Please cite this article in press as: Revankar et al., A Selective Ligand for Estrogen Receptor Proteins Discriminates Rapid and Genomic Signaling, Cell
Table 1. Summary of AB-1 Properties
E2
AB-1
Multiple approaches have been employed over the years to inves-
tigate mechanisms of rapid E2-mediated signaling, including the
generation of mutant forms of ERa (e.g., membrane- or nuclear-
pharmacological approaches employing novel ligands, such as
pathway preferential estrogens that exhibit exceptionally low af-
finity for ERa, purportedly resulting in the activation of non-
genomic signaling but not transcriptional activity (Madak-Erdogan
enhanced our understanding of rapid E2-mediated signaling
events in multiple cell types and tissues by selectively activating
ER-selective compound that displays no binding affinity or activity
toward GPER, enabling studies of ER-specific activities in the
absence of GPER signaling. Furthermore, the selective profile of
AB-1 with respect to ER activity, activating transcription while pre-
cluding ER-mediated rapid signaling, provides additional selec-
tivity that will further our understanding of ER function. It should
be noted that we only examined two aspects of ER-specific rapid
signaling, namely calcium mobilization and PI3K activation,
limiting our conclusions to these pathways. Because the mecha-
nisms of ER-mediated signaling are in general poorly understood,
it is possible that other aspects of rapid signaling may be pre-
served. Nevertheless, the ability of AB-1 to regulate ER-mediated
gene expression in a highly similar manner to E2, while having no
effect on ER-mediated signaling (thus acting as an antagonist of
these pathways), represents a previously unidentified pharmaco-
logical profile, analogous to the tissue-selective activities of
There has been mounting evidence that GPER expression and
activation by currently employed anti-estrogens, particularly
tamoxifen, play an important role in resistance to these drugs,
as suggested by the poor prognosis of breast cancer patients
GPER expression in breast cancer patient biopsies following
2010), inhibition of tamoxifen-resistant breast cancer cell growth
Based on such results, the development of highly ER-selective an-
tagonists that lack GPER cross-reactivity could be of significant
clinical benefit, lowering the occurrence of resistance seen with
ERa cell binding (IC50
ERb cell binding (IC50
ERa LBD binding (IC50
)
0.30 nM
0.65 nM
0.26 nM
0.47 nM
ꢀ8 nM
0.08 nM
0.3 pM
54
3 nM
)
26 nM
38 nM
24 nM
>>10 mM
15 nM
0.5 nM
52
)
ERb LBD binding (IC50
)
GPER cell binding
ERE expression (EC50
MCF-7 proliferation (EC50
ERa protein degradation (%)
Calcium signaling ERa (IC50
Calcium signaling ERb (IC50
Uterine imbibition (EC50
)
)
)
33 nM
75 nM
ꢀ90 mg
ꢀ30 mg
)
N/A
)
ꢀ3 ng
ꢀ5 ng
Uterine proliferation (EC50
aN/A, not applicable.
)
Our results demonstrated a high correlation between the
gene expression profiles of E2 and AB-1 in MCF-7 cells. Given
the lack of rapid signaling observed for AB-1, this would
suggest that rapid signaling has a minimal overall impact on
ERa-mediated transcriptional activity of the majority of genes.
There were, however, approximately 4%–5% of genes that
exhibited lower regulation (less activation or less repression
by 50% or more) by AB-1 as compared with E2, indicating a
contribution of rapid signaling to ‘‘maximal’’ transcription regu-
lation (defined as that induced by E2). A study employing an
E2-dendrimer conjugate (that cannot cross the plasma mem-
brane) that activates rapid (non-genomic) signaling pathways,
but not nuclear ER-mediated transcriptional (genomic) path-
ways revealed that approximately 25% of E2-regulated genes
Although this result suggests that rapid signaling alone can
recapitulate a portion of E2-regulated transcription, it does
not imply the converse, that the same genes require rapid
signaling. Downregulation of ERK2 (via siRNA) has also been
shown to alter the gene expression profile of E2 in MCF-7 cells
activated protein kinase signaling in transcriptional activity of
ERa. Interestingly, there were also unique genes that were
only regulated by E2 in the presence of ERK1/2 knockdown.
Overall, these results suggest the extreme complexity of
E2-mediated transcriptional regulation. In our gene expression
study, MCF-7 cells were deprived of E2 for a total of 4 days prior
to stimulation with either E2 or AB-1 for 24 h. Under these con-
ditions, basal levels of ERK2 activity are expected to be
decreased but perhaps not to the same extent as in the pres-
ence of ERK2 knockdown, suggesting that basal ERK2 activity
may be sufficient to support E2-mediated regulation of tran-
scription. Finally, the overall high concordance between E2-
and AB-1-mediated transcriptional regulation suggests that
the conformation of ERa induced by AB-1 is very similar to
that of E2, resulting in the similar recruitment of co-activators
and co-repressors.
SIGNIFICANCE
Cross-activation of G protein-coupled estrogen receptor
(GPER) by estrogen receptor (ER)-targeted therapeutic antag-
onists, such as tamoxifen and fulvestrant, has been implicated
in the development of endocrine resistance in breast cancer.
At present, truly ER-selective ligands lacking such GPER
cross-reactivity have not been identified. Here, Revankar
et al. report the identification and characterization of a small
The ability of E2 to mediate rapid (i.e., non-genomic) signaling
has been known for over 50 years, from early studies of E2-medi-
ated cAMP production and calcium (45Ca) mobilization (Pietras
8
Cell Chemical Biology 26, 1–11, December 19, 2019