Progesterone Receptor Protein Research Articles

Effect of 1,25(OH)2-vitamin D3 on expression and phosphorylation of progesterone receptor in cultured endometrial stromal cells of patients with repeated implantation failure

Repeated implantation failure (RIF) occurs in a condition when good quality embryos fail to implant in the endometrium following several in vitro fertilization (IVF) cycles. Suboptimal endometrial receptivity is one of the main underlying factors that causes this failure. Progesterone is the key regulator of endometrial receptivity which regulates gene expression through binding to its receptors in the endometrial stromal cells (eSC). The aim of this study was to evaluate the effect of 1,25(OH)2-vitamin D3 on progesterone receptor (PR) expression level and its phosphorylation on Ser294 residues in eSC of RIF patients and healthy fertile women. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, the cells were incubated with vitamin D3 and the expression level of PR mRNA, PR protein and phospho-Ser294 PR protein were evaluated after treatment. The results showed that vitamin D3 treatment increases PR mRNA and protein level and phospho-Ser294 PR protein level in the isolated eSC of both RIF patients and the control group. These results suggest that vitamin D3 may possibly play a key role during the embryo implantation process by affecting the expression pattern and regulatory modifications of the PR in the eSC.

Triple negative breast cancer: A thorough review of biomarkers.

Triple-negative breast cancer (TNBC) is defined as a type of breast cancer with lack of expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 protein. The tumorigenesis is not likely to be driven by hormonal or HER2 pathway. In comparison to other types of breast cancer, TNBC stands out for its aggressive behavior, more prone to early recurrence. Historically, TNBC has been considered a disease with poor response to molecular target therapy, requiring better validation of biomarkers. Recent issues related to tumor heterogeneity have been widely discussed suggesting the subdivision of TNBC into different molecular subtypes. Through a complete research on the main published trials databases and platforms of ongoing clinical studies, the current manuscript was carried out in order to present a critical view of the role of immunohistochemical and molecular biomarkers for the prognosis and response prediction of TNBC to traditional therapy and new molecular target agents.

The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & interleukin-1β

Increased expression of pro-labour genes that encode cyclooxygenase-2 (COX-2), oxytocin receptor (OTR) and connexin-43 (Cx43) at parturition is often attributed to P4 functional withdrawal, based on findings from animal models and human primary myometrial cells. However, the cause of reduced myometrial P4 responsiveness that promotes contractions at labour is not fully determined. Uterine stretch occurs with advancing gestation but most in vitro experimental models do not take this into consideration. We aimed to examine whether tissue-level myometrial stretch influences the ability of P4 to regulate pro-labour protein abundance by using myometrial biopsies from term gestation pregnant women to assess the impact of 24 h exposure to combinations of (i) stretch-mediated tension, (ii) P4 (100 nM) and (iii) an anti-progestin, RU-486 (1 μM). Firstly, we observed baseline COX-2 and Cx43 protein levels increased, whereas P4 content along with calponin-1 and progesterone receptor (PR) protein abundance decreased, in vehicle-treated tissues. P4 supplementation subtly reduced COX-2 levels in un-stretched tissues. Spontaneous and oxytocin-augmented contractility were unchanged by tissue culture exposure to P4 and/or RU-486. Interleukin-1β (IL-1β; 1 ng/ml) enhanced COX-2 protein and PGE2 content in un-stretched tissues. Overall, tissue stretch may, in part, regulate P4-sensitive pro-labour protein levels, but this is likely to be reliant on interaction with other in utero factors that were absent in our tissue cultures. More complex culture conditions should be evaluated in future to aid further development of a physiologically relevant model to improve our understanding of in utero myometrial P4 responsiveness.

The Correlation of Nottingham Prognostic Index with Immunoexpression of ER, PR, HER2, and Ki67 in Breast Cancer Tissue

