Synthetic Progestin Medroxyprogesterone Acetate Research Articles

In vitro effects of progesterone and the synthetic progestin medroxyprogesterone acetate on vascular remodeling

In this work we tested the hypothesis whether progesterone (Pg) or the synthetic progestin medroxyprogesterone acetate (MPA) could be involved in the regulation of events involved in vascular remodeling. Results revealed an enhancement in the capillary-like tubes formation induced by both progestogens. Unlike MPA, Pg acts through VEGF, nitric oxide, PI3K and ERK1/2 signaling pathways. However, the MPA effect depends on platelet activation. Under stress conditions, the proangiogenic action of Pg and MPA was sustained. The progestogens exhibit the ability to prevent vascular smooth muscle cells (VSMC) osteogenic transdifferentiation. Besides this antiosteogenic action, on bone cells the progestogens induced osteoblast maturation and mineralization. The mechanism of action of both steroids on vascular and bone cells involves the participation of progesterone receptor. The data presented in this work provide evidence that the progestogens reduce osteogenic-like transdifferentiation of VSMC and promote angiogenesis with a slight different mechanism of action elicited by each steroid.

Biological and clinical impact of imbalanced progesterone receptor isoform ratios in breast cancer.

There is a consensus that progestins and thus their cognate receptor molecules, the progesterone receptors (PR), are essential in the development of the adult mammary gland and regulators of proliferation and lactation. However, a role for natural progestins in breast carcinogenesis remains poorly understood. A hint to that possible role came from studies in which the synthetic progestin medroxyprogesterone acetate was associated with an increased breast cancer risk in women under hormone replacement therapy. However, progestins have been also used for breast cancer treatment and to inhibit the growth of several experimental breast cancer models. More recently, PR have been shown to be regulators of estrogen receptor signaling. With all this information, the question is how can we target PR, and if so, which patients may benefit from such an approach? PR are not single unique molecules. Two main PR isoforms have been characterized, PRA and PRB, that exert different functions and the relative abundance of one isoform respect to the other determines the response of PR agonists and antagonists. Immunohistochemistry with standard antibodies against PR do not discriminate between isoforms. In this review, we summarize the current knowledge on the expression of both PR isoforms in mammary glands, in experimental models of breast cancer and in breast cancer patients, to better understand how the PRA/PRB ratio can be exploited therapeutically to design personalized therapeutic strategies.

Abstract P3-05-03: A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts

Abstract Background: Progesterone (P) is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. P has been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. Progesterone receptors (PRs) are considered positive prognostic indicators yet potential targets for treatment, although the dilemma of positive or negative targeting persists. P can either inhibit or stimulate breast cancer cell growth in the absence of estrogens (E), and usually blocks E mediated growth. Recent studies using breast cancer cell lines have deciphered a mechanism by which P suppresses E dependent growth through modulation of the estrogen receptor (ER) cistrome through a physical ER/PR association. However, the ER/PR association is weak to undetectable in our ER+PR+ breast cancer patient-derived xenografts (PDX). We therefore hypothesize that PR can regulate E-dependent breast cancer growth independent of direct interference of ER transcription. The purpose of this study was to use unbiased genomics and proteomics of breast cancer PDX to uncover additional mechanisms of P repression of breast cancer growth. Methods: These studies used two luminal ER+PR+ PDX (UCD4 and UCD65) that contain high levels of ER and PR (>90%), and where P inhibits E-dependent growth. Tumors were grown in vivo in female NSG mice under continuous placebo, E (17b-estradiol), E plus P, or E plus the synthetic progestin medroxyprogesterone acetate (MPA) for 8-10 weeks. RNAseq, chromatin immunoprecipitation sequencing (ChIPseq), and rapid immunoprecipitation followed by mass spectrometry of endogenous proteins (RIME) were performed to analyze differential transcriptional, cistromic, and protein-protein interactions in E compared to E plus P or MPA treated tumors. Co-immunoprecipitation (co-IP) and ChIP were used to verify results. Results: Both P and MPA potently inhibited E-dependent growth of both tumor lines. Gene expression studies found that both hormones reversed transcription of over one third of the estrogen/ER transcriptome in both tumors. RIME for PR uncovered significant interactions between PR and multiple RNA polymerase III subunits (POLR3). Co-IP using POLR3A and POLR3B confirmed PR associates with the POLR3 holoenzyme. Furthermore, ChIPseq revealed that PR binds to one third of POLR3 regulated tRNA genes. PR also associated with Maf1 in one tumor, a known POLR3 suppressor. Conclusions: Here we describe a novel association of PR with POLR3 in two luminal breast cancer PDX, an interaction not described in breast cancer cell lines. Our data suggest this is a negative regulatory interaction that may occur through recruiting a POLR3 repressor. These data implicate multifaceted P control of E dependent tumor growth; in addition to antagonizing E regulated genes at the transcription level, P can regulate translation though depletion of amino acid carrying tRNAs, thus slowing protein synthesis. Identifying which tumors utilize this growth-suppressive mechanism may pinpoint appropriate candidates for progestin therapy and/or provide a prognostic tool for predicting tumor progression. Citation Format: Finlay-Schultz J, Gillen AE, Brechbuhl HM, Ivie J, Bentley DL, Kabos P, Sartorius CA. A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts [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 P3-05-03.

