Progesterone Receptor Activity Research Articles

Progesterone Receptor Signaling Promotes Cancer Associated Fibroblast Mediated Tumorigenicity in ER+ Breast Cancer.

Breast cancer progression involves intricate interactions between cancer cells and the tumor microenvironment (TME). This study elucidates the critical role of progesterone receptor (PR) signaling in mediating the pro-tumorigenic effects of cancer-associated fibroblasts (CAFs) on estrogen receptor-positive (ER+) luminal breast cancer cells. We demonstrate that CAFs produce physiologically relevant levels of estrogen and progesterone, which significantly contribute to breast cancer tumorigenicity. Specifically, CAF-conditioned media promoted PR-dependent anchorage-independent growth, tumorsphere formation/stem cell expansion, and CD44 upregulation. CAF cells formed co-clusters more frequently with PR+ breast cancer cells relative to PR-null models. While both PR isoforms mediated these actions, PR-A was a dominant driver of tumorsphere formation/stemness, while PR-B induced robust CD44 expression and CAF/tumor cell co-cluster formation. CD44 knockdown impaired CAF/tumor cell co-clustering. Fibroblast growth factor 2 (FGF2), also secreted by CAFs, phosphorylated PR (Ser294) in a MAPK-dependent manner and activated PR to enhance CD44 expression and breast cancer tumorigenicity. The FGFR inhibitor, PD173074, diminished CAF- and FGF2-dependent PR activation, tumorsphere formation, and co-clustering. In summary, this study reveals a novel mechanism through which stromal CAFs orchestrate elevated PR signaling in ER+ luminal breast cancer via secretion of both progesterone and FGF2, a potent activator of ERK1/2. Understanding tumor cell/TME interactions provides insights into potential therapeutic strategies aimed at disrupting PR- and/or FGF2/FGFR-dependent signaling pathways to prevent early metastasis in ER+ breast cancer patients.

Abstract 4390: IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells

Abstract Insulin-like growth factor binding protein-6 (IGFBP-6) is induced by progesterone (P4) in T-47D breast cancer cells. IGFBP-6 regulates insulin-like growth factor 2 (IGF2) by sequestering it to inhibit receptor binding and extend its extracellular half-life. IGF2 binds to insulin-like growth factor 1 receptor or insulin receptor A to promote breast cancer cell proliferation and survival. Progesterone is an ovarian steroid hormone which has complex roles in breast cancer through the actions of progesterone receptor A and B (PR-A and B). Progesterone antagonizes the actions of estradiol (E2), another steroid hormone which promotes proliferation and survival in breast cancer. T-47D cells treated with both P4 and E2 exhibit significantly reduced proliferation compared to treatment with E2 alone. To better understand the role of IGFBP-6 in progesterone signaling, T-47D cells which over-express IGFBP-6 were treated with P4, E2, or both. Overexpressing cells exhibited stronger antagonism compared to controls when co-treated with P4 and E2. RNA-interference was performed to knockdown IGFBP-6 induction in the presence of P4 and E2. Knockdown of IGFBP-6 in the presence of P4 and E2 decreases the antagonistic effects of P4. Expression of both PR-A and PR-B decreases by about 50% when IGFBP-6 is knocked down. These results indicate that IGFBP-6 increases PR expression in response to P4 stimulation. Citation Format: Francisco Javier Lariz, Pacha Botero, Kevin Houston. IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4390.

Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer

Abstract Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer. Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3584.

Ancient and modern mechanisms compete in progesterone receptor activation

The progesterone receptor is activated by ligands utilizing both ancestral and modern mechanisms.

