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. 2015 Feb;87(2):296–304. doi: 10.1124/mol.114.095554

Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

Cesar A Corzo 1, Yelenis Mari 1, Mi Ra Chang 1, Tanya Khan 1, Dana Kuruvilla 1, Philippe Nuhant 1, Naresh Kumar 1, Graham M West 1, Derek R Duckett 1, William R Roush 1, Patrick R Griffin 1,✉
PMCID: PMC4293447  PMID: 25473120

Abstract

The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1–expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.

Introduction

Liver receptor homolog-1 (LRH-1; NR5A2) is a member of the NR5A subfamily of nuclear receptors (NRs) for which there are two members (Fayard et al., 2004). LRH-1 and steroidogenic factor-1 (SF-1; NR5A1) both bind to identical DNA consensus sequences (response elements) in promoter regions of their target genes and both bind DNA as monomers (Krylova et al., 2005; Li et al., 2005; Solomon et al., 2005). LRH-1 and SF-1 are differentially expressed in tissues and thus are likely to have nonoverlapping, nonredundant functions. Whereas SF-1 is confined to steroidogenic tissues where it plays a critical role in the regulation of development, differentiation, steroidogenesis, and sexual determination (Broadus et al., 1999; Lavorgna et al., 1991; Luo et al., 1994), LRH-1 is highly expressed in tissues of endodermal origin and is essential for normal intestinal and pancreatic function. In the liver, LRH-1 regulates cholesterol metabolism and bile acid homeostasis (Francis et al., 2003). Most NRs require binding of ligand to become transcriptionally active, but LRH-1 appears to be constitutively active when expressed in cells. A number of laboratories have identified the presence of phospholipids in the ligand-binding pocket (LBP) of LRH-1; their presence leads to the recruitment of coactivators in vitro (Krylova et al., 2005; Li et al., 2005; Solomon et al., 2005), but whether phospholipids are endogenous ligands of LRH-1 remains unclear. Recently, the dietary phospholipid dilauroylphosphatidylcholine was shown to activate LRH-1 activity in mice (Lee et al., 2011), confirming that LRH-1 is capable of binding ligands (Musille et al., 2012) that can modulate its activity in vivo.

In addition to the normal physiologic processes already mentioned, LRH-1 also transcriptionally regulates pathways involved in cancer development. The aromatase cytochrome P450 (CYP19) regulates much of postmenopause estrogen synthesis, and higher amounts of estrogen in the blood are linked to an increased risk of breast cancer (Fabian, 2007; Key et al., 2011). Simpson and coworkers first reported high expression of LRH-1 in preadipocytes, and they associated LRH-1 expression with transcriptional activation of CYP19 (Clyne et al., 2002). In addition to aromatase regulation, LRH-1 was shown to contribute to cancer progression by promoting motility and cell invasiveness of estrogen receptor (ER)–positive and ER-negative breast cancer cell lines (Chand et al., 2010).

In intestinal and pancreatic cancer, LRH-1 has been implicated in the regulation of cellular proliferation with a tumor-promoting role. Overexpression of LRH-1 in murine pancreatic cells more than doubled their growth rates and induced rapid colony formation in soft agar. LRH-1 mediated these effects by acting synergistically with β-catenin to activate cyclin-D1 and E1 expression. Furthermore, these effects were reversed by introducing LRH-1 small-interfering RNA (siRNA) and by overexpression of small heterodimer partner (SHP), demonstrating LRH-1’s role in cell proliferation (Schoonjans et al., 2005; Benod et al., 2011).

These studies were extended to examine the tumor-promoting role of LRH-1 in two independent mouse models of intestinal cancer as LRH-1 expression is elevated in intestinal crypt cells. APCmin/+ mice that were haploinsufficient for LRH-1 displayed lower LRH-1 expression and significantly reduced tumor formation compared with wild-type mice. Mice lacking one LRH-1 allele that were treated with a chemical inducer of colon cancer also showed a reduced number of aberrant crypt foci compared with wild-type mice (Schoonjans et al., 2005). Collectively, these studies suggest that inhibition of LRH-1 signaling presents a great opportunity for the therapy of some cancers.

