Download Glucocorticoids induce CCN5/WISP

Biochem. J. (2012) 447, 71–79 (Printed in Great Britain)
71
doi:10.1042/BJ20120311
Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in
oestrogen receptor-negative human breast cancer cells
Nathalie FERRAND*†‡, Emilien STRAGIER‡§, Gérard REDEUILH*†‡ and Michèle SABBAH*†‡1
*Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Paris, France, †Institut National de la Santé et de la Recherche Médicale U938, Paris, France, ‡Université Pierre
et Marie Curie, Paris, France, §Centre de Psychiatrie et Neurosciences Site Pitié Salpêtrière, Paris, France, and Institut National de la Santé et de la Recherche Médicale U894, Paris,
France
CCN5 (cysteine-rich 61/connective tissue growth factor/
nephroblastoma overexpressed 5)/WISP-2 [WNT1 (winglesstype MMTV integration site family, member 1)-inducible
signalling pathway protein 2] is an oestrogen-regulated member
of the CCN family. CCN5 is a transcriptional repressor of genes
associated with the EMT (epithelial–mesenchymal transition)
and plays an important role in maintenance of the differentiated
phenotype in ER (oestrogen receptor)-positive breast cancer cells.
In contrast, CCN5 is undetectable in more aggressive ER-negative
breast cancer cells. We now report that CCN5 is induced in ERnegative breast cancer cells such as MDA-MB-231 following
glucocorticoid exposure, due to interaction of the endogenous glucocorticoid receptor with a functional glucocorticoid-response
element in the CCN5 gene promoter. Glucocorticoid treatment
of MDA-MB-231 cells is accompanied by morphological
alterations, decreased invasiveness and attenuated expression
of mesenchymal markers, including vimentin, cadherin 11 and
ZEB1 (zinc finger E-box binding homeobox 1). Interestingly,
glucocorticoid exposure did not increase CCN5 expression in
ER-positive breast cancer cells, but rather down-regulated ER
expression, thereby attenuating oestrogen pathway signalling.
Taken together, our results indicate that glucocorticoid treatment
of ER-negative breast cancer cells induces high levels of CCN5
expression and is accompanied by the appearance of a more
differentiated and less invasive epithelial phenotype. These
findings propose a novel therapeutic strategy for high-risk breast
cancer patients.
INTRODUCTION
lines such as MCF-7 compared with untransformed cells, which
expressed low levels of CCN5. In contrast, CCN5 is undetected
in the highly aggressive breast cancer cell lines [11,17]. When
introduced into poorly differentiated, oestrogen-independent and
invasive breast cancer cells, such as MDA-MB-231 cells, CCN5
was able to reduce their proliferative and invasive phenotypes [11].
Furthermore, in vivo studies have shown that CCN5 expression is
mainly detected in preneoplastic disorders, such as non-invasive
DCIS (ductal carcinoma in situ) and atypical ductal hyperplasia,
whereas expression levels were either minimal or undetectable in
invasive cancer cells [16,18]. These studies indicate that loss of
CCN5 activity may promote breast cancer progression [19].
We searched compounds that could elevate CCN5 expression
in metastatic human breast cancer cell lines. Previously, we have
cloned and characterized a fragment of the human CCN5 gene
promoter, which revealed the presence of a number of potential
binding sites for several transcription factors [9]. By using the
TRANSFAC database, a closer analysis revealed the presence of
an additional putative GRE (glucocorticoid-response element)binding site. There has been renewed interest in alternative
hormonal treatments, including glucocorticoids for use in the
advanced disease [ER- and PR (progesterone receptor)-negative]
when conventional therapies fail [20–22]. The purpose of the
present study was to examine the possibility of inducing the expression of CCN5 by glucocorticoid treatment in human
CCN5 {cysteine-rich 61/connective tissue growth factor/
nephroblastoma overexpressed 5; previously known as WISP-2
[WNT1 (wingless-type MMTV integration site family, member
1)-inducible signalling pathway protein 2]} is a member of the
CCN family [1,2]. The CCN family is composed of six members
grouped on the basis of structural analogies [3,4]. It has been
reported that CCN5 inhibits proliferation and motility of human
uterine smooth muscle cells [5], and loss of CCN5 signalling is
associated with the development of cancer in the pancreas [6]
and in the colon [7]. Previous studies have demonstrated that the
expression of CCN5 is induced by oestrogens in ER (oestrogen
receptor) α-positive breast cancer cells [8–10]. We found
that CCN5 knockdown induced an E2 (oestradiol)-independent
growth of these cells linked to a loss of ERα expression,
and promoted EMT (epithelial–mesenchymal transition) [11].
