Progesterone Receptor Protein Research Articles

Endometrial Cancer Risk Factors, Hormone Receptors, and Mortality Prediction.

Background: Endometrial tumors arise from a hormonally responsive tissue. Defining subtypes by hormone receptor expression might better inform etiology and prediction of patient outcomes. We evaluated the potential role of tumor estrogen receptor (ER) and progesterone receptor (PR) expression to define endometrial cancer subtypes.Methods: We measured semi-continuous ER and PR protein expression in tissue specimens from 360 endometrial primary tumors from the Nurses' Health Study. To explore the impact of different definitions of marker positivity, we dichotomized ER and PR expression at different cut points in increments of 5% positive cells. Logistic regression was used to estimate associations between endometrial cancer risk factors, such as body mass index, with dichotomous ER or PR status. Reclassification statistics were used to assess whether adding dichotomous ER or PR status to standard prognostic factors of stage, grade, and histologic type would improve endometrial cancer-specific mortality prediction.Results: Compared with not being obese, obesity increased the odds of having an ER-positive tumor at cut points of 0% to 20% [maximum OR, 2.92; 95% confidence interval (CI), 1.34-6.33] as well as the odds of having a PR-positive tumor at cut points of 70% to 90% (maximum OR, 2.53; 95% CI, 1.36-4.68). Adding dichotomous tumor ER or PR status to the panel of standard predictors did not improve both model discrimination and calibration.Conclusions: Obesity may be associated with greater endometrial tumor expression of ER and PR. Adding either marker does not appear to improve mortality prediction beyond the standard predictors.Impact: Body mass index might explain some of the biological variation among endometrial tumors. Cancer Epidemiol Biomarkers Prev; 26(5); 727-35. ©2017 AACR.

Androgen receptor status predicts development of brain metastases in ovarian cancers.

Brain metastases are uncommon localizations in epithelial ovarian cancer (EOC), their reported incidence is increasing and no predictive biomarkers have been identified yet. Goals of this study were: i) to define a possible association between Estrogen Receptor (ER), Progesterone Receptor (PR), Androgen Receptor (AR), human EGF receptor 2 (HER2) and brain progression in EOC patients, and ii) to identify differences in ER, PR, AR and HER2 protein expression from primary EOC and its matched resected brain metastasis. A retrospective series of 11 EOC with matched brain metastasis surgically removed was collected. For comparison, a “Control dataset” of 22 patients, without evidence of brain involvement after an adequate follow up was matched. ER, PR, AR and HER2 status were analyzed by means of immunohistochemistry forCases (both primary and metastatic lesions) and Controls.Univariate analysis showed that AR status was significantly associated with brain localization, both considered as discrete variable (cut-off: 10%, p=0.013) and as continuous one (p=0.035). Multivariate analysis confirmed this trend (p=0.053). When considered as continuous variables, ER and AR showed greater expression in primary tumors in comparison with brain metastases (p=0.013 and p=0.032, respectively).In our series, AR predicts brain involvement, with a 9.5 times higher propensity for AR-negative EOC. Moreover, brain dissemination is probably the result of progressive dedifferentiation of primary tumor, shown by reduction of ER and AR expression in metastases. Further studies are required, in order to anticipate and improve multimodal treatment of brain metastases.

HCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.

How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). N/A. This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.

Expression of β-defensins in bovine oviduct and uterus during follicular and luteal phases