Abstract A43: Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX)

Abstract Introduction: Progesterone (P4) and progesterone receptors (PR) play elusive roles in breast cancer. Synthetic progestins in hormone replacement therapy increase breast tumorigenesis, yet these same progestins at high doses can be an effective therapy for some advanced breast tumors. The literature supports that progestins can either stimulate or inhibit breast cancer cell growth, and an increasing body of evidence supports P4 promotes expansion of breast cancer stem cells. Interplay between PR and estrogen receptors (ER) is emerging as important in dictating growth and response to treatment. There is historical precedence for PR interference of ER dependent gene transcription; recent reports identify that PR modulate ER DNA binding events. Given the importance of PR as a biomarker and potential therapeutic target, and the uncertainty surrounding its context-dependent actions, we aimed resolve this using breast cancer PDX models. The purpose of this study was to evaluate the effects of natural and synthetic progestins on growth and estrogen-dependent gene expression in luminal breast tumors, and to define contexts that dictate their divergent effects. Experimental procedures: Six luminal breast cancer PDX developed at UCD-AMC were used for these studies. Most profiled as Luminal B subtype. Tumors generally fell into two types: a pure luminal phenotype and a mixed luminal/basal phenotype based on cytokeratin expression. Tumors varied in expression of ER, PR, and androgen receptors (AR) from low (<10% positive cells), moderate (30-50%), to high (>90%). Tumors were grown in vivo under conditions of continuous placebo, 17β-estradiol (estrogen), estrogen plus progesterone (P4), or estrogen plus the synthetic progestin medroxyprogesterone acetate (MPA) for several months and growth parameters monitored. Receptor content was measure by immunohistochemistry and western blots. Gene expression profiles of select tumors were determined using Affymetrix® GeneChip human gene microarrays and/or RNAseq and results analyzed using Partek Genomics Suite™ software, and Metacore™ pathway analysis. Summary: In two pure luminal PDX with high receptor levels (ER, PR, AR) both P4 and MPA significantly inhibited estrogen-dependent growth (growth-inhibited, GI). Two PDX (one pure luminal, one mixed) with moderate ER and low PR and AR showed no significant effect of P4 or MPA on estrogen-dependent growth (growth neutral, GN). One mixed luminal/basal PDX with low ER and PR and moderate AR showed no effect with P4, whereas MPA simulated tumor growth above that with estrogen alone (growth stimulated, GS). Study of one PDX is ongoing. In the GI tumors both P4 and MPA reversed expression of ~30% of estrogen regulated genes. In particular these tumors had high estrogen-dependent expression of the Notch pathway, which was blocked by both P4 and MPA. Progestins did not alter estrogen-dependent genes in the GN or GS tumors, which did not include the Notch pathway. MPA stimulated a unique set of genes in the GS tumor, and was able to mobilize AR to the nucleus whereas P4 did not. Conclusion: Progestins have mixed effects on breast cancer PDX tumor growth. Tumors that have high ER and PR and are highly estrogen-dependent are more likely to exhibit strong growth inhibition by progestins (both P4 and MPA). Tumors with mixed luminal/basal phenotype that have less ER and PR and are moderately estrogen-dependent are less likely to be progestin-inhibited. In tumors with low ER and PR but high AR content MPA can act as an AR agonist to stimulate growth. Tumor-specific downstream estrogen-dependent pathways such as Notch may indicate whether progestins are therapeutic. Additional tumors are needed to confirm these results with the long term goal of developing a molecular assay to define those tumors that may benefit from progestin treatment. Citation Format: Jessica Finlay-Schultz, Heather M. Brechbuhl, Rachel B. Rosen, Austin E. Gillen, Britta M. Jacobsen, Peter Kabos, Carol A. Sartorius. Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A43.

Luteolin inhibits progestin-dependent angiogenesis, stem cell-like characteristics, and growth of human breast cancer xenografts.