Moreover, in vitro studies suggest that loss of CCN5 signalling
may be a crucial event for EMT in pancreatic cancer [6]. EMT
is one of the key processes involved in tumour progression and
metastasis [12–15]. In ERα-positive cell lines, CCN5 inhibits the
expression of genes involved in the TGF-β (transforming growth
factor β) signalling cascade that is known to promote EMT [16].
Consistent with these results, further studies have shown that
CCN5 is highly expressed in less aggressive breast cancer cell
Key words: breast cancer, cysteine-rich 61/connective tissue
growth factor/nephroblastoma overexpressed 5 (CCN5)/WNT1
(wingless-type MMTV integration site family, member
1)-inducible signalling pathway protein 2 (WISP-2), epithelium,
glucocorticoid, mesenchyme, oestrogen.
Abbreviations used: CCN, cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed; ChIP, chromatin immunoprecipitation; DAPI,
4 ,6 -diamino-2-phenylindole; DEX, dexamethasone; DMEM, Dulbecco’s modified Eagle’s medium; E2, oestradiol; EMT, epithelial–mesenchymal transition;
ER, oestrogen receptor; FBS, fetal bovine serum; GR, glucocorticoid receptor; GRE, glucocorticoid-response element; PR, progesterone receptor; RT,
reverse transcription; TGF-β, transforming growth factor β; WISP-2, WNT1-inducible signalling pathway protein 2; ZEB1, zinc finger E-box binding
homeobox 1.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
72
N. Ferrand and others
ER-negative breast cancer cell lines that exhibit nearly
undetectable expression of CCN5.
In the present study, we show that the CCN5 mRNA transcript
level is up-regulated by glucocorticoids in highly invasive human
breast cancer cell lines. In ERα-positive cell lines, we observe
an anti-oestrogenic action of glucocorticoids due to, at least
in part, the down-regulation of the ERα mRNA [encoded by
the gene ESR1 (oestrogen receptor 1)] synthesis, leading to a
reduced ERα protein level and to the attenuation of the oestrogeninduced CCN5 expression. Furthermore, glucocorticoid treatment
of the poorly differentiated, hormone-independent and aggressive
MDA-MB-231 cells induced a high level of CCN5 and
significant morphological changes and growth as clusters, features
characteristic of epithelial cells. We also found that glucocorticoid
treatment reduced cell mobility and invasion. We hypothesize
that glucocorticoid-induced elevation of CCN5 expression in
micrometastatic tumour cells in breast cancer patients may inhibit
additional colonization and invasion, and prevent relapse.
EXPERIMENTAL
Cell culture and transfection
MDA-MB-231, MDA-MB-435, MCF-7, T47D and ZR75.1
human breast carcinoma cells were maintained in DMEM
(Dulbecco’s modified Eagle’s medium) or RPMI 1640
supplemented with 10 % (v/v) FBS (fetal bovine serum). For the
promoter activity assays, MDA-MB-231 and MCF-7 cells were
plated in six-well plates in Phenol-Red-free DMEM with 5 %
(v/v) dextran-charcoal stripped FBS, 24 h prior to transfection.
Cells were transfected with plasmids using LipofectamineTM
reagent (Invitrogen). After overnight incubation, cells were
treated with 10 nM DEX (dexamethasone) or vehicle and
harvested 24 h later for the determination of luciferase and βgalactosidase activities.
Plasmids and mutagenesis
The CCN5 reporter constructs used for luciferase assays were
described in [9]. The template for the mutagenesis was the
luciferase reporter plasmid -1919. Point mutations were created
by using PCR-based oligonucleotide-directed mutagenesis by
using QuikChange II site-directed mutagenesis (Agilent) and
a primer containing four point mutations in the GRE of the
CCN5 gene promoter 5 -GGTggGTACccTTCC-3 to yield 1919 mGRE. PCR-generated fragments were sequenced.
Real-time RT (reverse transcription)–PCR
Total RNA was extracted from all cell lines using the TRIzol®
RNA purification reagent. Total RNA (1 μg) from each sample
was reverse transcribed and real-time RT–PCR measurements
were performed as described previously [9] using an Mx3000P
apparatus (Agilent) with the corresponding SYBR Green kit.
Primers for the amplification of CCN5 and ERα genes are
described in [23].
