Nuclear Progesterone Receptor Research Articles

Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17β-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

Functions of Membrane Progesterone Receptors (mPRs, PAQRs) in Nonreproductive Tissues.

Gender differences in a wide variety of physiological parameters have implicated the ovarian hormones, estrogens and progesterone, in the regulation of numerous nonreproductive tissue functions. Rapid, nongenomic (nonclassical) progesterone actions mediated by membrane progesterone receptors (mPRs), which belong to the progestin and adipoQ receptor family, have been extensively investigated in reproductive and nonreproductive tissues since their discovery in fish ovaries 20 years ago. The 5 mPR subtypes (α, β, γ, δ, ε) are widely distributed in vertebrate tissues and are often expressed in the same cells as the nuclear progesterone receptor (PR) and progesterone receptor membrane component 1, thereby complicating investigations of mPR-specific functions. Nevertheless, mPR-mediated progesterone actions have been identified in a wide range of reproductive and nonreproductive tissues and distinguished from nuclear PR-mediated ones by knockdown of these receptors with siRNA in combination with a pharmacological approach using mPR- and PR-specific agonists. There are several recent reviews on the roles of the mPRs in vertebrate reproduction and cancer, but there have been no comprehensive assessments of mPR functions in nonreproductive tissues. Therefore, this article briefly reviews mPR functions in a broad range of nonreproductive tissues. The evidence that mPRs mediate progesterone and progestogen effects on neuroprotection, lordosis behavior, respiratory control of apnea, olfactory responses to pheromones, peripheral nerve regeneration, regulation of prolactin secretion in prolactinoma, immune functions, and protective functions in vascular endothelial and smooth muscle cells is critically reviewed. The ubiquitous expression of mPRs in vertebrate tissues suggests mPRs regulate many additional nonreproductive functions that remain to be identified.

Functional Implications of Estrogen and Progesterone Receptors Expression in Adenomyosis, Potential Targets for Endocrinological Therapy.

Adenomyosis is a common gynaecological disease associated with the presence of endometrial lesions in the uterine myometrium. Estrogens have been proven to be the crucial hormones driving the growth of adenomyosis. Little is known about the distinct mechanisms of progesterone action in adenomyosis. Hence, in this study, we decided to characterize the expression of all nuclear and membrane estrogen and progesterone receptors. Additionally, as a functional investigation, we monitored prolactin production and cell proliferation after estradiol and progesterone treatments. We confirmed the presence of all nuclear and membrane estrogen and progesterone receptors in adenomyotic lesions at gene and protein levels. The expression of membrane progesterone receptors α and β (mPRα, mPRβ) as well as estrogen receptor β (ERβ) was upregulated in adenomyosis compared to normal myometrium. Estradiol significantly increased adenomyotic cell proliferation. Progesterone and cAMP upregulated prolactin secretion in adenomyosis in the same pattern as in the normal endometrium. In the present study, we showed the functional link between estradiol action and adenomyotic cell proliferation, as well as progesterone and prolactin production. Our findings provide novel insights into the sex steroid receptor expression pattern and potential regulated pathways in adenomyosis, suggesting that all receptors play an important role in adenomyosis pathophysiology.

Association of the PROGINS PgR polymorphism with susceptibility to female reproductive cancer: A meta-analysis of 30 studies.

