Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen­induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL­dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro­apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro­apoptotic action in an antiestrogen­resistant breast cancer cell model. In addition, the present study identified a pro­survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen­resistant breast cancer cells survive EGFR targeting by pro­survival autophagy. These pre­clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro­survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL­dependent apoptosis.

New Generation of Meso and Antiprogestins (SPRMs) into the Osteoporosis Approach.

Receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) play key roles in bone metabolism and the immune system. The RANK/RANKL complex has also been shown to be critical in the formation of mammary epithelia cells. The female hormones estradiol and progesterone closely control the action of RANKL with RANK. Blood concentration of these sex hormones in the postmenopausal period leads to an increase in RANK/RANKL signaling and are a major cause of women's osteoporosis, characterized by altered bone mineralization. Knowledge of the biochemical relationships between hormones and RANK/RANKL signaling provides the opportunity to design novel therapeutic agents to inhibit bone loss, based on the anti-RANKL treatment and inhibition of its interaction with the RANK receptor. The new generation of both anti- and mesoprogestins that inhibit the NF-κB-cyclin D1 axis and blocks the binding of RANKL to RANK can be considered as a potential source of new RANK receptor ligands with anti-RANKL function, which may provide a new perspective into osteoporosis treatment itself as well as limit the osteoporosis rise during breast cancer metastasis to the bone.

Severe mammary fibroepithelial hyperplasia due to single injection of medroxyprogesterone acetate in two male cats / Hiperplasia fibroadenomatosa mamária felina secundária à aplicação única de acetato de medroxiprogesterona em dois felinos machos

ABSTRACT: Thirty and forty days after a 50 mg medroxyprogesterone acetate injection, respectively, two mixed-breed, 7 and 8-month-old entire male cats presented diffuse enlargement of thoracic and abdominal mammary glands, with ulceration, abscessation and necrosis. One patient was treated with 10 mg/kg aglepristone, antibiotic therapy, analgesic and non-steroidal anti-inflammatory compound; however a worse enlargement of mammary glands, necrosis and clinical condition was noted two days after antiprogestin injection. The second patient was submitted to surgical procedure without previous medical treatment. A partial bilateral mastectomy and conventional orchiectomy were performed, and both patients presented no clinical abnormalities 10 days after surgical treatment. In the male cat, the interruption of progesterone associated mammary fibroepithelial hyperplasia cannot be based in gonadectomy, being antiprogestin treatment the primary approach. Mastectomy can be a treatment option in selected cases, such as the two cases presented here, in case of antiprogestin treatment failure or in case of extensive ulceration, necrosis and risk of sepsis.

RESUMO: Trinta e quarenta dias após aplicação de acetato de medroxiprogesterona, dois gatos SRD machos não castrados, de sete e oito meses respectivamente, apresentaram aumento difuso de volume em glândulas mamárias torácicas e abdominais, com ulceração, abscedação e necrose. Um paciente foi tratado com 10 mg/kg de aglepristone, terapia antimicrobiana, analgésicos e anti-inflamatório não-esteroidal. Entretanto, apresentou piora dos sinais clínicos, da abscedação e necrose dois dias após a aplicação. O outro paciente foi submetido a procedimento cirúrgico sem tratamento clínico prévio. Mastectomia parcial bilateral e orquiectomia convencional foram realizadas e, ambos os pacientes se apresentaram sem alterações clínicas e receberam alta após 10 dias do procedimento cirúrgico. No felino macho, a interrupção da ação da progesterona associada à ocorrência de hiperplasia fibroadenomatosa mamária, não pode ser obtida por meio de gonadectomia, portanto, o tratamento de eleição deve ser realizado com utilização de antiprogestágeno. Em alguns casos, mastectomia parcial ou total é recomendada quando o tratamento clínico com antiprogestágeno falha ou, em casos de sepse, ulceração extensa e necrose, como nos pacientes deste relato.

Increased High Molecular Weight FGF2 in Endocrine-Resistant Breast Cancer.