THU570 Isoform Specific Gene Regulation By Progesterone Receptors Drives Expansion Of Breast Cancer Stem Cell Expansion In 3D Cell Culture Models

Abstract Disclosure: N.E. Gillis: None. T.H. Truong: None. C.A. Lange: None. Exposure to progesterone is a risk factor for breast cancer, and numerous PGR polymorphisms are linked to breast, endometrial, and ovarian cancer. Two progesterone receptor (PR) isoforms are expressed from PGR in breast tissues: full-length PR-B and truncated PR-A. In normal breast epithelia, PR isoforms are equally expressed and mostly function as A:B heterodimers. An imbalance of this ratio, and thus a predominance of homodimers, is frequent in ER+ breast cancer. Luminal A-type estrogen receptor positive (ER+) tumors often exhibit an elevated PR-A:PR-B ratio that predicts resistance to tamoxifen. It has recently been shown that PRs promote endocrine resistance and drive expansion of breast cancer stem-like cell (CSC) populations. However, the isoform-specific molecular mechanisms of PR action in ER+ breast cancers remain undefined. The goal of our current work is to map the progesterone-driven gene expression in CSC cells through transcriptomic profiling of T47D cells grown in spheroid-culture conditions that express exclusively PR-A or PR-B. This transcriptomic data has been integrated with phenotypic studies of T47D and MCF7 cells that express a single isoform of PR. Our data shows PR-A is a regulator of the cell cycle and of senescence, while PR-B mediates cell metabolism and intracellular signaling. We further integrated our cell line data with transcriptomes from patient tumors that have been classified based on their PR-A:PR-B ratio and their response to treatment with anti-progestins. Our results are a first step towards re-defining the role of PRs in gene regulation within the CSC population. Presentation Date: Thursday, June 15, 2023

Progesterone Receptor Activation Regulates Sensory Sensitivity and Migraine Susceptibility

Women develop chronic pain during their reproductive years more often than men, and estrogen and progesterone regulate this susceptibility. We tested whether brain progesterone receptor (PR) signaling regulates pain susceptibility. During the estrous cycle, animals were more sensitive to mechanical stimulus during the estrus stage than in the diestrus stage, suggesting a role for reproductive hormones, estrogen, and progesterone. Progesterone treatment of ovariectomized and estrogen-primed mice caused a delayed reduction in the mechanical threshold. Segesterone, a specific agonist of PRs replicated this effect, whereas, the segesterone-induced reduction in mechanical threshold was blocked in the mice lacking PRs in the nervous system. Segesterone treatment also did not alter mechanical threshold in adult male and juvenile female mice. PR activation increased the cold sensitivity but did not affect the heat and light sensitivity. We evaluated whether PR activation altered experimental migraine. Segesterone and nitroglycerin when administered sequentially, reduced the pain threshold but not when given separately. PRs were expressed in several components of the migraine ascending pain pathway, and their deletion blocked the painful effects of nitroglycerin. PR activation also increased the number of active neurons in the components of the migraine ascending pain pathway. These studies have uncovered a pain-regulating function of PRs. Targeting PRs may provide a novel therapeutic avenue to treat chronic pain and migraine in women. PerspectiveThis article provides evidence for the role of progesterone receptors in regulating pain sensitivity and migraine susceptibility in females. Progesterone receptors may be a therapeutic target to treat chronic pain conditions more prevalent in women than men.

TRIM28 modulates nuclear receptor signaling to regulate uterine function

Estrogen and progesterone, acting through their cognate receptors the estrogen receptor α (ERα) and the progesterone receptor (PR) respectively, regulate uterine biology. Using rapid immunoprecipitation and mass spectrometry (RIME) and co-immunoprecipitation, we identified TRIM28 (Tripartite motif containing 28) as a protein which complexes with ERα and PR in the regulation of uterine function. Impairment of TRIM28 expression results in the inability of the uterus to support early pregnancy through altered PR and ERα action in the uterine epithelium and stroma by suppressing PR and ERα chromatin binding. Furthermore, TRIM28 ablation in PR-expressing uterine cells results in the enrichment of a subset of TRIM28 positive and PR negative pericytes and epithelial cells with progenitor potential. In summary, our study reveals the important roles of TRIM28 in regulating endometrial cell composition and function in women, and also implies its critical functions in other hormone regulated systems.