Whitby et al. (2006, 2011) described the development of small molecule agonists for both LRH-1 and SF-1. These compounds induced the activation of the small ubiquitin-like modifier–dependent transrepression function of LRH-1 and subsequent inhibition of induction of proinflammatory genes in the liver acute-phase response (Venteclef et al., 2010). More recently, synthetic repressors of LRH-1 activity have been reported (Busby et al., 2010; Rey et al., 2012; Benod et al., 2013). Here we report the characterization of SR1848 [ML180; CID 3238389; 6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione] (Fig. 1) as an effective repressor of LRH-1 activity. SR1848 inhibits LRH-1–dependent transactivation of the CYP19 aromatase gene in a promoter-reporter assay and reduces the expression of LRH-1 target genes. Interestingly, treatment of cells with SR1848 causes rapid loss of the LRH-1 message in an LRH-1–dependent manner. SR1848 triggers cytoplasmic translocation of LRH-1 from the nucleus, which ultimately abrogates its ability to induce transcription of its targets and its potentiation of cell proliferation. SR1848 was well tolerated when administered to mice, and the treatment resulted in a reduction of LRH-1 target gene expression. Taken together, these studies suggest that SR1848 represents an anticancer strategy for malignancies in which LRH-1 plays a critical role.

Fig. 1.

Fig. 1.

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Activity of SR1848 in luciferase assays. (A) HEK293T cells were cotransfected with CYP19 promoter driving luciferase in the absence or presence of 5 µM SR1848 without or with SHP as a positive control. A statistically significant difference was observed with either SR1848 treatment or SHP when compared with the DMSO group (P < 0.05). (B) HEK293T cells were transfected with UAS-luc and Gal4-VP16 plasmids in the absence (DMSO) or presence of 5 µM SR1848. No statistically significant difference was observed. (C) HEK293T cells were cotransfected with StAR promoter driving luciferase with empty vector (pS6), SF-1, or LRH-1. Data were fit using a nonlinear regression curve and IC50 values were calculated. (D) JEG3 cells were transfected with CYP19 promoter driving luciferase and treated at the indicated concentrations of SR1848. Luciferase activity was determined using BriteLite Plus. For all panels, each data point was performed in triplicate.

Materials and Methods

Chemicals.

SR1848 [ML180; CID 3238389] was synthesized as previously described by Busby et al. (2010).

Cell Culture and Transcriptional Assays.

Human embryonic kidney 293T (HEK293T), hepatoma Huh-7, and ovarian adenocarcinoma SK-OV-3 cells were obtained from the American Type Culture Collection (Manassas, VA). The HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium; Huh-7 and SK-OV-3 were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum, and the cells were maintained by incubation at 37°C under 5% CO2. Transient transfections were performed in bulk by plating 3 × 106 cells per 10-cm plate and 9 μg of total DNA [1:1 ratio of receptor to reporter and FuGene6 (Roche Applied Science, Indianapolis, IN) in a 1:3 DNA-to-lipid ratio]. After 24 hours, the cells were replated in 384-well plates at a density of 10,000 cells/well. After 4 hours, the cells were treated with the indicated concentration of compound or dimethylsulfoxide (DMSO) as control. Luciferase levels were assayed after an additional 20-hour incubation period by a one-step addition of BriteLite Plus (PerkinElmer Life and Analytical Sciences, Waltham, MA) and read using an Envision plate reader (PerkinElmer). Data were normalized as fold change over DMSO-treated cells.

Animals.

All animal experiments were performed according to procedures approved by the Scripps-Florida Institutional Animal Care and Use Committee. We obtained 8-week-old C57Bl/6J mice from the Jackson Laboratory (Jackson ImmunoResearch Laboratories, West Grove, PA), and they were fed a regular chow diet throughout the experiments. The mice were dosed by intraperitoneal injection with 30 mg kg−1 SR1848 formulated in 10% DMSO and 10% Tween 80 in phosphate-buffered saline for 10 days. A total of five animals per group were studied. Tissues were immediately preserved in RNAlater (Qiagen, Valencia, CA) for gene expression analysis after QIAzol (Qiagen) extraction

Proliferation and Cytotoxicity Assays.

The cells were seeded in 96-well plates at a density of 5 × 103 cells/ml 24 hours before treatment. After 24 hours, the cells were treated with various concentrations of the SR1848 or DMSO. At 48 hours after treatment with SR1848 compound, cell proliferation was determined with the CellTiter-Glo luminescent assay (Promega, Madison, WI) using the manufacturer’s instructions. Cytotoxicity was determined 24 hours after treatment using the CytoTox-Glo Citotoxicity Assay Reagent (Promega).

Gene Expression Analysis.

Huh-7 and SK-OV-3 cells were treated with the indicated amounts of SR1848. After 24 hours, RNA was isolated following the Qiagen RNeasy miniprep manufacturer’s protocol. We prepared cDNA from total RNA using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) following the manufacturer’s protocol, which we analyzed with the Applied Biosystems 7900HT real-time polymerase chain reaction (PCR) instrument. Glyceraldehyde 3-phosphate dehydrogenase was used as the housekeeping gene. The primer sequences used are listed in Table 1.