The progesterone response of the nuclear progesterone receptor plays an important role in the female reproductive system. Changes in the function of the progesterone receptor gene may increase the risk of reproductive cancer. The present study performed a meta-analysis to examine whether the progesterone receptor gene PROGINS polymorphism was a susceptibility factor for female reproductive cancer. We searched the PubMed, Cochrane Library, Web of Science and EMBASE databases for literature on PROGINS polymorphisms and female reproductive cancer published before September 2020. We evaluated the risk using odds ratios [ORs] and 95% confidence intervals via fixed effects models and random-effects models, which were calculated for all five genetic models. We grouped the analyses by race, cancer, and HWE. Thirty studies comprised of 25405 controls and 19253 female reproductive cancer cases were included in this meta-analysis. We observed that the Alu insertion polymorphism and the V660L polymorphism were significantly associated with female reproductive cancer in the allele and dominant genetic models. The allele genetic model and (Alu-insertion polymorphism: OR = 1.22, 95% CI = 1.02-1.45; V660L polymorphism: OR = 1.02, 95% CI = 1.00-1.13) dominant genetic model (Alu-insertion polymorphism: OR = 1.27, 95% CI = 1.03-1.58; V660L polymorphism: OR = 1.10, 95% CI = 1.011.19) demonstrated a significantly increased risk of female reproductive cancer. A subgroup analysis according to ethnicity found that the Alu insertion was associated with female reproductive cancer incidence in white (Allele model: OR = 1.21, 95% CI = 1.00-1.45; Heterozygous model: OR = 3.44, 95% CI = 1.30-9.09) and Asian (Dominant model: OR = 3.12, 95% CI = 1.25-7.79) populations, but the association disappeared for African and mixed racial groups. However, the V660L polymorphism was significantly associated with female reproductive cancer in the African (Allele model: OR = 2.52, 95% CI = 1.14-5.56; Heterozygous model: OR = 2.83, 95% CI = 1.26-6.35) and mixed racial groups (Dominant model: OR = 1.28, 95% CI = 1.01-1.62). Subgroup analysis by cancer showed that the PROGINS polymorphism increased the risk of cancer in the allele model, dominant mode and heterozygous model, but the confidence interval for this result spanned 1 and was not statistically significant. This sensitivity was verified in studies with HWE greater than 0.5. Our meta-analysis showed that the progesterone receptor gene Alu insertion and the V660L polymorphism contained in the PROGINS polymorphism were susceptibility factors for female reproductive cancer.

Rapid Actions of the Nuclear Progesterone Receptor through cSrc in Cancer

The nuclear progesterone receptor (PR) is mainly known for its role as a ligand-regulated transcription factor. However, in the last ten years, this receptor’s extranuclear or rapid actions have gained importance in the context of physiological and pathophysiological conditions such as cancer. The PR’s polyproline (PXPP) motif allows protein–protein interaction through SH3 domains of several cytoplasmatic proteins, including the Src family kinases (SFKs). Among members of this family, cSrc is the most well-characterized protein in the scenario of rapid actions of the PR in cancer. Studies in breast cancer have provided the most detailed information on the signaling and effects triggered by the cSrc–PR interaction. Nevertheless, the study of this phenomenon and its consequences has been underestimated in other types of malignancies, especially those not associated with the reproductive system, such as glioblastomas (GBs). This review will provide a detailed analysis of the impact of the PR–cSrc interplay in the progression of some non-reproductive cancers, particularly, in GBs.

Progesterone Signaling and Mammalian Ovarian Follicle Growth Mediated by Progesterone Receptor Membrane Component Family Members.

How progesterone influences ovarian follicle growth is a difficult question to answer because ovarian cells synthesize progesterone and express not only the classic nuclear progesterone receptor but also members of the progestin and adipoQ receptor family and the progesterone receptor membrane component (PGRMC) family. Which type of progestin receptor is expressed depends on the ovarian cell type as well as the stage of the estrous/menstrual cycle. Given the complex nature of the mammalian ovary, this review will focus on progesterone signaling that is transduced by PGRMC1 and PGRMC2 specifically as it relates to ovarian follicle growth. PGRMC1 was identified as a progesterone binding protein cloned from porcine liver in 1996 and detected in the mammalian ovary in 2005. Subsequent studies focused on PGRMC family members as regulators of granulosa cell proliferation and survival, two physiological processes required for follicle development. This review will present evidence that demonstrates a causal relationship between PGRMC family members and the promotion of ovarian follicle growth. The mechanisms through which PGRMC-dependent signaling regulates granulosa cell proliferation and viability will also be discussed in order to provide a more complete understanding of our current concept of how progesterone regulates ovarian follicle growth.

Maternal-fetal conflict averted by progesterone- induced FOXP3+ regulatory T cells

SummaryPregnancy stimulates an intricately coordinated assortment of physiological changes to accommodate growth of the developing fetus, while simultaneously averting rejection of genetically foreign fetal cells and tissues. Despite increasing evidence that expansion of immune-suppressive maternal regulatory T cells enforces fetal tolerance and protects against pregnancy complications, the pregnancy-associated signals driving this essential adaptation remain poorly understood. Here we show that the female reproductive hormone, progesterone, coordinates immune tolerance by stimulating expansion of FOXP3+ regulatory T cells. Conditional loss of the canonical nuclear progesterone receptor in maternal FOXP3+ regulatory T cells blunts their proliferation and accumulation, which is associated with fetal wastage and decidual infiltration of activated CD8+ T cells. Reciprocally, the synthetic progestin 17α-hydroxyprogesterone caproate (17-OHPC) administered to pregnant mice reinforces fetal tolerance and protects against fetal wastage. These immune modulatory effects of progesterone that promote fetal tolerance establish a molecular link between immunological and other physiological adaptions during pregnancy.