Abstract Background In the underdeveloped and developing countries, the application of immunohistochemistry (IHC) to assess the status of oestrogen receptor (ER), progesterone receptor (PR), HER2 receptor, and Ki67 proteins in formalin-fixed, paraffin-embedded tissue sections of primary invasive breast carcinoma is utterly hindered by high cost, long procedure, and need for both skilled operator and pathologist. The objective of this study was to evaluate the correlation of Nottingham prognostic index (NPI) with immunoexpression of ER, PR, HER2, and Ki67 proteins in the formalin-fixed, paraffin-embedded tissue sections of primary invasive breast carcinoma. The ethical approval reference number is IRBRTA 306/2560. Methods Two hundred and forty primary invasive breast carcinoma cases were determined their NPI which was calculated as [0.2 × maximum tumour diameter in cm] + tumour grade + axillary lymph node score. Tumour grade was given a score 1 to 3 based on the Nottingham Combined Histologic Grade system (Elston-Ellis modification of Scarff-Bloom-Richardson grading system) of primary invasive breast carcinoma. Axillary lymph node metastasis (ALNM) was given a score 1 if no ALNM, a score 2 if 1 to 3 metastatic nodes, and a score 3 if at least 4 nodal involvement. The correlation between NPI and the IHC status of ER, PR, HER2, and Ki67 was analysed at the 95% confidence interval. Results The NPI had a significant association with the immunoexpression of ER, PR, and Ki67 proteins. There was no correlation with HER2 immunohistochemical reactivity. The proposed NPI score less than 4.5 will significantly correlate with positive immunoexpression of ER and PR. The NPI score greater than or equal to 4.5 will correlate with high Ki67. Conclusion The NPI might have a role in predicting the results of ER, PR and Ki67 expression in the formalin-fixed, paraffin-embedded tissue sections of primary invasive breast carcinoma, if immunohistochemical method is unavailable.

Clinical significance of evaluating hormone receptor and HER2 protein using cell block against metastatic breast cancer: a multi-institutional study.

Hormone receptor and human epidermal growth factor receptor 2 (HER2) protein tests in metastatic breast cancer tissue are recommended in the guidelines of the American Society of Clinical Oncology/American Pathology Association. As part of a multi-institutional study by the National Hospital Organization, we conducted an investigation to examine these molecular markers, using cytological specimens as a substitute for tissue specimens from breast cancer metastasis. To confirm the usefulness of receptors tested in metastatic lesions, the treatment course of registered metastatic breast cancer patients was analyzed. During the April 2015 to March 2016 registration period, there were 62 registrations. Types of metastatic lesions include pleural fluid (44 samples), ascites (14 samples), lymph nodes (2 samples), pericardial fluid (1 sample), and dorsal subcutaneous mass (1 sample). A stable test result was obtained by adopting the receptor examination method, using cell block for immunostaining cytological specimens. The discordance rates of estrogen receptor (ER), progesterone receptor (PR), and HER2 protein expression were 18.2% (95% confidence interval (CI): 7.9–28.8%), 36.4% (95% CI: 23.7–49.1%), and 8.2% (95% CI: 0.1–16.3%), respectively, between the primary tumor and metastatic lesion. Patients who changed from primary negative to metastatic positive ER status had taken a significantly longer time for metastatic foci to appear. Patients with positive ER status in metastatic lesions had significantly better prognosis than ER-negative cases (P = 0.030) by the Log-Rank test. The ER status of the metastatic lesion and the metastatic site were independent prognostic factors by Cox multivariate analysis. Receptor examination with cytological specimens in metastatic lesions has been useful as it provides guidance for the treatment of metastatic breast cancer.

Effects of COH on the expression of connexin43 in endometrial stromal cells

ObjectiveThe purpose of this study was to investigate the effect of controlled ovarian hyperstimulation (COH) on gap junction, and to induce the effect of an estrogen level overdose on gap junction in vitro by COH. Here, we mainly focus on connexion43 (Cx43), progesterone receptor (PR) and prolactin-related protein (PRP), and CyclinD3 genes expression, as well as the expression of Cx43 protein, were investigated. Materials and methodsMature BDF-1 mice were divided into different COH, and the mouse uterus was isolated, Paraffin sections evaluate the effect of COH on mouse uterine endometrial morphology. The other part was used for the extraction of mouse uterine endometrial stromal cells (ESC), some related gene changes are detected. Human ESC were isolated from human endometrium by primary culture, the estrogen concentrations 10−6 mol/L, 10−7 mol/L were added, the changes of Cx43 gene and related proteins were detected, too. Results(1) HE staining showed that in the ovulatory endometrium of mice in the high super ovulation group, uterine glands in the stromal layer were significantly increased, the relative vascular tissues was less abundant. (2) In three groups of COH mice, the expression of Cx43, PR, and PRP genes in ESC was significantly different (P < 0.05). (3) In vitro ESC in the COH group showed significant differences in Cx43, PR, and CyclinD3 gene expression (P < 0.05), and showed an obvious dose effect. In addition, Western blot analysis showed that the Cx43 protein and Cx43 gene expression were similar. Conclusions(1) Animal experiments study showed that Cx43 gene expression in ESC was significantly decreased in hyper COH, in addition, the advance in gene expression was significantly earlier, suggesting decidualization appeared significantly earlier. (2) In vitro COH demonstrated when the estrogen concentration used was higher, the expression level of Cx43 gene and protein was lower. Combined with animal experiments, the endometrium decidualization was advanced in mice that were underwent hyper COH, which may reflect the endometrial receptivity.

