anti-proliferation activity of a small molecule repressor of lrh-1 · 2014. 12. 3. · 1 can bind...

MOL #95554

1

Anti-proliferation activity of a small molecule repressor of LRH-1

Cesar Corzo, Yelenis Mari, Mi Ra Chang, Tanya Khan, Dana Kuruvilla, Philippe Nuhant, Naresh

Kumar, Graham M. West, Derek R. Duckett, William R. Roush, and Patrick R. Griffin

Departments of Molecular Therapeutics (C.C., Y.M., M.R.C., T.K., D.K., N.K., G.M.W., D.R.D., P.R.G.) and Chemistry (P.N., W.R.R.)

The Scripps Research Institute, Scripps Florida

Jupiter, FL33458, USA

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on December 3, 2014 as DOI: 10.1124/mol.114.095554

at ASPE

T Journals on July 31, 2021

molpharm

.aspetjournals.orgD

ownloaded from

http://molpharm.aspetjournals.org/

MOL #95554

2

Running Title: LRH1 repression in cancer

Corresponding Author: Patrick R. Griffin, The Scripps Research Institute, Scripps, Florida,

130 Scripps Way, Jupiter, FL 33458, [email protected]

The number of text pages - 13

Number of tables - 1

Number of Figures - 6

Number of References - 31

Number of words in the:

Abstract - 184

Introduction – 748

Discussion – 584

Abbreviations: Liver receptor homolog 1 (LRH1), chromatin

immunoprecipitation (ChIP), nuclear receptor (NR), cytochrome p450 19 (Cyp19).


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

3

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 ER positive

and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-

1 can bind phospholipids and the dietary phospholipid DLPC activates LRH-1 activity in rodents.

Here we characterize the activity of a novel synthetic non-phospholipid small molecule

repressor of LRH-1, SR1848. In co-transfection 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 CyclinD1 and E1 resulting in inhibition of cell proliferation. The

cellular effects of SR1848 treatment are recapitulated following transfection of cells with siRNAs

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 impacting 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.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

4

INTRODUCTION

Liver receptor homologue-1 (LRH-1; NR5A2) is a member of the NR5A subfamily of nuclear

receptors 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 or

REs) 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 non-overlapping, non-redundant 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 nuclear receptors (NRs)

require binding of ligand to become transcriptionally active, however 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 and 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 DLPC 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 physiological processes already mentioned, LRH-1 also

transcriptionally regulates pathways involved in cancer development. The aromatase

cytochrome p450 (CYP19) regulates much of the post-menopause 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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

5

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 CyclinD1 and E1 expression.

Furthermore, these effects were reversed by introducing LRH-1 siRNA and by overexpression

of SHP, demonstrating LRH-1’s role in cell proliferation (Benod et al, 2011; Schoonjans et al,

2005). These studies were extended to examine the tumor-promoting role of LRH1 in two

independent mouse models of intestinal cancer as LRH1 expression is elevated in intestinal

crypt cells; APCmin/+ mice that were haplo-insufficient for LRH-1 displayed lower LRH-1

expression and significantly reduced tumor formation than wild-type mice, and mice lacking one

LRH-1 allele that were treated with a chemical inducer of colon cancer, also showed reduced

number of aberrant crypt foci than 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 and coworkers (Whitby et al, 2006; Whitby et al, 2011) described the development of

small molecule agonists for both LRH-1 and SF-1. These compounds induced the activation of

the SUMO-dependent transrepression function of LRH-1 and subsequent inhibition of induction

of pro-inflammatory genes in the liver acute phase response (Venteclef et al, 2010). More

recently, synthetic repressors of LRH-1 activity have been reported (Benod et al, 2013; Busby et

al, 2010; Rey et al, 2012). Here we report the characterization of SR1848 [ML180; CID

3238389] (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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

6

expression of LRH-1 target genes. Interestingly, treatment of cells with SR1848 causes rapid

loss of 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 treatment resulted in a reduction of LRH-1 target gene

expression. Taken together, these studies suggest that SR1848 represents an anti-cancer

strategy for malignancies in which LRH-1 plays a critical role.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

7

MATERIALS AND METHODS:

Chemicals. SR1848 [ML180; CID 3238389] was synthesized as previously described (Busby et

al, 2010).