ChIP (chromatin immunoprecipitation) assay
MDA-MB-231 cells were grown in DMEM supplemented
with 5 % (v/v) dextran-charcoal stripped FBS for 3 days
and then treated for 45 min with vehicle or 10 nM DEX.
ChIP assays were performed largely as described previously
[23]. Briefly, a small portion (1 %) of the cross-linked,
sheared chromatin solution was saved as input DNA, and
c The Authors Journal compilation c 2012 Biochemical Society
the remainder was used for immunoprecipitation by specific
antibodies against GR (glucocorticoid receptor; Santa Cruz
Biotechnology) or non-specific IgG. Immunoprecipitated DNA
was deproteinized, precipited by ethanol and resuspended in
30 μl of water. DNA was then subjected to PCR using the
following primer pairs of the CCN5 gene promoter amplification:
forward, 5 -TCCCCAAATCTTGGTGATGT-3 ; and reverse, 5 GAGAGGGCAGAATGTTCCAG-3 .
Immunoblotting
Cell lysates were made in RIPA lysis buffer (50 mM Tris/HCl,
pH 8, 150 mM NaCl, 1 % Nonidet P40, 0.1 % SDS and
0.5 % sodium deoxycholate) containing 0.5 mM PMSF, 1 μM
leupeptin and 1 μM aprotinin and cleared by centrifugation
(20 min at 10 000 g). Protein (50 μg) was separated by
SDS/PAGE (7.5 or 12% gels) and transferred on to nitrocellulose
membranes. The membranes were blocked with saturating buffer
for 1 h at room temperature (25 ◦ C). The membranes were
probed with the following specific antibodies: anti-ERα (Ab15, Neomarkers), anti-GR, anti-actin, anti-(cadherin 11), antiZEB1 (zinc finger E-box binding homeobox 1), anti-CCN5
(Santa Cruz Biotechnology) and anti-vimentin (Sigma) overnight
at 4 ◦ C. Membranes were then washed and incubated with
horseradish peroxidase-conjugated secondary antibodies for 2 h.
Membranes were washed extensively and developed with an
enhanced chemiluminescence kit (GE Healthcare).
Microscopic imaging
MDA-MB-231 cells were cultured in DMEM supplemented
with 5 % (v/v) dextran-charcoal stripped FBS with or without
various concentrations of DEX for various times. The cells
are photographed by System Time-relapse imaging microscopy
(Biostation IM-Q, Nikon) at ×20 magnification. Alternatively,
cells were fixed in 4 % (w/v) paraformaldehyde/PBS and then
treated briefly with 0.1 % Triton X-100 in PBS. After rinses with
PBS, cells were incubated with fluorescein phalloidin in the dark
for 40 min at room temperature or with primary antibodies against
CCN5 for 1 h at room temperature, rinsed and incubated for 1 h
with FITC-conjugated secondary antibody (Invitrogen). Nuclei
were stained with DAPI (4 ,6 -diamino-2-phenylindole).
Images were obtained with a Leica DMR microscope equipped
with a fluorescence imaging system (×63 objective).
In vitro migration and invasion assays
Cell migration and invasion were determined with Boyden
chambers or modified Boyden chambers coated with Matrigel
(BD Biosciences) according to the protocol provided by the
manufacturer. Briefly, 100 000 MDA-MB-231 cells treated or not
with DEX were placed on 8.0-μm pore size membrane inserts
in 24-well plates in serum-free media. Medium with 10 % FBS
was placed in the bottom wells as a chemoattractant. After an
incubation period of 24 h, cells on the lower side of the membrane
were stained with Diff-Quick (Harleco) and the cells on each
insert were counted at ×10 magnification by System Time-relapse
imaging microscopy (Biostation IM-Q, Nikon).
Data analysis
Data are shown as the means+
−S.D. of results of at least three
independent experiments. Differences between test and control
conditions were assessed by Student’s t test. Significance is
indicated by: *P < 0.05, **P < 0.01 and ***P < 0.001.
Regulation of CCN5 gene expression by glucocorticoids
Figure 1
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DEX-induced CCN5 mRNA expression in ERα-negative breast cancer cell lines
RNA was isolated from human breast cancer cell lines treated with 100 nM DEX for 8 h and 24 h, RU486 at 1 μM was added in combination with 100 nM DEX for 24 h and CCN5 (A) and G0S8
(B) expression were analysed by real-time RT–PCR. The result after normalization represents the relative CCN5 and G0S8 mRNA transcript levels and is the means+
−S.D. of triplicate experiments.