This study was conducted to examine the expression of β-defensins of cow’s reproductive tract and their relationship with estrus cycle using quantitative PCR and ELISA methods. Six kinds of β-defensins and the receptors of estrogen and progesterone were studied in cows’ reproductive tract during the follicular and luteal phases. The results show that besides LAP, TAP, BNBD4, and BNBD5, BNBD1 and BNBD10 were also found in cow oviduct, uterine horn, and uterine body. During the follicular phase, the uterine body had the highest expressions of BNBDI, TAP, LAP1, BNBD4, and BNBD5. However, the highest expression of BNBD10 was observed in the uterine horn at the same phase. The highest expressions of BNBD1, TAP, LAP1, BNBD4, BNBD5, and BNBD10 were observed in the uterine horn during the luteal phase, while the oviduct had the least expression. A significant higher expression of BNBD1, TAP, LAP1, BNBD4, and BNBD5 was observed in the uterine horn in the luteal phase than in the follicular phase. However, BNBD10 expression of the follicular phase was significantly higher than luteal phase. Significant expression of estrogen receptor protein was observed in the uterine body, the oviduct, and the uterine horn during the follicular phase, while a significant progesterone receptor protein (PGR) expression was detected in same portions of the reproductive tract during the luteal phase. BNBD10 was found to perform key function in the uterine horn natural defense process during the follicular phase. The production of bovine uterus defensins did not influenced the expression of estrogen receptor α and progesterone receptor.

Abstract P3-04-10: Utility of the orally bioavailable selective estrogen receptor degrader AZD9496 in ESR1 mutant preclinical models of estrogen receptor positive breast cancer

Abstract Approximately 70% of breast cancers express estrogen receptor alpha (ERα) and anti-hormonal therapies which either block the production of estrogen (e.g. anastrozole) or directly block ERα function (e.g. tamoxifen) remain the mainstay of treatment for these patients. While these therapies are highly effective resistance can occur. A common resistance mechanism to anti-hormonal agents is the mutation of ESR1, the gene that encodes ERα, which leads to ligand independent activity. Evidence is emerging that the selective estrogen receptor degrader (SERD) fulvestrant is effective in patients with ESR1 mutations. However, given the low bioavailability of fulvestrant and the still detectable levels of ER in clinical samples after treatment with fulvestrant it is hypothesised that SERDs with improved pharmacokinetic properties which are able to drive greater degradation of ERα may provide additional clinical benefit. We have previously described the discovery and characterisation of the orally bioavailable SERD AZD9496, which is currently in phase I clinical trials in ER+ breast cancer patients. Here we report the preclinical activity of AZD9496 in cell line and patient derived xenograft models expressing clinically relevant ESR1 mutations. We have engineered MCF-7 cells to express Y537S ESR1 which confers the ability to proliferate in the absence of estradiol, consistent with ligand independent activation of ER signalling. AZD9496 is able to inhibit proliferation of these cells and downregulate progesterone receptor protein expression at low nanomolar concentrations. Furthermore, when MCF-7 Y537S ESR1 cells are implanted as xenografts they grow in the absence of exogenous estradiol, are as sensitive to AZD9496 as parental MCF7 xenografts and demonstrate downregulation of ER dependent biomarkers. AZD9496 also has anti-tumour and pharmacodynamic efficacy in patient derived xenograft models expressing D538G ESR1. Taken together, these data strengthen the body of data suggesting that SERDs may be active in patients with tumours containing ESR1 mutations and supports the inclusion of this patient population in AZD9496 clinical trials. Citation Format: Morrow CJ, Lawson M, Ladd B, de Almeida C, James N, Curwen JO, Hanson L, Bell K, Goeppert A, Fisher D, Gangl E, De Savi C, Scott JS, D'Cruz C, Klinowska T, Weir HM. Utility of the orally bioavailable selective estrogen receptor degrader AZD9496 in ESR1 mutant preclinical models of estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-10.

Can the androgen receptor blocking become the basis of a new method of treatment of triple negative breast cancer?

Triple-negative breast cancer (TNBC) represents approximately 15% –20% of all diagnosed breast cancers. This tumor subtype characterized by the absence of expression of the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor (HER2) protein. Tumor heterogeneity of triple negative breast cancer is the main barrier in the treatment of this tumor subtype. Although estrogen receptor (ER) and human epidermal growth factor receptor (HER2) are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR) is evolving as a molecular target for cancers that have developed resistance to conventional treatments.

Correlation between the Expression of Topo IIα and Ki67 in breast cancer and its clinical Pathological characteristics.