Cell Culture and transcriptional Assays. The human embryonic kidney HEK293T, hepatoma

Huh-7, and ovarian adenocarcinoma SK-OV-3 cells were obtained from American Type Culture

Collection (ATCC). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium

(DMEM); Huh-7 and SK-OV-3 were maintained in RPMI 1640. All media was supplemented with

10% fetal bovine serum (FBS) and cells maintained by incubation at 37 °C under 5% CO2.

Transient transfections were performed in bulk by plating 3x106 cells per 10 cm plate and 9μg of

total DNA (1:1 ratio of receptor to reporter and FuGene6 (Roche) in a 1:3 DNA to lipid ratio).

After 24 hours, cells were re-plated in 384 well plates at a density of 10,000 cells/well. After 4h,

cells were treated with the indicated concentration of compound or DMSO as control. Luciferase

levels were assayed following an additional 20h incubation period by a one-step addition of

BriteLite Plus (Perkin Elmer) and read using an Envision plate reader (Perkin Elmer). Data was

normalized as fold change over DMSO treated cells.

Animals. All animal experiments were performed according to procedures approved by the

Scripps-Florida IACUC Committee. 8-week old C57Bl/6J mice were obtained from the Jackson

Laboratory and fed a regular chow diet through experiments. Mice were dosed by

intraperitoneal (i.p.) injection with 30mg kg-1 SR1848 formulated in 10% DMSO and 10% Tween

80 in PBS for 10 days. A total of 5 animals per group were studied. Tissues were immediately

preserved in RNAlater (Qiagen) for gene expression analysis after QIAzol (Qiagen) extraction

Proliferation and Cytotoxicity Assays. Cells were seeded in 96-well plates at a density of

5x103 cells/ml 24h prior to treatment. After 24h, cells were treated with various concentrations of

the SR1848 or DMSO. Cell proliferation was determined with the CellTiter-Glo (Promega)


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

8

luminescent assay 48 hours after treatment with SR1848 compound using manufacturer’s

instructions. Cytotoxicity was determined 24h after treatment using the CytoTox-Glo Citotoxicity

Assay Reagent (Promega).

Gene Expression Analysis Huh-7 and SK-OV-3 cells were treated with indicated amounts of

SR1848. After 24 hours RNA was isolated following the Qiagen RNeasy mini prep

manufacturer's protocol. cDNA was prepared from total RNA using the Applied Biosystems high

capacity cDNA archive kit following the manufacturer's protocol and analyzed with the Applied

Biosystems 7900HT real-time PCR instrument. GAPDH was used as the housekeeping gene.

Sequences of primers used are listed in Table 1 of supplementary figures.

Western Blot Analysis. For the detection of endogenous LRH-1 protein in Huh-7 and SK-OV-

3 cell lines, cells were harvested at the indicated time points by adding iced-cold PBS and

scraping them off the plates followed by centrifugation to collect the cell pellet. Cells were then

lyzed and nuclear extracts were collected using either the Qproteome Cell Compartment Kit or

the Qproteome Nuclear Protein Kit (QIagen) as indicated following manufacturer’s directions.

Protein concentration was determined by BCA protein assay (Pierce). Membranes were probed

with anti-human LRH-1 mouse monoclonal antibody (R&D Systems). Lamin A/C (Cell Signaling

Technology) was used as the nuclear loading control.

Chromatin immunoprecipitation (ChIP) assay. Immunoprecipitations were performed using

mouse monoclonal anti-LRH-1 antibody (R&D Systems, cat.# PP-H2325-00). Preparation of

chromatin-DNA and ChIP assay were performed with ChIP-IT Express Enzymatic Kit (Active

Motiff, cat.#53009) per the manufacturer's instructions, using antibody against LRH-1, normal

mouse IgG (Santa Cruz Biotechnology), and protein G agarose/salmon sperm DNA (EMD

Millipore). After reversal of cross-linking, purified DNA was subjected to PCR with the following