Endocrine resistance may develop as a consequence of enhanced growth factor signaling. Fibroblast growth factor 2 (FGF2) consists of a low and several high molecular weight forms (HMW-FGF2). We previously demonstrated that antiprogestin-resistant mammary carcinomas display lower levels of progesterone receptor A isoforms (PRA) than B isoforms (PRB). Our aim was to evaluate the role of FGF2 isoforms in breast cancer progression. We evaluated FGF2 expression, cell proliferation, and pathway activation in models with different PRA/PRB ratios. We performed lentiviral infections of different FGF2 isoforms using the human hormone-responsive T47D-YA cells, engineered to only express PRA, and evaluated tumor growth, metastatic dissemination, and endocrine responsiveness. We assessed FGF2 expression and localization in 81 human breast cancer samples. Antiprogestin-resistant experimental mammary carcinomas with low PRA/PRB ratios and T47D-YB cells, which only express PRB, displayed higher levels of HMW-FGF2 than responsive variants. HMW-FGF2 overexpression in T47D-YA cells induced increased tumor growth, lung metastasis, and antiprogestin resistance compared to control tumors. In human breast carcinomas categorized by their PRA/PRB ratio, we found nuclear FGF2 expression in 55.6% of tumor cells. No differences were found between nuclear FGF2 expression and Ki67 proliferation index, tumor stage, or tumor grade. In low-grade tumor samples, moderate to high nuclear FGF2 levels were associated to carcinomas with low PRA/PRB ratio. In conclusion, we show that HMW-FGF2 isoforms are PRB targets which confer endocrine resistance and are localized in the nuclei of breast cancer samples. Hence, targeting intracellular FGF2 may contribute to overcome tumor progression.

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs.

BACKGROUND: Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. METHODS: To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. RESULTS: Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. CONCLUSIONS: We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Development of Recombinant HSV-Based Vaccine Vectors.

Herpes simplex virus (HSV) causes significant morbidity on the human population through such clinical syndromes as cold sores, genital herpes, herpes stromal keratitis, and encephalitis. Attempts to generate efficacious vaccines to date have failed. We have recently described the use of a conditionally replication-competent HSV-1 vector to immunize mice against a lethal challenge of HSV-1. The unique feature of this vaccine vector is that its replication is tightly controlled and can only occur in the presence of local heat and the presence of a small molecule inducer (an antiprogestin). This gives it the safety advantage of a replication-defective vaccine vector as well as the advantage of a replication-competent vector in that it is able to stimulate innate and adaptive aspects of the immune response in a natural context that a replication-defective vector cannot. In this chapter we provide a brief overview of HSV vaccines followed by the methodology used to propagate and utilize replication-conditional HSV vectors as vaccines.

Estrogen-dependent estrous behavior in rabbits is antagonized by the antiprogestin RU486.

Estrogen receptor-alpha (ERα) dimerizes with unliganded progesterone receptor (PR) in target tissues to trigger genomic and non-genomic effects. In ovariectomized rats the antiprogestin RU486 or antisense nucleotides against PR antagonize estradiol-induced sexual receptivity. We determined the relevance of unliganded PR for the expression of estrogen-dependent scent-marking (chinning) and sexual receptivity by injecting RU486 to: a) ovariectomized (ovx) rabbits given estradiol benzoate (EB; 5µg/day); b) intact rabbits. Chinning and lordosis were quantified in ovx animals before (5days; baseline) and during hormonal treatments: EB+RU486 (20mg/day; n=18) or EB+vehicle (n=18). On treatment day 4 LQ (lordosis quotient) increased in both groups, relative to baseline (mean±se): LQ=1±5 (baseline) vs 25±21 (EB+RU486) and 2±6 (baseline) vs 37±29 (EB+vehicle). On day 9 LQ values were: 22±23 (EB+RU486) and 54±39 (EB+vehicle). Chinning increased only in the EB+vehicle group (day 9=55±46 vs baseline=17±20 marks/10min). In intact rabbits one RU486 injection: reduced the LQ from 72±7to 36±8 five hrs later, increased the latency to receive first ejaculation from 45 to 98s, and decreased the number of ejaculations received in the test from 3 to 2 but did not modify mounting latency or chinning. Results support a participation of unliganded PR for the induction (ovx) and maintenance (intact) of rabbit estrous behavior by estrogens.

Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes.

BACKGROUND: The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. METHODS: We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. RESULTS: TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. CONCLUSIONS: By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.

Progesterone plays a critical role in canine oocyte maturation and fertilization.