TABLE 1.

List of quantitative PCR primers used

Target Sense Antisense
hLRH-1 5′-CTGATAGTGGAACTTTTGAA-3′ 5′-CTTCATTTGGTCATCAACCTT-3′
hCyp19 5′-TCACTGGCCTTTTTCTCTTGGT-3′ 5′-GGGTCCAATTCCCATGCA-3′
hSHP 5′-AAAGGGACCATCCTCTTCAAC-3′ 5′-CTGGTCGGAATGGACTTGAC-3′
hCycD1 5′-GTTCGTGGCCTCTAAGATGAAG-3′ 5′-GTGTTTGCGGATGATCTGTTTG-3′
hCycE1 5′-GCTTCGGCCTTGTATCATTTC-3′ 5′-CTGTCTCTGTGGGTCTGTATG-3′
hCyp8b1 5′-GGCAAGAAGATCCACCACTAC-3′ 5′-CTTCATTTGGTCATCAACCTT-3′
hGATA3 5′-GAACTGTCAGACCACCACAA-3′ 5′-CTGGATGCCTTCCTTCTTCATA-3′
hGATA4 5′-TCTCGATATGTTTGACGACTTCT-3′ 5′-GGTTGATGCCGTTCATCTTG-3′
hGAPDH 5′-GAAATCCCATCACCATCTTCCAGG-3′ 5′-GAGCCCCAGCCTTCTCCATG-3′
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Western Blot Analysis.

For the detection of endogenous LRH-1 protein in the Huh-7 and SK-OV-3 cell lines, cells were harvested at the indicated time points by adding ice-cold phosphate-buffered saline and scraping them off the plates followed by centrifugation to collect the cell pellet. The cells were then lysed, and the nuclear extracts were collected using either the Qproteome Cell Compartment Kit or the Qproteome Nuclear Protein Kit (Qiagen) as indicated, following the manufacturer’s directions. The protein concentration was determined by the BCA protein assay (Pierce/Thermo Scientific, Rockford, IL). The membranes were probed with anti-human LRH-1 mouse monoclonal antibody (R&D Systems, Minneapolis, MN). Lamin A/C (Cell Signaling Technologies, Danvers, MA) was used as the nuclear loading control.

Chromatin Immunoprecipitation Assay.

Immunoprecipitations were performed using mouse monoclonal anti–LRH-1 antibody (R&D Systems). Preparation of chromatin-DNA and the chromatin immunoprecipitation (ChIP) assay were performed with ChIP-IT Express Enzymatic Kit (Active Motif, Carlsbad, CA) per the manufacturer’s instructions, using antibody against LRH-1, normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and protein G agarose/salmon sperm DNA (EMD Millipore, Billerica, MA). After reversal of cross-linking, we subjected purified DNA to PCR with the following primers spanning potential LRH-1 binding sites in the SHP promoter: forward 5′-GACACCTGCTGATTGTGCAC-3′; reverse, 5′-GGCACTGATATCACCTCAGTCAAT-3′.

Reduction of Endogenous Gene Expression by Small-Interfering RNAs.

Huh-7 cells were transfected with ON-TARGETplus Human NR5A2 siRNA (GE Dharmacon, Lafayette, CO) following instructions for the DharmaFECT transfection reagent. Briefly, cells were seeded onto six-well plates (2 × 105 cells/well) overnight in complete 10% fetal bovine serum medium. The next day, the medium was removed and replaced with RPMI media containing a final siRNA concentration of 25 nM and 1 μl DharmaFECT transfection reagent per 1 ml of medium. The cells were harvested, and the RNA and nuclear protein were isolated. To evaluate cell proliferation after LRH-1 knockdown, we plated 2 × 103 cells in 96-well plates and transfected following the previously described protocol. Proliferation was evaluated at the indicated time points after transfection.

Immunocytochemistry.

Huh-7 cells were serum starved for 16 hours and treated with 5 μM SR1848. Cells were then fixed in 4% paraformaldehyde for 30 minutes at room temperature and processed for immunocytochemistry. Briefly, the fixed cells were incubated with 0.25% Triton X-100 to permeabilize the cells, and they were incubated with monoclonal anti-human LRH-1 (R&D Systems) for 1 hour at 4°C. Alexa488 anti-mouse IgG was used as the secondary antibody and incubated for 1 hour at 4°C. The samples were mounted in Gold prolong with 4,6-diamidino-2-phenylindole (Invitrogen/Life Technologies, Carlsbad, CA) and analyzed by confocal microscopy. The negative controls, incubated without primary antibody, showed no staining. Quantification of the mean fluorescence intensity in cytoplasm and nucleus regions was performed by use of ImageJ software (http://imagej.nih.gov/ij/). For the image analysis, at least three fields from two independent experiments were examined.