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

9

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, cat #: L-003430-00-

0005) following instructions for DharmaFECT transfection reagent. Briefly, cells were seeded

onto 6 well plates (2X105 cells/well) overnight in complete 10% FBS media. The following day,

media was removed and replaced with RPMI containing a final siRNA concentration of 25nM

and 1uL DharmaFECT transfection reagent per 1ml of media. Cells were harvested and RNA

and nuclear protein were isolated. To evaluate cell proliferation after LRH-1 knockdown, 2X103

cells were plated in 96 well plates and transfected following the above 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% TritonX-

100 to permeabilize the cells and incubated with monoclonal anti-human LRH-1 (R & D

Systems) for 1 hour at 4oC. Alexa488- anti mouse IgG was used as the secondary antibody and

incubated for 1 hour at 4oC. The samples were mounted in Gold prolong with DAPI (Invitrogen)

and analyzed by confocal microscopy. Negative controls, incubated without primary antibody,

showed no staining. Quantification of the mean fluorescence intensity in cytoplasm and nucleus

regions was carried out by using the ImageJ software. For image analysis, at least three fields

from two independent experiments were examined.

Statistical Data Analysis. Results were analyzed using GraphPad Prism software. 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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

10

comparisons a 1-way ANOVA was used. When significant differences were determined with an

ANOVA, post-hoc analysis was conducted using a Dunnett Test to compare all treatment

groups or time points to a control group.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

11

RESULTS

SR1848 inhibits LRH-1 activity in luciferase-reporter assays. The first indications

that repressors could be identified for the NR5A family of nuclear receptors was revealed by a

publication from the Scripps Florida MLPCN center describing a HTS campaign for SF-1

(Madoux et al, 2008). In this report, chemically tractable small molecule repressors of SF-1

were discovered in a HTS campaign where the primary assay was a Gal4-SF-1/UAS 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 non-specific transcriptional modulation

and cytotoxicity. Eight compounds were selected based on selectivity for SF-1 activity (at least

10–fold difference compared to 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). Fig. 1B demonstrates that treatment of

these cells with 5 μM SR1848 did not affect the ability of the Gal4 VP-16 fusion protein to

transactivate the UAS luciferase reporter. Thus, the effects of SR1848 were not due to non-

specific transcriptional modulation or cytotoxicity, but require 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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

12

reduction in receptor-mediated transcription in HEK293T cells following 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 if 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 reveals the presence of

such binding site (CCAAGGCCA) 89bp 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 alpha

(PPARα; NR1C1) (Pineda Torra et al, 2002). Accordingly, SR1848 inhibited LRH-1 mRNA

expression in a dose-dependent manner (Fig. 2B). Similar effects on 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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

13

targets of LRH-1, including CYP19 and the nuclear receptor SHP. As shown in Fig. 2C and 2D,

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 if SR1848 could

repress LRH-1 signaling in vivo. Compound or vehicle only (10%DMSO, 10% Tween 80) was

administered daily (intraperitoneal) for 10 days to 8-week old male C57Bl/6 mice with vehicle. A

dose of 30mg/kg was selected as it gave an average plasma concentration of 12.4 μM 8h post

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, therefore

we evaluated expression of genes regulated by LRH-1 (Fig. 2 E) in these organs. Livers of

SR1848-treated animals showed significant repression of SHP and CYP7A1. In adrenal glands

and pancreatic tissue we observed significant decrease of both LRH-1 and SHP mRNA.

Furthermore, blood analysis of mice treated with SR1848 showed no significant difference in

key physiological analytes (Supplemental Figure 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 anti-proliferative action we utilized 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 sub-cellular 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 cyclins 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. 3C-D),

but had little effect on repression in SK-OV-3 cells. To assess the anti-proliferative effects of


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

14

SR1848, Huh-7 and SK-OV-3 cells were treated with SR1848 or DMSO for 48h and proliferation

was measured using CellTiter-Glo (Promega) luminescent assay. Compared to DMSO-treated

controls, 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 is LRH-1 dependent (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 pre-treatment time-point was equal to the

amount of cells 48h after SR1848 treatment (5 μM) range (Supplemental Figure 2). However, to

ensure that the decrease in proliferation was not caused by general toxicity of the compound,

cytotoxicity was measured using the CytoTox-Glo citotoxicity assay, which measures the activity

of proteases after cells lose membrane integrity. Results show that the percentage of dead Huh-

7 cells in culture after 24h was almost identical in 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, percentage 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 biological effects of SR1848. The effects of