Objective:To investigate the expression of Topo IIα and Ki67 and its clinical significance.Methods:The clinical pathological data of one hundred and sixteen invasive breast cancer patients who were admitted into our hospital from July 2013 to December 2015 and underwent radical mastectomy were retrospectively analyzed. The expression of topoisomerase (Topo) IIα and Ki67 was detected using immunohistochemical method, and the correlation between the two kinds of proteins and the general clinical pathological characteristics of the patients was analyzed.Results:The positive expression rates of Topo IIα and Ki67 in breast cancer were 58.6% and 75% respectively. The expression of Topo IIα was in no apparent correlation with the age, tumor size, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) (P>0.05), but in a correlation with the number of metastatic lymph glands (P<0.05). The expression of Ki67 was in no apparent correlation with the age, tumor size, EP and HER-2, but in a correlation with the number of metastatic lymph glands and PR (P<0.05). The multi-factor logistic regression analysis results suggested that the number of metastatic lymph glands was the independent predictive factor of Topo IIα positive expression and the number of metastatic lymph glands and PR protein expression state are the independent predictive factors of Ki67 positive expression.Conclusion:Topo IIα and Ki67 can be regarded as the indicators for reflecting the proliferation activity of tumor cells, and the detection of Topo IIα and Ki67 expression is of great significance to the prognosis evaluation of breast cancer patients and clinical treatment.

Vertebrate-Type Steroid Profile in Different Tissues of Wild and Endocrinologically Manipulated Female Brood Stocks of &lt;i&gt;Penaeus monodon&lt;/i&gt;

The endocrine mechanism regulating reproduction of tiger shrimp, Penaeus monodon , is a field of topical interest, which is to be addressed in detail as control of reproduction in captivity is conditional for domestication. In this study, the titres of estradiol and progesterone in hemolymph, ovary and hepatopancreas during various reproductive phases of females were quantified using ELISA. Circulating concentration of hemolymph estradiol and progesterone in the endocrinologically-induced (eyestalk ablated) females was also quantified. Additionally, the evidence for estradiol and progesterone receptors was provided using immunohistochemistry. Levels of progesterone in hemolymph and ovary were highest in the vitellogenic and ripe phases (P > 0.05). Conversely, in hepatopancreas significantly higher progesterone level was detected at immature phase (184.3 ± 2.3 pg/ml). Estradiol was highest in hemolymph (65.97 ± 0.8 pg/ml) and ovary (58.47 ± 0.7 pg/ml) at the early vitellogenic phase. In hepatopancreas, however, lowest level of estradiol was detected at the early vitellogenic phase (121 ± 0.38 pg/ml). Progesterone and estradiol receptor protein signals were predominantly found in the pre vitellogenic oocytes. These results suggest the possible involvement of vertebrate-type steroid in the reproductive maturation of P. monodon .

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

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

RNPC1 enhances progesterone receptor functions by regulating its mRNA stability in breast cancer.

Progesterone receptor (PR) could activate transcriptional process involved in normal mammary gland proliferation and breast cancer development. Moreover, PR expression is an important marker of luminal breast cancer, which is associated with good prognosis and indicates better responding to endocrine therapies. The regulation of PR expression was studied mainly on its post-translational levels. In this study, we found PR was positively regulated by RNA-binding region-containing protein 1 (RNPC1), a RNA-binding protein, in PR positive breast cancer. Overexpression of RNPC1 increased, whereas knockdown of RNPC1 decreased, the level of PR protein and transcripts. Additionally, we demonstrated that RNPC1 could bind to PR mRNA via AU-rich elements (AREs) within PR 3′-untranslated region (3′-UTR) and then enhance PR mRNA stability. Moreover, we proved that progesterone-dependent PR functions which could induce breast cancer proliferation were enhanced by RNPC1, both in vitro and in vivo. Conclusively, we revealed a novel mechanism by which PR could be regulated by RNPC1 via stabilizing its mRNA.

MiR-196a overexpression activates the MEK/ERK signal and represses the progesterone receptor and decidualization in eutopic endometrium from women with endometriosis.

