Agonist R5020 Research Articles

Effects of Xihuang Pills on proliferation and apoptosis of prostate cancer LNCaP cells based on AR/m TOR signaling pathway

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.

Rapid and reagent-free bioassay using autobioluminescent yeasts to detect agonistic and antagonistic activities of bisphenols against rat androgen receptor and progesterone receptor

Bisphenol A (BPA) and its analogues have been classified as endocrine disruptors via binding to nuclear receptors. Two novel bioassays, BLYrARS and BLYrPRS, were developed for rapid detection of agonistic and antagonistic activities of BPA and five of its analogues binding rat androgen receptor (rAR) and rat progesterone receptor (rPR). The reporter bioassay was based on two autonomously bioluminescent strains of the yeast Saccharomyces cerevisiae, recombined with a bacterial luciferase reporter gene cassette (lux) that can produce autofluorescence, regulated by the corresponding hormone response element acting as the responsive promoter. The bioluminescent signal is autonomous and continuous without cell lysis or addition of exogenous reagents. The AR agonist R1881 could be detected at 4 h with a half-maximal effective concentration (EC50) of ~9.4 nM. The PR agonist progesterone could be determined at 4 h with an EC50 of ~2.74 nM. None of the sixteen bisphenols presented agonistic activities against rAR and rPR. However, thirteen BPs were rAR antagonists and eleven BPs acted as rPR antagonists with different potency. The BLYrARS and BLYrPRS bioassay characterized by automated signal acquisition without additional manipulations or cost can be applied for simple and rapid detection of agonistic and antagonistic activities of BPs and other compounds acting as agonists or antagonists of rAR and rPR. Based on data derived by use of this bioassay endocrine-disrupting activities of some BPA analogues are more potent than BPA.

Abstract 743: Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer

Abstract Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies display intrinsic or acquired therapy resistance. One-third of patients with acquired resistance present with metastatic tumors containing ERα-Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. Previous research suggests that ERα and the progesterone receptor (PR) engage in complex interactions involving reciprocal regulation of ERα and PR transcription factor activity known as ERα/PR crosstalk. My central hypothesis is that ERα-Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. While previous research has assessed the impact of the ERα-Y537S mutation on ERα-associated chromatin binding and gene expression, here I assess the effect of the resistance-associated ERα-Y537S mutation on ERα and PR activity and crosstalk. Simultaneous analysis of transcription factor binding activity (ChIP-seq), gene expression (RNA-seq), and proximity-based interaction (NanoBRET) indicates that active PR binding across the genome is increased in the context of ERα-Y537S, suggesting that ERα/PR crosstalk is significantly altered. Importantly, PR chromatin binding in the context of ERα-Y537S appears to be elevated regardless of the stimulation of PR activity with the agonist R5020. These findings, along with identification of candidate genes of potential therapeutic value, will be applied using patient-derived tumor organoids to determine the efficacy of simultaneously targeting ERα and PR to overcome endocrine therapy resistance in breast cancer. Citation Format: Rosemary J. Huggins. Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 743.

Identification of retinoid acid induced 16 as a novel androgen receptor target in prostate cancer cells

BackgroundRetinoid acid induced 16 (RAI16) was reported to enhance tumorigenesis in hepatocellular carcinoma (HCC). The androgen receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in several cancer progressions. However, whether RAI16 is a candidate AR target gene that may involve in prostate cancer progression was unclear. Materials & methodsRAI16 expression was detected in prostate cancer cells with or without the AR agonist R1881 treatment by quantitative RT-PCR and Western blot. Direct AR binding to the RAI16 promoter was tested using AR chromatin immunoprecipitation (ChIP) and luciferase assay. Cell viability and colony formation assays in response to R1881 were analyzed in cells with RAI16 knockdown by specific siRNA. ResultsThe expression of RAI16 was high in LNCaP(AI), LNCaP(AD), C4-2 expressing AR, but low in Du145 and Pc-3 cells without AR expressing. In addition, the expression of RAI16 could be induced by 10 nM R1881 treatment LNCaP(AD) and C4-2 cells, but inhibited by AR specific siRNA treatment. Furthermore, AR binds directly to ARE3 (−2003~-1982bp) of RAI16 promoter region by ChIP and luciferase assay. RAI16 knockdown inhibited the enhancement of cell viability and colony formation of AR stimulation. ConclusionsWe demonstrate for the first time that RAI16 is a direct target gene of AR. RAI16 may involved in cell growth of prostate cancer cells in response to AR signaling.