PurposeClinical trials and epidemiological evidence have shown that combined estrogen/progestin hormone replacement therapy, but not estrogen therapy alone, increases breast cancer risk in post-menopausal women. Previously we have shown that natural and synthetic progestins, including the widely used synthetic progestin medroxyprogesterone acetate (MPA), increase production of a potent angiogenic factor, vascular endothelial growth factor (VEGF), in human breast cancer cells, potentially providing an explanation for progestin’s mechanism of action. Here, we tested the effects of luteolin (LU), a flavonoid commonly found in fruits and vegetables, on inhibiting progestin-dependent VEGF induction and angiogenesis in human breast cancer cells, inhibiting stem cell-like characteristics, as well as breast cancer cell xenograft tumor growth in vivo and expression of angiogenesis markers.MethodsViability of both T47-D and BT-474 cells was measured using sulforhodamine B assays. Enzyme-linked immunosorbent assays were used to monitor VEGF secretion from breast cancer cells. Progestin-dependent xenograft tumor growth was used to determine LU effects in vivo. CD31 immunohistochemistry was used to determine blood-vessel density in xenograft tumors. CD44 expression, aldehyde dehydrogenase activity, and mammosphere-formation assays were used to monitor stem cell-like characteristics of breast cancer cells.ResultsLuteolin treatment reduced breast cancer cell viability, progestin-dependent VEGF secretion from breast cancer cells, and growth of MPA-dependent human breast cancer cell xenograft tumors in nude mice. LU treatment also decreased xenograft tumor VEGF expression and blood-vessel density. Furthermore, LU blocked MPA-induced acquisition of stem cell-like properties by breast cancer cells.ConclusionsLuteolin effectively blocks progestin-dependent human breast cancer tumor growth and the stem cell-like phenotype in human breast cancer cells.

Synthetic progestins medroxyprogesterone acetate and dydrogesterone and their binary mixtures adversely affect reproduction and lead to histological and transcriptional alterations in zebrafish (Danio rerio).

Medroxyprogesterone acetate (MPA) and dydrogesterone (DDG) are synthetic progestins widely used in human and veterinary medicine. Although aquatic organisms are exposed to them through wastewater and animal farm runoff, very little is known about their effects in the environment. Here we provide a comprehensive analysis of the responses of zebrafish (Danio rerio) to MPA, DDG, and their binary mixtures at measured concentrations between 4.5 and 1663 ng/L. DDG and both mixtures impaired reproductive capacities (egg production) of breeding pairs and led to histological alterations of ovaries and testes and increased gonadosomatic index. Transcriptional analysis of up to 28 genes belonging to different pathways demonstrated alterations in steroid hormone receptors, steroidogenesis enzymes, and specifically, the circadian rhythm genes, in different organs of adult zebrafish and eleuthero-embryos. Alterations occurred even at environmentally relevant concentrations of 4.5-4.8 ng/L MPA, DDG and the mixture in eleuthero-embryos and at 43-89 ng/L in adult zebrafish. Additionally, the mixtures displayed additive effects in most but not all parameters in adults and eleuthero-embryos, suggesting concentration addition. Our data suggest that MPA and DDG and their mixtures induce multiple transcriptional responses at environmentally relevant concentrations and adverse effects on reproduction and gonad histology at higher levels.

Abstract A19: Progesterone receptor signaling inhibits cervical cancer

Abstract Human papillomavirus (HPV) is the major etiological factor for cervical cancer, which remains third most frequent and fourth most deadly malignancy in women worldwide. However, evidence indicates that other non-viral cofactors are also required for the disease. The uterine cervix is sensitive to female hormones, which have been implicated in cervical carcinogenesis. A transgenic mouse model expressing HPV oncogenes E6 and/or E7 is relevant to human cancer. We use this model to study a mechanism of hormone actions in the context of this common malignancy. Estrogen and estrogen receptor α (ERα) are required for the development of cervical cancer in this mouse model. ERα is known to upregulate expression of the progesterone receptor (PR), which, upon activation by its ligands, either promotes or inhibits carcinogenesis depending on the tissue context. In the present study, we demonstrate that progesterone-PR signaling inhibits cervical and vaginal epithelial cell proliferation in a ligand-dependent manner. We also demonstrate that synthetic progestin medroxyprogesterone acetate (MPA) promotes regression of cancers and precancerous lesions in the female lower reproductive tracts (i.e., cervix and vagina). Our results provide the first experimental evidence supporting that the progesterone signaling is inhibitory for cervical cancer in vivo. Our results also suggest that cervical cancer recurs if MPA treatment is ceased. They imply that MPA efficiently get rid of bulk of disease, yet cancer progenitor/stem cells remain. Our mouse model provides a unique opportunity to interrogate mechanism of PR and PR ligands in cervical cancer. Citation Format: Fabiola Mehta, Young A Yoo, Jieun Son, Francesco J. DeMayo, John P. Lydon, Sang-Hyuk Chung. Progesterone receptor signaling inhibits cervical cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A19.