Significant differences: *P < 0.05; **P < 0.01.
RESULTS
DEX increases CCN5 mRNA in human ERα-negative breast cancer
cell lines
CCN5 mRNA expression has been reported to be overexpressed
in cancerous cells of human breast tissue and in breast cancer
cell lines characterized by a more differentiated phenotype
[11,16,18,24]. In the experiment shown in Figure 1, five human
breast cancer cell lines were first cultured in hormone-free
medium for 2 days and then treated with 10 − 7 M DEX in the
absence or presence of 10 − 6 M RU486 (a potent synthetic
antiglucocorticoid). We found that in the ERα-negative, highly
invasive and metastasizing cell lines MDA-MB-231 and MDAMB-435, the level of CCN5 mRNA was increased 8-fold and
3-fold respectively after a 24 h incubation with DEX. A 2-fold
elevation of mRNA expression in response to DEX was observed
in the ERα-positive, poorly invasive cell line MCF-7 and only a
slight, but reproducible, increase in the two similar ERα-positive
cell lines T47D and ZR-75.1. The DEX-mediated increase in
the CCN5 mRNA transcript level was antagonized by RU486,
confirming the specificity of the induction. The DEX-specific
induction of G0S8 mRNA observed in the MCF-7 and ZR-75.1
cells served as a positive control [25] and suggested that
CCN5 could be regulated differently by DEX in these cells.
Furthermore, DEX regulation of CCN5 expression appears to be
explained by transcriptional activation of its gene rather than RNA
stabilization, since accumulation of CCN5 mRNA is blocked by
the RNA synthesis inhibitor actinomycin D (results not shown).
Inhibition of protein synthesis with cycloheximide increased
the accumulation of CCN5 mRNA by DEX treatment (results
not shown), suggesting that a labile protein may act to repress
transcription of the CCN5 gene.
The GRE motif in the CCN5 promoter confers glucocorticoid
inducibility
To identify the glucocorticoid-responsive region in the CCN5
gene promoter, we used the serial deletion constructs described
previously [9]. Constructs containing varying lengths of the CCN5
promoter upstream of the luciferase reporter gene (Figure 2A)
were transiently transfected in ERα-negative MDA-MB-231 cells
or ERα-positive MCF-7 cells, and the cells were incubated
with 10 nM of DEX. The reporter MMTV-Luc, which is
strongly induced by glucocorticoid, was used as a control
(Figures 2B and 2C, inset). As shown in Figure 2(B), the
promoter constructs -1919 and -1200 were inducible by
DEX (factor of induction ∼4) and also produced a high basal
and induced absolute level of expression in MDA-MB-231
cells, whereas a very slight increase was observed in MCF-7
cells. In contrast, additional promoter deletion constructs (808, -520 and -422) prevented glucocorticoid induction. We
conclude that the − 1200 to − 808 region of the promoter is
responsible for the glucocorticoid stimulation. Since one putative
GRE (5 -GGTACGTACTGTTCC-3 ) resides in this region, we
hypothesized that this site was required to confer glucocorticoid
up-regulation to the CCN5 gene promoter. To confirm this
hypothesis, we modified the GRE by site-directed mutagenesis
with an oligonucleotide containing four point mutations (5 GGTggGTACccTTCC-3 ; mutations in lower-case) reported to
lead a loss of glucocorticoid responsiveness [26]. Mutation of
the GRE site in the -1919 construct totally prevented the
glucocorticoid induction (Figure 2C). These results demonstrate
that the presence and the integrity of the GRE are necessary to
confer glucocorticoid inducibility to the CCN5 gene promoter.
Although the motif of the GRE identified in the present study is
imperfect when compared with the classical sequence of GRE (5 GGTACANNNTGTTCT-3 ), it is a functional GRE responsible
for the glucocorticoid-induced stimulation of the CCN5 gene
promoter.
To address the question of whether endogenous GR is recruited
to the CCN5 gene promoter in vivo, we performed a ChIP assay
using a GR-specific antibody. The recruitment of endogenous
GR to the CCN5 gene promoter was examined in MDA-MB231 cells after a 45 min DEX treatment. The results show that
DEX induces a significant increase in GR occupancy of the
− 1150 to − 969 region of the CCN5 gene promoter containing
the GRE (Figure 2D). As expected, we did not observe any
significant increase in the GR occupancy of the coding region
of the CCN5 gene used as a negative control. In addition, a
ChIP assay performed using non-immune IgG failed to show
any amplification of the CCN5 gene promoter region containing
the GRE (Figure 2D). These results show that endogenous GR
is recruited to the CCN5 gene promoter in a glucocorticoiddependent manner, confirming the implication of this region in
the DEX regulation of the CCN5 gene promoter.