Statistical Data Analysis.

The results were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA). Statistical differences were determined using unpaired t tests to compare treatment groups and were considered significant if P < 0.05 (*P < 0.05, **P < 0.01, and ***P < 0.001). For multiple group comparisons, a one-way analysis of variance was used. When a statistically significant difference was determined via the one-way analysis of variance, we conducted a post-hoc analysis using Dunnett’s test to compare all treatment groups or time points to a control group.

Results

SR1848 Inhibits LRH-1 Activity in Luciferase-Reporter Assays.

The first indication that repressors could be identified for the NR5A family of NRs was revealed by a publication from the Scripps Florida Molecular Libraries Probe Centers Network describing a high-throughput screen campaign for SF-1 (Madoux et al., 2008). In this report, chemically tractable small molecule repressors of SF-1 were discovered in a high-throughput screen campaign where the primary assay was a Gal4-SF-1/UAS (upstream activation sequence) luciferase reporter assay using the constitutively active herpes virus protein 16 fused to Gal4 (Gal4-VP16) and the yeast UAS luciferase reporter as a control for nonspecific transcriptional modulation and cytotoxicity. Eight compounds were selected based on selectivity for SF-1 activity (at least 10-fold difference compared with Gal4-VP16).

To assess whether these compounds could modulate LRH-1 activity, we performed a luciferase reporter assay where HEK293T cells were cotransfected with full-length LRH-1 and a luciferase reporter driven by the CYP19 aromatase promoter. As a control for repression of LRH-1’s constitutive activity, we also cotransfected the nuclear corepressor SHP along with the full-length receptor and CYP19 luciferase reporter construct. Of the eight compounds evaluated, one of them (SR1848) was able to inhibit LRH-1–dependent transactivation of the CYP19 luciferase reporter by 50%, which was similar to that observed with overexpression of the SHP (Fig. 1A). Figure 1B demonstrates that the treatment of these cells with 5 μM SR1848 did not affect the ability of the Gal4-VP16 fusion protein to transactivate the UAS luciferase reporter. Thus, the effects of SR1848 were not due to nonspecific transcriptional modulation or cytotoxicity, but rather required the presence of LRH-1.

To determine whether SR1848 could also inhibit SF-1–dependent transactivation using native reporters and DNA-binding domains, we performed a dose-response experiment on HEK293T cells expressing either full-length SF-1 or full-length LRH-1 cotransfected with a luciferase reporter gene driven by the promoter for StAR (steroidogenic acute regulatory protein), a known downstream target of both LRH-1 and SF-1. Both LRH-1 and SF-1 displayed a dose-dependent reduction in receptor-mediated transcription in HEK293T cells after increasing doses of SR1848 (Fig. 1C).

Finally, we evaluated the ability of this compound to inhibit endogenously expressed LRH-1–dependent gene expression. Unlike HEK293T cells that express moderate levels of SF-1 and very low levels of LRH-1, JEG3 endometrial cancer cells express high levels of LRH-1 and very low levels of SF-1 (Lin et al., 2009). Therefore, to determine whether SR1848 affected endogenously expressed LRH-1–dependent transactivation of aromatase, JEG3 cells were transfected with the CYP19 aromatase reporter (Fig. 1D). Consistent with our previous observations, increasing doses of SR1848 led to a dose-dependent decrease in endogenous LRH-1–dependent transactivation of the CYP19 luciferase reporter. This result indicates that SR1848 is capable of repressing CYP19 aromatase expression by endogenously expressed LRH-1 in JEG3 cells to the same degree as it repressed CYP19 aromatase expression by overexpressed LRH-1 in HEK293T cells.

SR1848 Inhibits Endogenous LRH-1 Signaling in Hepatic Cell Lines.

LRH-1 is highly abundant in hepatic cells. We therefore evaluated the effects of SR1848 in the hepatocellular carcinoma cell line Huh-7. Quantitative PCR analysis confirmed significant expression of endogenous LRH-1 mRNA in this cell line (Fig. 2A). The consensus LRH-1 response element is (T/C) CAAGG (T/C) C (A/G). Analysis of the human LRH-1 promoter revealed the presence of a binding site (CCAAGGCCA) 89 bp upstream of the LRH-1 start codon. LRH-1 has been shown to bind to this promoter region and regulate transcription of its own promoter (Hadizadeh et al., 2008). This phenomenon has been documented for other NRs, such as liver X receptor (LXRα; NR1H3) (Laffitte et al., 2001) and peroxisome proliferator-activated receptor α (PPARα; NR1C1) (Pineda Torra et al., 2002).