The Contraceptive MPA, Unlike NET, Modulates Expression of Immune Function Genes and Increases HIV-1 Infection in Cervical Tissue Explants and PBMCs

Background: The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN) are widely used in developing countries as injectable contraceptives where disease burden is high. Some studies suggest that MPA unlike NET increases HIV-1 acquisition in women. Whether MPA and NET differentially affect HIV-1 infection and the expression of key genes relevant to HIV-1 acquisition via differential molecular mechanisms is key to understanding choice of progestin contraceptive for HIV-1 prevention. Methods: Regulation of selected genes was investigated in cervical tissue explants and peripheral blood mononuclear cells (PBMCs) by qRT-PCR western blotting and Luminex assays in response to physiologically relevant doses of progestogens. Infection assays were performed in the absence and presence of HIV-1 using HIV-1BAL-RENILLA or HIVpNL4.3 IMCs. The GR specific antagonist RU486 or GR siRNA knockdown were used to determine the role of the GR in modulating ligand-specific effects. Results: In PBMCs MPA like dexamethasone (DEX a GR specific agonist) showed anti-inflammatory effects decreasing pro-inflammatory IL6 IL8 and RANTES levels and increasing anti-inflammatory GILZ gene expression levels while NET and progesterone (P4) did not. In primary cervical tissue explants DEX and MPA repressed IL6 and IL8 and increased GILZ gene expression levels. Differential gene expression by MPA versus NET and P4 were mediated via the GR in PBMCs. Similarly MPA and DEX unlike NET and P4 increased HIV-1 replication in viable PBMCs. In primary cervical explants MPA but not NET increased HIV-1 replication. Conclusions: Collectively the data suggest that NET unlike MPA would be a safer choice of injectable progestin contraceptive in young women in high risk areas for HIV-1 infection. The molecular basis for this choice most likely involves differential effects of MPA as compared to NET and P4 on transcription of immunomodulatory genes due to their differential actions via the ubiquitous GR.

Synthetic gestagens exert differential effects on arterial thrombosis and aortic gene expression in ovariectomized apolipoprotein E-deficient mice.

Combined hormone replacement therapy with oestrogens plus the synthetic progestin medroxyprogesterone acetate (MPA) is associated with an increased risk of thrombosis. However, the mechanisms of this pro-thrombotic effect are largely unknown. The purpose of this study was to: (i) compare the pro-thrombotic effect of MPA with another synthetic progestin, norethisterone acetate (NET-A), (ii) determine if MPA's pro-thrombotic effect can be antagonized by the progesterone and glucocorticoid receptor antagonist mifepristone and (iii) elucidate underlying mechanisms by comparing aortic gene expression after chronic MPA with that after NET-A treatment. Female apolipoprotein E-deficient mice were ovariectomized and treated with placebo, MPA, a combination of MPA + mifepristone or NET-A for 90 days on a Western-type diet. Arterial thrombosis was measured in vivo in a photothrombosis model. Aortic gene expression was analysed using microarrays; GeneOntology and KEGG pathway analyses were conducted. MPA's pro-thrombotic effects were prevented by mifepristone, while NET-A did not affect arterial thrombosis. Aortic gene expression analysis showed, for the first time, that gestagens induce similar effects on a set of genes potentially promoting thrombosis. However, in NET-A-treated mice other genes with potentially anti-thrombotic effects were also affected, which might counterbalance the effects of the pro-thrombotic genes. The pro-thrombotic effects of synthetic progestins appear to be compound-specific, rather than representing a class effect of gestagens. Furthermore, the different thrombotic responses elicited by MPA and NET-A might be attributed to a more balanced, 'homeostatic' gene expression induced in NET-A- as compared with MPA-treated mice.

Progesterone Signaling Inhibits Cervical Carcinogenesis in Mice

Human papillomavirus is the main cause of cervical cancer, yet other nonviral cofactors are also required for the disease. The uterine cervix is a hormone-responsive tissue, and female hormones have been implicated in cervical carcinogenesis. A transgenic mouse model expressing human papillomavirus oncogenes E6 and/or E7 has proven useful to study a mechanism of hormone actions in the context of this common malignancy. Estrogen and estrogen receptor α are required for the development of cervical cancer in this mouse model. Estrogen receptor α is known to up-regulate expression of the progesterone receptor, which, on activation by its ligands, either promotes or inhibits carcinogenesis, depending on the tissue context. Here, we report that progesterone receptor inhibits cervical and vaginal epithelial cell proliferation in a ligand-dependent manner. We also report that synthetic progestin medroxyprogesterone acetate promotes regression of cancers and precancerous lesions in the female lower reproductive tracts (ie, cervix and vagina) in the human papillomavirus transgenic mouse model. Our results provide the first experimental evidence that supports the hypothesis that progesterone signaling is inhibitory for cervical carcinogenesis in vivo.