P-515 EHD1 impaired endometrial decidualization linked to recurrent implantation failure through SENP1-mediated progesterone resistance

Abstract Study question Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness to progesterone, an intractable clinical problem with elusive mechanisms. Summary answer EHD1 overexpression promoted the SUMOylation and ubiquitinated degradation of the PRB protein leading to decreased transcriptional activity and responsiveness of endometrial stromal cells to progesterone. What is known already Abnormal decidualization is a significant cause of infertility in RIF patients. A previous study has demonstrated that EH homeodomain 1 (EHD1) is significantly elevated in the endometrium of RIF patients and plays an essential role in the decidualization of human endometrial stromal cells (HESCs). However, far less is known about the function of EHD1 in the endometrium during embryo implantation. Study design, size, duration After approval from the Ethics Committee of Nanjing Drum Tower Hospital (2013-081-01), endometrial specimens were collected from women who received treatment at the Reproductive Center of Nanjing Drum Tower Hospital from January 2020 to December 2021. Twelve RIF women and Twelve fertile normal control women were enrolled in the present study. Endometrium in the middle and late stages of secretion was obtained by aspiration and curettage 5-7 days after ovulation. Participants/materials, setting, methods The expression and location of EH homeodomain 1 (EHD1) in endometrium were evaluated by IHC, qRT-PCR and Western blotting. Transcriptomic analysis was performed to predict the mechanism by which EHD1 is mediated in human endometrial stromal cells (HESCs) decidualization. Luciferase reporter assays and Western blotting were used to detect the protein activity and stability of progesterone receptor B (PRB) regulated by EHD1. The potential mechanisms were further confirmed through immunoprecipitation, nucleocytoplasmic separation and immunofluorescence experiments. Main results and the role of chance EHD1 was abnormally elevated in RIF patients and linked to aberrant endometrial decidualization. EHD1 overexpression inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness of HESCs to progesterone. Indeed, EHD1 overexpression promoted the SUMOylation of PRB, leading to ubiquitinated degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. In addition, the expression of PRL and IGFBP1 was rescued by interfering with the expression of EHD1 in HESCs from RIF patients. Limitations, reasons for caution In our study, we provided novel insight into the molecular scaffold of EHD1 to mediate the ubiquitination of PRB for degradation. However, our data do not account for the specific mechanisms by which EHD1 promotes the ubiquitination of PRB, and The specific enzymes involved remain to be elucidated. Wider implications of the findings We demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF. Trial registration number not applicable

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis.

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.

Reevaluating the Role of Progesterone in Ovarian Cancer: Is Progesterone Always Protective?

Ovarian cancer (OC) represents a collection of rare but lethal gynecologic cancers where the difficulty of early detection due to an often-subtle range of abdominal symptoms contributes to high fatality rates. With the exception of BRCA1/2 mutation carriers, OC most often manifests as a post-menopausal disease, a time in which the ovaries regress and circulating reproductive hormones diminish. Progesterone is thought to be a "protective" hormone that counters the proliferative actions of estrogen, as can be observed in the uterus or breast. Like other steroid hormone receptor family members, the transcriptional activity of the nuclear progesterone receptor (nPR) may be ligand dependent or independent and is fully integrated with other ubiquitous cell signaling pathways often altered in cancers. Emerging evidence in OC models challenges the singular protective role of progesterone/nPR. Herein, we integrate the historical perspective of progesterone on OC development and progression with exciting new research findings and critical interpretations to help paint a broader picture of the role of progesterone and nPR signaling in OC. We hope to alleviate some of the controversy around the role of progesterone and give insight into the importance of nPR actions in disease progression. A new perspective on the role of progesterone and nPR signaling integration will raise awareness to the complexity of nPRs and nPR-driven gene regulation in OC, help to reveal novel biomarkers, and lend critical knowledge for the development of better therapeutic strategies.