Membrane Progesterone Receptors (mPRs/PAQRs) Differently Regulate Migration, Proliferation, and Differentiation in Rat Schwann Cells.

Several studies in the last decade demonstrated that progestogens play an important role in the biology of Schwann cells, the main neuroglial cells of the peripheral nervous system. Since a recent study showed that the S42 rat Schwann cell line expressed membrane progesterone receptors (mPRs), members of the PAQR family, in this study, we examined mPR expression in a more physiological model, primary rat Schwann cells. We demonstrated that primary rat Schwann cells show a different pattern of mPR expression compared to the previously studied model; mPRα (PAQR7) and β (PAQR8) isoforms were the major mPR members identified, with different sub-cellular localizations. Activation of the nuclear progesterone receptor (PR) with the specific agonist R5020 upregulated mPR expression, while activation of mPRs with the specific agonist Org OD 02-0 changed their sub-cellular localization. An in-depth analysis revealed additional effects of mPR activation, such as AKT activation, reduced expression of the myelin-associated glycoprotein (MAG), morphological changes, altered expression of several Schwann cell differentiation markers, and increased Schwann cell migration and proliferation. In conclusion, we identified mPRα and mPRβ in primary rat Schwann cells, and our findings suggest a putative role for mPRs in the regulation of Schwann cell migration, proliferation, and differentiation. Therefore, mPRs are a potential pharmacological target for Schwann cell-related disorders and neurodegenerative diseases, especially those in which Schwann cell-mediated axon remyelination is desirable.

Ethanolic extract of Algerian propolis decreases androgen receptor transcriptional activity in cultured LNCaP cells

Antiandrogens have a peculiar place in the treatment of metastatic prostate cancer by blocking the androgen receptor (AR). Unfortunately, aggressive tumors could rapidly develop into a castration resistant state. It is therefore essential to look for new molecules that are more effective, affecting not only the androgen signaling and with minimum undesirable effects. Natural products are an interesting source of new therapeutics, especially for cancer therapy as 70% of them have botanical origin. Based on an ethnobotany screening, we evaluated the effects of ethanolic extract of propolis (EEP) from Algeria on LNCaP cells. Results pointed out that EEP reduces the survival of LNCaP cells with an IC50 of 0.04 mg/ml, induces the apoptosis and blocks the cell cycle at G0/G1 phase. Interestingly, EEP decreased the accumulation of AR suggesting some anti-androgen activity. Indeed, secreted amount of the androgen target protein PSA was decreased when LNCaP cells were incubated with EEP, starting after 4 h of treatment. This anti-androgen activity was also shown on the androgen target genes Fkbp5 and Sgk1. Finally, the capacity of EEP to block AR functioning was demonstrated in transient transfections with human AR and the reporter gene ARE-tk-Luc. Propolis antagonizes the induction of the luciferase activity induced by the natural androgen DHT (10−8M) or the synthetic AR agonist R1881 (10-7M). Altogether, these results highlight the potential pharmacological effects of EEP in future treatments of prostate cancer.

Androgen induces olfactory expression of prostaglandin E2 receptor Ep1 in the burrow-living fish Bostrychus sinensis

It is well documented that androgens modify olfactory processing in vertebrates. In fish, several lines of evidence indicate that androgens increase olfactory sensitivity to prostaglandin pheromone, but the molecular mechanism is still unclear. Our previous studies showed that prostaglandin E2 (PGE2) is a sex pheromone in the burrowing-living fish Chinese black sleeper (Bostrychus sinensis) and that the PGE2 receptor 1 (Ep1) in the olfactory rosette is a candidate receptor for sensing sex pheromone PGE2. In the present study, we found that testosterone (T) and 11-ketotestosterone (11-KT) exhibited stimulatory effects on the expression of ep1 in the olfactory rosette in vivo and ex vivo. Moreover, the androgen receptor (Ar) agonist R1881 had similar effects to 11-KT on the expression of ep1 ex vivo, suggesting the up-regulatory effect is mediated by Ar. The amount of arα transcripts (˜1500 copies/100 ng total RNA) was greater than that of arβ (˜300 copies/100 ng total RNA) in the olfactory rosette, and the expression levels of arα increased with spermatogenesis and peaked at late meiosis stage. Moreover, activated Arα but not Arβ transactivated a 2k bp ep1 promoter in HEK293T cell, and some OSNs exhibited co-localization of arα mRNA and Ep1 protein signals. Taken together, our results suggest that Arα, but not Arβ, plays a crucial role in mediating the androgen-induced up-regulation of ep1 expression in B. sinensis. The present study is the first to shed light on the molecular mechanisms whereby androgens enhance responsiveness to prostaglandin sex pheromones in teleosts.

