Results: Using the ER(+) T47D cell series that express no PR (T47D-Y), endogenous PR (T47D CO), or either exclusively PR-A or PR-B isoforms, we observed that CAF conditioned media (CM) increased primary and secondary tumorsphere formation relative to controls. In soft agar assays, CAF CM increased adhesion-independent proliferation compared to non-CM. Previously, our group reported that phosphorylation of PR Ser294 is required for PR-induced cancer stem cell phenotypes. Phospho-mutant S294A T47D PR-A or PR-B cell lines attenuated the response to CAF CM in tumorsphere assays. Conversely, in soft agar assays, S294A PR-A+ cells did not differ from WT PR-A+ cells, though S294A PR-B+ cells formed significantly fewer colonies than WT PR-B+ cells. Western blots showed that CAF CM induced rapid phosphorylation of PR S294 and activation of AKT and MAPK/ERK kinases; the MEK inhibitor U0126 blocked CAF-induced PR S294 phosphorylation whereas the AKT inhibitor, LY294006, was without effect. Two candidate cytokines, SDF1 and FGF2, were tested for their ability to induce PR activity, tumorsphere formation, and anchorage independent proliferation. Treatment of cells with FGF2, but not SDF1, mimicked the patterns of phosphorylation and the phenotypes observed for CAF CM. Lastly, physical interaction between CAFs and PR expressing cells as measured by in vitro clustering assays demonstrated that T47D CO, PR-A and PR-B cells preferentially cluster with CAF cells relative to PR null (T47D-Y) cells. Conclusion: We have shown that CAF-derived paracrine factors, possibly FGF2, activate MAPK/ERK, which in turn phosphorylates PR on Ser294 to initiate tumorsphere formation and adhesion independent proliferation. Our study of the interactions between PR (+) breast cancer cells with neighboring CAFs either in the primary tumor or in the circulation (as CAF/CTC clusters) may shed light on alternative treatment strategies, particularly when endocrine therapies have failed. Citation Format: Angela Spartz, Caroline Diep, Benjamin Troness, Carol A. Lange, Dorraya El-Ashry. Convergence of cancer associated fibroblasts (CAFs) and progesterone receptor (PR) signaling in ER+ luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2513.

Abstract 743: Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer

Abstract Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies display intrinsic or acquired therapy resistance. One-third of patients with acquired resistance present with metastatic tumors containing ERα-Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. Previous research suggests that ERα and the progesterone receptor (PR) engage in complex interactions involving reciprocal regulation of ERα and PR transcription factor activity known as ERα/PR crosstalk. My central hypothesis is that ERα-Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. While previous research has assessed the impact of the ERα-Y537S mutation on ERα-associated chromatin binding and gene expression, here I assess the effect of the resistance-associated ERα-Y537S mutation on ERα and PR activity and crosstalk. Simultaneous analysis of transcription factor binding activity (ChIP-seq), gene expression (RNA-seq), and proximity-based interaction (NanoBRET) indicates that active PR binding across the genome is increased in the context of ERα-Y537S, suggesting that ERα/PR crosstalk is significantly altered. Importantly, PR chromatin binding in the context of ERα-Y537S appears to be elevated regardless of the stimulation of PR activity with the agonist R5020. These findings, along with identification of candidate genes of potential therapeutic value, will be applied using patient-derived tumor organoids to determine the efficacy of simultaneously targeting ERα and PR to overcome endocrine therapy resistance in breast cancer. Citation Format: Rosemary J. Huggins. Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 743.

Limbic progesterone receptor activity enhances neuronal excitability and seizures.