Progesterone inhibitory role on gastrointestinal motility.

Progesterone is a steroidal hormone that is produced from the corpus luteum of the ovaries and from the placenta. The main function of progesterone is to promote the secretory differentiation in the endometrium of the uterus and to maintain pregnancy by inhibiting uterine contractions throughout pregnancy. Progesterone performs its actions by activating the classical progesterone nuclear receptors that affect gene transcription and by the non-classical activation of cell surface membrane receptors that accounts for the rapid actions of progesterone. Besides the reproductive roles of progesterone, it exerts functions in many tissues and systems such as the nervous system, the bone, the vascular system, and the gastrointestinal (GI) tract. This review will summarize the recent literature that investigated the role of progesterone in GI tract motility. Most literature indicates that progesterone exerts an inhibitory role on gut smooth muscle cells in part by elevating nitric oxide synthesis which induces relaxation in smooth muscle. Moreover, progesterone inhibits the signaling pathways that lead to contraction such as Rho kinase inhibition. These data serve as a quick resource for the future directions of progesterone research that could lead to better understanding and more effective treatment of gender-related GI tract motility disorders.

Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy.

Progesterone is a gonadal pro-gestational hormone that is absolutely necessary for the success of pregnancy. Most notable actions of progesterone are observed in the female reproductive organs, the uterus and the ovary. Acting through the nuclear progesterone receptor (PGR), progesterone prepares the endometrium for implantation of the embryo. Interestingly, the maternal thymus also is a known expressor of Pgr; its absence is associated with murine pregnancy complications. However, the localization of its expression and its functional importance were not known. Here, we used a transgenic dual fluorescent reporter mouse model and genetic deletion of Pgr in Foxn1+ thymic epithelial cells (TEC) to demonstrate TEC-specific Pgr expression in pregnancy, especially in the cortex where thymocyte maturation occurs. Using our TEC-specific Pgr deletion mouse model, we demonstrate that TEC-specific Pgr is necessary for pregnancy-induced thymic involution in pregnancy. Our investigation reveals that PGR expression is upregulated in the cortical thymic epithelial cells during pregnancy, and that PGR expression is important for thymic involution during murine pregnancy.

Complexes of ortho-Phthalic Acid in Spontaneous Animal Tumors.

The presence of diethyl-, dibutyl-, and 2-ethylhexyl phthalates in spontaneous animal tumors was determined for the first time. Small-cell breast cancer of the dog and Ehrlich ascites carcinoma with the cells are particularly rich of these compounds. It is assumed that the level of phthalates in these cells is due to the activity of expression of nuclear receptors for estrogens and progesterone, as well as the peculiarities of the metabolism of xenobiotics. The accumulation of these compounds in tumor cells can increase their malignancy. The data obtained can be used for development of new diagnostic and therapeutic technologies.

Antigestagens Mediate the Expression of Decidualization Markers, Extracellular Matrix Factors and Connexin 43 in Decidualized Dog Uterine Stromal (DUS) Cells.