Progesterone receptor integrates the effects of mutated MED12 and altered DNA methylation to stimulate RANKL expression and stem cell proliferation in uterine leiomyoma

Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.

Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)β1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFβ1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFβ1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFβ1 in the lactotroph population.

Structural Changes Due to Antagonist Binding in Ligand Binding Pocket of Androgen Receptor Elucidated Through Molecular Dynamics Simulations.

When a small molecule binds to the androgen receptor (AR), a conformational change can occur which impacts subsequent binding of co-regulator proteins and DNA. In order to accurately study this mechanism, the scientific community needs a crystal structure of the Wild type AR (WT-AR) ligand binding domain, bound with antagonist. To address this open need, we leveraged molecular docking and molecular dynamics (MD) simulations to construct a structure of the WT-AR ligand binding domain bound with antagonist bicalutamide. The structure of mutant AR (Mut-AR) bound with this same antagonist informed this study. After molecular docking analysis pinpointed the suitable binding orientation of a ligand in AR, the model was further optimized through 1 μs of MD simulations. Using this approach, three molecular systems were studied: (1) WT-AR bound with agonist R1881, (2) WT-AR bound with antagonist bicalutamide, and (3) Mut-AR bound with bicalutamide. Our structures were very similar to the experimentally determined structures of both WT-AR with R1881 and Mut-AR with bicalutamide, demonstrating the trustworthiness of this approach. In our model, when WT-AR is bound with bicalutamide, Val716/Lys720/Gln733, or Met734/Gln738/Glu897 move and thus disturb the positive and negative charge clumps of the AF2 site. This disruption of the AF2 site is key for understanding the impact of antagonist binding on subsequent co-regulator binding. In conclusion, the antagonist induced structural changes in WT-AR detailed in this study will enable further AR research and will facilitate AR targeting drug discovery.

Androgen suppresses protein kinase D1 expression through fibroblast growth factor receptor substrate 2 in prostate cancer cells.

In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment.

Androgen Receptor Overexpression in Mantle Cell Lymphoma: Potential Novel Therapeutic Implications