Emerging evidence raises the possibility that progesterone receptor (PR) signaling may contribute to the reproductive hormone fluctuation-linked seizure precipitation, called catamenial epilepsy. Therefore, we studied PR isoform expression in limbic regions involved in temporal lobe epilepsy and the effect of PR activation on neuronal activity and seizures. We evaluated PR expression in the limbic regions, entorhinal cortex (EC), hippocampus, and amygdala in female rats using quantitative real-time polymerase chain reaction (qRT-PCR). A selective agonist, Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) activated PRs, and the effect on excitability and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission of EC neurons was studied using electrophysiology. Finally, we assessed PR regulation of epileptic seizures and status epilepticus (SE) induced by lithium-pilocarpine in female rats with the global deletion of PRs (PR knockout; PRKO) using video electroencephalography (-EEG). Limbic regions EC, hippocampus, and amygdala robustly expressed PR messenger RNA (mRNA). Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) treatment reduced the action potential threshold of layer II/III EC neurons and increased the frequency of AMPA receptor-mediated synaptic currents of ovariectomized and estrogen-primed female rats. Female rats lacking PRs (PRKO) experienced a shorter duration, less intense, and less fatal SE than wild-type (WT) animals. Furthermore, Nestorone treatment caused seizure exacerbation in the WT epileptic animals, but not in the PRKO epileptic animals. Activation of PRs expressed in the EC and hippocampus increased neuronal excitability and worsened seizures. These receptors may play a role in catamenial epilepsy.

226-LB: Elimination of the Progesterone Receptor in ß Cells Improves ß-Cell Function and Glucose Tolerance during Pregnancy and Diet-Induced Insulin Resistance in Female Mice

We showed that the gonadal hormones estradiol and testosterone influence islet β-cell function and survival in a sex-specific manner via estrogen and androgen receptors expressed in islet β-cells. The progesterone receptor (PR) is a ligand-activated transcription factor and is believed to be involved in the β-cell adaptation to pregnancy. Leveraging public single-cell RNA-seq datasets we observe PR mRNA expression in α- β- and γ-cells in male and female human pancreatic endocrine cells. Using immunohistochemistry, we observe that, unlike in the uterus where PR is nuclear, PR is localized in the cytoplasm of β-cells in male and female human and mouse islets. We investigated the role of progesterone and PR in β-cell physiology in male and female mice with conditional deletion of PR in β-cells (βPRKO) by crossing PR-Lox/lox and MIP-Cre transgenic mice. Male βPRKO mice exhibited no alteration in glucose homeostasis on normal chow (NC) or following western-diet (WD)-induced insulin resistance and obesity compared to littermate PR-Lox/lox controls. Similarly, NC-fed female βPRKO mice exhibited no abnormality of glucose or insulin homeostasis compared to littermate PR-Lox/lox controls. However, during pregnancy or WD feeding, female βPRKO mice developed enhanced IP-glucose-stimulated insulin secretion, associated with improved IP glucose tolerance compared to littermate PR-Lox/lox controls. These results reveal that PR is a cytosolic receptor in β-cells, and suggest that in conditions of high insulin demand, such as pregnancy and diet-induced insulin resistance, elimination of PR action in β-cells of females improves the ability of β-cells to produce insulin and lowers blood glucose. PR may be involved in the pathogenesis of gestational diabetes. Disclosure P. Mota de sa: None. M. Qadir: None. F. Mauvais-jarvis: None. Funding National Institutes of Health (DK074970, DK107444); U.S. Department of Veterans Affairs (BX003725)

Mechanism of Action of Irilone as a Potentiator of Progesterone Receptor Signaling

Progesterone signaling and its proper regulation is important for reproductive function. When progesterone signaling is dysregulated, gynecological diseases can occur, for example endometriosis, uterine fibroids, and endometrial cancer. While these diseases are treated with progestin therapy, progestins can bind to multiple steroid receptors, exerting side effects of weight gain, immunosuppression, cardiovascular disease, and stroke. Discovering an alternative progestin that is selective for the progesterone receptor (PR) is ideal. One potential source of such an alternative is botanical dietary supplements, which have become increasingly popular among consumers with sales reaching $9.6 billion in 2019. Although botanical supplements are popular, the chemical structures and biological action of botanical supplements would benefit from deeper scientific investigation. Studies of Trifolium pratense L. (red clover), primarily used for the treatment of menopausal symptoms, identified phytoestrogen compounds as the chemicals that mitigate those symptoms. Interestingly, irilone, identified from red clover, potentiated progesterone signaling via a progesterone response element luciferase (PRE/Luc) assay. Potentiation is when a compound has no activity by itself but when combined with another molecule, i.e. progesterone, that compound enhances PR activity. Prior to irilone, a natural compound with the ability to potentiate progesterone signaling had not been previously reported. The purpose of this study was to determine the mechanism of action of irilone. We hypothesized that irilone was potentiating PR by blocking PR degradation and by altering PR post-translational modifications. Irilone was found to potentiate 5 nM P4 using a PRE-luciferase assay in both T47D and Ishikawa PR expressing cells. Since PR is a downstream target gene of ER, we investigated if irilone also had ER activity. Irilone increased expression of an ERE-luciferase reporter gene. Next, we investigated if irilone could stabilize PR degradation and if irilone altered PR phosphorylation via western blot. Irilone was found to increase PR protein levels, but when ER was blocked, this was mitigated. In the presence of P4, irilone did not increase phosphorylation of serine 294 on PR. Future studies will determine if irilone is altering sumoylation of PR, and if irilone can potentiate PR signaling in vivo. Determining how irilone is potentiating progesterone will help us understand PR biology and could be an effective treatment for gynecological diseases by enhancing endogenous progesterone action.