Effects of Progesterone and Medroxyprogesterone on Actin Remodeling and Neuronal Spine Formation

Sex steroids are important regulators of neuronal cell morphology, and this is critical for gender differences in brain function and dysfunction. Neuronal morphology is controlled by multiprotein complexes including moesin (a member of the ezrin/radixin/moesin family), focal adhesion kinase (FAK), or the Wiskott-Aldrich syndrome protein-family verprolin homologous (WAVE1) protein, controlling dynamic remodeling of the cytoskeleton and cell membrane. We investigated the actions of natural progesterone (P) and of the synthetic progestin medroxyprogesterone acetate (MPA) on actin remodeling, focal adhesion complex formation, and actin branching in rat cortical neurons. Treatment with P and, to a lesser extent, MPA, increases the number and density of dendritic spines. P increases the phosphorylation of moesin, FAK, and WAVE1, and their redistribution toward cell membrane sites where spines are formed. Signaling to moesin is achieved by PR via a Gα/Gβ-dependent signaling to the small GTPase Ras homolog gene family, member A and its related kinase, Rho-associated kinase-2. In parallel, WAVE1 recruitment is triggered by a Gαi/Gβ-dependent signaling of PR to c-Src, FAK, and Rac1 GTPase. Rac1 recruits cyclin-dependent kinase-5, which phosphorylates WAVE1. Silencing of moesin, FAK, or WAVE1 abrogates the increase in dendritic spines induced by progesterone. In all applications, MPA is found to act similar to P, albeit with a lower efficacy. In conclusion, our findings indicate that the control of actin polymerization and branching and focal adhesion complex formation via moesin, FAK, and WAVE1 is a key function of progesterone receptor in neurons, which may be relevant for the regulation of dendritic spine turnover and neuronal plasticity.

Investigating the anti-mineralocorticoid properties of synthetic progestins used in hormone therapy

A more detailed understanding of the affinities and efficacies for transcriptional regulation by the synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) via the mineralocorticoid receptor (MR) is required, to better understand their relative risk profiles. Both MPA and NET-A bind to the MR, although with about 100-fold lower affinities than that of Prog. MPA and NET-A exhibit no agonist activity, but NET-A, unlike MPA, has similar antagonistic efficacy to Prog on the endogenous mineralocorticoid/glucocorticoid response element (MRE/GRE)-containing genes, α-glycolytic protein or orosomucoid-1 (Orm-1) and plasminogen activator inhibitor-1 (PAI-1). This study is the first to show that NET-A, but not MPA, can dissociate between transrepression and transactivation via the MR. Given the relatively low affinity and potency of MPA and NET-A for the MR, our results suggest that these progestins are unlikeley to exert significant effects via the MR at doses used in hormonal therapy. However, considering their relative free concentrations compared to endogenous hormones, the possibility that NET-A may exhibit significant MR antagonist activity, with some possible cardiovascular protective benefits, should not be excluded.

Progestin drives breast cancer growth by inducing p21CIP1 expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2

Cell cycle regulator p21CIP1 has controversial biological effects in breast cancer since in spite of its role as cell cycle inhibitor and promoter of cellular senescence, it also induces cell proliferation and chemoteraphy resistance. We here explored the molecular mechanisms involved in progestin regulation of p21CIP1 expression. We also investigated the biological effects of p21CIP1 in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) upregulates p21CIP1 protein expression via c-Src, signal transducer and activator of transcription 3 (Stat3) and ErbB-2 phosphorylation. Notably, we also found that ErbB-2 nuclear function plays a key role in MPA-induction of p21CIP1 expression. Interestingly, we determined that progestin drives p21CIP1 transcriptional activation via a novel nonclassical transcriptional mechanism in which progesterone receptor is recruited along with Stat3 and ErbB-2 to a Stat3 binding site at p21CIP1 promoter. Our findings revealed that ErbB-2 functions as a coactivator of Stat3 in progestin induction of p21CIP1 transcriptional activation. Furthermore, we demonstrated that blockage of p21CIP1 expression strongly inhibited in vitro and in vivo progestin-induced breast cancer cell proliferation. These results further support the hypothesis that according to cell context and type of stimulus, p21CIP1 is capable of inducing cell cycle progression. Moreover, we provided evidence that Stat3 and nuclear ErbB-2 are key players in progestin-induced p21CIP1 regulation.