Embryo-maternal interactions revealed by phosphoproteomic analysis of endometrial organoids exposed to embryo secretome

The equine embryo remains mobile and without physical attachment to the endometrium for a prolonged period in early pregnancy. During this time, the embryo only communicates with the maternal system via secreted compounds in its immediate environment. These interactions are poorly understood, and a definitive signal for maternal recognition of pregnancy remains elusive. In this study, we sought to develop a model to examine embryo-maternal interactions at close range in vitro, demonstrating that incubation of endometrial organoids with embryo-conditioned medium results in detectable changes in the endometrial phosphoproteome. Mare endometrial organoids were derived from isolated endometrial glands of uteri obtained immediately post-mortem. Embryos were recovered by uterine lavage from Standardbred mares at day 8-9 post-ovulation, washed and pre-cultured in BSA-containing medium under oil, and cultured in protein-free medium under oil. Embryo-conditioned and control media were collected at 12 h, centrifuged to remove debris and stored at −80°C until further analysis. For co-incubation experiments, organoids were primed with 10nM estradiol (48 h), then 10nM estradiol + 1 µM progesterone (48 h). Embryo-conditioned (n=3) or control (n=3) media were added at a dilution of 1:100 in organoid medium and co-incubated for 30 minutes. Organoids were then recovered and processed to remove media and secretome components (on ice and in the presence of phosphatase inhibitors). Cell pellets were frozen at -80˚C until protein extraction. Proteins were extracted with urea-based extraction buffer, reduced,alkylated, and digested with trypsin. Phosphopeptides were isolated using titanium dioxide before LC tandem mass spectrometry (MS/MS) using a Q-Exactive Plus hybrid quadrupole-Orbitrap MS system (Thermo Fisher Scientific, Bremen, Germany). Endometrial organoids were successfully cultured in vitro and their functional mimicry of in vivo endometrium was confirmed via multiple routes including histological analysis of gland-like structures, epithelial and endometrial markers, and RNA sequencing alongside isolated endometrial glands. Phosphoproteomic mass spectrometry of endometrial organoids revealed a profile of 181 phosphopeptides mapping to 90 protein IDs. Of these, 10 phosphopeptides were decreased (log2[fold-change] < -0.8) in co-incubated organoids, while 3 were uniquely detected in secretome-exposed organoids only. Most notably, we observed a decrease in the phosphorylated peptides of PGRMC1 (progesterone receptor membrane component 1). This protein forms part of a non-classical system of progesterone action, located in the cell membrane. A change in PGRMC1 phosphorylation suggests that an alternative system of progesterone receptor activation participates in equine embryo-maternal signaling. There were also changes in phosphorylation status of chaperone proteins essential to progesterone receptor assembly (e.g., HSP90AB1). In summary, this study describes the immediate response of mare endometrial organoids to compounds released by embryos, identifying novel mechanisms possibly implicated in signaling between the embryo and maternal system.

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.

Association of Val660Leu, progesterone receptor polymorphic variant, with susceptibility to RRMS disease

ABSTRACT Background Multiple sclerosis is an inflammatory, autoimmune, and progressive neurodegenerative disease of the central nervous system with an unknown etiology. Based on the gender differences in epidemiological, clinical, and pathological features of multiple sclerosis, the role of sex hormones and their receptors in this disease has been considered. A single nucleotide polymorphism located in the exon 4 of progesterone receptor, rs1042838 (G/T -Val660Leu), was associated with reduced progesterone receptor activity. We aimed to investigate the association of this polymorphism with the risk of multiple sclerosis. Method A total of 426 individuals were included in the present study, including 200 patients and 226 age and sex adjusted healthy controls in Iranian population. The target SNP was genotyped using PCR-RFLP, and statistical analysis was performed using SPSS 21.0 and by ꭓ2 and logistic regression tests. Results The results showed that the allele T acts as a risk allele, so that the genotypes TG and TT significantly increase RRMS susceptibility compared to the genotype GG. Conclusion Our data suggest that Val660Leu polymorphism might be a risk factor for the development of RRMS.