Tibolone induces lordosis behavior, but not concurrent or sequential inhibition, in Sprague Dawley rats

Activation of progesterone receptor (PR) facilitates lordosis 40 hr after estradiol treatment, but induces concurrent inhibition (CI) when given with estradiol, or sequential inhibition (SI) when given subsequent to the faciliatory time interval. Tibolone (TBL) is a broad spectrum gonadal steroid agonist that facilitates lordosis when given after estradiol and in place of progesterone (P). The present experiment examined whether it can also induce CI or SI of lordosis behavior in rats as a means of determining its dominant receptor mechanism of action. Subcutaneous (SC) injections of estradiol benzoate (EB), TBL, or P were varied in time to examine whether P induced CI in females pre-treated with TBL or EB, or whether P or TBL induced CI when injected prior to EB (Experiment 1); whether P or TBL induced SI after EB treatment (Experiment 2); and whether P induced SI after TBL treatment (Experiment 3). In Experiment 1, P injected 1 h before EB induced CI after a second P administration 40 h later. However, the same treatment of P to females primed with TBL did not induce CI. In Experiment 2, injections of P or TBL 40 h after EB or TBL induced lordosis within 4 h (facilitation test); however, a second injection of P, 24 h later, induced significant lordosis in rat pretreated with TBL, but not in rats pretreated with P (inhibition test). In Experiment 3, P injected 40 hs after different doses of TBL induced intense lordosis behavior (facilitation test); however, a second dose of P injected 64 h later induced SI, but not in females primed with the highest dose of TBL (inhibition test). Unlike P, TBL did not induce CI or SI. This suggests that TBL likely induces its facilitation of lordosis by an action that is independent of PR.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

Mechanical stiffness augments ligand-dependent progesterone receptor B activation via MEK 1/2 and Rho/ROCK–dependent signaling pathways in uterine fibroid cells

To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. Laboratory studies. Translational science laboratory. Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). Progesterone-responsive reporter activation. Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.

Abstract PO020: Approval for the proliferative marker Ki-67 as an integrated biomarker for endometrial cancer in NRG study GY011

Abstract Introduction: NRG Study GY011 is a surgical window trial of medroxy-progesterone acetate (MPA) +/- entinostat (E), a histone deacetylase inhibitor hypothesized to upregulate PR levels through epigenetic modifications. The principal goal of this trial was to determine the impact of treatment on the level as well as the activity of progesterone receptors (PR) in women with endometrial cancer. However, a reliable marker for PR activity was needed, and the most promising choice was Ki-67. To be approved for inclusion in the study as an integrated biomarker, Ki-67 had to first undergo rigorous evaluation by the participating pathologists with oversight by the NIH Biomarker Review Committee. Methods: Fifty patients were enrolled on GY011, and specimen collection was planned by biopsy prior to receiving study drugs and by tissue sample at the time of the hysterectomy. There were 42 pre-drug administration specimens with tumor for analysis and 1 without and 43 hysterectomy specimens with tumor. Ki-67 immunohistochemistry performed on pre and post drug specimens was evaluated by three experienced reviewers (MS, KC and AR) using a qualitative approach, blinded to treatment arm. Pre and post-drug samples for each individual patient were compared and scored as increased, decreased, or unchanged. The Fleiss kappa statistic was chosen as the measure of concordance as it allows for more than two reviewers and more than two categories. Percent agreement and standard weighted kappa statistics were calculated for individual pairs of reviewers. Results: There was very good concordance between reviewers using the qualitative comparison of post to pre-drug Ki-67 staining with Fleiss’ Kappa of 0.91 and weighted Kappa’s ranging between 0.85 and 0.95. Reviewers 1 and 2 had the highest level of concordance. Reviewer agreement ranged from 0.92 to 0.98. The reviewer-specific (marginal) score distributions were similar. Each reviewer scored about 80% (79.5%-82.5%) of pairs as decreasing, with the remainder split similarly between no change (7.5%-10%) and increasing (10%-12.8%). Conclusion: Based upon the high concordance between reviewers, the NIH Biomarker Review Committee approved Ki-67 as an integrated biomarker for GY011, setting the stage for its use as an approved biomarker of therapeutic effectiveness in future endometrial cancer trials. Citation Format: Megan I. Samuelson, Virginia Filiaci, Kelley Carrick, Anand Rajan KD, William H. Rodgers, Kristina W. Thiel, Linda R. Duska, Kimberly K. Leslie. Approval for the proliferative marker Ki-67 as an integrated biomarker for endometrial cancer in NRG study GY011 [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO020.