BACKGROUND: Mantle cell lymphoma (MCL) represents approximately 5-10% of all non-Hodgkin lymphoma (NHL) diagnosed annually in the US and Europe. In contrast to the slight male predominance of other non-Hodgkin lymphomas, most series report that approximately 80% of MCL patients are male. While the pathobiology underlying this gender-distribution imbalance is unknown, sex-related differences in androgen receptor (AR) expression in the hematopoietic system have been described, leading us to hypothesize that the gender imbalance in MCL may be mediated via differential sex-related effects on AR activity. Enzalutamide is a potent inhibitor of the AR axis, binding to the AR with high selectivity, and targeting multiple steps in AR signaling, including inhibition of AR ligand binding, AR nuclear translocation, and AR-mediated DNA binding.OBJECTIVE: To evaluate AR expression in MCL cell lines and human specimens and explore the effect of AR-axis blockade with enzalutamide on MCL tumor cell proliferation.METHODS: MCL cell lines (JVM-13, Maver-1, Jeko-1, Rec-1, JVM-2, Granta and Mino), non-MCL cell lines (SUDHL4, FL18 and Ramos) and prostate cancer cell lines (LNCaP, VCaP, PC3 and Du-145) were obtained from American Type Culture Collection (Rockville, Maryland, USA) and cultured in accordance with supplier recommended methods. Buffy coat specimens from three MCL patients with circulating tumor cells were utilized for RNA isolation and analysis of AR transcript expression. Additionally, formalin fixed samples from nine archival MCL tumor specimens were obtained for immunohistochemistry (IHC) analysis of AR staining. RNA was isolated and prepared for quantitative real time PCR (qRT-PCR) using primers for AR and PSA. All qRT-PCR experiments were performed in triplicate, and the housekeeping gene RPL13A was used as an endogenous control. Proliferation assays were carried out with cells brought up in serum free media, incubated under standard culture conditions for 24 hours, then plated in triplicate with the AR antagonist enzalutamide (10uM), the synthetic AR agonist R1881 (1nM), or the combination for 96 hours. Following the incubation period, cells were transferred to a flat-bottomed 96-well plate and proliferation was quantified using the CyQUANTTM Assay Kit.RESULTS: The median expression of AR transcript was significantly higher in MCL lines than in non-MCL B-NHL lines (normalized to the difference in cycle threshold (dCT) of the housekeeping gene RPL13A). The median dCT for AR was -12 in MCL lines (range -11 to -15) vs. -18 in non-MCL lines (p=0.006; Figure 1A), representing a 64 fold difference in transcript expression. Additionally, the tight correlation between the canonical AR-regulated target gene PSA and AR (r=.715, p=0.001; Figure 1B) suggests that AR expression within these MCL lines is functionally active and capable of driving transactivation of target genes. These findings were also observed in a limited number of frozen, patient-derived MCL tumors; where 2 of 3 samples demonstrated AR and PSA transcripts at levels similar to those observed in the MCL cell lines (Figure 1C). Staining of formalin fixed human MCL tumor cells for AR expression by IHC demonstrated a spectrum of expression; including tumors with no staining, tumors with predominantly cytoplasmic AR staining, and tumors with evidence of scattered nuclear AR staining. Proliferation assays of four different AR+ MCL lines (Granta, Jeko, Maver, Rec-1) with the AR agonist R1881, enzalutamide, or the combination demonstrated somewhat limited stimulation by agonist but consistent and statistically significant suppression of proliferation by enzalutamide (Figure 2). The magnitude of suppression in several of the MCL lines (eg, Granta and Jeko) was comparable to that observed in the AR+ LNCaP PCa cell line.CONCLUSION: Functional AR transcript is overexpressed in both MCL cell lines and human tumor samples. Blockade of the AR-axis by enzalutamide effects consistent and reproducible suppression of MCL tumor growth, even in the presence of AR agonist R1881. These results have led to the conception and recent initiation of an NCCN funded phase II clinical trial evaluating the safety and clinical efficacy of enzalutamide as a novel therapy for patients with relapsed or refractory MCL (NCT02489123). [Display omitted] [Display omitted] DisclosuresGopal:Paid Consultancy- Gilead, Janssen, Seattle Genetics, Spectrum, Research funding- Gilead, Janssen, Pfizer, BMS, Merck, Teva, Takeda, Spectrum, Seattle Genetics: Consultancy, Honoraria, Research Funding.

Silibinin inhibitis progesterone induced RANKL expression in human uterine leiomyoma cells

Silibinin is a natural compound that was reported to inhibit the effects of RANKL; the underlying mechanism however is unclear. We previously demonstrated that progesterone stimulates receptor activator of nuclear factor kappa-B ligand (RANKL) expression in leiomyoma. Here we investigate whether silibinin inhibits progesterone induced RANKL expression, cell cycle proteins and extracellular matrix deposition. Molecular analysis in freshly isolated leiomyoma tissue and cells. Fresh leiomyoma tissue explants (n= 3 patients) were treated with vehicle or silibinin alone, as well as in the presence or absence of synthetic progesterone agonist R5020. Total RNA was extracted and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to asses fold change of RANKL mRNA levels. Next, we isolated leiomyoma smooth muscle cells from fresh fibroid tissues and maintained them in primary culture (n=3). The cells were treated with vehicle or varying doses of silibinin (30, 60, or 90 mM) for 24, 48, or 72 hours (h) respectively. Total RNA was extracted and RT-PCR was used to measure mRNA levels of RANKL, Cyclin D1 (a downstream target of RANKL and a cell cycle protein increased with cell proliferation) and Col1A2 and Col3A1 (markers of extracellular matrix deposition). Treatment of tissue explants with R5020 led to a significant increase in RANKL expression (p<0.001), which was significantly decreased by treatment with silibinin (p<0.01). In primary cells, silibinin reduced RANKL expression in a dose-dependent manner with the most significant change observed at 60 mM (up to 60% decrease, p<0.05). Silibinin significantly decreased cyclin D1, Col1A2 and Col3A1 mRNA levels in a dose-dependent manner by up to 80% change (p<0.05). Inhibition of RANKL, Cyclin D1, Col1A2 and Col3A1 expression by silibinin was observed as early as 24 h, peaked at 48 h and persisted till 72 h of treatment. Silibinin is an effective inhibitor of RANKL expression. Silibinin also decreased proliferative activity and extracellular matrix deposition in leiomyoma smooth muscle cells. Thus, silibinin may serve as a novel medical treatment option with no known side effects for uterine leiomyoma.