Expression of membrane progestin receptors (mPRs) α, β and γ in the bovine uterus during the oestrous cycle and pregnancy

Progesterone (P4) affects cell function through the nuclear progesterone receptor and membrane-bound progesterone binding proteins, including the membrane progestin receptors (mPRs) alpha (mPRα), beta (mPRβ) and gamma (mPRγ), which belong to the progestin and adipoQ receptor family (PAQR7, 8 and 5, respectively). The aim of this study was to determine the mRNA and protein expression levels of mPRα, mPRβ and mPRγ through real-time PCR and Western blot analyses, respectively, and to determine the cellular localization of these proteins in the bovine endometrium and myometrium on days 2–5, 6–10, 11–16 and 17–20 of the oestrous cycle and weeks 3–5, 6–8 and 9–12 of pregnancy (n = 5/each time period). The resulting data showed the highest (P < 0.05) mPRα and mPRβ mRNA expression in the endometrium on days 11–16 of the oestrous cycle compared to the other stages. In the myometrium, the level of mPRα mRNA was the lowest (P < 0.05) on days 6–16 of the oestrous cycle, while mPRβ was the lowest on days 11–16. There were no changes (P > 0.05) in mPRγ mRNA expression in the endometrium and myometrium during the oestrous cycle. During pregnancy, in the endometrium and myometrium, the levels of mPRα and mPRβ mRNA were comparable with those observed during the oestrous cycle. However, mPRγ mRNA expression was the highest (P < 0.001) during all stages of pregnancy compared with that observed during the oestrous cycle in both uterine tissues. The mPRα protein level only changed in the myometrium and was the highest (P < 0.05) during weeks 9–12 of pregnancy. However, in the endometrium, the expression of mPRβ protein was higher (P < 0.05) on days 6–10 of the oestrous cycle than during weeks 6–8 of pregnancy. Strong positive immunoreactions for all mPR proteins were observed in the luminal and glandular epithelium but were less evident in the stromal cells and myocytes. In addition, all proteins were also localized in the endothelial cells of blood vessels in the uterus, suggesting that P4 may affect blood flow in this organ through mPRs. The presence of mPR receptors in the uterus indicates their participation in the regulation of uterine functions.

MicroRNA-375 regulates oocyte in vitro maturation by targeting ADAMTS1 and PGR in bovine cumulus cells

MicroRNAs (miRNAs) have attracted increasing attention for their function in oocyte in vitro maturation (IVM). In this study, we aimed to explore the functional role and underlying mechanism of miR-375 in oocyte IVM. Cumulus-oocyte complexes (COCs) were cultured in standard cell culture conditions until they reach metaphase II (MII) stage. MiR-375 overexpression or knockdown was achieved by corresponding lentiviral transduction. Levels of miR-375, disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) mRNA and progesterone receptor (PGR) mRNA were detected by qRT-PCR. Western blotting was used to assess the expression of ADAMTS1 and PGR protein. The targeted interaction between miR-375 and ADAMTS1 or PGR was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results demonstrate that miR-375 is downregulated, and ADAMTS1 and PGR are upregulated in cumulus cells during COC maturation. MiR-375 negatively regulates COC maturation. Moreover, ADAMTS1 and PGR are two targets of miR-375 in cumulus cells. ADAMTS1 or PGR knockdown represses COC maturation and miR-375 inhibits the expression levels of ADAMTS1 and PGR in cumulus cells. Additionally, miR-375 overexpression-mediated suppressive effect on COC maturation is abated by ADAMTS1 or PGR expression restoration. In conclusion, our study suggests that miR-375 represses oocyte IVM at least partially through targeting ADAMTS1 and PGR in cumulus cells, providing a novel insight for the involvement and underlying mechanism of miR-375 in oocyte IVM.

The antiestrogenic effects of black cohosh on BRCA1 and steroid receptors in breast cancer cells.