Do microRNAs (miRNAs) contribute to aberrant progesterone receptor (PGR) expression in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: miR-196a upregulates MEK/ERK signalling, mediating a downregulation of PGR expression in the eutopic endometrium of women with minimal or mild endometriosis. Implantation failure is strongly suggested as an underlying cause for the observed infertility in minimal or mild endometriosis. Progesterone resistance, which is mainly caused by aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key factor of decreased endometrial receptivity; thus far, epigenetics, but not miRNA, has been shown to affect PGR expression in the endometrium. Microarray analysis was used to analyse the eutopic endometrium. The differential expression of miR-196a was validated. Bioinformatics analysis predicted that miR-196a targets the 3'-untranslated region (UTR) of the PGR. The relationship between the miR-196a level and PGR expression was studied and the role of the MEK/ERK signal pathway was investigated. Total RNA was extracted from eutopic endometrium samples in three infertile women with mild/minimal endometriosis and three disease-free control subjects. The miRNA and mRNA expression levels were analysed by microarray analysis. The miR-196a expression was validated by qRT-PCR [endometriosis (n=22) and control (n=20)], while functional analysis utilised in vitro transfection of endometrial stromal cells (ESCs), induction of decidualization of ESCs, and luciferase reporter assays in 293 T cell lines. A total of 66 dysregulated miRNAs and 357 dysregulated mRNAs were screened by microarray analysis. miR-196a and P-MEK/P-ERK were both found to be significantly upregulated in the eutopic endometrium in patients with mild/minimal endometriosis. PGR and PGR-B mRNA were inhibited by miR-196a overexpression and upregulated by miR-196a inhibition. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR. Transfection of ESCs with a miR-196a mimic led to an increase in the P-MEK/P-ERK protein levels, decrease in the PGR protein levels, and atypical decidualization. Following miR-196a inhibition, the P-MEK/P-ERK protein was downregulated and the PGR protein was upregulated. Inhibition of P-MEK/P-ERK also increased PGR expression. Data are presented in Supplementary Tables SI and SII. This study focused on the role of miR-196a, and therefore does not involve other miRNAs; hence, it is possible that other miRNAs may also be responsible for progestin resistance in endometriosis. Our data revealed altered miRNA expression and activated MEK/ERK signalling in the eutopic endometrium in minimal or mild endometriosis. We showed that the miR-196a level is associated with reduced expression of PGR isoforms through MEK/ERK, suggesting that miR-196a and MEK/ERK are both potential biomarkers of endometriosis. These results provide a novel approach to target the mechanisms behind progesterone resistance in endometriosis. This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.

Factors related to completeness of medical abortion with mifepristone and misoprostol

BackgroundMedical abortion that occurs in early pregnancy is generally safe and successful, but incomplete medical abortion can result in complications. This study aimed to examine factors related to completeness of medical abortion with mifepristone and misoprostol, and then to provide a new direction for research into establishing complete abortion with mifepristone and misoprostol. MethodsSixty-three patients with early pregnancy requesting medical abortion with mifepristone and misoprostol were selected. Immunohistochemistry was used to detect the expression and location of progesterone receptor, estrogen receptor, insulin-like growth factor-1, and vascular endothelial growth factor in chorionic villi among these women. Reverse transcriptase polymerase chain reaction was then used to determine the expression of insulin-like growth factor-1 and vascular endothelial growth factor mRNA. ResultsAccording to the outcome of medical abortion, the women were divided into either the incomplete medical abortion group (n=34) or the complete medical abortion group (n=29). Immunohistochemical analysis showed that progesterone receptor and estrogen receptor protein expression was not detected in chorionic villi in the two groups. However, compared with the complete abortion group, there was a marked decrease in the expression of insulin-like growth factor-1 and a significant increase in the expression of vascular endothelial growth factor (p<0.05) in the incomplete abortion group. There was no significant difference in mRNA expression between the incomplete and complete abortion groups. ConclusionThe expression of insulin-like growth factor 1 protein and vascular endothelial growth factor protein in chorionic villi may be related to the outcome of medical abortion with mifepristone and misoprostol.

Expression of progesterone receptor in the porcine uterus and placenta throughout gestation: correlation with expression of uteroferrin and osteopontin

Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo or fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine and placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR.