Canine oocyte maturation and fertilization take place within the oviducts under increasing plasma levels of progesterone (P4). In order to investigate the role of P4 in these processes, 51 beagle bitches were treated with the P4 receptor antagonist aglepristone at the end of proestrus and 32 females were kept untreated. Fifteen treated and 13 control bitches were inseminated at Days +1 and +2 after ovulation (Day 0). Stages of oocyte maturation and embryo development were determined after ovariectomy at different time points after ovulation. Aglepristone did not prevent ovulation but delayed the resumption of oocyte meiosis and inhibited its progression: first metaphase I (MI) stage was observed at 173 h postovulation and 39% of oocytes reached MII as late as 335 h postovulation in treated females whereas first MI occurred at 76 h and 100% of oocytes were in MII at 109 h postovulation in controls. Aglepristone extended the stay of morphologically normal oocytes within the oviducts: first signs of oocyte degeneration were observed at 335 h in treated versus 100- to 110-h postovulation in control bitches. In inseminated females, aglepristone prevented sperm progression toward the oviducts and fertilization, although motile spermatozoa were observed in the uterine tip flush and within the cranial uterine glands. A proteomic analysis of the tubal fluid from treated and control noninseminated bitches at Day +4 found evidence of 79 differential proteins potentially involved in the oocyte phenotype. In conclusion, P4 plays key roles in postovulatory canine oocyte maturation, aging, and in fertilization.

Development of a technique to detect the activated form of the progesterone receptor and correlation with clinical and histopathological characteristics of endometrioid adenocarcinoma of the uterine corpus.

OBJECTIVE: Hormonal therapy is generally reserved for patients with endometrial cancers that fail cytotoxic chemotherapy, but there is a lack of sufficiently sensitive diagnostics to identify potential responders. We sought to develop a diagnostic technique to detect activated progesterone receptors (APR) in endometrial cancers using routine immunohistochemistry (IHC) and to correlate the presence of APR with other histopathological features and clinical disease stage. METHODS: Seventy-two tumor block specimens from patients with endometrial cancer were processed with conventional IHC methods for estrogen receptor-α (ERα), progesterone receptor (PR) and Ki67, a marker of proliferation. Tumor specimens were analyzed for the PR nuclear distribution patterns in individual tumor cells: APR positive (APR(pos)) tumors were prospectively defined as any tumor with >5% countable malignant cells with an aggregated nuclear pattern. Tumor APR status was analyzed against other biomarkers including ERα expression, Ki67 and tumor grade. RESULTS: Fifty-six of 72 samples were endometrioid. Twenty-six of 49 PR-positive endometrioid tumors (53%; 95% CI 39-67%) were APR(pos). Percent of ER(pos) cells correlated with % PR(pos) malignant cells (p=0.001, rho=0.44). APR positivity did not correlate with % PR(pos) cells in a given tumor, nor did it correlate with % Ki67 positivity; APR positivity was independent of disease stage and tumor grade (p=NS). CONCLUSIONS: In this study, approximately half of endometrioid tumors were APR(pos). APR is independent of histopathological and other known risk factors. Refining conventional PR detection has the potential to prospectively identify patients with endometrial cancer who may benefit from anti-progestin therapy.

Progestin and antiprogestin responsiveness in breast cancer is driven by the PRA/PRB ratio via AIB1 or SMRT recruitment to the CCND1 and MYC promoters.

There is emerging interest in understanding the role of progesterone receptors (PRs) in breast cancer. The aim of this study was to investigate the proliferative effect of progestins and antiprogestins depending on the relative expression of the A (PRA) and B (PRB) isoforms of PR. In mifepristone (MFP)-resistant murine carcinomas antiprogestin responsiveness was restored by re-expressing PRA using demethylating agents and histone deacetylase inhibitors. Consistently, in two human breast cancer xenograft models, one manipulated to overexpress PRA or PRB (IBH-6 cells), and the other expressing only PRA (T47D-YA) or PRB (T47D-YB), MFP selectively inhibited the growth of PRA-overexpressing tumors and stimulated IBH-6-PRB xenograft growth. Furthermore, in cells with high or equimolar PRA/PRB ratios, which are stimulated to proliferate in vitro by progestins, and are inhibited by MFP, MPA increased the interaction between PR and the coactivator AIB1, and MFP favored the interaction between PR and the corepressor SMRT. In a PRB-dominant context in which MFP stimulates and MPA inhibits cell proliferation, the opposite interactions were observed. Chromatin immunoprecipitation assays in T47D cells in the presence of MPA or MFP confirmed the interactions between PR and the coregulators at the CCND1 and MYC promoters. SMRT downregulation by siRNA abolished the inhibitory effect of MFP on MYC expression and cell proliferation. Our results indicate that antiprogestins are therapeutic tools that selectively inhibit PRA-overexpressing tumors by increasing the SMRT/AIB1 balance at the CCND1 and MYC promoters.