Abstract IA020: Chromatin regulation of the endometrium: What are our models telling us?

Abstract ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. ARID1A is a SWI/SNF chromatin-remodeling complex subunit. ARID1A loss in the mouse endometrial epithelium leads vaginal bleeding when combined with PI3K pathway activation. Sorted mutant endometrial epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT), inflammation and estrogen signaling in vivo. ARID1A loss leads to the expression of EMT genes, suggesting ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates. In contrast to its repressive roles, ARID1A is required for the activation estrogen responsive genes, including the progesterone receptor (PGR). Estrogen responsive genes are marked by bivalent chromatin domains containing both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). High H3K27me3 correlates with low PGR expression, suggesting Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 normally silences PGR. In support of this, pharmacological inhibition of PRC2 partially rescues PGR activation is estrogen treated, ARID1A-deficient cells. Given the known antagonistic relationship between SWI/SNF and PRC2 complexes, we propose that SWI/SNF normally opposes PRC2 mediated silencing of the PGR promoter in response to estrogen, leading to PGR expression. In the ARID1A mutant endometrium, PGR promoter silencing by PRC2 is maintained. In the absence of PGR, progesterone cannot oppose the actions estrogen in the endometrium, leading to unopposed estrogen signaling. Our data demonstrate ARID1A has both activating and repressive roles in the endometrium, and ARID1A loss contributes to multiple aspects of disease pathogenesis. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Chromatin regulation of the endometrium: What are our models telling us? [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA020.

The Influence Mechanism of Abnormal Immunophilin FKBP52 on the Expression Levels of PR-A and PR-B in Endometriosis Based on Endometrial Stromal Cell Model in Vitro

ABSTRACT As a chaperone protein of progesterone receptor (PR), FK-506 Binding Protein 52 (FKBP52) can enhance the activity of PR, but the mechanism of FKBP52 affecting PR expression levels is difficult to clarify. Here, we report a novel in vitro model of ectopic endometrial stromal cells (ESCM) established through the primary culture method of endometrial stromal cells, which is used to study the details of relationship between FKBP52 abnormality and PR expression level in endometriosis (Ems). At the same time, the clinical study of the relationship between FKBP52 and PR expression levels in endometriosis patients was used to verify our conclusions. The results showed that the expression levels of PR-A mRNA and protein in endometriosis are positively correlated with FKBP52 and the abnormality of FKBP52 leads to the decrease of PR-B mRNA and protein expression. When FKBP52 was deleted or reduced, the expression levels of m RNA and protein of PR-A and PR-B have decreased leading to the proliferation of ectopic endometrium cells (ESC) and the occurrence of endometriosis, which is consistent with the expression levels of clinical endometriosis patients and fully confirms our conclusions and reliability of the model, and has great guiding significance for the research of Ems disease occurrence mechanism and clinical treatment.