Simple SummaryAdequate embryo-maternal communication is essential for a successful pregnancy. In the dog, this interaction is intimately associated with maternal stroma-derived decidual cells, the only cell population in the canine placenta expressing the nuclear progesterone receptor (PGR) and, therefore, sensitive to the circulating progesterone levels. Prepartum decrease of progesterone or clinical application of PGR blockers (antigestagens, e.g., aglepristone and mifepristone) induce placental release of luteolytic factors and terminate pregnancy. However, the importance of progesterone for decidual cells functionality has not been fully elucidated. Therefore, we investigated the effects of PGR blockers on the expression of markers of decidualization and cellular viability, as well as on epithelial and mesenchymal factors in in vitro decidualized dog uterine stromal (DUS) cells. Decidualization increased the expression of the respective markers, including factors involved in cell growth and prostaglandin synthesis. Their expression was suppressed by the application of antigestagens. Additionally, the expression of factors involved in tissue remodeling and cell-cell communication was increased, and antiproliferative and proapoptotic effects were induced in decidualized cells. Altogether, progesterone signaling appears to be crucial for modulating decidual cells physiology and biological activity, and thus for the maintenance of pregnancy.Feto-maternal communication in the dog involves the differentiation of stromal cells into decidual cells. As the only placental cells expressing the nuclear progesterone (P4) receptor (PGR), decidual cells play crucial roles in the maintenance and termination of pregnancy. Accordingly, to investigate possible PGR-mediated mechanisms in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with functional PGR-blockers, mifepristone and aglepristone. Effects on decidualization markers, epithelial and mesenchymal factors, and markers of cellular viability were assessed. Decidualization increased the expression of PTGES, PGR, IGF1, and PRLR, along with ECM1, COL4 and CX43, but downregulated IGF2. DUS cells retained their mesenchymal character, and the expression of COL4 indicated the mesenchymal-epithelial transformation. Antigestagen treatment decreased the availability of PTGES, PRLR, IGF1 and PGR. Furthermore, antigestagens decreased the mRNA and protein expression of CX43, and transcriptional levels of ECM1 and COL4. Additionally, antigestagens increased levels of activated-CASP3 (a proapoptotic factor), associated with lowered levels of PCNA (a proliferation marker). These data reveal important aspects of the functional involvement of PGR in canine decidual cells, regarding the expression of decidualization markers and acquisition of epithelial-like characteristics. Some of these mechanisms may be crucial for the maintenance and/or termination of canine pregnancy.

Comparison of Estrogen and Progesterone Receptors Status between Cell-Blocks of Fine Needle Aspiration and Tissue-Blocks of Corresponding Mastectomy Specimen in Invasive Ductal Breast Cancer

Background: Breast cancer is one of the common causes of mortality in female population, world-wide. Pakistan has highest incidence among Asian countries. Majority of breast cancers belong to invasive ductal carcinoma type. Estrogen and Progesterone nuclear receptors are important expression tools for molecular categorization of invasive breast carcinoma. Aim: To correlate status of estrogen and progesterone receptor expression between FNA cell-blocks and tissue blocks from mastectomy specimen in invasive ductal carcinoma of female breast. Methods: In current study, carried out at Pathology Department of Niazi Medical and Dental College, Sargodha from August, 2018 to July,2020, 125 cell blocks of FNA and tissue blocks of corresponding mastectomy specimens of invasive ductal breast carcinoma were included. Routine H & E staining protocol was adopted for microscopic diagnosis.24 Estrogen and Progesterone receptor expression was evaluated by immunohistochemistry25. Data was analyzed by using SPSS-20 version. Results: Spearman’s correlation showed a strong positive relationship between status of ER (rs = .959, p<.001) and PR expression (rs = .958, p < .001) observed in fine needle aspirate cell-blocks and in corresponding tissue blocks of mastectomy specimen. Conclusion: The study concluded that as there is concordance in ER and PR expression status, hence, FNA cell-blocks are reliable for planning hormonal treatment strategies instead of tissue blocks in invasive ductal breast carcinoma. Keywords: Estrogen receptor, Progesterone receptor, FNA cell block.

Molecular modeling, mutational analysis and steroid specificity of the ligand binding pocket of mPRα (PAQR7): Shared ligand binding with AdipoR1 and its structural basis