The rapid immunosuppression in phytohemagglutinin-activated human T cells is inhibited by the proliferative Ca2+ influx induced by progesterone and analogs

Progesterone, an endogenous immunomodulator, suppresses human T-cell activation during pregnancy. A sustained Ca2 + influx is an important signal for T-cell proliferation after crosslinking of T-cell receptor/CD3 complexes by anti-CD3 antibodies or phytohemagglutinin (PHA). Progesterone targets cell membrane sites inducing rapid responses including elevated intracellular free calcium concentration ([Ca2+]i) and suppressed T-cell PHA-activated proliferation. Interestingly, both PHA and progesterone induce [Ca2+]i elevation, but it remains unclear whether the PHA-induced Ca2+ influx is affected by progesterone leading to T-cell immunosuppression. Primary T-cells were isolated from human peripheral blood and the quench effect on intracellular fura-2 fluorescence of Mn2+ was used to explore the responses to Ca2+ influx with cell proliferation being determined by MTT assay. PHA-stimulated Ca2+ influx was dose-dependently suppressed by progesterone and its agonist R5020, which correlated with PHA-activated T-cell proliferation inhibition. A similar dose-dependent suppression effect on cellular Ca2+ influx and proliferation occurred with the TRPC channel inhibitor BTP2 and selective TRPC3 channel inhibitor Pyr3. In addition, two progesterone analogs, Org OD 02-0 and 20α-hydroxyprogesterone (20α-OHP), also produced dose-dependent suppression of Ca2+ influx, but had no effect on proliferation. Finally, inhibition of PHA-activated T-cell proliferation by progesterone is further suppressed by 20α-OHP, but not by Org OD 02-0. Overall, progesterone and R5020 are able to rapidly decrease PHA-stimulated sustained Ca2+ influx, probably via blockade of TRPC3 channels, which suppresses T-cell proliferation. Taken together, the roles of progesterone and its analogs regarding the rapid response Ca2+ influx need to be further explored in relation to cytokine secretion and proliferation in activated T-cells.

Mifepristone inhibits extracellular matrix formation in uterine leiomyoma

To characterize the efficacy of mifepristone treatment on extracellular matrix (ECM) production in leiomyomas. Laboratory study. University research laboratory. None. Treatment of human immortalized two-dimensional (2D) and three-dimensional (3D) leiomyoma and myometrial cells with mifepristone and the progestin promegestone (R5020). Expression of COL1A1, fibronectin, versican variant V0, and dermatopontin in treated leiomyoma cells by Western blot analysis and confirmatory immunohistochemistry staining of treated 3D cultures. Treatment with progestin stimulated production of COL1A1, fibronectin, versican, and dermatopontin. Mifepristone treatment inhibited protein production of these genes, most notably with versican expression. Combination treatment with both the agonist and antagonist further inhibited protein expression of these genes. Immunohistochemistry performed on 3D cultures demonstrated generalized inhibition of ECM protein concentration. Our study demonstrated that the progesterone agonist R5020 directly stimulated extracellular matrix components COL1A1, fibronectin, versican, and dermatopontin production in human leiomyoma cells. Progesterone antagonist mifepristone decreased protein production of these genes to levels comparable with untreated leiomyoma cells.