Fig. 2.

Fig. 2.

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SR1848 inhibits LRH-1 target genes in vitro and in vivo. (A) LRH-1 mRNA expression analysis of Huh-7 cells at the indicated time points. One-way analysis of variance shows a statistically significant difference when the 2- and 6-hour time points are compared (P < 0.05). There was no difference when the other time points were compared versus 2 hours. (B, C, and D) Dose-dependent inhibition of LRH-1 (B), CYP19 (C), and SHP (D) mRNA expression. Each data point was performed in triplicate. Data are represented as mean ± S.E.M. ***Statistically significant difference between DMSO and SR1848 treatment as determined by t test (P < 0.05). (E) Gene expression in tissues from C57Bl/6 mice dosed with SR1848 or vehicle for 10 days (n = 5 per group).

Accordingly, SR1848 inhibited LRH-1 mRNA expression in a dose-dependent manner (Fig. 2B). Similar effects on the LRH-1 message by SR1848 treatment were also observed in HepG2 cells, which express moderate LRH-1 levels (data not shown). We also assessed the effect of SR1848 on the expression of well-established targets of LRH-1, including CYP19 and the NR SHP. As shown in Fig. 2, C and D, analysis of mRNA levels in Huh-7 cells demonstrated that SR1848 inhibits the expression of both CYP19 and SHP in a dose-dependent manner. These results indicate that SR1848 leads to a rapid decrease of LRH-1 expression and efficiently represses endogenous LRH-1 signaling.

Inhibition of LRH-1 Signaling Pathways In Vivo.

Next we investigated whether SR1848 could repress LRH-1 signaling in vivo. Compound or vehicle only (10% DMSO, 10% Tween 80) was administered daily (intraperitoneally) for 10 days to 8-week-old male C57Bl/6 mice with vehicle. A dosage of 30 mg/kg was selected as it gave an average plasma concentration of 12.4 μM at 8 hours after administration, a concentration above that required to observe repression of target genes in Huh-7 cells.

LRH-1 is strongly expressed in the liver, pancreas, and adrenal glands, so we evaluated expression of genes regulated by LRH-1 (Fig. 2E) in these organs. Livers of SR1848-treated animals showed statistically significant repression of SHP and CYP7A1. In adrenal glands and pancreatic tissue we observed a statistically significant decrease of both LRH-1 and SHP mRNA. Furthermore, blood analysis of mice treated with SR1848 showed no statistically significant difference in key physiologic analytes (Supplemental Fig. 1). These results demonstrate the safety and efficacy of SR1848 as an LRH-1 inhibitor in vivo.

SR1848 Inhibits Proliferation of LRH-1 Expressing Cell Lines.

As a means to establish whether SR1848 requires LRH-1 for its antiproliferative action, we used a cell line that lacks expression of LRH-1. The ovarian adenocarcinoma cell line SK-OV-3 expresses very little LRH-1 message relative to Huh-7 cells (Fig. 3A). Using subcellular fractionation, LRH-1 can be detected in the nuclear compartment of Huh-7 cells, but there is no evidence of LRH-1 protein in SK-OV-3 cells (Fig. 3B). LRH-1 plays a significant role in cancer cell proliferation through the upregulation of cyclin-D1 and -E1, (Benod et al., 2011; Schoonjans et al., 2005). SR1848 showed a significant inhibition of cyclin-D1 and cyclin-E1 expression in hepatic cells (Fig. 3, C and D), but had little effect on repression in SK-OV-3 cells.

Fig. 3.

Fig. 3.

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SR1848 inhibits cell proliferation in an LRH-1–dependent manner. (A and B) Endogenous LRH-1 expression comparison between Huh-7 and SKOV-3 cells: mRNA levels (A) and nuclear protein (B). Figures represent three separate experiments with similar results. (C and D) Dose-dependent inhibition of cyclin-D1 (C) and cyclin-E1 (D) expression in Huh-7 versus SKOV-3 cells. ****Statistically significant difference between DMSO and SR1848 treatment as determined by t test (P < 0.05). Each data point was performed in triplicate. (E) Antiproliferative effects of SR1848 in a dose-dependent manner. Data were fit using a nonlinear regression curve, and IC50 values were calculated. Each data point was performed in triplicate. (F) CytoTox-Glo cytotoxicity assay of SR1848 dose response in Huh-7 and SK-OV-3 cells. Each data point was performed in triplicate. A statistically significant difference as compared with control was noted at 10 and 30 µM SR1848 doses (P < 0.05).