Allopregnanolone's attenuation of the lordosis-inhibiting effects of restraint is blocked by the antiprogestin, CDB-4124.

A brief restraint experience reduces lordosis behavior in ovariectomized females that have been hormonally primed with estradiol benzoate. The addition of progesterone to the priming prevents the lordosis inhibition. Based on prior studies with an inhibitor of progesterone metabolism, we have implicated the intracellular progesterone receptor, rather than progesterone metabolites, as responsible for this protection. However, the progesterone metabolite, allopregnanolone (3α-hydroxy-5α-pregnan-20-one), also prevents lordosis inhibition after restraint. In a prior study, we reported that the progestin receptor antagonist, RU486 (11ß-(4-dimethylamino)phenyl-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), attenuated the effect of allopregnanolone. Because RU486 can also block the glucocorticoid receptor, in the current studies, we evaluated the effect of the progestin receptor antagonist, CDB-4124 (17α-acetoxy-21-methoxy-11ß-[4-N,N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione), which is relatively devoid of antiglucocorticoid activity. Ovariectomized, Fischer rats were injected with 10 µg estradiol benzoate. Two days later, rats received either 60 mg/kg CDB-4124 or 20% DMSO/propylene glycol vehicle 1 h before injection with 4 mg/kg allopregnanolone. After a pretest to confirm sexual receptivity, rats were restrained for 5min and immediately tested for sexual behavior. Lordosis behavior was reduced by the restraint and attenuated by allopregnanolone. Pretreatment with CDB-4124 reduced allopregnanolone's effect. These findings support prior suggestions that allopreganolone reduces the response to restraint by mechanisms that require activation of the intracellular progesterone receptor.

Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 2. Gene expression analysis and in vitro activity.

Here, we analyzed the transcriptional effects of the antiprogestin mifepristone (MIF, RU486) and progesterone (P4) in zebrafish as well as their in vitro activities in yeast-based reporter gene assays. This study is associated with the reproduction study in adult zebrafish and embryos exposed for 21 days to 5, 39, 77 ng/L MIF, and 25 ng/L P4 (Blüthgen et al., 2013a). The in vitro activities of MIF and P4 were investigated using a series of recombinant yeast-based assays (YES, YAS, YPS) and compared to transcriptional alterations obtained in fish tissues and embryos from the exposure study. MIF elicited antiestrogenic, androgenic and progestogenic activities in recombinant yeast, similar to P4, and no antiprogestogenic activity in vitro. The transcriptional alterations of steroid hormone receptors were similar in adult males and females, and more pronounced in embryos. MIF tended to transcriptionally down-regulate the androgen (ar), progesterone (pgr) and glucocorticoid (gr) receptors in adult fish and embryos. Transcripts of the estrogen receptor (esr1) and vitellogenin (vtg1) were not significantly altered. A trend for down-regulation was observed for transcripts of genes belonging to steroidogenic enzymes including 17ß-hydroxysteroid dehydrogenase type 3 (hsd17b3), 3 ß-hydroxysteroid dehydrogenase (hsd3b), P450 aromatase A (cyp19a) and 11ß-hydroxylase (cyp11b). P4 resulted in similar transcriptional alterations as MIF. The data indicate that gene expression changes (here and later gene expression is taken as synonym to gene transcription) and in vitro activities match only in part including the lack of antiprogestogenic activity of MIF. Additionally, effects on reproduction and gonad histology described in the associated report (Blüthgen et al., 2013a) can only partly be explained by gene expression data presented here.

Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 1. Reproductive and early developmental effects.