SR1848 on LRH-1 protein were first tested by treating Huh-7 cells with a moderate (5 µM)


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

15

concentration of SR1848. Following 24 hours of treatment, cells were harvested and the nuclear

fraction analyzed. As expected, 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 targets CYP19, SHP and CYP8B1 (Fig,

4C). In addition, the anti-proliferative effect of SR1848 treatment in Huh-7 cells was reproduced

by siRNA against LRH-1. 96h after transfection, LRH-1 siRNA transfected cells were unable to

proliferate as efficiently as control-transfected cells (Fig. 4D). These results show that

pharmacological repression of the LRH-1 pathway with SR1848 achieves similar biological

effects as knock down of the receptor and support the compound’s efficacy is mediated via

LRH-1 signaling.

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 two hours of SR1848

treatment the mRNA levels of LRH-1 receptor and its downstream targets (CYP19, GATA3 and

GATA4) are 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).

We hypothesized that the ability of LRH-1 to bind chromatin might be altered by SR1848. In

chromatin immunoprecipitation 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 1h after treatment

(Fig. 5C). Immunohistochemistry assays conducted to visualize LRH-1 within the nucleus (Fig.

5D) show that 2 hours following SR1848 treatment, LRH-1 translocates from nucleus to the

cytoplasm. From these results it appears that SR1848 interferes with LRH-1 chromatin


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

16

interaction in part by inducing nuclear export of the receptor to the cytoplasm which results in

reduced transcriptional activation of LRH-1 target genes. A cartoon depicting this mechanism of

action for SR1848 is shown in Fig. 6. In the top panel of Fig. 6, transcription is constitutively on

when LRH1 binds to the response elements in the promoter region of its target genes. SR1848

treatment leads to dissociation of LRH1 from chromatin and subsequently, inhibiting

transcription of the target gene (bottom panel Fig. 6).

While it is plausible that SR1848 represses LRH-1 activity by binding to the ligand binding

pocket (LBP) within its LBD to facilitate recruitment of co-repressors thus acting as a classical

inverse agonist, our efforts to detect direct binding of SR1848 to the LBD of LRH-1 expressed

and purified from E. coli has not proven fruitful. As described above, the LRH-1 LBD isolated

from bacterial expression systems contains phospholipids in the LBP (Musille et al, 2012).

Therefore, it is possible that a non-lipid like 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 of 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.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

17

DISCUSSION

The data presented in this paper characterizes 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 VP-16 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 siRNAs directed against LRH-1 is sufficient to induce

biological effects similar to 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 begin to diminish four hours after treatment with

SR1848, whereas SR1848 starts to repress certain target genes in as early as two 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 if post-translational modifications (i.e.

sumoylation, phosphorylation, acetylation) known to be associated with LRH-1 regulation (Lee

et al, 2006); Chalkiadaki & Talianidis, 2005) are involved in SR81848’s mechanism of action.

Our initial studies indicate that SR1848 has anti-proliferative 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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

18

expressing Huh-7 cells. The anti-proliferative 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

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. While most nuclear receptors require binding of ligand to become transcriptionally

active, LRH-1 appears to be constitutively active when expressed in cells. The data presented

here demonstrates that SR1848 is a non-lipid-like synthetic repressor of LRH-1 activity and a

potential therapeutic agent for targeting LRH-1 dependent cancers.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

19

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


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

20

References

Benod C, Carlsson J, Uthayaruban R, Hwang P, Irwin JJ, Doak AK, Shoichet BK, Sablin EP, Fletterick RJ

(2013) Structure-based discovery of antagonists of nuclear receptor LRH-1. The Journal of biological

chemistry 288: 19830-19844

Benod C, Vinogradova MV, Jouravel N, Kim GE, Fletterick RJ, Sablin EP (2011) Nuclear receptor liver

receptor homologue 1 (LRH-1) regulates pancreatic cancer cell growth and proliferation. Proc Natl Acad

Sci U S A 108: 16927-16931

Botrugno OA, Fayard E, Annicotte JS, Haby C, Brennan T, Wendling O, Tanaka T, Kodama T, Thomas W,

Auwerx J, Schoonjans K (2004) Synergy between LRH-1 and beta-catenin induces G1 cyclin-mediated cell

proliferation. Molecular cell 15: 499-509

Bouchard MF, Taniguchi H, Viger RS (2005) Protein Kinase A-Dependent Synergism between GATA

Factors and the Nuclear Receptor, Liver Receptor Homolog-1, Regulates Human Aromatase (CYP19) PII

Promoter Activity in Breast Cancer Cells. Endocrinology 146: 4905-4916

Broadus J, McCabe JR, Endrizzi B, Thummel CS, Woodard CT (1999) The Drosophila beta FTZ-F1 orphan

nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone.