To assess the antiproliferative effects of SR1848, we treated Huh-7 and SK-OV-3 cells with SR1848 or DMSO for 48 hours and measured proliferation using a CellTiter-Glo luminescent assay. Compared with the DMSO-treated controls, the SR1848-treated cells showed diminished capacity to proliferate at concentrations above 1 μM, the EC50 being roughly 2.8 μM in Huh-7 (Fig. 3E). Inhibition of proliferation was not observed in SK-OV-3 cells, suggesting that SR1848-mediated repression of proliferation in Huh-7 cells depends on LRH-1 (Fig. 3E).

SR1848 appears to behave as a cytostatic compound, as in our proliferation assays we observed that the amount of Huh-7 cells at the pretreatment time point was equal to the amount of cells 48 hours after SR1848 treatment (5 μM) (Supplemental Fig. 2). However, to ensure that the decrease in proliferation was not caused by general toxicity of the compound, we measured cytotoxicity using the CytoTox-Glo cytotoxicity assay, which measures the activity of proteases after cells lose membrane integrity. The results showed that the percentage of dead Huh-7 cells in culture after 24 hours was almost identical when comparing DMSO-treated cells to cells treated with SR1848 up to a 10 μM concentration (Fig. 3F, DMSO 3.3%; 5 μM SR1848 3.9%). The SK-OV-3 cell line was equally unaffected by SR1848, and the percentage of dead cells was not significantly changed up to a 10 μM concentration (Fig. 3F). Beyond 10 μM, the percentage of cell death dramatically increased in both cell lines, explaining the reduced proliferation of SK-OV-3 after 10 μM concentrations (Fig. 3F).

These data suggest that SR1848 limits the proliferative ability of LRH-1 expressing cells without inducing cytotoxic side effects in non–LRH-1 expressing cells. The lack of inhibition in SK-OV-3 suggests that LRH-1 is necessary to mediate the effects of SR1848, as transcriptional repression of similarly expressed targets was only observed in the LRH-1 expressing Huh-7 cells.

Knockdown of LRH-1 Reproduces the Biologic Effects of SR1848.

The effects of SR1848 on LRH-1 protein were first tested by treating Huh-7 cells with a moderate (5 µM) concentration of SR1848. After 24 hours of treatment, we harvested the cells and analyzed the nuclear fraction. As expected, the LRH-1 protein levels were significantly reduced (Fig. 4A). Transfection of cells with siRNAs directed against LRH-1 efficiently knocked down LRH-1 protein expression (Fig. 4B). Knocking down LRH-1 resulted in the significant reduction of LRH-1 mRNA expression, and the expression level of LRH-1 targeted CYP19, SHP, and CYP8B1 (Fig. 4C). In addition, the antiproliferative effect of SR1848 treatment in Huh-7 cells was reproduced by siRNA against LRH-1. At 96 hours after transfection, LRH-1 siRNA transfected cells could not proliferate as efficiently as the control-transfected cells (Fig. 4D). These results show that pharmacologic repression of the LRH-1 pathway with SR1848 achieves similar biologic effects as knockdown of the receptor, which supports that the compound’s efficacy is mediated via LRH-1 signaling.

Fig. 4.

Fig. 4.

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LRH-1 knockdown reproduces the effects of SR1848. (A) Huh-7 cells were treated in culture with either DMSO or SR1848 (5 µM) for 24 hours followed by subcellular fractionation and detection of LRH-1 protein by anti–LRH-1 antibody. Lamin A/C was used as a nuclear loading control. This figure represents three separate experiments with same result. (B–D) Huh-7 cells were transfected with either siRNA control (siControl) or siRNA directed against LRH-1 (siLRH-1). (B) Knockdown of LRH-1 protein 48 hours after transfection. This figure represents two individual experiments with similar results. (C) Expression of LRH-1 target genes by quantitative PCR 48 hours after transfection. The data represent two independent experiments performed in triplicate. A statistically significant difference was noted when comparing siControl with siLRH-1 for all genes tested (P < 0.05). (D) Evaluation of cell proliferation 96 hours after siRNA transfection. Each data point was performed in triplicate. A statistically significant difference between siControl and siLRH-1 was noted at the 96- and 120-hour time points (P < 0.05).

Mechanism of Action of SR1848.