The 7-transmembrane architecture of adiponectin receptors (AdipoRs), determined from their X-ray crystal structures, was used for homology modeling of another progesterone and adipoQ receptor (PAQR) family member, membrane progesterone receptor alpha (mPRα). The mPRα model identified excess positively charged residues on the cytosolic side, suggesting it has the same membrane orientation as AdipoRs with an intracellular N-terminus. The homology model showed identical amino acid residues to those forming the zinc binding pocket in AdipoRs, which strongly implies that zinc is also present in mPRα. The homology model showed a critical H-bond interaction between the glutamine (Q) residue at 206 in the binding pocket and the 20-carbonyl of progesterone. Mutational analysis showed no progesterone binding to the arginine (R) 206 mutant and modeling predicted this was due to the strong positive charge of arginine stabilizing the presence of an oleic acid (C18:1) molecule in the binding pocket, as observed in the X-rays of AdipoRs. High Zn2+ concentrations are predicted to form a salt with the carboxylate group of the oleic acid, thereby eliminating its binding to the free fatty acid (FFA) binding pocket, and allowing progesterone to bind. This is supported by experiments showing 100 µM Zn2+ addition restored [3H]-progesterone binding of the Q206R mutant to levels in WT mPRα and increased [3H]-progesterone binding to mPRγ and AdipoR1 which have arginine residues in this region. The model predicts hydrophobic interactions of progesterone with amino acid residues surrounding the binding pocket, including valine 146 in TM3, which when mutated into a polar serine resulted in a complete loss of [3H]-progesterone binding. The mPRα model showed there is no hydrogen bond donor in the vicinity of the 3-keto group of progesterone and ligand structure-activity studies with 3-deoxy steroids revealed that, unlike the nuclear progesterone receptor, the 3-carbonyl oxygen is not essential for binding to mPRα. Interestingly, the small synthetic AdipoR agonist, AdipoRon, displayed binding affinity for mPRα and mimicked progesterone signaling, whereas D-e-MAPP, a ceramidase inhibitor, blocked progesterone signaling. Thus, critical residues around the binding pocket and steroid structures that bind mPRα, as well as similarities with AdipoRs, can be predicted from the homology model.

Abstract P1-02-17: Artificial intelligence-based whole slide scoring of nuclear breast cancer IHC markers Ki67, ER, and PR matches performance of manual clinical scoring

Abstract Background: 2.1 million breast cancers are newly diagnosed each year. Current guidelines endorse routine testing for estrogen receptor (ER) and progesterone receptor (PR), while use of the Ki67 biomarker can provide additional prognostic value. All three biomarkers currently require quantitative evaluation using manual review of a glass slide, resulting in reproducibility issues across labs due to interpretative and scoring variabilities. Current on-market image analysis algorithms only offer limited field-of-view (FOV) that analyze a tiny fraction of the entire tissue. Whole slide image (WSI) analysis, in comparison, analyzes the entire tissue and, therefore, more closely mimics how pathologists assess these slides in clinical practice. In this work, we developed three deep learning artificial intelligence (AI) based algorithms for WSI analysis (IA) of digitized images from Ki67, ER, and PR stained slides that address these variabilities and allow pathologists across labs to consistently score at the same accuracy as selected expert labs. The complete software solution delivers high throughput analyzing whole-slide images in less than 2 minutes during pre-computation on conventional computer hardware and returning results on user-provided annotations in milliseconds. Methods: We assembled a benchmark validation data set of 312 breast cancer cases (100 Ki67, 102 ER, and 110 PR slides, stained at multiple sites) representative of breast cancer subtypes (i.e. ductal, lobular, mucinous, medullary, tubular), score (i.e. 0%-100% positivity), tumor grade (i.e. well, moderately, and poorly differentiated), and specimen type (i.e. biopsy and resection). Three pre-clinical validation studies were performed using the Roche uPath enterprise software and each of the ER, PR and Ki67 image analysis algorithms. A total of 6 pathologists participated in the study split into expert (n=3) and study (n=3) readers. A non-inferiority Ground-Truth (GT) study design was implemented in which the study and expert readers performed manual read (MR) followed by AI-assisted scoring. The expert manual scores were used as GT to which the readers’ manual and AI scores were compared for each marker and case. Results: The overall concordance rates between AI scores and expert GT was as follows: For Ki67, OPA=97.2% (95% CI: 94.0, 99.7), NPA=97.8% (95% CI: 93.4,100), and PPA=96.7% (95% CI: 91.3, 100), for ER, OPA=95.4% (CI:91.4,98.4), NPA=96.4% (CI:92.5,99.4), and PPA=94.4% (CI:87.4,100), and for PR, OPA=96.1% (95% CI:92.7,99.1), NPA=96.7% (95% CI:92.5,100), and PPA=95.6% (95% CI:89.9,100). The differences between AI and MR overall concordance rates (AI-MR) when compared to the expert GT were: for Ki67: OPA-diff=1.4% (2-sided 95% CI:-0.7,3.7), NPA-diff=3.8% (CI:0.6,7.8), PPA-diff=-1.0% (CI:-3.5,0.0), for ER: OPA-diff=-0.9% (CI:-3.3,1.0), NPA-diff=-0.1% (CI:-3.1,3.0), PPA-diff=-1.8% (CI:-6.2,0.0), and for PR: OPA-diff=-1.5% (CI:-3.9,0.6), NPA-diff=-2.4% (CI:-6.8,1.2), PPA-diff= -0.7%(CI:-2.8,1.1) using the cutoffs 20% (Ki67), 1% (ER), and 1% (PR) respectively. Conclusion: Our preliminary feasibility data shows that pathologists using WSI analysis assisted scoring was equivalent to manual scoring and an expert panel GT using a truly representative benchmark data set. Additionally, image analysis algorithms are known to provide high reproducibility and precision. We will provide those numbers at a later stage as they were not fully available at time of submission. Our results show the value and potential of deep learning technologies to improve the diagnosis and care of patients with breast cancer. Citation Format: Monesh Kapadia, Mehrnoush Khojasteh, Margarita Kouzova, Carol Jones, Xiao-Meng Xu, Matthew T. Olson, Sarah Gladden, Nancy Sapanara, Shalini Singh, Chen Chun Chen, Isaac Bai, Jim Ranger-Moore, Landon J. Inge, Uday Kurkure, Ipshita Bhattacharya, Margaret Zhao, Karel Zuiderveld, Chandana Chintakindi, Bryan Lopez, Christoph Guetter. Artificial intelligence-based whole slide scoring of nuclear breast cancer IHC markers Ki67, ER, and PR matches performance of manual clinical scoring [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-17.