Progesterone, estrogen, and androgen receptors in the corpus luteum of the domestic cat, Iberian lynx (Lynx pardinus) and Eurasian lynx (Lynx lynx)

In contrast to the species studied, the corpus luteum (CL) of Iberian and Eurasian lynx physiologically persists in the ovary for more than 2 years and continues to secrete progesterone. Such persistent CL (perCL) transition into the next cycle and are present in the ovary together with the freshly formed CL (frCL) of a new ovulation. To date, the mechanisms supporting such CL persistence are not known. We analyzed the potential receptivity of feline CL to sex steroids through mRNA measurements of progesterone receptor (PGR), progesterone receptor membrane components (PGRMC) 1 and 2, estrogen receptor (ESR) 1 and ESR2, G protein–coupled estrogen receptor 1 (GPER1), and androgen receptor (AR). All receptors were present in domestic cat CL during pregnancy and the nonpregnant luteal phase, in frCL and perCL of post-mating Iberian lynx and in perCL of pre-mating Eurasian lynx. Mass spectrometry detected the presence of PGRMC1 protein in frCL and perCL of the Iberian lynx. In both domestic cat and lynx CL, PGR, PGRMC1, and ESR1 proteins were localized in luteal cells by immunohistochemistry. The mRNA levels of PGR, PGRMC1, PGRMC2, ESR1, and AR changed significantly throughout the domestic cat luteal phase. This may indicate involvement of these receptors in the processes of formation, maintenance, and regression of feline CL. In Iberian lynx, expression of PGRMC1, PGRCM2, ESR1, GPER1, and AR was significantly higher in perCL compared with frCL, whereas ESR2 was reversed. High mRNA amounts of these receptors in perCL suggest that physiological persistence of lynx CL may be partly regulated by actions of sex steroids through their nuclear and/or membrane receptors.

Exploration of mechanisms of α-linolenic acid in reducing the growth of oestrogen receptor positive breast cancer cells (MCF-7)

The n-3 fatty acid α-linolenic acid (ALA) inhibits oestrogen receptor (ER) positive MCF-7 cell growth in vitro and in vivo. Our objective was to explore potential mechanisms. MCF-7 cells were treated with 100 µM ALA and 1 nM oestradiol (E2) then analysed for fatty acid profile, mRNA and protein biomarkers compared to an E2-containing control. The independent and combined effects of ALA, ER activator (E2) and antagonist (ICI-182,780, fulvestrant) on cell growth were determined. ALA was incorporated into cells but not further metabolized. ALA modulated the mRNA expression of targets regulated by or that regulate the ER including caveolin-1, survivin and progesterone receptor (PGR). Lower cyclin-D1, PGR, caveolin-1 and higher ERα protein expression were seen in ALA-treated cells. Co-incubation with ICI-182,780 negated the growth-reducing effect of ALA. In conclusion, ALA may reduce MCF-7 growth by modulating ER-related signalling, thus suggesting a plausible mechanism for ALA independent of its metabolites.

Insulin Induced Down Regulation of the Progesterone Receptor Number in Neutrophils in the Synthesis of Maspin in Breast Cancer