DEX counteracts the stimulatory effect of E2 on transcription
As oestrogens can regulate CCN5 expression [8–10], we
examined whether, in MCF-7 cells, E2 and DEX could
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 2
N. Ferrand and others
The GRE motif in the CCN5 promoter confers glucocorticoid inducibility
(A) Schematic representation of the different CCN5 promoter luciferase (LUC) reporter constructs. (B) MDA-MB-231 and MCF-7 cells were transiently co-transfected with 0.5 μg of the luciferase
reporter pGL3-Basic vector or each CCN5 promoter-luciferase reporter and 0.1 μg of plasmid expressing β-galactosidase as an internal control. The inset represents the MMTV-Luc reporter that is
strongly induced by DEX. (C) MDA-MB-231 cells were transiently co-transfected with 0.5 μg -1919 or -1919mGRE and 0.1 μg plasmid expressing β-galactosidase as an internal control. After
transfection, cells were treated with vehicle or 10 nM DEX for 24 h and assayed for luciferase and β-galactosidase activities. The results represent the means for three independent experiments assayed
in triplicate. *P < 0.05; **P < 0.01. (D) Crosslinked sheared chromatin, from MDA-MB-231 cells treated with vehicle or DEX for 45 min, was immunoprecipated with the indicated antibodies. DNA
was analysed by PCR using primers to amplify the CCN5 promoter region or a coding region of the CCN5 gene. Results shown are representative of three independent experiments. ORF, open
reading frame.
synergistically activate the transcription downstream of the CCN5
gene promoter. For this purpose, we evaluated the CCN5 mRNA
transcript level by real-time RT–PCR in MCF-7 cells in response
to E2 or DEX alone or together. As shown in Figure 3(A),
DEX was 50 % as efficient as E2 in stimulation of transcription
of the CCN5 gene. The presence of oestrogen together with
DEX did not increase the promoter activity compared with DEX
alone. Furthermore, the oestrogen-mediated increase of the CCN5
mRNA decreased in the presence of DEX and could be restored by
c The Authors Journal compilation c 2012 Biochemical Society
the antiglucocorticoid RU486. DEX induction of CCN5 mRNA
was unaffected by co-treatment with the anti-oestrogen ICI
182780. Other endogenous specific oestrogen-regulated genes,
such as those encoding pS2 or PR, were also down-regulated
by co-incubation of oestrogen and DEX (Figure 3A). Thus, in
breast cancer cells, glucocorticoids exert inhibitory effects on
the ERα-dependent transcription of endogenous oestrogen target
genes, including CCN5, even when these genes are not directly
regulated by glucocorticoids.
Regulation of CCN5 gene expression by glucocorticoids
Figure 3
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Glucocorticoids inhibit oestrogen-dependent transcription in MCF-7 cells
(A) MCF-7 cells were grown in Phenol Red- and hormone free-medium supplemented with 5 % (v/v) dextran-charcoal stripped FBS for 48 h. Cells were treated with vehicle, 10 nM E2, 100 nM DEX
alone or in combination with or without 1 μM RU486 or 1 μM ICI 182780 for 24 h. mRNA levels were detected by real-time PCR and were normalized to 36B4 mRNA. Fold induction in mRNA
expression was calculated as compared with the corresponding vehicle control conditions. Results are the means+
−S.D. for three independent experiments. (B) MDA-MB-231 cells were transiently
co-transfected with 0.5 μg of -1919 and 0.1 μg of plasmid expressing ERα or GR and 0.1 μg of plasmid expressing β-galactosidase as an internal control. After transfection, cells were treated
with vehicle, 10 nM E2, 10 nM DEX or 10 nM E2 with 10 nM DEX for 24 h and assayed for luciferase and β-galactosidase activities. The results represent the means+
−S.D. for three independent
experiments assayed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001.