Abstract 3084: Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2

Abstract Late recurring ER+ breast cancer emerges in post-menopausal women despite low/no hormone conditions. Cancer associated fibroblasts (CAFs) represent a major player in the promotion of metastasis, stemness and chemotherapy resistance in breast cancer. Progesterone receptors (PR) are emerging as drivers of stemness and metastasis in ER+ breast cancers. We propose that CAFs provide a mechanism to turn on ligand-independent PR signaling pathways through the secretion of paracrine factors. Methods: Tumorsphere assays were used to measure stem cell expansion. Cell clustering assays were used to measure CAF/tumor cell interactions. ChIP assays were used to measure PR direct binding to stemness gene regulatory elements. Results: Using the ER(+) T47D cell series (i.e., PR-null variants or cells expressing either endogenous PR (T47DCO) or PR-A or PR-B isoforms), we observed that CAF conditioned media (CM) increased tumorsphere formation in PR+ cells relative to PR-null and controls. Similar results were obtained with MCF7L cell models. CAF CM activated MAPK/ERK kinases and induced rapid phosphorylation of PR at Ser294. Physical interaction between CAFs and PR expressing T47D and MCF7L cells in clustering assays demonstrated PR driven co-cluster formation. CAF-derived FGF2 increased PR dependent tumorsphere and co-cluster formation. Blockage of FGF2 with the inhibitor, PD173074, decreased CAF CM-induction of tumorspheres and co-cluster formation. Previous data demonstrated that the cell adhesion molecule, CD44, is implicated in CAF/BC co-cluster formation. Both FGF2 and progestins increased CD44 expression in ER+ breast cancer cell models. Chromatin immunoprecipitation (ChIP) assays demonstrated that both PR isoforms are recruited to PRE-containing regions in the CD44 gene in response to progestin. Finally, we compared the gene expression profiles of breast cancer cells from T47DCO mono-clusters and CAF/T47DCO co-clusters using Single Cell RNA Seq. Among the differentially expressed genes identified were two genes implicated in tamoxifen resistance, BAMBI and NR2F2. Conclusion: We propose a model in which clustering between CAFs and ER+/PR+ breast cancer cells is promoted by the CAF secreted factor, FGF2. FGF2 promotes PR activation and upregulation of CD44 expression, which may further increase physical cell-cell interactions. Importantly, CAF-derived FGF2 can also act through PR to initiate MAPK/ERK signaling pathways that drive stemness and endocrine therapy resistance. Our studies of the interactions between PR+ breast cancer cells with CAFs either in the primary tumor or in the circulation (as CAF/CTC clusters) suggest the need to include anti-progestins as a component of endocrine therapy in late recurring ER+ breast cancer. Citation Format: Angela K. Spartz, Caroline H. Diep, Carol A. Lange, Dorraya El-Ashry. Progesterone receptor mediated crosstalk between cancer associated fibroblasts (CAFs) and ER+/PR+ breast cancer cells through CAF-secreted FGF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3084.

Abstract OT2-01-11: ctDNA-guided Adaptive Therapy Escalation in ER+ MBC: A Phase 1b Study with Letrozole, Palbociclib and Onapristone ER