Abstract 274: The combined actions of DHT and MPA lead to altered AR signaling in normal and malignant post-menopausal breast epithelial cells

Abstract Consistent with several observational studies examining combined hormone replacement therapy (cHRT: conjugated equine estrogen in conjunction with the synthetic progestin medroxyprogesterone acetate, MPA) in postmenopausal women, a re-analysis of the placebo-controlled randomized Women's Health Initiative clinical trial demonstrated a markedly increased breast cancer risk in newly menopausal women following ≥ 5 years of cHRT (Hazard Ratio, 3.05; 95% Confidence Interval, 1.62-5.70) [1]. We have previously demonstrated that androgen receptor (AR)-mediated effects of MPA impede 5α-dihydrotestosterone (DHT)-induced AR signaling in normal and malignant breast epithelial cells (AACR abstract 2010). The current study aimed to further investigate the biological actions of DHT and/or MPA on steroid receptor expression and cancer-related intracellular signaling pathways. Immunohistochemical analysis of estrogen receptor alpha (ERα), progesterone receptor (PR) and AR expression was performed on histologically normal human post-menopausal breast tissues and measured by image analysis in tissues cultured ex vivo with vehicle (0.1% ethanol control), DHT (1nM), MPA (1nM) or the AR antagonist, bicalutamide (Bic;1uM) for 48 hr, either alone or in combination. Microarray analysis and qRT-PCR validation were performed using the ERα positive breast cancer cell line, ZR-75-1 to determine changes in gene expression in key intracellular signaling pathways. The microarray data was analyzed with Ingenuity Gene Pathway Analysis and Gene Ontology software. Statistical tests included both Wilcoxon matched pairs test and one-way ANOVA (p<0.05). DHT treatment increased AR expression in cultured breast tissues compared to vehicle control (p<0.05), and co-treatment with either MPA or Bic impeded this effect. No change in ERα or PR protein levels was induced by the hormone treatments. Microarray studies revealed that DHT or MPA treatment for 6 hr altered the expression of 439 and 858 genes, respectively, whereas co-treatment altered 1494 genes (p<0.05). Only 114 genes were uniquely regulated by DHT, and the expression of 32% (41% induced and 27% repressed) of these genes was abrogated by MPA. Similarly, the expression of 38% (51% induced and 24% repressed) of the 690 genes uniquely regulated by co-treatment with DHT and MPA was altered compared to DHT alone (p<0.05). Examples of genes that were regulated by DHT (p<0.05) and this effect of DHT was antagonised by co-treatment with DHT and MPA (p<0.05) are FGFR2, OLR1 and C1ORF116. Co-treatment with DHT and MPA altered the expression of genes involved in cell growth, cell cycle, cell death, cancer and intracellular signaling pathways compared to individual treatments (p<0.05). Collectively, these findings suggest an AR-mediated mechanism for the action of MPA in breast cancer. 1.Prentice, R.L., et al. Am J Epidemiol, 2009 170(1): 12-23. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 274. doi:1538-7445.AM2012-274

Abstract 2305: miR-16 is a tumor suppressor in progestin-induced breast cancer

Abstract Breast cancer is the most common type of cancer among women in the US and the second leading cause of death associated to cancer. microRNA (miRNA) are short ribonucleic acids with important regulatory functions and with an increasingly acknowledged role in cancer. We recently found that progestins modulate miRNA expression in a progestin-dependent murine breast cancer model. In particular, miR-16 was found to be downregulated by progestins. We here demonstrate that the treatment of T47D and BT-474 human breast cancer cells with the synthetic progestin medroxyprogesterone acetate (MPA) results in a significant miR-16 decrease and a concomitant cell proliferation augment. Interestingly, MPA treatment of T47D-Y cells, a variant of T47D lacking progesterone receptor (PR) expression, did not modify miR-16 levels. Furthermore, transfection of T47D-Y with PR-mPRO, a mutant PR which lacks the ability to activate nongenomic signaling pathways (but retains the transcriptional activity of PR), or with PR-C587A, a variant with the capacity to activate nongenomic pathways but uncapable of binding to DNA, did not recover the ability of progestins to downregulate miR-16, suggesting that both PR actions are necessary for miR-16 dowregulation by progestins. In addition, MPA induced a potent increase in the levels of the oncogenic transcription factor c-Myc, a previously reported suppressor of miR-16. Silencing of the signal transducer and activator of transcription 3 (Stat3) by siRNA, abrogated MPA capacity to induce c-Myc expression. Cyclin E, a cell cycle promoter with a major role in breast cancer progression, has been shown to be a miR-16 target. In this study, we not only demonstrate that MPA induces Cyclin E upregulation (both at the mRNA and protein level), but also that this can be prevented by the inhibition of Stat3 and/or c-Myc. We then wondered whether this regulation would occur in vivo. Thus, we injected 2 x 107 BT-474 cells in nude mice carrying a 17α-Estradiol (E2) pellet, in the absence of matrigel. As previously reported, the tumors first grew but started to regress soon afterwards. At that time point (7 days post-injection), half of the animals were administered MPA. The tumors in the group treated with MPA started to grow at a higher rate than the group supplied with E2 alone. Seven days after MPA supply, we excised some of the tumors and measured miR-16 levels, which showed that the treatment with MPA in vivo, produced a profound downregulation of miR-16. Furthermore, these tumors exhibited a higher level of cyclin E by western blot, validating cyclin E as a target of miR-16 in vivo. Our results are the first ones to identify miR-16 as a tumor suppressor modulated by progestins in human breast cancer cells, demonstrating as well that this regulation is relevant for the proliferative biological effect of progestins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2305. doi:1538-7445.AM2012-2305