Abstract PR08: ARV-330: An androgen receptor PROTAC degrader for prostate cancer

Abstract Patients with prostate cancer who progress on therapy often have enhanced Androgen Receptor (AR) signaling due to several mechanisms: increased androgen production, increased AR expression, and/or specific AR mutations that render current therapies ineffective. A novel approach to block AR signaling is to specifically target AR for degradation. To do this, we have created AR PROTACs (PROtein-TArgeting Chimeras), bi-functional molecules that have an AR binding moiety on one end and an E3 ligase-recruiting element on the other end, which leads to AR ubiquitination and degradation. We have applied this technology to determine whether it could address mechanisms of resistance to current therapy in prostate cancer models. Our lead AR PROTAC, ARV-330, degrades AR in LNCaP and VCaP cells with 50% degradation concentrations (DC50s) &amp;lt; 1nM. AR degradation had functional consequences in cells, suppressing the AR target gene PSA, inhibiting proliferation, and potently inducing apoptosis in VCaP cells, with maximal apoptosis observed around 20 nM, versus 1 uM for enzalutamide. While both ARV-330 and enzalutamide block proliferation of VCaP cells in response to 0.1 nM of the AR agonist R1881, enzalutamide lost antiproliferative potency with increasing R1881 concentrations, whereas ARV-330 maintained antiproliferative effects. In cells containing the ARF876L mutation, enzalutamide was ineffective; however, ARV-330 maintained complete effectiveness. In mice, ARV-330 exhibited good pharmacokinetic properties, with t1/2 values of several hours and bioavailability of &amp;gt;80% after sc injection. Treatment of mice with ARV-330, at doses ranging from 0.3 to 10 mg/kg, resulted in reduction of AR protein levels and prostate involution in normal mice and, in mice implanted with VCaP tumors, reduction in plasma PSA and blockade of tumor growth. In summary, the AR PROTAC ARV-330 removes AR from prostate cancer cells in a potent manner and produces therapeutic effects as a result. This cellular efficacy has translated into biomarker activity and efficacy in animal models, and ARV-330 is now in preclinical development. Thus, targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed. Citation Format: James D. Winkler, Meizhong Jin, Andy P. Crew, AnnMarie K. Rossi, Ryan R. Willard, Hanqing Dong, Kam Siu, Jing Wang, Deborah A. Gordon, Xin Chen, Caterina Ferraro, Craig M. Crews, Kevin Coleman, Taavi K. Neklesa. ARV-330: An androgen receptor PROTAC degrader for prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR08.

Abstract LB-097: Targeted degradation of the androgen receptor in prostate cancer

Abstract Progression of prostate cancer in patients treated with anti-androgen therapy usually involves several mechanisms of enhanced Androgen Receptor (AR) signaling, including increased intratumoral androgen synthesis, increased AR expression and AR mutations. We have developed a protein degradation technology called PROTACs (PROteolysis TArgeting Chimera), which uses bi-functional molecules that simultaneously bind a target of choice and an E3 ligase. PROTACs, via induced proximity, cause ubiquitination and degradation of the targeted, pathological protein. As opposed to traditional target inhibition, which is a competitive process, degradation is a progressive process. As such, it is less susceptible to increases in endogenous ligand, target expression, or mutations in the target. Thus this technology seems ideal for addressing the mechanisms of AR resistance in patients with prostate cancer. AR PROTACs were shown to degrade AR in LNCaP and VCaP cells, with low nM to pM potency, and had a &amp;gt;85% reduction in AR concentration (Dmax). Degradation was rapid, with 50% of AR lost within 15 minutes and maximal degradation observed by 4 hours. The degradation process in cells was specific, as the PROTAC activity can be competed with excess E3 ligand and PROTACs with an inactive epimer for E3 ligase binding did not degrade AR. AR PROTACs induced rapid apoptosis and cell death in VCaP cells. In LNCap and VCaP cell systems, AR PROTACs were anti-proliferative under conditions in which enzalutamide was inactive, such as increasing concentrations of the AR agonist R1881 and cells containing the ARF876L mutation. AR PROTACs typically exhibited good pharmacokinetic properties, with t1/2 values of several hours and bioavailability of &amp;gt;50% after ip or sc injection. In mice, AR PROTACs demonstrate in vivo activity, including reduction of AR protein levels, prostate involution and tumor growth inhibition. In summary, PROTACs designed to degrade AR are potent, specific, active in vitro and in vivo, and have cellular efficacy superior to enzalutamide. Targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed. Citation Format: Meizhong Jin, James D. Winkler, Kevin Coleman, Andrew P. Crew, AnnMarie K. Rossi, Ryan R. Willard, Hanqing Dong, Kam Siu, Jing Wang, Deborah A. Gordon, Xin Chen, Caterina Ferraro, Craig M. Crews, Taavi K. Neklesa. Targeted degradation of the androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-097. doi:10.1158/1538-7445.AM2015-LB-097