To further elucidate the mechanism of action of SR1848 on LRH-1 signaling, Huh-7 and HepG2 cells were treated with DMSO or SR1848, and LRH-1 mRNA was analyzed in a time-dependent manner. Within 2 hours of SR1848 treatment, the mRNA levels of LRH-1 receptor and its downstream targets (CYP19, GATA3, and GATA4) were rapidly and significantly decreased (Fig. 5A). Similar observations have been shown in breast cancer cells where GATA factors and LRH-1 regulate human aromatase (CYP19) PII promoter activity (Bouchard et al., 2005). However, during the early stages of SR1848 treatment, no changes in the level of LRH-1 protein were observed (Fig. 5B).

Fig. 5.

Fig. 5.

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SR1848 treatment leads to export of LRH-1 from the nucleus. (A) LRH-1 mRNA expression analysis by quantitative PCR from Huh-7 treated with DMSO or SR1848 (5 μM) for various time points. The data represent three different experiments performed in triplicate. A statistically significant difference was observed when comparing DMSO and time points >1 hour (P < 0.05). (B) Huh-7 cells were treated in culture with either DMSO or SR1848 (5 µM) for 2 hours followed by subcellular fractionation and detection with anti–LRH-1 antibody. (C) Chromatin was extracted from Huh-7 cells followed by precipitation with either anti–LRH-1 antibody or control mouse IgG. PCR was performed with primers specific for promoter regions of the SHP gene. Input: PCR reaction performed with DNA isolated from nuclear extract without immunoprecipitation. (D) Immunocytochemistry analysis of Huh-7 cells treated in culture with DMSO or SR1848 (5 µM) for 30 minutes or 2 hours. Cells were fixed with 4% paraformaldehyde and incubated with anti–LRH-1 antibody for 1 hour followed by incubation with AlexaFluor488 anti-mouse antibody for 1 hour. 4,6-Diamidino-2-phenylindole (DAPI) was used for counterstaining, and cells were visualized by fluorescence microscopy. Quantification of the mean fluorescence intensity from cytoplasm and nucleus is shown. The figure represents two separate experiments with similar results.

We hypothesized that the ability of LRH-1 to bind chromatin might be altered by SR1848. In ChIP assays of Huh-7 cells, treatment with SR1848 reduced the interaction of LRH-1 with the promoter regions of the SHP gene as early as 1 hour after treatment (Fig. 5C). Immunohistochemistry assays conducted to visualize LRH-1 within the nucleus (Fig. 5D) showed that 2 hours after SR1848 treatment LRH-1 translocated from nucleus to the cytoplasm. From these results, it appears that SR1848 interferes with LRH-1 chromatin interaction in part by inducing nuclear export of the receptor to the cytoplasm, which results in reduced transcriptional activation of LRH-1 target genes. An illustration depicting this mechanism of action for SR1848 is shown in Fig. 6. In the top panel of Fig. 6, transcription is constitutively on when LRH-1 binds to the response elements in the promoter region of its target genes. SR1848 treatment led to dissociation of LRH-1 from chromatin, subsequently inhibiting transcription of the target gene (Fig. 6, bottom panel).

Fig. 6.

Fig. 6.

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Proposed mechanism of SR1848. Top panel: LRH-1 binding to the promoter region of its target genes leads to transcription activation. Bottom panel: SR1848 treatment causes release of LRH-1 from chromatin, resulting in reduced LRH-1 interaction with promoter regions of target genes and subsequent decreasing or turning off transcription. DBD: DNA binding domain

Although it is plausible that SR1848 represses LRH-1 activity by binding to the LBP within its ligand-binding domain (LBD) to facilitate recruitment of corepressors, thus acting as a classic inverse agonist, our efforts to detect direct binding of SR1848 to the LBD of LRH-1 expressed and purified from Escherichia coli have not proven fruitful. As described earlier, the LRH-1 LBD isolated from bacterial expression systems contains phospholipids in the LBP (Musille et al., 2012). Therefore, it is possible that a nonlipidlike compound such as SR1848 is not capable of competing out lipid within the LBP of the recombinant protein. It is also plausible that SR1848 binds to domains of the receptor outside the LBD or that SR1848 impacts LRH-1 binding to chromatin indirectly. Further detailed proteomic analysis or biophysical studies on the full-length receptor are required to elucidate this.

Discussion

The data presented in this study characterize SR1848 as an effective repressor of LRH-1 activity. In our initial reporter assays, SR1848 inhibited LRH-1–dependent transactivation of the CYP19 aromatase gene, an established LRH-1 target gene. However, the transactivating ability of a Gal4-VP16 fusion protein was unaffected by SR1848 when cotransfected with a UAS luciferase reporter in the same HEK293T cell line. SR1848 also inhibits endogenous LRH-1 signaling, decreasing the mRNA levels of target proteins (CYP19 and SHP) and causing the rapid loss of LRH-1 message. Furthermore, treatment of mice with SR1848 is well tolerated and results in efficient repression of LRH-1 responsive genes in vivo.