Abstract ctDNA-guided Adaptive Therapy Escalation in ER+ MBC: A Phase 1b Study with Letrozole, Palbociclib and Onapristone ER Background: Extended release onapristone (onapristone ER) is a progesterone receptor (PR) antagonist that inhibits hormone-mediated PR activation and stabilizes PR association with corepressors, resulting in an antineoplastic effect when applied alone or in combination with antiestrogen therapy to breast cancer cells in vitro. Recent preclinical studies further suggest that onapristone adds to inhibition of cell proliferation when combined with CDK4/6 inhibitors and fulvestrant. Elevations in ctDNA can precede overt disease progression by a matter of months in metastatic breast cancer and may represent an opportunity for proactive therapeutic intervention. Trial Design: This is an investigator initiated open-label, single institution phase 1b study of onapristone ER added as escalation therapy in patients with ER+, PR+, HER2 negative MBC, who have detectable ctDNA after six months of treatment with letrozole and palbociclib in the first line. The study is supported by Context Therapeutics and involves two stages. Stage 1 is a dose escalation/de-escalation phase of 18 patients maximum, in which the safety and recommended phase 2 dose (RP2D) will be established for onapristone ER when used in combination with letrozole and palbociclib. In stage 2, the dose expansion phase, the RP2D of onapristone will be combined with letrozole and palbociclib in 10 patients to further explore the tolerability of the regimen. ctDNA will be collected serially while patients are on this triplet therapy. Eligibility Criteria: This study is enrolling patients with radiologically measurable or evaluable metastatic or unresectable ER+/PR+/HER2-negative MBC in whom a tumor-derived somatic mutation can be detected in ctDNA at a variant allele fraction of 0.5% or greater after 6 months (+/- 4 weeks) of treatment with first line letrozole and palbociclib without progression, using our in house CLIA certified MSK-ACCESS assay. Adequate organ function and functional status for enrollment are stipulated in the protocol. Specific Aims: The primary objective of this study is to define the safety, tolerability, and recommended phase 2 dose of onapristone ER used in combination with letrozole and palbociclib. Secondary objectives include to investigate ctDNA response rate of the triplet therapy regimen, to gather early data regarding the 6-month clinical benefit rate, overall response rate, and progression free survival of this triplet escalation therapy regimen in high risk ctDNA+ patients, and to evaluate the pharmacokinetics of Onapristone ER when used in combination with letrozole and Palbociclib. Exploratory objective include to describe ctDNA dynamics during antiprogestin therapy escalation in ER+ MBC, as well as the molecular features present in responders vs. non-responders using ctDNA and pre-treatment tissue. Target Accrual: The total planned cohort for the phase I dose escalation is a maximum of 18 patients across 3 dose levels, and the total planned cohort for the dose expansion is 10 patients, with an anticipated maximum total of 28 patients. We will allot for 5 additional patients to account for inevaluability during the dose escalation and expansion portions of the trial. The trial is currently open to enrollment at MSKCC. Contact Information: Dragoj@mskcc.org; Jhaverik@mskcc.org Citation Format: Joshua Drago, Elaine Walsh, Mithat Gonen, Michael Berger, Mark E. Robson, Sarat Chandarlapaty, Pedram Razavi, Komal Jhaveri. ctDNA-guided Adaptive Therapy Escalation in ER+ MBC: A Phase 1b Study with Letrozole, Palbociclib and Onapristone ER [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-01-11.

Progesterone Receptor-Mediated Regulation of Cellular Glucose and 18F-Fluorodeoxyglucose Uptake in Breast Cancer.

Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor-positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.

Activation of progesterone receptor is essential for folic acid-regulated cancer cell proliferation and migration

We previously demonstrated that activation of progesterone receptor (PR) is essential for folic acid (FA)-inhibited proliferation in colorectal cancer cell lines. In the present study, we further investigated whether the requirement of PR activation for the FA-regulated cell proliferation and migration is a general phenomenon for all cancer cell lines or specific for colorectal cancer cell lines only. Initially, we examined the expression of PR in various cancer cell lines using Western blot analyses and RT-PCR technique, and then investigated the effects of FA on these cancer cell lines. Our data showed that the effects of FA on proliferation and migration only occurred in the PR positive (+) cancer cell lines, but not the PR negative (-) cancer cell lines, and these effects were abolished by pre-treatment with the PR specific inhibitor, Org 31710. On the other hand, FA significantly reduced the proliferation and migration in the PR (-) cancer cell lines transfected with PR pcDNA. However, FA did not significantly affect the proliferation and migration in the PR-transefected Hep-3B cell line, which does not express endogenous PR and FA receptor (FR). Since we previously showed that FA-regulated proliferation in colorectal and breast cancer cell lines through the cSrc-mediated pathway, we conducted immunoprecipitation assay to demonstrate that PR formed a complex with FR and cSrc, but FR did not directly associate with cSrc. Taken together, these findings suggest that the requirement of PR activation for the FA-regulated cell proliferation and migration is a general phenomenon for all cancer cell lines.