Synergistic interaction between steroid hormone receptors or
between steroid hormone receptors and other transcription factors
has been observed previously [27–30]. Furthermore, the existence
of a negative HRE (hormone-response element), at least in the
case of GR, has also been reported [31]. Thus, to examine
how GR and ERα could exert mutually negative effects,
we measured the -1919 promoter activity in the MDAMB-231 cells co-transfected with expression vectors for GR
and ERα and treated with E2 or DEX alone or together
(Figure 3B). In contrast with the results obtained from
endogenous gene expression, the results obtained by transient
transfection experiments show a synergistic activation of
the promoter when cells were treated with E2 and DEX
together, suggesting that DEX could inhibit endogenous ERα
activity.
Effect of DEX on ERα mRNA and protein expression
In order to analyse the effects of DEX on ERα activity, MCF-7
cells were treated for 24 h with E2, DEX or both together
in the presence or absence of RU486 or ICI 182780. ERα
mRNA transcripts levels were quantified by real-time RT–PCR
and ERα and GR proteins by Western blotting. As expected
(Figure 4A), E2 inhibited the expression of its mRNA and
protein [32,33]. DEX treatment decreased both basal and E2repressed levels of ERα. This effect was prevented specifically by
the antiglucocorticoid RU486, but not by the anti-oestrogen ICI
182780 (Figure 4A). DEX reduced the levels of the ERα mRNA
transcripts in a dose-dependent manner (Figure 4B). The decrease
in ERα expression following DEX treatment was blocked by
the transcriptional inhibitor actinomycin D, demonstrating that
regulation of ERα expression was inhibited as the result of a
decrease in transcription (Figure 4C). In the presence of
cycloheximide, a potent translational inhibitor, DEX inhibited
ERα expression, suggesting that ERα repression by DEX does
not require protein synthesis and is therefore direct (Figure 4C).
The effect of DEX on ERα expression was mediated by the nuclear
effects of GR, given that RU486 and actinomycin D completely
blocked DEX-repressed ERα mRNA expression.
DEX treatment induces morphological and biological changes in
MDA-MB-231 cells
Our previous work [11] led us to propose that the CCN5 protein
is an important regulator involved in the maintenance of a
differentiated phenotype in breast tumour epithelial cells. As
DEX treatment of MDA-MB-231 cells induced the expression of
CCN5, we investigated whether these cells underwent significant
morphological changes. Parental MDA-MB-231 cells are spindle
shaped and grow as dispersed cells. In contrast, the MDA-MB-231
cells treated with 10 − 7 M DEX for 24 h appear well spread and
grow as groups of cells, features that are consistent with epithelial
cell growth (compare Figures 5A and 5M). The cell spreading was
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 4
N. Ferrand and others
DEX decreased ERα mRNA and protein expression
(A) Left-hand panel, MCF-7 cells were grown in Phenol Red- and hormone free-medium supplemented with 5 % (v/v) dextran-charcoal stripped FBS for 48 h then cells were treated with vehicle,
10 nM E2, 100 nM DEX alone or in combination with or without 1 μM RU486 or 1 μM ICI 182780 for 24 h, and ERα expression was analysed by real-time RT–PCR. Right-hand panel, 50 μg of
proteins extracted from MCF-7 cells treated as described above were separated by SDS/PAGE and blotted with the indicated antibodies. Actin was used as a loading control. (B) Dose-dependent
inhibition of ERα mRNA by DEX. MCF-7 cells were grown as described above and then cells were treated with vehicle or various concentrations of DEX for 24 h. (C) Effect of cycloheximide (CHX)
and actinomycin D (ActD) on the repression of ERα mRNA by DEX. MCF-7 cells were grown as described above and then cells were treated with vehicle or 10 μg/ml of cycloheximide or 1 μg/ml of
ActD with or without 100 nM DEX for 3 or 6 h. (A–C) ERα mRNA levels were detected by real-time PCR and were normalized to 36B4 mRNA. Fold induction in mRNA expression was calculated as
compared with the corresponding vehicle control conditions. Results are means+
−S.D. for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
very prominent after 48 h of treatment (Figure 5N). This effect was
also observed at lower concentrations of DEX (10 − 8 M) after 72 h
of treatment (compare Figures 5C and 5K), but no morphological
changes were observed at the lowest concentration of DEX
used, even after 6 days (Figures 5E–5H). Thus treatment of the
MDA-MB-231 cells with DEX produces marked changes in
the cellular morphology identical to those previously observed
in the MDA-MB-231-derived cell lines expressing CCN5 [11].