Molecular cell 3: 143-149

Busby S, Nuhant P, Cameron M, Mercer BA, Hodder P, Roush WR, Griffin PR (2010) Discovery of Inverse

Agonists for the Liver Receptor Homologue-1 (LRH1; NR5A2). In Probe Reports from the NIH Molecular

Libraries Program. Bethesda (MD)

Chand AL, Herridge KA, Thompson EW, Clyne CD (2010) The orphan nuclear receptor LRH-1 promotes

breast cancer motility and invasion. Endocr Relat Cancer 17: 965-975

Clyne CD, Speed CJ, Zhou J, Simpson ER (2002) Liver Receptor Homologue-1 (LRH-1) Regulates

Expression of Aromatase in Preadipocytes. J Biol Chem 277: 20591-20597

Fabian CJ (2007) The what, why and how of aromatase inhibitors: hormonal agents for treatment and

prevention of breast cancer. Int J Clin Pract 61: 2051-2063

Fayard E, Auwerx J, Schoonjans K (2004) LRH-1: an orphan nuclear receptor involved in development,

metabolism and steroidogenesis. Trends in Cell Biology 14: 250-260

Francis GA, Fayard E, Picard F, Auwerx J (2003) Nuclear receptors and the control of metabolism. Annual

review of physiology 65: 261-311


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

21

Hadizadeh S, King DN, Shah S, Sewer MB (2008) Sphingosine-1-phosphate regulates the expression of

the liver receptor homologue-1. Mol Cell Endocrinol 283: 104-113

Key TJ, Appleby PN, Reeves GK, Roddam AW, Helzlsouer KJ, Alberg AJ, Rollison DE, Dorgan JF, Brinton LA,

Overvad K, Kaaks R, Trichopoulou A, Clavel-Chapelon F, Panico S, Duell EJ, Peeters PH, Rinaldi S,

Fentiman IS, Dowsett M, Manjer J, Lenner P, Hallmans G, Baglietto L, English DR, Giles GG, Hopper JL,

Severi G, Morris HA, Hankinson SE, Tworoger SS, Koenig K, Zeleniuch-Jacquotte A, Arslan AA, Toniolo P,

Shore RE, Krogh V, Micheli A, Berrino F, Barrett-Connor E, Laughlin GA, Kabuto M, Akiba S, Stevens RG,

Neriishi K, Land CE, Cauley JA, Lui LY, Cummings SR, Gunter MJ, Rohan TE, Strickler HD (2011) Circulating

sex hormones and breast cancer risk factors in postmenopausal women: reanalysis of 13 studies. Br J

Cancer 105: 709-722

Krylova IN, Sablin EP, Moore J, Xu RX, Waitt GM, MacKay JA, Juzumiene D, Bynum JM, Madauss K,

Montana V, Lebedeva L, Suzawa M, Williams JD, Williams SP, Guy RK, Thornton JW, Fletterick RJ, Willson

TM, Ingraham HA (2005) Structural Analyses Reveal Phosphatidyl Inositols as Ligands for the NR5

Orphan Receptors SF-1 and LRH-1. Cell 120: 343-355

Laffitte BA, Joseph SB, Walczak R, Pei L, Wilpitz DC, Collins JL, Tontonoz P (2001) Autoregulation of the

human liver X receptor alpha promoter. Mol Cell Biol 21: 7558-7568

Lavorgna G, Ueda H, Clos J, Wu C (1991) FTZ-F1, a steroid hormone receptor-like protein implicated in

the activation of fushi tarazu. Science (New York, NY 252: 848-851

Lee JM, Lee YK, Mamrosh JL, Busby SA, Griffin PR, Pathak MC, Ortlund EA, Moore DD (2011) A nuclear-

receptor-dependent phosphatidylcholine pathway with antidiabetic effects. Nature 474: 506-510