Environmental water extracts differentially activate zebrafish and human nuclear progesterone receptors

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20β-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment.

RF15 | PMON305 Membrane Progesterone Receptor α (mPRα/PAQR7) Promotes Survival and Axonal Sprouting in Human Neuronal Cells Lines Through Direct and Human Schwann-Cell Like Differentiated Adipose Stem Cells-Mediated Mechanisms

Abstract Injuries to peripheral nerves affect millions of people worldwide. These injuries can be significantly detrimental to the patients’ quality of life. While central nervous system neurons have extremely limited regenerative potential, peripheral nerves are able to regenerate after injury. However, this regeneration process often leads to unsatisfactory regenerative outcomes. Improving peripheral nerve regeneration is therefore a strong medical need. Schwann cells, the glial myelinating cells of the peripheral nervous system, are known to play an important role in promoting peripheral nerve regeneration. However, their limited in vitro expansion capability and limited availability has led several research groups to investigate the possibility of using differentiated stem cells as a potential tool to promote nerve regeneration, in association with other tools such biomaterial tubes. The activation of membrane progesterone receptor α (mPRα, PAQR7) with the selective agonist 19-ethenyl-progesterone (02-0) was recently shown to elicit potentially pro-regenerative effects in a model of human Schwann cell-like (SCL) differentiated adipose stem cells (ASC). Indeed, mPRα activation led to increased SCL-ASC migration, proliferation and neurotrophin (specifically BDNF) release. All these outcomes are expected to be beneficial for nerve regeneration. In this project, we investigated the effect of mPRα activation in SCL-ASC on neuronal cell lines to demonstrate that the positive effects previously observed in SCL-ASC have a beneficial effect in neuronal cells. We first investigated how the conditioned medium of untreated SCL-ASC (CM-) and SCL-ASC treated with the selective mPR agonist 02-0 (CM+) affected cell survival and neuronal sprouting in two different neuronal cell lines, IMR-32 and SH-SY-5Y. In both cases, CM- had a beneficial effect, reducing cell death, as assessed in starvation experiments, and promoting axonal sprouting, with the presence of longer cell processes in the two cell models compared to control. In both cases, CM+ had an even stronger effect than CM-, suggesting that mPRα activation may lead to the release of specific molecules that promote nerve regeneration. Moreover, CM- and CM+ treatments were effective in increasing the gene expression of selected peripheral regeneration markers (CREB3, ATF3 and GAP46) in SH-SY-5Y cells. Since mPR receptors are present in neuronal cells, we performed another series of cell death and axonal sprouting experiments in SH-SY-5Y6 cells, comparing CM- and CM+ effects to an experimental group in which 02-0 was added to CM- after incubation with SCL-ASC. These experiments showed that the effect on cell death was mostly due to direct activation of mPR receptors in neuronal cells. On the other hand, the effect on axonal sprouting depended on mPRα activation in SCL-ASC and subsequent release of active molecules in the medium. Our results support the hypothesis that mPRα activation in SCL-ASC may represent a promising pharmacological target to promote peripheral nerve regeneration. Presentation: Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

MECHANICAL AUGMENTATION OF PROGESTERONE ACTION IN UTERINE FIBROID CELLS REQUIRES PROGESTERONE RECEPTOR B (PRB) AND IS ASSOCIATED WITH PRB PHOSPHORYLATION

Progesterone responsive genes are implicated in fibroid growth, but the mechanisms that integrate mechanical signaling with progesterone receptor activation are not fully understood. We previously reported that a mechanically stiff substrate promoted progesterone-dependent, progesterone receptor-mediated gene activation in fibroid cells. Here we investigated whether altered mechanical stiffnesses directly influenced phosphorylation of progesterone receptor B (PRB), and used siRNA directed against PRB to test whether this isoform was required to transduce mechanical signals in fibroid cells.