We then examined the expression and the subcellular distribution
of CCN5 in MDA-MB-231 cells treated with DEX (Figures 6A
and 6B). Immunofluorescence microscopy using anti-CCN5
antibodies revealed the presence of CCN5 in discrete nuclear
structures in addition to a cytoplasmic staining (Figure 6B). To
determine whether the flattened morphology induced by DEX was
associated with the formation of new stress fibers, filamentous
actin was visualized by FITC–phalloidin. Whereas the MDAMB-231 cells lack stress fibers, DEX treatment reorganizes the
cytoskeleton, as evident from the emergence of stress fibres
(Figure 6C).
(Figure 7A). Then we performed migration experiments and
we observed a 2.5-fold decrease in the cellular migration of
the DEX-treated cells (Figure 7B). These glucocorticoid effects
are reversible, since after withdrawal of DEX, the behaviour of
the cells is again the same as that of control cells (Figures 7A and
7B).
MDA-MB-231 cells do not express E-cadherin, but they do
express cadherin 11, which is associated with the increased
motility and invasive potential of some breast cancer cells
[34,35]. Treatment of MDA-MB-231 cells with DEX decreased
the expression of cadherin 11 (Figures 7C and 7D), but did
not lead to re-expression of E-cadherin (results not shown).
Furthermore, DEX treatment had no effect on Snail and Twist
expression (results not shown), but decreased the expression of the
mesenchymal marker vimentin and the epithelial–mesenchymal
transcription factor ZEB1 (Figures 7C and 7D). These effects are
observed in a time- and dose-dependent manner and may account,
at least in part, for the reduced invasiveness associated with these
treatments.
DEX treatment reduces the aggressive phenotype of human highly
invasive breast cancer cells.
DISCUSSION
We have previously shown that CCN5 is able to reduce the overall
aggressiveness of breast cancer cells [11]. Thus we evaluated the
invasive phenotype and motility of MDA-MB-231 cells treated or
not with DEX. In the matrigel invasion assay, cells treated by
DEX were approximately 4-fold less invasive than control cells
c The Authors Journal compilation c 2012 Biochemical Society
We provide evidence that DEX, a synthetic glucocorticoid and an
agonist ligand for the GR, elevates CCN5 expression in highly
invasive ER-negative human breast carcinoma cell lines in vitro.
The effect of the glucocorticoid is mediated via a transcriptional
GR-based mechanism. The glucocorticoid antagonist RU486 did
not increase CCN5 gene expression. Addition of RU486
Regulation of CCN5 gene expression by glucocorticoids
Figure 5
DEX induced morphological changes in MDA-MB-231 cells
Cells were treated with vehicle or various concentrations of DEX for different periods of time
before they were viewed by System Time-relapse imaging microscopy at magnification ×20
(Biostation IM-Q, Nikon).
simultaneously with DEX completely blocked the glucocorticoidinduced stimulation. These results indicate that the induction
of the CCN5 gene promoter requires agonist-activated GR. We
demonstrate that the glucocorticoid-induced expression of CCN5
is associated with the interaction of GR with a functional GRE
present in the CCN5 gene promoter. The importance of the
CCN5 putative site as a functional GRE was established as
follows: (i) mutation of this element abolished the glucocorticoid
response of the CCN5 gene promoter; and (ii) the GR association
to this DNA element was demonstrated in vivo using a ChIP
assay. Furthermore, glucocorticoid regulation of the CCN5 gene
promoter is abolished by actinomycin D, an inhibitor of RNA
polymerase II-dependent transcription. Thus the regulation of
the CCN5 gene by DEX is mediated, at least in part, at the
transcriptional level, clearly as a result of a direct interaction
of GR with the promoter region of the CCN5 gene.
Figure 6
77
In the ER-positive, poorly invasive and low-metastasizing
human breast cancer cell lines, DEX treatment was 50 %
as efficient as E2 in stimulation of transcription of the CCN5
gene. Furthermore, in these cells, DEX inhibited the stimulatory
transcription effect of oestrogen, possibly by down-regulation
of mRNA transcripts of the ER in a dose-dependent manner. A
similar effect of glucocorticoid on ER levels has been described
previously [36]. It has also been reported that pre-treatment
of MCF-7 cells with E2 produced significant decreases in GR
concentration [37]. It appears that oestrogens and glucocorticoids
exert inhibitory effects on the actions of each other in breast
cancer cells [30]. Hall et al. [38] have reported that ERα-negative
cell lines expressed more GR mRNA than ERα-positive lines and
showed an inverse relationship between ER and GR mRNA levels
in cells derived from normal or cancerous breast epithelium. This
mechanism could account for the side effects of glucocorticoids on
oestrogen target cells. The down-regulation of ERα expression is
inhibited by the presence of actinomycin D, but not in the presence
of cycloheximide, suggesting that the synthesis of RNA is implied
in this process.