BackgroundBlack cohosh (BC) is an herbal remedy often used by women to treat symptoms associated with menopause. Research has shown that the molecular activity of BC is associated with estrogen receptor alpha (ER-α) regulation. Progesterone receptor (PR) expression is found to be consistent with ER expression and mutations in the BRCA1 gene, a tumor-suppressor gene, are known to be responsible for about 40%–45% of hereditary breast cancers.PurposeThe objective of this study was to determine the effects of BC alone, as well as in combination with hormones and antihormones, on cell viability and expression of ER-α, PR, and BRCA1 in both T-47D and MCF-7 cell lines.MethodsCells were cultured in charcoal-stripped serum prior to their treatment and subsequent protein extraction. Western blot analyses were performed following a Bio-Rad Bradford protein assay and SDS-PAGE gel electrophoresis, with ECL luminescence and Image Studio Lite software. Cellular viability assays were performed using propidium iodine (PI) staining, and the distribution of fluorescent structures was evaluated through confocal microscopy. RT-qPCR analysis was performed on extracted cellular RNA. All statistical analyses were performed using SPSS software, and data was subjected to Kruskal-Wallis testing, followed by post-hoc analysis using the Mann-Whitney U-test to determine the statistical significance of all findings.ResultsWestern blot analysis displayed significant alterations of ER-α, PR, and BRCA1 protein levels after 24-hour treatment with 80–500 μM BC. BC displayed a concentration-dependent decrease on ER-α and BRCA1 expression, with an 87% reduction of ER-α expression and a 43% of BRCA1 expression in T-47D cells compared to control. After six days of treatment with 400 μM BC, a 50% decrease in cell proliferation was observed. Following 24 hours of co-treatment with 400 μM BC and 10 nM E2, ER-α was downregulated by 90% and BRCA1 expression was reduced by 70% compared to control. The expression of PR, following the same treatment, exhibited similar effects. The proliferative effect of E2 was reduced in the presence of BC.ConclusionBlack Cohosh demonstrates substantial anti-cancer properties, and this study may significantly aid in the understanding of the molecular effects of BC on ER-α, PR, and BRCA1 in breast cancer cells.

Activin-like kinase 5 (ALK5) inactivation in the mouse uterus results in metastatic endometrial carcinoma

The endometrial lining of the uterine cavity is a highly dynamic tissue that is under the continuous control of the ovarian steroid hormones, estrogen and progesterone. Endometrial adenocarcinoma arises from the uncontrolled growth of the endometrial glands, which is typically associated with unopposed estrogen action and frequently occurs in older postmenopausal women. The incidence of endometrial cancer among younger women has been rising due to increasing rates of obesity, a major risk factor for the disease. The transforming growth factor β (TGFβ) family is a highly conserved group of proteins with roles in cellular differentiation, proliferation, and cancer. Inactivating mutations in the genes encoding the TGFβ cell surface receptors (TGFBR1/ALK5 and TGFBR2) have been detected in various human cancers, indicating that a functional TGFβ signaling pathway is required for evading tumorigenesis. In this study, we present a mouse model with conditional inactivation of activin receptor-like kinase 5 (ALK5) in the mouse uterus using progesterone receptor cre ("Alk5 cKO") that develops endometrial adenocarcinoma with metastasis to the lungs. The cancer and metastatic lung nodules are estrogen dependent and retain estrogen receptor α (ERα) reactivity, but have decreased levels of progesterone receptor (PR) protein. The endometrial tumors develop only in Alk5 cKO mice that are mated to fertile males, indicating that TGFβ-mediated postpartum endometrial repair is critical for endometrial function. Overall, these studies indicate that TGFβ signaling through TGFBR1/ALK5 in the endometrium is required for endometrial homeostasis, tumor suppression, and postpartum endometrial regeneration.

Investigating Estrogen and Progesterone Receptors in the Lacrimal Sacs of Individuals With and Without Chronic Dacryocystitis.

Objectives:Chronic dacryocystitis is usually seen in middle-aged or older women, suggesting that decreased estrogen and progesterone serum levels may be a causative factor in the disease pathology. However, the occurrence of the disease in premenopausal females and males suggests that there may be more to the explanation than the level of female sex hormones. The purpose of the present study was to investigate estrogen and progesterone receptor positivity in the lacrimal sacs of individuals with and without chronic dacryocystitis.Methods:The study group included 50 female and 20 male patients diagnosed with chronic dacryocystitis. Lacrimal sac samples were taken during a dacryocystorhinostomy. The control group comprised 29 cadavers with no evidence of lacrimal system pathology in the health records. The samples were obtained transconjunctivally. Lacrimal sac samples from both groups were stained with the estrogen and progesterone receptor protein antigen. Fisher’s exact test and a chi-square test were used to compare the receptor positivity results of premenopausal and postmenopausal women, and samples of those with dacryocystitis and cadaver sacs without the disease.Results:In the control group, estrogen receptor positivity was observed in the samples of 2 premenopausal females. In the study group, estrogen receptor positivity was seen in 4 premenopausal females. There was no significant difference in estrogen receptor positivity between the premenopausal and postmenopausal female groups (p=0.41). A similar result was not established between the premenopausal and postmenopausal females in case group (p=0.056). No comparison was made of the progesterone receptor because only 1 example of progesterone receptor positivity was found in a premenopausal female in the dacryocystitis group.Conclusion:Estrogen receptor positivity did not seem to be a factor in chronic dacryocystitis physiopathology.