Immune modulatory effects of progesterone on oxLDL-induced trained immunity in monocytes.

Atherosclerotic cardiovascular diseases (CVD) are among the leading causes of death in the world. Monocyte‐derived macrophages are key players in the pathophysiology of atherosclerosis. Innate immune memory following exposure of monocytes to atherogenic compounds, such as oxidized low‐density lipoproteins (oxLDL), termed trained immunity, can contribute to atherogenesis. The current study aimed to elucidate intracellular mechanisms of oxLDL‐induced trained immunity. Using untargeted intracellular metabolomics in isolated human primary monocytes, we show that oxLDL‐induced trained immunity results in alterations in the balance of intracellular steroid hormones in monocytes. This was reflected by a decrease in extracellular progesterone concentrations following LPS stimulation. To understand the potential effects of steroid hormones on trained immunity, monocytes were costimulated with oxLDL and the steroid hormones progesterone, hydrocortisone, dexamethasone, β‐estradiol, and dihydrotestosterone. Progesterone showed a unique ability to attenuate the enhanced TNFα and IL‐6 production following oxLDL‐induced trained immunity. Single nucleotide polymorphisms in the nuclear glucocorticoid, progesterone, and mineralocorticoid receptor were shown to correlate with ex vivo oxLDL‐induced trained immunity in 243 healthy volunteers. Pharmacologic inhibition experiments revealed that progesterone exerts the suppression of TNFα in trained immunity via the nuclear glucocorticoid and mineralocorticoid receptors. Our data show that progesterone has a unique ability to suppress oxLDL‐induced trained immunity. We hypothesize that this effect might contribute to the lower incidence of CVD in premenopausal women.

Abstract PO-101: CmP signaling network unveils novel biomarkers for triple negative breast cancer in African American women