Lee YK, Choi YH, Chua S, Park YJ, Moore DD (2006) Phosphorylation of the hinge domain of the nuclear

hormone receptor LRH-1 stimulates transactivation. The Journal of biological chemistry 281: 7850-7855

Li Y, Choi M, Cavey G, Daugherty J, Suino K, Kovach A, Bingham NC, Kliewer SA, Xu HE (2005)

Crystallographic identification and functional characterization of phospholipids as ligands for the orphan

nuclear receptor steroidogenic factor-1. Molecular cell 17: 491-502

Lin BC, Suzawa M, Blind RD, Tobias SC, Bulun SE, Scanlan TS, Ingraham HA (2009) Stimulating the GPR30

estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell

proliferation. Cancer Res 69: 5415-5423

Luo X, Ikeda Y, Parker KL (1994) A cell-specific nuclear receptor is essential for adrenal and gonadal

development and sexual differentiation. Cell 77: 481-490


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

22

Madoux F, Li X, Chase P, Zastrow G, Cameron MD, Conkright JJ, Griffin PR, Thacher S, Hodder P (2008)

Potent, selective and cell penetrant inhibitors of SF-1 by functional ultra-high-throughput screening. Mol

Pharmacol 73: 1776-1784

Musille PM, Pathak MC, Lauer JL, Hudson WH, Griffin PR, Ortlund EA (2012) Antidiabetic phospholipid-

nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation. Nature

structural & molecular biology 19: 532-537, S531-532

Pineda Torra I, Jamshidi Y, Flavell DM, Fruchart JC, Staels B (2002) Characterization of the human

PPARalpha promoter: identification of a functional nuclear receptor response element. Mol Endocrinol

16: 1013-1028

Rey J, Hu H, Kyle F, Lai CF, Buluwela L, Coombes RC, Ortlund EA, Ali S, Snyder JP, Barrett AG (2012)

Discovery of a new class of liver receptor homolog-1 (LRH-1) antagonists: virtual screening, synthesis

and biological evaluation. ChemMedChem 7: 1909-1914

Schoonjans K, Dubuquoy L, Mebis J, Fayard E, Wendling O, Haby C, Geboes K, Auwerx J (2005) Liver

receptor homolog 1 contributes to intestinal tumor formation through effects on cell cycle and

inflammation. Proceedings of the National Academy of Sciences 102: 2058-2062

Solomon IH, Hager JM, Safi R, McDonnell DP, Redinbo MR, Ortlund EA (2005) Crystal structure of the

human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity.

Journal of molecular biology 354: 1091-1102

Venteclef N, Jakobsson T, Ehrlund A, Damdimopoulos A, Mikkonen L, Ellis E, Nilsson LM, Parini P, Janne

OA, Gustafsson JA, Steffensen KR, Treuter E (2010) GPS2-dependent corepressor/SUMO pathways

govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response. Genes &

development 24: 381-395

Whitby RJ, Dixon S, Maloney PR, Delerive P, Goodwin BJ, Parks DJ, Willson TM (2006) Identification of

Small Molecule Agonists of the Orphan Nuclear Receptors Liver Receptor Homolog-1 and Steroidogenic

Factor-1. J Med Chem 49: 6652-6655

Whitby RJ, Stec J, Blind RD, Dixon S, Leesnitzer LM, Orband-Miller LA, Williams SP, Willson TM, Xu R,

Zuercher WJ, Cai F, Ingraham HA (2011) Small molecule agonists of the orphan nuclear receptors

steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2). Journal of

medicinal chemistry 54: 2266-2281

Yumoto F, Nguyen P, Sablin EP, Baxter JD, Webb P, Fletterick RJ (2012) Structural basis of coactivation of

liver receptor homolog-1 by beta-catenin. Proc Natl Acad Sci U S A 109: 143-148


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

23

FOOTNOTES

This work was supported in part by the by the Intramural Research Program of 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]

COI statement: None of the authors have conflicts of interest with the work presented in this

manuscript.


