L. Zhang et al. / European Journal of Pharmacology 789 (2016) 439–448
447
apoptosis (Roshan-Moniri et al., 2014). However, almost half of the
currently found NRs are orphan receptors whose endogenous li-
specific inverse agonist, transient transfection luciferase reporter
assay and TR-FRET assay were also carried out. After 24 h, HSP1604
gands have not been identified. ERR
α
is originally thought to
inhibited the interaction between ERR
dose dependent manner which was in according with the positive
control of ERR M).
The therapeutic focus of HSP1604 and other related compounds
is the treatment of solid tumors such as breast, ovary and cervical
carcinomas. Notably, recent investigations have suggested the pi-
α
-LBD with PGC-1
α
in a
regulate energy homeostasis (Giguere, 2008), including lipid
handing, mitochondrial respiration and biogenesis, gluconeogen-
esis and glycolysis, by interacting with various coactivators (Kallen
α
(IC50¼0.8270.11 μ
et al., 2004). Moreover, ERR
α
is one of the several important
transcription factors that act in concert to maintain the energy
homeostasis and meet variable energy demands of an organism.
Nonetheless, previous studies demonstrate that overexpression of
votal role of ERR
α
in breast cancers, independent of ER status
(Manna et al., 2016; Stein et al., 2008; Wu et al., 2016). HSP1604,
which inhibits the proliferation of various cancer cells and the
migration of human MDA-MB-231 cells, also significantly inhibits
the growth of MDA-MB-231 human breast cancer xenografts in
nude mice.
ERR
α
has been associated with poor clinical prognosis in breast
(Stein et al., 2008), ovarian, colon, adrenocortical carcinoma (Ca-
saburi et al., 2015) and other types of tumors patients. Moreover,
regulating the activity of ERR would presumably not only impede
α
primary hormone dependent breast or ovary cancers, but also
those which have acquired a resistance to anti-hormonal therapy
accompanied with a pessimistic prognosis (Valsecchi et al., 2015).
Our previous research reported that downregulation of ERR
α
inhibited angiogenesis in human umbilical vein endothelial cells
through regulating VEGF production and PI3K/Akt/STAT3 signaling
pathway (Zhang et al., 2015). Taken with all of these findings,
HSP1604 may be a potential therapeutic means to cure tumors in
part by suppressing the angiogenesis of cells.
Therefore, it is reasonable to speculate that suppressing ERR
activity would be a promising therapeutic strategy for the design
of alternative treatments of cancers.
α
It was found that ERR
homologies of their amino acid of DNA-binding domain (DBD) and
sequence identity with estrogen receptors (ERs) (Bonnelye and
Aubin, 2005), is the closest relatives to ER
other member of those nuclear hormone receptors. Recent bio-
chemical discoveries also suggest that ERR exhibits cross-talk
with estrogen receptors and it has also share common target
genes, such as pS2 (Lu et al., 2001), aromatase (Yang et al., 1998),
osteopontin (Gallet and Vanacker, 2010), lactoferrin and other
several co-regulatory proteins. Furthermore, ERR
milarity with classical estrogen receptors (ER
mary sequence, structural levels, and response elements (Zhang
and Teng, 2000), but it is classified as an orphan nuclear receptor
because it does not bind any natural identified endogenous ligands
(Giguere et al., 1988). Therefore, unlike those of estrogen receptors
α
, which shares the highest (70%)
In summary, through in vitro and in vivo studies, HSP1604 were
identified as a ERR
α
specific inverse agonist. ERR
α
suppressing
effect of HSP1604 were investigated by cell based reporter gene
assay, quantitative real-time PCR and Western blot assay. Mean-
while, HSP1604 demonstrated inhibitory effect on the growth of
ER-negative MDA-MB-231 human breast cancer xenografts and a
reduction in the migration of human MDA-MB-231 cells in a dose
dependent manner. These experimental results further evidence
α
and ER
β
than any
α
that HSP1604 acts as an ERR
therapeutic potential to inhibit the growth of tumors. The next
step was to explore the effect of HSP1604 on the growth of breast
α
specific inverse agonizts and has
α
shares high si-
α
and ER ) at pri-
β
cancer xenografts overexpressing ERR
In conclusion, discovery the inverse agonist specific to ERR
may be helpful to elucidate the function of ERR in the process of
cancer development and metastasis. There are many advantages in
the use of agent targeting to ERR as a strategy for future clinical
treatments (May, 2014). Therefore, through the identification of
novel ERR specific inverse agonist such as HSP1604, it is likely
α
.
α
α
ER
added ligand and 17
ERR can bind to selective synthetic ligands. Specially, compound
A and synthetic compounds XCT790 which induces ubiquitin
proteasome dependent ERR degradation, have been shown to be
ERR specific inverse agonist, while synthetic estrogen diethyl-
stilbestrol acts as an inverse agonist for all ERR subfamily (ERR
ERR and ERR ). Notably, it was found that XCT790 suppressed the
growth of triple negative breast cancer cells (Wu et al., 2016). In
our study, we presented findings that a novel ERR selective in-
α
and ER
β, ERR
α
are constitutively active without binding any
α
β-estradiol (E2) (Giguere, 2002). However,
α
α
that these ligands may eventually lead to the discovery of new
α
efficacious drugs as alternative strategy to cure cancer and other
α
ERR
α
related diseases.
α
,
β
γ
Conflicts of interest
α
verse agonist HSP1604 would be a promising candidate drug for
breast cancer patients.
The authors indicated no potential conflicts of interest.
In the present study, HSP1604 is found to have significant in-
hibitory effects on the ERR
MDA-MB-231 cells. Meanwhile, it functioned as a selective mod-
ulator of ERR in vitro. We drew these conclusions on the base of
following experiment results: (1) HSP1604 inhibited the con-
stitutive transcriptional activity of ERR in transient transfection
α
dependent gene expression in human
Acknowledgments
α
This study was supported by grants from the National Natural
Science Foundation of China (81302853 and 81202389) and
Shanghai Municipal Commission of Health and Family Planning
(201540261).
α
assays that transfected an ERRE-dependent luciferase reporter
gene in human MDA-MB-231 cells. (2) HSP1604 selectively dis-
rupted the interaction between ERR
vator PGC-1 in the ERR TR-FRET coactivator assay. (3) HSP1604
treatment reduced the amount of ERR protein in human MDA-
MB-231 cells. (4) HSP1604 treatment decreases the expression of
ERR -regulated target genes. Taken together, HSP1604 treatment
strongly suppressed ERR activity in a dose dependent manner
(IC50¼1.9270.10 M) while showing a slight inhibitory effects of
ERR and ERR with the value of IC50420 M. Moreover,
HSP1604 had no suppressing effect of ER , confirming that
HSP1604 acted as a selective ERR inverse agonist.
To extend the finding that HSP1604 functions as an ERR
α
LBD and its nuclear coacti-
α
α
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