MicroRNA Dysregulation and Steroid Hormone Receptor Expression in Uterine Tissues of Rats with Endometriosis during the Implantation Window.

Background:Estrogen receptor (ER) and progesterone receptor (PR) are involved in endometriosis, but the involvement of microRNAs (miRNAs) is unknown. The aim of the study was to explore the correlation between miRNA and ER/PR in uterine tissues of rats with endometriosis during the implantation window.Methods:Twenty female Sprague-Dawley rats were randomized in three groups: endometriosis (n = 7), fat tissue control (n = 6), and normal (n = 7) groups. The female rats were mated and sacrificed on day 5 (implantation). Uterine tissues were obtained for hematoxylin-eosin staining, immunohistochemistry, and miRNA expression. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the expression of rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p.Results:The 475 miRNAs were found to differentially express between the endometriosis and normal control groups, with 127 being upregulated and 348 being downregulated. Expression of five miRNAs (rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p) were validated by RT-PCR and found to be differentially expressed among the three groups. Expression of ER and PR proteins (immunohistochemistry) in the glandular epithelium and endometrial stroma was significantly different among the three groups (all P < 0.05). Five miRNAs were involved in pathways probably taking part in implantation and fertility.Conclusions:The results suggested that miRNAs, ER, and PR could play important roles in the embryo implantation period of rats with endometriosis. These miRNAs might play a role in endometrial receptivity in endometriosis.

Targeting Breast Cancer Stem Cells to Overcome Treatment Resistance.

Despite advances in breast cancer diagnosis and treatment, many patients still fail therapy, resulting in disease progression, recurrence, and reduced overall survival. Historically, much focus has been put on the intrinsic subtyping based in the presence (or absence) of classical immunohistochemistry (IHC) markers such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-related protein (HER2). However, it is widely understood that tumors are composed of heterogeneous populations of cells with a hierarchical organization driven by cancer stem cells (CSCs). In breast tumors, this small population of cells displaying stem cell properties is known as breast CSCs (BCSCs). This rare population exhibit a CD44+/CD24−/low phenotype with high ALDH activity (ALDH+), and possesses higher tolerability to chemotherapy, hormone therapy, and radiotherapy and is able to reproduce the bulk of the tumor after reduction of cell populations sensitive to first-line therapy leading to disease relapse. In this review, we present special attention to BCSCs with future directions in the establishment of a therapy targeting this population. Drugs targeting the main BCSCs signaling pathways undergoing clinical trials are also summarized.

Study on the regulatory mechanism of electroacupuncture based on thyroid pathway for mammary gland hyperplasia rats