Inhibition of progesterone receptor activity during development increases reelin-immunoreactivity in Cajal-Retzius cells, alters synaptic innervation in neonatal dentate gyrus, and impairs episodic-like memory in adulthood

Progesterone receptor (PR) is expressed in Cajal-Retzius (CR) cells of the dentate gyrus (DG) molecular layer during the postnatal period (P1–28), a critical stage of development for the dentate gyrus and its circuitry. CR cells secrete the glycoprotein, reelin, which is required for typical development of the DG and its connections, particularly afferent input from the perforant path. This pathway regulates the processing of sensory information arriving from entorhinal cortex and integrates this information to form episodic memories. To assess the potential role of PR activity on the development of these connections and associated behavior, rats were treated daily from P1 to 7 with the PR antagonist, RU486. RU486 treatment increased the number of reelin-ir cells, suggesting an accumulation of reelin, and implicating PR in the regulation of a principle developmental function of CR cells. RU486 also altered the synaptic bouton marker, synaptophysin-ir, in a sex-specific manner, suggesting a role for PR activity in the development of perforant path innervation of the molecular layer (MOL). Finally, both control and RU486 treated rats spent significantly more time with a temporally distant object in the Relative Recency task, suggesting an intact associative memory for object identity and temporal order in both groups. In contrast, the same RU486 treated rats were impaired in an episodic-like memory task compared to controls, failing to integrate object identity (‘what’), time (‘when’), and object position (‘where’). These findings reveal a novel role for PR in regulating CR cell function within the MOL, thereby altering development of DG connectivity and behavioral function.

Особливості плацентарних структур у жінок з передчасними пологами

The objective: study of histochemical, morpho-functional features of placentas in premterm birth at different gestational ages. Materials and methods. We examined 172 pregnant women, who were hospitalized and gave birth in the Perinatal Center of Kyiv during 2016–2018 years at 24–33 weeks of gestation. The control group included 40 women with a physiological course of singleton pregnancy at 38–41 weeks of gestation. Patients of the experimental group were divided into three subgroups: the first consisted of 46 pregnant women at 22–27 weeks of gestation, the second one – of 44 pregnant women at 28–33 weeks, the third one – of 42 pregnant women at 34–37 weeks. Clinical and statistical analysis of exchange cards of pregnant women (form 113/y) and birth histories (form 096/0) of mothers of all groups was performed. For morphofunctional assessment of placenta in the study histological (staining with hematoxylin-eosin after Van-Gizon) and immunohistochemical (indirect streptavidin-peroxidase – for detection of progesterone receptor’s expression level) methods were used. Results. No significant age difference was found between the study groups. More than 66.5% of patients had a second pregnancy. Obstetric and gynecological history of women are burdened in most cases by the following conditions: previous premature birth, miscarriage, inflammatory diseases, extragenital pathology (diseases of the urinary and endocrine systems). When analyzing the results of histological examination of the placenta in patients whose pregnancies resulted in childbirth at 22–27 weeks, inflammatory changes in the placenta were found in 84.8%, and in women in labor at 28–33 weeks – only 56.8%. At 34–37 weeks of gestation, the percentage of inflammatory changes in placental structures do not have a significant difference with the control group (30.9% and 27.5%, respectively; p≤0.05). With gestational age increase the percentage of involutive-dystrophic changes in the placenta decreases, the number of circulatory disorders increases, and accelerated chorionic maturation is observed. Analysis of the data of monoclonal antibodies’ to progesterone receptors expression in the structures of the placenta depending on gestational age revealed the greatest expression in decidual membranes (55%) in very early preterm birth, which had a significant difference with the control group (14%), (p&lt;0.05) . In women of group II, the level of expression of progesterone receptors was reduced (30%), but had no significant difference with group III of the studied placentas (26%), (p&gt;0.05). Immunohistochemical examination of the progesterone receptors (PR) shows a reaction in the epithelium and stem cells of the stroma, intermediate and terminal villi; in amniotic membranes and extravillous cytotrophoblast; in the vascular endothelium. Conclusion. In very early preterm birth, inflammatory changes and significant expression activity of progesterone receptors predominate in placental structures. During childbirth at 33–37 weeks of gestation the most common disorders are maturation of the villous chorion, combined with circulatory and involutive-dystrophic changes. Keywords: preterm labor, placenta, histological changes, immunohistochemistry, progesterone receptors.

Exploring the Genetic Background of the Differences in Nest-Building Behavior in European Rabbit.