Purpose: The binding of either progesterone or insulin to their specific receptors on neutrophils has been reported to stimulate nitric oxide (NO) induced maspin synthesis in these cells. Experiments were carried out to determine the role of progesterone receptor interaction in the nitric oxide induced maspin synthesis in neutrophils that was preincubated with insulin. Methods: progesterone receptor positive (PR+), progesterone receptor negative (PR–) neutrophils were isolated from the blood cancer subjects. Maspin was determined by enzyme linked immunosorbent assay after in vitro translation of maspin mRNA. NO was determined by methemoglobin method. Results: Immunohistochemical studies of progesterone receptor (PR) demonstrated the presence of progesterone receptor in the normal peripheral neutrophils and less in number in PR+ breast cancer neutrophils. In contrast, PR– breast cancer neutrophils lacked the progesterone receptor, suggesting pathophysiological defects in the synthesis of PR protein in peripheral PR– neutrophils. It was also found that as a result of incubation of neutrophils with insulin the binding affinity for progesterone to its receptor in normal neutrophils remained essentially unchanged which demonstrated Kd = 47.619 nM compared to Kd of the binding of progesterone is 46.08 nM in the normal neutrophils that were not pretreated with insulin. The progesterone receptors which were 11.5×1010/cell in the untreated cells was found to be decreased to 8.2×1010/cell (p<0.005, n=6) after the same cell were treated with 200μU of insulin. The reduction of PR number on normal neutrophils due to the pretreatment with insulin resulted in the decreased NO induced maspin synthesis from 2.329 ± 0.012 nM to 1.410 ± 0.002 nM. Decreased PR number in PR+ breast cancer neutrophils due to disease condition and pretreatment with insulin reduced the maspin synthesis from 1.138 ± 0.024 nM to 0.555 ± 0.003 nM compared to normal control. Conclusion: These results suggested that insulin down regulated maspin synthesis in normal and in breast cancer neutrophils by decreasing the progesterone receptor number in both cases.

Abstract P6-03-14: Expression of genes for aromatase inhibitor targets to discriminate invasive lobular from invasive ductal carcinomas of the breast using LCM-procured cells to complement endocrine biomarkers

Abstract Background: The BIG 1-98 trial and later the ABCSG 8 study reported that patients with invasive lobular carcinoma (ILC) exhibited better response to aromatase inhibitors (AIs) compared to those with invasive ductal carcinoma (IDC). Aromatase cytochrome P450 (CYP19) synthesizes estrogen from androgens and is the target of AI's. CYP19 substrates are generated by upstream enzymes including estrone sulfatase (SULT1E1) and 3b-hydroxysteroid dehydrogenase type 1 (HSD3B1). Enzymes of the aromatase pathway have been reported to be expressed in intact tissue biopsies of breast cancer. To learn more about the pathogenic mechanisms that may underlie the survival benefit of ILCs treated with AIs, we analyzed expression levels of key enzymes related to the aromatase pathway in ILC and IDC. Unlike previous studies, we determined gene expression levels directly on pure populations of carcinoma cells procured by laser capture microdissection, eliminating the contribution of non-cancerous cells. Methods: Using an IRB-approved biorepository and database, total RNA was extracted from carcinoma cells of 247 de-identified biopsies to perform microarray analyses of 22,000 genes. Of the 247 samples, 16 were ILC, 13 were low grade IDC, 55 were intermediate grade IDC and 85 were high grade IDC, and 107 of these were hormone receptor positive. CYP19, HSD3B1 and SULT1E1 expression was directly detected in LCM-procured breast carcinoma cells of ILC and of IDC. Expression of other genes generally associated with the aromatase pathway, e.g., NADPH-cytochrome P450 reductase (POR), ATP-binding cassette gene (ABCG2), catechol-o-methyltransferase (COMT) and uridine-5'-diphosphate glucuronosyltransferase (UGT1A3 &amp; UGT1A9) as well as HSD17B2 were assessed with LCM-procured cells. Estrogen receptor (ER) and progesterone receptor (PR) proteins were quantified by radio-ligand binding and EIA, and gene expression was validated by qPCR. Results: Univariate Cox regression analyses indicated that ABCG2, HSD17B2and UGTA3 independently predicted disease free and/or overall survival of breast carcinomas. We found that CYP19 expression in carcinoma cells, as well as SULT1E1, COMT, POR, HSD17B2 and UTG1A3 expression, decreased as either ER or PR protein increased. HSD3B1 appeared to be over-expressed in ILC compared to IDC, however this difference did not approach statistical significance, likely due to the small sample size. No differences were seen in expression levels of CYP19 and SULT1E1 between ILC and IDC. Conclusions: An inverse relationship between CYP19 and ER and PR expression levels was observed and suggests that synthesis of estrogens by breast cancer cells in situ plays a significant role in defining tumor biology. Our results also indicate overexpression of HSD3B1 in ILC, although not statistically significant. This finding suggests that HSD3B1 may be a key contributor to the increased benefit of AI therapy seen in ILC. Collectively our results suggest a comprehensive study is warranted to ascertain the molecular basis for differences in expression of genes directing estrogen synthesis in situ in relationship to AI therapeutic responses of histologic subtypes of breast carcinomas. Citation Format: Sanders MAG, Daniels MW, Wittliff JL. Expression of genes for aromatase inhibitor targets to discriminate invasive lobular from invasive ductal carcinomas of the breast using LCM-procured cells to complement endocrine biomarkers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-14.