To study the regulating effect of thyroid pathway on electroacupuncture (EA) for mammary gland hyperplasia (MGH) so as to provide new research ideas for the mechanism of EA for MGH and to provide the evidence for clinical application. Sixty adult female SD rats were randomly divided into a blank group, a model group, an EA group, an EA with thyroidectomy group, an EA with sham operation group, 12 rats in each one. Except the blank group, the MGH model was established. Thyroid ablation was performed in the EA with thyroidectomy group, and sham operation was used in the EA with sham operation group, exposing thyroid without excision, 1 day after model establishment. EA was applied in the EA, the EA with thyroidectomy, and the EA with sham operation groups on the 4th day after model establishment, and not used in the other groups, but catching, routine disinfection and fixation were all the same as the above groups. The acupoints in the group A were bilateral "Tianzong" (SI 11), "Ganshu" (BL 18) and "Zusanli" (ST 36); and those in the group B were bilateral "Wuyi" (ST 15), "Hegu" (LI 4) and "Danzhong" (CV 17). The two groups of points were alternately used. EA, continuous wave, 2 Hz and 1 mA, was connected at "Tianzong" (SI 11) and "Ganshu" (BL 18), "Wuyi" (ST 15) and "Hegu" (LI 4) at the same side, 2 pairs EA a time, 20 min a time, once a day. All the intervention was given for 4 courses, 5 times as 1 course with 2 days between courses. After intervention, the height and diameter of the rat papilla were measured. Estrogen (E2) and progestational hormone (P) in the serum were detected by enzyme linked immunosorbent assay (ELISA), and the contents and protein expression of estrogen receptor α (ERα) and progesterone receptor (PR) in the mammary glands were detected by immunofluorescence and Western-blot. (1) The height and diameter of papilla in the model group increased compared with those in the blank group (both P<0.01). The height and diameter of papilla in the EA, EA with sham operation groups reduced compared with those in the model group (all P<0.01). Those in the EA with thyroidectomy group were lower than those in the model group, without statistical significance (both P>0.05). (2) Compared with the blank group, E2 increased and P decreased in the model group (both P<0.01). Compared with the model group, E2 decreased and P increased in the EA and EA with sham operation groups (all P<0.01). The contents of E2 and P had no statistical significance between the model and the EA with thyroidectomy groups (both P>0.05). (3) Compared with the blank group, the ERα content and protein expression increased and the PR content and protein expression decreased in the model group (all P<0.01). Compared with the model group, the ERα content and protein expression decreased and the PR content and protein expression increased in the EA and EA with sham operation groups (all P<0.01). The ERα and PR content and protein expression had no statistical significance between the model and the EA with thyroidectomy groups (all P>0.05). The effect of EA for MGH may be closely related to the regulation of thyroid.

Abstract B58: Progesterone receptor (PR) isoforms drive distinct cell-cell interactions and gene expression programs in human fallopian tube models of early HGSOC

Abstract High-grade serous ovarian carcinoma (HGSOC), the most prevalent and aggressive form of ovarian cancer (OC), contains abundant receptors for the ovarian steroid hormones, estrogen (ER; 76%) and progesterone (PR; 35%). These receptors contribute to the development of breast and reproductive tract cancers via complex mechanisms. Estrogen, acting through ERalpha, is well accepted as a stimulatory hormone while progesterone is thought to be a protective factor that reduces OC risk. Our understanding, however, of the molecular and cell-type specific mechanisms of PR isoform action in the initiation and progression of OC is limited. Therefore, to investigate altered PR isoform signaling in early-stage HGSOC, we utilized p53-dominant negative mutant fallopian tube epithelial (FTE) cells to generate stable cell lines expressing either PR-A or PR-B. Progestin (R5020) treatment of 2D adherent cultures revealed functional PR signaling through MAPK-dependent PR S294 phosphorylation as well as isoform specific expression of PR target genes encoding adhesion molecules (e.g., HEF1), cell cycle regulators (e.g., FOXO1), and glucocorticoid signaling proteins (e.g., CRISPLD2, NDRG1). In order to mimic early dissemination of FTE cells from serous tubal intraepithelial carcinoma (STIC) lesions, a precursor for the majority of HGSOCs, we established a 3D spheroid culture model. Interestingly, we observed that PR expression greatly increased the formation, size, and number of total spheroids compared to PR-negative controls. Progestin treatment resulted in additional enhancement of spheroid size with distinct morphologic differences observed between PR-A and PR-B expressing lines. Furthermore, progestin-treated cells grown as 3D spheroids exhibited increased basal PR protein and PR S294 phosphorylation levels relative to cells maintained in 2D adherent cultures, suggesting that hyperactivation of PRs occurs via cell-cell interactions, in part conferred by 3D architecture. Both PR-A and PR-B spheroids secreted matrix proteins such as fibronectin and other EMT biomarkers (e.g., integrin alpha 5) and progestin treatment modulated the PR isoform-specific expression of these markers. Preliminary RNA-seq analyses, comparing PR-A and PR-B spheroids, revealed differential expression of genes encoding pathways that regulate cell adhesion and cell movement as well as cytokine signaling and cell metabolism. Our results highlight the importance of cell context in understanding PR action and suggest that activation of PR signaling promotes and enhances the formation of nonadherent 3D structures in an isoform-specific manner. Such effects of progesterone are predicted to influence the shedding, aggregation, and dissemination of early FTE lesions. These preliminary findings demonstrate the importance of understanding the impact of steroid hormone receptors, including PR isoforms, on early OC development. Citation Format: Megan I. Seibel, Angela Spartz, Nuri Temiz, Laura J. Mauro, Carol A. Lange. Progesterone receptor (PR) isoforms drive distinct cell-cell interactions and gene expression programs in human fallopian tube models of early HGSOC. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B58.