Simple SummaryThe rabbit is one of the genetically most diverse farm animals, where domestication has resulted in a change primarily in the genes responsible for behavior. The elements of intensive production technology (e.g., nursing, change of nest material) can have a significant effect on maternal behavior. Its individual variability is evident in the timing of nest building and nest composition. As a result that the hormone progesterone strongly influences the steps of nest building, we investigated the association of genetic mutations in the progesterone receptor gene with nest-building behavior in wild-type rabbit does. In addition to the already described point mutations in domesticated lines, we detected a new mutation in our wild type rabbits. However, the timing of nest building (hay carrying) was related to an already described point mutation. The heritability of this trait was low, but it confirms the genetic determination of the behavior besides the environmental factors. One of the genotypes was responsible for the two days earlier initiation of the hay carrying behavior. The early initiation of this process allows animals to construct a high-quality nest resulting in better survival of the offsprings in nature.Once a day, nursing and absentee mothering make the wild rabbit (Oryctolagus cuniculus) an ideal model animal for measuring differences in maternal behavior. Behavioral events and their hormonal regulation leading to parturition are well documented; however, the genetic background behind individual differences in this complex process is unknown. Decreased progesterone hormone level and the reduction of progesterone receptor activity are crucial to initiating the collection of nest material. The progesterone receptor gene is a likely candidate affecting nest-building behavior. In addition to several known point mutations in the progesterone receptor gene of the European wild rabbit, we have found a new mutation in the promoter region of the gene at 2682 T > C. Although this new single nucleotide polymorphism (SNP) was not involved in the formation of the nest-building behavior, an SNP (2464G > A) already described in the promoter region showed an association with individual differences in the initiation of hay carrying. The distribution of this SNP delivered an opposite result compared to domestic rabbits. Genotype (GG) with high uterine capacity was most frequent; the hereditary value of the trait was h2 = 0.10. Thus, progesterone receptor gene polymorphism may manifest in individual differences affecting breeding success in this species.

COMPARATIVE ANALYSIS OF FK506-BINDING PROTEINS 51 (FKBP51) AND 52 (FKBP52) IN UTERINE FIBROIDS COMPARED TO NORMAL MYOMETRIUM

FK506-binding protein 51 (FKBP51) and 52 (FKBP52) are immunophilins and common co-regulators of transcription meditated by the glucocorticoid receptor (GR), progesterone receptor (PR) and androgen receptor (AR). While FKBP51-PR or -GR binding inhibits transcriptional activity, FKBP51-AR binding induces AR-mediated transcription. FKBP52 has the opposite effect on PR and GR transcriptional activity, enhancing transcription. Although all three steroid receptors are expressed in both uterine myometrial and fibroid cells, the steroid-induced proliferative response of fibroid cells differ from that in adjacent myometrial cells, indicating aberrant steroid signaling in fibroids.

Abstract 4378: Contribution of ERα/PR crosstalk to endocrine therapy resistance in breast cancer

Abstract Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies display intrinsic or acquired therapy resistance. One-third of patients with acquired resistance present with metastatic tumors containing ERα-Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. ERα/PR crosstalk, referring to the complex relationship of ERα and progesterone receptor (PR) interaction, offers therapeutic value in targeting both nuclear receptors simultaneously in ERα/PR-positive breast cancers. Previous research identified a synergistic effect of treating ERα-WT cancer cells with ERα/PR antagonism, where combined treatment results in tumor regression in vivo and increased cell death in vitro. Conversely, preliminary data suggests that treatment of ERα-Y537S-containing tumors with combined ERα and PR antagonists results in significantly increased proliferation in vivo and reduced cell death in vitro. My central hypothesis is that ERα-Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. Using a three-dimensional culture system of patient-derived xenografts grown as organoids (PDxO), I have observed a synergistic effect of certain combinations of ERα/PR antagonists in the context of ERα-WT similar to previous results in two-dimensional cell culture. At the level of transcription, PR-associated transcriptional activity appears to be significantly altered in homozygous ERα-Y537S T47D cells compared to ERα-WT cells. Further understanding the role of ERα-Y537S in altering gene expression and reducing the response to SERM and SPRM treatment will provide understanding as to why these tumors are resistant to standard-of-care treatment and will suggest alternative targets for treatment as well. Citation Format: Rosemary J. Huggins. Contribution of ERα/PR crosstalk to endocrine therapy resistance in breast cancer [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 4378.