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

24

Figure Legends

Figure 1. Activity of SR1848 in luciferase assays. A) HEK293T cells were co-transfected with

Cyp19 promoter driving luciferase in the absence and presence of 5 µM SR1848 without or with

SHP as positive control. Significant difference was observed with either SR1848 treatment or

SHP when compared to 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

significant difference was observed. C) HEK293T cells were co-transfected 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 transfected with CYP19

promoter driving luciferase and treated at the indicated concentrations of SR1848. Luciferase

activity was determined using Brite-lite plus. For all panels, each data point was performed in

triplicate.

Figure 2. 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 ANOVA analysis

shows a statistical significant difference when 2hr and 6hr time points are compared, p < 0.05.

There is no difference when the other time points are compared vs. 2 hours. B, C, D) Dose

dependent inhibition of LRH-1 (B), CYP19 (C) and SHP (D) mRNA expression. Each data point

was performed in triplicate. Data is represented as mean +/- SEM. Significant difference

between DMSO and SR1848 treatment as determined by t-test, p < 0.05, is depicted by

asterisks (***).E) Gene expression in tissues from C57Bl/6 mice dosed with SR1848 or vehicle

for 10 days (n=5 per group).

Figure 3. SR1848 inhibits cell proliferation in an LRH-1-dependent manner. A, B)

Endogenous LRH-1 expression comparison between Huh-7 and SKOV-3 cells; mRNA levels (A)

and nuclear protein (B). Figures are representative of 3 separate experiments with similar


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

25

results. C, D) Dose dependent inhibition of cyclin D1 (C) and cyclin E1 (D) expression in Huh-7

versus SKOV-3 cells. Significant difference between DMSO and SR1848 treatment as

determined by t-test, p < 0.05, is depicted by asterisks (****). Each data point was performed in

triplicate. E) Anti-proliferative 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. Significant difference as

compared to control is noted at 10 and 30 µM SR1848 doses, p < 0.05.

Figure 4. LRH-1 knockdown reproduces the effects of SR1848. A) Huh-7 cells were treated

in culture with either DMSO or SR1848 (5 µM) for 24h followed by sub-cellular fractionation and

detection of LRH-1 protein by anti-LRH-1 antibody. Lamin A/C was used as a nuclear loading

control. Figure is representative of 3 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 48h after transfection. Figure is representative of 2

individual experiments with similar result C) Expression of LRH-1 target genes by qPCR 48h

after transfection. Data shown are representative of two independent experiments performed in

triplicate. Significant difference was noted when comparing siControl to siLRH-1 for all genes

tested, p < 0.05. D) Evaluation of cell proliferation 96h after siRNA transfection. Each data point

was performed in triplicate. Significant difference between siControl and siLRH-1 was noted at

96 and 120 hour time points, p < 0.05.

Figure 5. SR1848 treatment leads to export of LRH-1 from the nucleus. A) LRH-1 mRNA

expression analysis by QPCR from Huh-7 treated with DMSO or SR1848 (5 μM) for various

time points. Data represents 3 different experiments performed in triplicate. Significant

difference was observed when comparing DMSO and time points > 1hr, p < 0.05. B) Huh-7 cells

were treated in culture with either DMSO or SR1848 (5 µM) for 2 hours followed by sub-cellular


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

26

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 1hr followed by incubation with AlexaFluor488-anti mouse antibody for 1hr.

Dapi was used for counter staining and cells were visualized by fluorescence microscopy.

Quantification of the mean fluorescence intensity from cytoplasm and nucleus is shown. Figure

is representative of 2 separate experiments with similar results.

Figure 6. Proposed mechanism of SR1848. Top panel) LRH1 binding to the promoter region

of its target genes leads to transcription activation. Bottom panel) SR1848 treatment causes

release of LRH1 from chromatin resulting in reduced LRH1 interaction with promoter regions of

target genes and subsequently, decreasing or turning off transcription


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


MOL #95554

27

Table 1. List of QPCR primers used

Target Sense Anti-sense

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’


at ASPE


molpharm

.aspetjournals.orgD

ownloaded from



at ASPE


molpharm

.aspetjournals.orgD

ownloaded from


anti-proliferation activity of a small molecule repressor of lrh-1 · 2014. 12. 3. · 1 can bind...

Documents