Histological Characteristics of Thymus Assessed with Stereological Parameters after Medroxyprogesterone Acetate Application

Aim: The purpose of this research was to examine the effect of the synthetic progestin medroxyprogesterone acetate (MPA) on the morphological characteristics of the thymus. Material and Methods: A total of 24 female Wistar rats were divided into two groups. The control group received saline, and the second group was administered medroxyprogesterone acetate in the therapeutic dose of 30 mg/kg bw. The substances were applied daily, intramuscularly for a period of 7 days. Paraffin sections of thymus were dyed with the following methods: hematoxylin - eosin and elastica Van Gieson. Results: Histological analysis of the samples obtained after the application of MPA, showed a reduction in the thymic parenchyma and increasing of the stroma. Stereological analysis and statistical data processing (Student t-test) showed that the volume density (% per mm3 tissue) of thymic parenchyma was 89.94 ± 0.85% (average value ± standard deviation) in the control group of rats, while significantly decreased to 18.07 ± 2.20% in the group of rats treated with MPA (p <0.01). It was due to significant reduction of the cortex (from 71.37 ± 1.33% to 11.81 ± 1.31%, p <0.01) and significant reduction of the medulla (from 18.56 ± 1.07% to 6.23 ± 0.93%, p <0.01). To account thymic parenchyma, intense presence of increased stroma and its bulk density of 10.06 ± 0.85% in control group of rats significantly increased to 81.93 ± 2.20% after the treatment with MPA. Conclusion: The results showed that major morphological characteristic of the thymus is the atrophy of its parenchyma after the application of medroxyprogesterone acetate.

Cognitive-impairing effects of medroxyprogesterone acetate in the rat: independent and interactive effects across time.

The synthetic progestin medroxyprogesterone acetate (MPA), widely used in hormone therapy (HT) and as the contraceptive Depo Provera, is implicated in detrimental cognitive effects in women. Recent evidence in aged ovariectomized (Ovx) rodents shows that short-term MPA treatment impairs cognition and alters the GABAergic system. Using rats, we evaluated the long-lasting cognitive and GABAergic effects of MPA administered in young adulthood (Early-MPA), modeling contraception, and how this early exposure interacts with later MPA treatment (Late-MPA), modeling HT. Early-MPA treatment involved weekly anti-ovulatory MPA injections (3.5 mg) from 4 to 8 months of age in ovary-intact rats. At 10 months old, rats were Ovx and weekly MPA injections were re-initiated and continued throughout testing for Late-MPA treatment. On the water radial-arm maze, all MPA-treated groups showed working memory impairment compared to Controls (p < 0.05); Early + Late-MPA rats were impaired on multiple dimensions of working memory (p < 0.05). On the Morris maze, Late-MPA rats showed greater overnight forgetting compared to Controls (p < 0.05). At study conclusion, MPA was detected in serum in all MPA-treated groups except Early-MPA, confirming treatment and clearance from serum in Early-MPA rats. In animals with detectable serum MPA, higher MPA levels were associated with less dorsal-hippocampal glutamic acid decarboxylase, the synthesizing enzyme for GABA (p = 0.0059). Findings suggest that MPA treatment leads to long-lasting cognitive impairments in the rodent, even in the absence of circulating MPA in animals given prior MPA treatment, which may relate to the GABAergic system. Further research defining the parameters of the negative impact of this widely used progestin on brain and cognition is warranted.

Abstract 2174: Progesterone receptor, AP-1 and Stat3 cooperative transcriptional complex in breast cancer

Abstract We have previously demonstrated that progestin induces the rapid activation of AP-1 transcription factor and its nuclear interaction with the progesterone receptor (PR) by tethering in C4HD cells an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice and in the human breast cancer line T47D. In this work, we demonstrated that MPA induces AP-1 transcriptional activation through the activation of MAPKs signaling pathways. Our recent findings have demonstrated that MPA induces the recruitment of Stat3 and PR to the GAS site in the cyclin D1 promoter. Here, we identified a novel PR/AP-1/Stat3 transcriptional complex in breast cancer cells. Cyclin D1 is a cancer-related protein whose promoter contains AP-1 binding sites (TRE sites) and Stat3 binding sites (GAS sites) in a very close proximity and lacks progesterone response elements (PRE sites). By using quantitative chromatin immunoprecipitation (ChIP) and sequential ChIP, we demonstrated that progestin induces the recruitment of c-jun, c-fos and PR to the TRE site and this recruitment is abolished when we pretreat the cells with the MAPKs inhibitor U0126. When cells were transfected with c-jun and c-fos Dominant Negatives Forms (TAM-67 and A-Fos respectively) none of these proteins are able to activate cyclin D1 promoter or are recruited to the TRE site and there is not MPA-induced Stat3 recruitment to the GAS site. In the same way when we pre-treat the cells with siRNAs against Stat3 neither c-jun, c-fos, PR or Stat3 are recruited to the AP-1 or GAS siteS in the cyclin D1 promoter. Inhibition of MPA-induced AP-1 transcriptional activation by transfection of breast cancer cells with TAM-67 and A-Fos resulted in complete abrogation of progestin-induced in vitro and in vivo breast cancer growth. Our findings reveal a novel MPA-induced PR/AP-1/Stat3 cooperative transcriptional complex in breast cancer cells. Interestingly the blockage of any of the members of the complex prevents its assembly and inhibits the MPA-induced cyclin D1 up-regulation and cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2174. doi:10.1158/1538-7445.AM2011-2174