Sensitivity and Isoform Specificity of 18F-Fluorofuranylnorprogesterone for Measuring Progesterone Receptor Protein Response to Estradiol Challenge in Breast Cancer.

The purpose of this study was to evaluate the ability of 21-18F-fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione (18F-FFNP) to measure alterations in progesterone receptor (PR) protein level and isoform expression in response to estradiol challenge. Methods: T47D human breast cancer cells and female mice-bearing T47D tumor xenografts were treated with 17β-estradiol (E2) to increase PR expression. 18F-FFNP uptake was measured using cell uptake and tissue biodistribution assays. MDA-MB-231 breast cancer clonal cell lines were generated that express the A or B isoforms of human PR. PR protein levels, transcriptional function, and subcellular localization were determined. In vitro 18F-FFNP binding was measured via saturation and competitive binding curves. In vivo 18F-FFNP uptake was measured using tumor xenografts and positron emission tomography. Statistical significance was determined using analysis of variance and t-tests. Results: After 48 and 72 h of E2, 18F-FFNP uptake in T47D cells was maximally increased compared to both vehicle and 24 h E2 treatment (p<0.0001 vs ethanol; P = 0.02 and P = 0.0002 vs 24 h for 48 and 72 h, respectively). T47D tumor xenografts in mice treated with 72 h E2 had maximal 18F-FFNP uptake compared to ethanol-treated mice (11.3±1.4 vs 5.2±0.81 %ID/g; P = 0.002). Corresponding tumor-to-muscle uptake ratios were 4.1±0.6, 3.9±0.5, and 2.3±0.4 for 48 h E2, 72 h E2, and ethanol-treated mice, respectively. There was no significant preferential 18F-FFNP binding or uptake by PR-A versus PR-B in the PR isoform-specific cell lines and tumor xenografts. Conclusion:18F-FFNP is capable of measuring estrogen-induced shifts in total PR expression in human breast cancer cells and tumor xenografts with equivalent isoform binding.

Abstract 1811: O-GlcNAc-dependent regulation of progesterone receptor function in breast cancer

Abstract Emerging clinical trial data implicate progestins in the development of breast cancer. While the role for the progesterone receptor (PR) in this process remains controversial, it is clear that PR, a steroid-activated nuclear receptor, alters the transcriptional landscape of breast cancer. PR interacts with many different types of proteins, including transcriptional co-activators and co-repressors, transcription factors, nuclear receptors and proteins that post-translationally modify PR (i.e. kinases and phosphatases). Herein, we identify a novel interaction between PR and OGT, the enzyme that catalyzes the addition of a single N-acetylglucosamine sugar, referred to as O-GlcNAc, to acceptor serines and threonines in target proteins. This interaction between PR and OGT leads to the post-translational modification of PR by O-GlcNAc. Moreover, we show that O-GlcNAcylated PR is more transcriptionally active on PR-target genes, despite the observation that PR mRNA and protein levels are decreased when O-GlcNAc levels are high. O-GlcNAcylation in breast cancer is clinically relevant, as we show that O-GlcNAc levels are higher in breast cancer as compared to matched normal tissues, and PR-positive breast cancers have higher levels of OGT. These data predict that under conditions where O-GlcNAc levels are high (breast cancer), PR, through an interaction with the modifying enzyme OGT, will exhibit increased O-GlcNAcylation and potentiated transcriptional activity. Therapeutic strategies aimed at altering cellular O-GlcNAc levels may have profound effects on PR transcriptional activity in breast cancer. Citation Format: Gloria M. Trinca, Merit L. Goodman, Evangelia K. Papachristou, Clive S. D'Santos, Prabhakar Chalise, Rashna Madan, Chad Slawson, Christy R. Hagan. O-GlcNAc-dependent regulation of progesterone receptor function in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1811.

Notch-1 Signaling Activation and Progesterone Receptor Expression in Ectopic Lesions of Women With Endometriosis.