Abstract Breast cancer is the most commonly diagnosed cancer worldwide and remains the second leading cause of cancer death. While breast cancer mortality has steadily declined over the past decades through medical advances, an alarming disparity in breast cancer mortality has emerged between African American women (AAW) and Caucasian American women (CAW); and new evidence suggests more aggressive behavior of triple-negative breast cancer (TNBC) in AAW may contribute to racial differences in tumor biology and mortality. Progesterone (PRG) is capable of exerting its cellular effects through either its classic, non-classic or combined responses through binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs), warranting both pathways an equally important status in PRG-mediated signaling. In our previous report, we demonstrated that the CCM signaling complex (CSC) consisting of CCM1, CCM2, and CCM3 proteins can couple both nPRs and mPRs signaling cascades to form a CSC-mPRs-PRG-nPRs (CmPn) signaling network in nPR positive(+) breast cancer cells. In this report, we furthered our research by establishing the CSC-mPRs-PRG (CmP) signaling network in nPR(-) breast cancer cells, demonstrating that a common core mechanism exists, regardless of nPR(+/-) cell type. This is the first report stating that inducible expression patterns exist between CCMs and major mPRs in TNBC cells. Furthermore, we firstly show mPRs in TNBC cells are localized in the nucleus and participate in nucleocytoplasmic shuttling in a coordinately synchronized fashion with CCM proteins under steroid actions, following the same cellular distribution as other well-defined steroid hormone receptors. Finally, for the first time, we deconvoluted the CmP signalosome by using multi-omics approaches, which helped us understand key factors within the CmP network, and identify 21 specific biomarkers with potential clinical applications associated with AAW-TNBC tumorigenesis. These novel biomarkers could have immediate clinical implications to dramatically improve health disparities among AAW-TNBCs. Citation Format: Johnathan Abou-Fadel, Jun Zhang. CmP signaling network unveils novel biomarkers for triple negative breast cancer in African American women [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-101.

Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells†.

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air–liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen–progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.

New Insights as to Why Progesterone Receptor Modulators, such as Mifepristone, Seem to Be More Effective in Treating Cancers that Are Devoid of the Classical Nuclear Progesterone Receptor.

Mifepristone treatment for advanced cancer has demonstrated considerable improvement in both length and quality of life in patients who no longer have any other treatment options. The target is the progesterone induced blocking factor (PIBF), which helps the tumor to invade the normal tissue and proliferate and suppress cellular immunity. Most of the benefit has been observed in cancers not associated with the classical nuclear progesterone receptor (nPR). There are data showing that the presence of a nPR may be associated with a better prognosis. Membrane PRs seem to be responsible for PIBF secretion. Mifepristone, possibly fails to block another P associated protein that enables the tumor to proliferate, e.g., the progesterone receptor membrane component-1 (PGRMC-1) protein. One hypothesis is that the nPR helps to inhibit tumor production of PGRMC-1 protein. Thus, mifepristone may inhibit tumor spread by suppressing PIBF, but this may be negated by blocking the nPR, allowing PGRMC-1 levels to increase.

Anticancer effects of mifepristone on human uveal melanoma cells

BackgroundUveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.MethodsThe effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR.ResultsMF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor.ConclusionThis study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Mifepristone May Be the Best Single Pharmaceutical Agent for Treatment of a Variety of Advanced Cancers

Rationale: To provide evidence to support the provocative statement that the progesterone receptor modulator (antagonist) mifepristone may be the best single anticancer drug to use for advanced metastatic cancer. Objective: A summary of cancer cell line studies, controlled treatment trials of several types of spontaneous murine cancers, and anecdotal human case reports that support the premise of this review, is provided. The concept suggests that the mechanism of action involves the suppression of an immunosuppressive protein called the progesterone induced blocking factor (PIBF) that requires stimulation of a membrane progesterone receptor for its production. Findings: Cell line studies show that mifepristone can down-regulate PIBF production by many different leukemia cell lines and inhibit proliferation of cancer cell lines, e.g., ovarian cancer and glioblastoma multiform stage IV. Controlled murine studies found improved longevity and quality of life as monitored by body conditioning scores following oral gavage of mifepristone including leukemia, lung, testicular, and prostate cancer. Most importantly, mifepristone anecdotally has been found to provide extension of life and improved quality of life in patients with a large majority of advanced cancers that were no longer responding to any available anticancer drug options. These cancers include colon, kidney, small cell and non- mall cell lung, pancreatic, thymic epithelial cell, transitional cell of the renal pelvis, grade IV glioblastoma multiform, fibrous osteogenic sarcoma, and leiomyosarcoma cancers. Of note, the majority of the cancers treated are not known to be associated with the presence of classical nuclear progesterone receptor. Conclusions: Related to additional benefits of easy oral administration, low cost, low risk of short-term side effects, or immediate, severe, or delayed severe complications, and its efficacy despite starting it, in general, in late stages of advanced metastatic disease in patients previously treated with standard or experimental drugs, and its beneficial effects in a large variety of cancers, mifepristone may be the best single agent anticancer drug for the treatment of advanced cancers.