Abstract 2281: GATA3 and progestin interaction in mammary cancer

Abstract GATA3 is a Master Transcription Factor crucial in mammary gland development and differentiation. Due to the role of progesterone in mammary gland development and in the etiology and progression of breast cancer, we explored the regulation of GATA3 by the synthetic progestin medroxyprogesterone acetate (MPA) in breast cancer cells. The experiments were performed in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D. Our results indicate that MPA produces GATA3 downregulation, as measured by Western Blot (WB) experiments, and this effect is abolished by the progestin antagonist RU486. In order to assess MPA regulation of GATA3 mRNA levels we performed Real Time PCR, and observed that MPA induced GATA3 transcriptional downregulation. By performing in silico analysis, we detected that GATA3 aminoacidic sequence contains a PEST sequence, recognized as a site for protein degradation. This finding lead us to explore whether MPA affects GATA3 post-translational regulation. For that purpose we treated T47D cells with cicloheximide, and a reduction of GATA3 half-life was observed with MPA treatment. To evaluate whether the B isoform of Progesterone Receptor (PR-B) was sufficient to maintain GATA3 downregulation, we used T47D – YB cell line, which expresses only PR-B. As assessed by WB analysis, PR-B proved to be sufficient for MPA-induced GATA3 downregulation. Remarkably, MPA treatment of T47D-Y- DBD cells, which express a mutant form of PR that is unable to bind to DNA or to tether with other transcription factors, was able to cause GATA3 downregulation. This result suggests that GATA3 downregulation occurs via PR citoplasmatic signaling. In order to dissect the signaling pathway involved in MPA regulation of GATA3 expression, cells were incubated with U0126 + MPA. We found that the addition of U0126 abrogated GATA3 dowregulation by MPA. This result suggests that ERK 1/2 phosphorylation is necessary to maintain this regulation. However, when Src phosphorylation was prevented, together with Src downstream target ERK 1/2 phosphorylation, GATA3 downregulation persisted. This result points to an off-target effect of U0126 inhibitor, probably preventing ERK 5 phosphorylation, as it was previously reported. Finally, we performed in silico analysis of cyclin D1 gene proximal promoter and found three potential GATA3 binding sites. To assess the effect of GATA3 on cyclin D1 expression, we overexpressed human GATA3 in T47D cells, and measured cyclin D1 levels by WB. Overexpression of GATA3 resulted in lower cyclin D1 levels. This study shows for the first time GATA3 downregulation by MPA in breast cancer cells, both at transcriptional and post-translational levels. It also sheds light over the PR mechanism involved in this process and the signaling pathway potentially implicated. Finally, we demonstrate that GATA3 overexpression affects cyclin D1 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2281. doi:10.1158/1538-7445.AM2011-2281

Novel role of signal transducer and activator of transcription 3 as a progesterone receptor coactivator in breast cancer

Interactions between progesterone receptor (PR) and signal transducer and activator of transcription 3 (Stat3)-mediated signaling pathways have already been described. In the present study, we explored the capacity of Stat3 to functionally interact with progesterone receptor (PR) and modulate PR transcriptional activation in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) induced the association of a PR/Stat3 complex in which Stat3 acts as a coactivator of PR. We demonstrated that Stat3 activation is required for MPA modulation of the endogenous genes bcl-X and p21 CIP1 which are involved in MPA-induced cell cycle regulation. Stat3 activity as a coactivator of PR was observed in both the classical and nonclassical ligand activated-PR transcriptional mechanisms, since the effects described were identified in the bcl-X promoter which contains a progesterone responsive element and in the p21 CIP1 promoter which carries Sp1 binding sites where PR is recruited via the transcription factor Sp1. The data herein presented identifies a potential therapeutic intervention for PR-positive breast tumors consisting of targeting Stat3 function or PR/Stat3 interaction which will result in the inhibition of PR function.