Effects of synthetic progestins have recently been reported in fish, but potential effects of the synthetic antiprogestin mifepristone (MIF), also called RU486, have not been studied. The present study provides first insights into reproductive effects of MIF in zebrafish in comparison to the progesterone receptor agonist, progesterone (P4). We carried out a reproductive study using breeding groups of adult zebrafish. After a 14 day pre-exposure, zebrafish were exposed for 21 days to 5, 39, 77 ng/L MIF, 25 ng/L P4 and water and solvent controls. In addition, embryos originating from exposed adult fish were continuously exposed to 3, 15, 26 ng/L MIF, and 254 ng/L P4, respectively, for 96 h post fertilization. We found a significant U-shaped increase in egg production after exposure to 5 and 77 ng/L MIF, but no effects at 25 ng/L P4. Levels of sex steroid hormones in blood plasma of adult males (11-ketotestosterone) and females (17 ß-estradiol) were not altered. In addition to an increase of mature vitellogenic oocytes in ovaries of females exposed to MIF and P4, we observed several histopathological changes in ovaries, including post-ovulatory follicles, atretic follicles and proteinaceous fluid. Male gonads showed no or less alterations and no histopathological effects. Fertility of eggs and hatching success of embryos (F1 generation) was not affected at 3-26 ng/L MIF and 254 ng/L P4, respectively. The data lead to the conclusion that trace quantities of MIF affect reproduction of zebrafish and ovaries of female zebrafish. Effects on transcriptional changes in adult and embryonic zebrafish of this study in comparison to in vitro effects are reported in the associated report (Blüthgen et al., 2013a).

Randomized phase II study of lonaprisan as second-line therapy for progesterone receptor-positive breast cancer.

BACKGROUND: The progesterone-receptor (PR) antagonists onapristone (type I) and mifepristone (type II) showed modest activity in hormone-receptor-positive breast cancer; however, onapristone in particular was associated with hepatotoxicity. Lonaprisan is a novel, type III PR antagonist that was well tolerated in phase I studies. PATIENTS AND METHODS: This randomized, open-label, phase II study evaluated the efficacy and tolerability of lonaprisan as second-line endocrine therapy in postmenopausal women with stage IV, PR-positive, HER2-negative, metastatic breast cancer. RESULTS: Patients received once-daily lonaprisan 25 mg (n = 34) or 100 mg (n = 34). The primary objective was not met (≥ 35% clinical benefit rate: complete/partial responses at any time until month 6 or stable disease [SD] for ≥ 6 months from start of treatment). There were no complete/partial responses. In the 25 mg and 100 mg groups, 6 of 29 patients (21%) and 2 of 29 patients (7%), respectively, had SD ≥ 6 months. Overall, 61 of 68 patients (90%) had ≥ 1 adverse event (AE), the most frequent (≥ 10% overall) being fatigue, hot flush, dyspnoea, nausea, asthenia, headache, constipation, vomiting, and decreased appetite; 33 patients had serious AEs. CONCLUSION: Lonaprisan showed limited efficacy as second-line endocrine therapy in postmenopausal women with PR-positive metastatic breast cancer.

Synthesis and antiprogestational properties of novel 17-fluorinated steroids.

Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit progesterone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-dependent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211, RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-transactivation assays to establish the antiprogestational activity of these molecules. The representative antiprogestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by a colony formation assay at 1 and 10nM concentrations. The potent in vivo antiprogestational activity of EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may demonstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.

Synergistic lethality of mifepristone and LY294002 in ovarian cancer cells.

We have previously shown that the antiprogestin and antiglucocorticoid mifepristone inhibits the growth of ovarian cancer cells. In this work, we hypothesized that cellular stress caused by mifepristone is limited to cytostasis and that cell killing is avoided as a consequence of the persistent activity of the PI3K/Akt survival pathway.To investigate the role of this pathway in mifepristone-induced growth inhibition, human ovarian cancer cells of various histological subtypes and genetic backgrounds were exposed to cytostatic doses of mifepristone in the presence or absence of the PI3K inhibitor, LY294002. The activation of Akt in ovarian cancer cells, as marked by its phosphorylation on Ser473, was not modified by cytostatic concentrations of mifepristone, but it was blocked upon treatment with LY294002. The combination mifepristone/LY294002, but not the individual drugs, killed ovarian cancer cells via apoptosis, as attested by genomic DNA fragmentation and cleavage of caspase-3, and the concomitant down-regulation of anti-apoptotic proteins Bcl-2 and XIAP. From a pharmacological standpoint, when assessing cell growth inhibition using a median-dose analysis algorithm, the interaction between mifepristone and LY294002 was synergistic. The lethality caused by the combination mifepristone/LY294004 in two dimensional cell cultures was recapitulated in organized, tri-dimensional spheroids. This study demonstrates that mifepristone and LY294002, when used individually, cause cell growth arrest, yet when combined, they cause lethality.