Effects of low dose of tibolone on steroid receptors and Bcl-2 on the postmenopausal endometrium.

A prospective randomized controlled trial was conducted to evaluate the effect of low dose of tibolone on the histology, expression of estrogen (ER) and progesterone receptors (PR) and Bcl-2 protein, in endometrium of postmenopausal women. Forty postmenopausal women consented to treatment and were allocated into two groups of 20 women: Group 1 (Control) without hormone replacement therapy (HRT); Group 2 (Tibolone) treatment at the dose of 1.25 mg/day of oral tibolone administered for a 24-week period. The effect on the endometrium was assessed by histology and the apoptosis marker Bcl-2. The immunoexpression of ER and PR were also measured. Tibolone group showed higher expression of ER, PR and Bcl-2 protein in glandular epithelium and stroma compared to control group. Tibolone in a daily dose of 1.25 mg during 24 weeks demonstrated endometrial action that resulted in low proliferation and was shown to lead to atrophic endometrium. It had favorable effects on the postmenopausal endometrium due to its higher immunoexpression of PR and Bcl-2 protein in endometrial glandular epithelium, thereby creating a balance between pro-apoptotic and anti-apoptotic actions.

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

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

Tamoxifen impairs prepubertal mammary development and alters expression of estrogen receptor α (ESR1) and progesterone receptors (PGR)

Research has shown that prepubertal heifers experience allometric mammary growth that is influenced by the ovaries. Our purpose was to determine the role of estrogen in prepubertal mammary gland development. Sixteen Holstein calves were randomly assigned to 1 of 2 treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were administered the antiestrogen tamoxifen (0.3 mg kg1 d1) or placebo from 28 to 120 d of age. At 120 d, calves were euthanized and udders removed. Weight and DNA content of trimmed parenchymal tissue were halved (P ≤ 0.0001) in TAM compared with CON calves. Parenchymal samples from 3 zones of the left rear mammary gland (lower, middle, and outer regions) were processed for immunohistochemical staining for estrogen receptor α (ESR1) and progesterone receptor (PGR), Ki67-positive cells, and 5-bromo-2'-deoxyuridine label retaining cells (LRCs). Overall, neither the percentage nor location within the epithelial tissue layer of either ESR1- or PGR-positive cells was impacted by TAM treatment. However, image analysis indicated a 6.2-fold lower (P = 0.0001) level of ESR1 protein expression in TAM calves. Similarly, messenger RNA expression of ESR1 was also reduced (P = 0.0001) in TAM heifers. In contrast, expression of PGR protein was greater by 43% (P = 0.03) in TAM calves, but messenger RNA expression did not differ between treatments. Overall, TAM calves had a higher (P ≤ 0.03) percentage and density (cells per tissue area) of Ki67-positive cells. Irrespective of treatment, there were also more Ki67-labeled cells in the outer zones of the mammary gland (P ≤ 0.001). We were able to effectively use multispectral imaging to identify positive cells and quantify the expression of ESR1 and PGR protein. We also identified and counted the proportion of label retaining cells (LCR) (putative epithelial stem cells). We noted an overall 2.9-fold greater number of LRCs in TAM heifers and more LRCs in the outer sampling zones. This suggests that a cohort of LCR cells in TAM remained inactivated in comparison with CON heifers, which exhibited markedly increased growth of the mammary parenchymal tissue over the treatment period. These results suggest that the impacts of ovariectomy are partially explained by loss of ESR1 expression and/or estrogen receptor signaling in the prepubertal bovine mammary gland. The significance of mammary expression of PGR in control of prepubertal bovine mammary development remains unresolved.