Silencing LRH-1 expression, using siRNA directed against LRH-1 is sufficient to induce biologic effects similar to the treatment of cells with SR1848. Transfection of siRNA targeting LRH-1 inhibited known LRH-1 target genes and resulted in a reduced ability of cells to proliferate. Protein levels in the nucleus began to diminish 4 hours after treatment with SR1848, whereas SR1848 started to repress certain target genes as early as 2 hours. The initial mechanism of action of SR1848 suggested by ChIP and immunocytochemistry studies is the release of LRH-1 from chromatin and translocation to the cytoplasm, which ultimately abrogates its ability to induce transcription of its targets and its potentiation of cell proliferation. The upstream signaling cascade events that lead to LRH-1 removal from chromatin remain to be elucidated; further studies are needed to determine whether posttranslational modifications (i.e., SUMOylation, phosphorylation, acetylation) known to be associated with LRH-1 regulation (Chalkiadaki and Talianidis, 2005; Lee et al., 2006) are involved in SR81848s mechanism of action.

Our initial studies indicated that SR1848 has antiproliferative properties. SR1848, at moderate concentrations, behaves as a cytostatic compound, inhibiting cell proliferation through the repression of cyclin-D1 and cyclin-E1 expression, without inducing cell death in the LRH-1–expressing Huh-7 cells. The antiproliferative properties of SR1848 were not observed in the LRH-1–deficient SK-OV-3 cell line. The regulation of cyclin expression raises the possibility that SR1848 may regulate β-CATENIN and the WNT signaling pathway. β-CATENIN is a direct LRH-1 target gene (Yumoto et al., 2012) and in pancreatic cancer, increased proliferation has been shown to be mediated through LRH-1 and β-CATENIN interaction, ultimately impacting cyclin expression (Botrugno et al., 2004). Therefore, the data presented in this study strongly propose SR1848 as a novel potential therapy for targeting LRH-1 signaling in the context of cancer.

LRH-1 has been implicated in the growth deregulation and metastasis of ER-positive breast cancers and the more difficult to treat ER-negative breast cancers. Aromatase inhibitors are used clinically in breast cancer treatment, but resistance to these drugs is an emerging problem. In addition, these drugs reduce estrogen production in all tissues, giving rise to adverse effects. As such, repressors of LRH-1–dependent activation of aromatase could represent a strategy for the development of novel breast-specific tumor therapies. Additionally, overexpression of LRH-1 in both ER-positive and ER-negative breast cancer cell lines was shown to promote motility and cell invasiveness (Chand et al., 2010). This suggests that repression of LRH-1 could be useful in treating not only ER-positive breast cancer subtypes, but perhaps could provide a treatment option for the more aggressive triple-negative breast cancer subtype where therapies are limited.

Although most NRs require the binding of ligand to become transcriptionally active, LRH-1 appears to be constitutively active when expressed in cells. The data presented here demonstrate that SR1848 is a nonlipidlike synthetic repressor of LRH-1 activity and a potential therapeutic agent for targeting LRH-1–dependent cancers.

Supplementary Material

Data Supplement
supp_87_2_296__index.html (1.2KB, html)

Abbreviations

ChIP

chromatin immunoprecipitation

DMSO

dimethylsulfoxide

ER

estrogen receptor

HEK293T

human embryonic kidney 293T cells

LBD

ligand-binding domain

LBP

ligand-binding pocket

NR

nuclear receptor

PCR

polymerase chain reaction

SHP

small heterodimer partner

siRNA

small-interfering RNA

SR1848

6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione

UAS

upstream activation sequence

Authorship Contributions

Participated in research design: Corzo, Mari, Chang, Duckett, Roush, Griffin.

Conducted experiments: Corzo, Mari, Chang, Khan, Kuruvilla, Kumar, West.

Contributed new reagents or analytic tools: Nuhant, Roush.

Performed data analysis: Corzo, Mari, Chang, Kumar, Griffin.

Wrote or contributed to the writing of the manuscript: Corzo, Mari, Chang, Duckett, Roush, Griffin.

Footnotes

This work was supported by the National Institutes of Health National Institute of Mental Health [Grant U54-MH074404] and by the National Institutes of Health National Cancer Institute [Grant R01-CA134873].

Inline graphicThis article has supplemental material available at molpharm.aspetjournals.org.

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Associated Data

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Supplementary Materials

Data Supplement
supp_87_2_296__index.html (1.2KB, html)
supp_mol.114.095554_Supplemental_Data_95554.pdf (219.4KB, pdf)

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