ContextProgesterone (P) resistance is a hallmark of endometriosis, but the underlying mechanism(s) for loss of P sensitivity leading to lesion establishment remains poorly understood.ObjectiveTo evaluate the association between Notch-1 signaling activation and P resistance in the progression of endometriosis.DesignCase control study; archived formalin-fixed, paraffin-embedded tissues.SettingUniversity hospitals (United States, Taiwan).PatientsWomen with endometriosis; human endometrial stromal cell line (HESC).InterventionEutopic endometria (EU) and ectopic lesions (ECs) were collected from surgically diagnosed patients. Archived tissue sections of EU and ECs were identified. HESCs were treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and valproic acid (VPA) to, respectively, suppress and induce Notch-1 activation.Outcome MeasuresTissues were analyzed for Notch Intra-Cellular Domain 1 (NICD1) and progesterone receptor (PGR) protein expression by immunohistochemistry and for transcript levels of NICD1 target genes HES1, PGR, and PGR-B by quantitative reverse transcription polymerase chain reaction. DAPT- or VPA-treated HESCs with and without P cotreatment were evaluated for cell numbers and for PGR, HES1, and PGR target gene DKK1 transcript levels.ResultsNuclear-localized stromal NICD1 protein levels were inversely associated with those of total PGR in EU and ECs. Stromal ECs displayed higher HES1 and lower total PGR and PGR-B transcript levels than EU. In HESCs, DAPT reduction of NICD1 decreased cell numbers and increased PGR transcript and nuclear PGR protein levels and, with P cotreatment, maintained P sensitivity. Conversely, VPA induction of NICD1 decreased PGR transcript levels and, with P cotreatment, abrogated P-induced DKK1 and maintained HES1 transcript levels.ConclusionsAberrant Notch-1 activation is associated with decreased PGR that contributes to P resistance in endometriosis.

Evaluation of the HER2 and Hormone Receptor Status in Metastatic Breast Cancer Using Cell Blocks: A Multi-Institutional Study

Objective: We explore the problems associated with the cell block (CB) method for receptor analysis in breast cancer metastases and propose a method for reporting the results. Study Design: Nine institutions used the CB method for the analysis of hormone receptors (HRs) and HER2 (human epidermal growth factor receptor 2) protein in cytological specimens of breast cancer metastases in routine practice. The stained slides were independently evaluated by 8 pathologists. Dual in situ hybridization assay was performed in cases of discordant results for HER2 protein. Based on the results, we propose a method for receptor scoring in the CB method. Results: Of 61 specimens, 57 contained tumor cells. Two or more pathologists disagreed on the results for the estrogen receptor, progesterone receptor, and HER2 protein in 3 (5.3%), 13 (22.8%), and 19 (33.3%) cases, respectively. The discrepant results for the HRs were attributed to the presence of a few positive cells or faintly stained cells. The high interobserver discordance rate for HER2 protein was explained by interobserver differences in the scoring criteria. Conclusion: The use of categorical scoring into positive and negative is recommended for evaluating the HR expressions. Use of strict criteria for HER2 protein 2+ and 3+ cases is recommended, as HER2-positive cases should not be missed.

MiR-194-3p Represses the Progesterone Receptor and Decidualization in Eutopic Endometrium From Women With Endometriosis.

Progesterone resistance in the eutopic endometrium (EuE) is suggested to be a critical factor for decreased endometrial receptivity and implantation failure in reproductive-aged women with endometriosis. Altered expression of miRNAs has been reported to play an important role in the pathophysiology of endometriosis-associated infertility. However, the underlying mechanisms of aberrant progesterone receptor (PR) and deficient decidualization regulated by miRNAs in endometriosis have not been thoroughly elucidated. The goal of this study was to explore the regulation and roles of miR-194-3p in aberrant PR expression and impaired decidualization in endometrial stromal cells (ESCs) from the EuE of women with mild or minimal endometriosis. Using a series of studies, we observed decreased PR mRNA expression and an increasing PR-A/PR-B mRNA ratio trend in the midsecretory phase of the EuE of women with minimal or mild endometriosis (n = 19) compared with controls (n = 14); the increased expression of miR-194-3p in the endometriosis group was consistent with previous microarray analysis. We also found that PR protein levels were inhibited by the transfection of ESCs with an miR-194-3p mimic and upregulated by miR-194-3p inhibition. As predicted by the bioinformatic analysis, the 3'-untranslated region luciferase assay indicated the direct regulation of PR expression by miR-194-3p. Furthermore, miR-194-3p overexpression inhibited the in vitro decidualization of ESCs via both cellular morphological changes and prolactin levels. Therefore, our study demonstrated that miR-194-3p contributes to progesterone resistance in endometriosis, which hinders fertility by repressing the levels of PR and decidualization in the EuE. Thus, miR-194-3p regulation is a future therapeutic strategy for endometriosis.