Incubation of MDA-MB-231cells with DEX induces not only
a high level of CCN5, but also marked changes in the cellular
morphology; the DEX-treated MDA-MB-231 cells grow as
groups of flattened cells, consistent with normal epithelial cell
growth and in correlation with the reduction of cell motility and
invasion. Indeed, these cells behave similarly to the MDA-MB231-derived cell lines expressing CCN5 [11]. Furthermore, we
observed that DEX treatment not only repressed cadherin 11
expression as previously reported during bone cell differentiation
[39], but also vimentin and ZEB1 expresssion, two other
mesenchymal markers. Decreased expression of cadherin 11
without re-expression of E-cadherin has been shown in MDAMB-231 cells treated with EGFR inhibitors and induced a similar
phenotypic conversion [40].
Interestingly, it has been reported that treatment of two human
breast carcinoma cell lines, MDA-MB-231 or MDA-MB-435,
with glucocorticoids elevates the cell contents of a metastasis
suppressor protein Nm23-H1 [41,42]. Thus treatment of ERαnegative breast cancer with glucocorticoids could have two
important positive consequences: first, the re-establishment of
CCN5 expression could repress the genes associated with the
TGF-β signalling pathway concomitant with EMT as observed
previously [16]; and secondly, the induction of Nm23-H1, which
plays a role in regulating tumour cell metastasis. Therefore such
treatment may constitute a therapeutic strategy for high-risk breast
cancer patients. We hypothesize that treatment of ERα-negative
Immunostaining of CCN5 and phalloidin in MDA-MB-231 cells
(A) Protein extracts of cells treated with vehicle or 100 nM DEX were tested by Western blotting for CCN5 expression. Actin was used as a loading control. (B) Cells were fixed, permeabilized, and
then incubated with anti-CCN5 and fluorescein-labelled secondary antibody or (C) fluorescein–phalloidin to identify actin filaments. Nuclear DNA was stained with DAPI. Images were obtained by
microscopy at magnification ×63. Scale bars, 25 μm.
c The Authors Journal compilation c 2012 Biochemical Society
78
N. Ferrand and others
wrote the paper, and Nathalie Ferrand, Gérard Redeuilh and Michèle Sabbah were involved
in critical reading of the paper prior to submission.
ACKNOWLEDGEMENTS
We thank J. Mester and A.K. Larsen for critically reviewing the manuscript prior to
submission.
FUNDING
This work was supported by Institut National de la Santé et de la Recherche Médicale,
Centre National de la Recherche Scientifique and the Groupement d’Entreprises Françaises
dans la Lutte contre le Cancer (GEFLUC).
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Figure 7 Effect of DEX treatment on matrix invasion and mobility of the
MDA-MB-231 cells
(A) MDA-MB-231 cells treated or not with 100 nM DEX were plated on Matrigel-coated filters,
and the number of cells that migrated through the membrane after 24 h was determined. Error
bars indicate S.D. of the results from ten independent fields. (B) Boyden chamber assays were
performed to compare the migration of MDA-MB-231 treated or not with 100 nM DEX. Error
bars indicate S.D. of the results from ten independent fields. (C and D) Protein extracts of
MDA-MB-231 cells treated or not with various concentrations of DEX for 3 days (C) or 6 days
(D) were tested by Western blotting for ZEB1, vimentin and cadherin 11 expression. Actin was
used as a loading control. *P < 0.05; ***P < 0.001.
breast cancer patients with glucocorticoids could have beneficial
effects. Indeed, inhibition of EMT and/or reversion of breast
cancer cells to a more differentiated epithelial phenotype may
improve the prognosis and sensitize the tumour to conventional
therapy.
AUTHOR CONTRIBUTION
Gérard Redeuilh and Michèle Sabbah conceived and design the study, Nathalie Ferrand
and Emilien Stragier performed the experiments, Nathalie Ferrand, Gérard Redeuilh and
Michèle Sabbah analysed and interpreted the data, Gérard Redeuilh and Michèle Sabbah
c The Authors Journal compilation c 2012 Biochemical Society
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Received 20 February 2012/5 July 2012; accepted 5 July 2012
Published as BJ Immediate Publication 5 July 2012, doi:10.1042/BJ20120311
c The Authors Journal compilation c 2012 Biochemical Society