Abstract 3452: Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer

Abstract Castration resistant prostate cancer (CRPC) still relies on androgens for progression. Abiraterone, an FDA approved CYP17A inhibitor, blocks androgen synthesis and prolongs survival. Abiraterone metabolites are generally thought to be inactive. We have identified a novel abiraterone metabolite, D4A, which inhibits three steroidogenic enzymes and androgen receptor (AR) itself. After abiraterone acetate administration, plasma from mice and patients were analyzed with liquid chromatography-mass spectrometry (LC-MS). The LC-MS results indicate that abiraterone is converted to D4A at detectable concentrations in both mice and patients. To test the function of D4A, the androgen synthesis pathway was analyzed in cell lines and xenografts. After incubation with [3H] labeled androgens, metabolites from cell lines and xenografts were analyzed by HPLC. We found that D4A inhibits activity of the steroidogenic enzymes CYP17A, 3β-hydroxysteroid dehydrogenase (3[[Unsupported Character - Symbol Font ]]HSD) and 5α-reductase. Furthermore D4A competes with the AR agonist R1881 and binds to AR directly. D4A inhibits DHT-induced AR occupancy on the PSA enhancer as detected by chromatin immunoprecipitation and reduces PSA expression. Our findings suggest that D4A is an abiraterone metabolite and a potent inhibitor of the androgen pathway. Citation Format: Zhenfei Li, Nima Sharifi. Conversion of abiraterone to D4A drives antitumor activity in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2015-3452

CCAAT/Enhancer binding protein β controls androgen-deprivation-induced senescence in prostate cancer cells.

The processes associated with transition to castration independent prostate cancer growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) β function is frequently deregulated in human malignancies and interestingly, androgen dependent prostate cancer cells express primarily the LIP isoform. We found that C/EBPβ expression is negatively regulated by androgen receptor activity and that treatment of androgen dependent cell lines with anti-androgens increases C/EBPβ mRNA and protein levels. Accordingly, we also find that C/EBPβ levels are significantly elevated in primary prostate cancer samples from castration resistant compared with therapy naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the androgen receptor to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPβ is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPβ promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPβ expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPβ under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci, and increased numbers of Ki67 positive cells. Ectopic expression of C/EBPβ caused pronounced morphological changes, reduced PC cell growth, and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to prostate cancer cell survival under androgen deprivation, and C/EBPβ deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that up-regulation of C/EBPβ is critical for complete maintenance of androgen deprivation induced senescence and that targeting C/EBPβ expression may synergize with anti-androgen or chemotherapy in eradicating prostate cancer.

The mTOR and canonical Wnt signaling pathways mediate the mnemonic effects of progesterone in the dorsal hippocampus.

Although much is known about the neural mechanisms responsible for the mnemonic effects of 17β-estradiol (E2 ), very little is understood about the mechanisms through which progesterone (P4 ) regulates memory. We previously showed that intrahippocampal infusion of P4 in ovariectomized female mice enhances object recognition (OR) memory consolidation in a manner dependent on activation of dorsal hippocampal ERK and mTOR signaling. However, the role of specific progesterone receptors (PRs) in mediating the effects of progesterone on memory consolidation and hippocampal cell signaling are unknown. Therefore, the goals of this study were to investigate the roles of membrane-associated and intracellular PRs in mediating hippocampal memory consolidation, and identify downstream cell signaling pathways activated by PRs. Membrane-associated PRs were targeted using bovine serum albumin-conjugated progesterone (BSA-P), and intracellular PRs (PR-A, PR-B) were targeted using the intracellular PR agonist R5020. Immediately after OR training, ovariectomized mice received bilateral dorsal hippocampal infusion of vehicle, P4 , BSA-P, or R5020. OR memory consolidation was enhanced by P4 , BSA-P, and R5020. However, only P4 and BSA-P activated ERK and mTOR signaling. Furthermore, dorsal hippocampal infusion of the ERK inhibitor U0126 blocked the memory-enhancing effects of BSA-P, but not R5020. The intracellular PR antagonist RU486 blocked the memory-enhancing effects of R5020, but not BSA-P. Interestingly, P4 robustly activated canonical Wnt signaling in the dorsal hippocampus, which is consistent with our recent findings that canonical Wnt signaling is necessary for OR memory consolidation. R5020, but not BSA-P, also elicited a modest increase in canonical Wnt signaling. Collectively, these data suggest that activation of ERK signaling is necessary for membrane-associated PRs to enhance OR, and indicate a role for canonical Wnt signaling in the memory-enhancing effects of intracellular PRs. This study provides the first evidence that membrane and intracellular PRs may employ different molecular mechanisms to enhance hippocampal memory.