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Page 8 of 16 Lei et al. J Cancer Metastasis Treat 2019;5:38 I http://dx.doi.org/10.20517/2394-4722.2019.12

Despite such in-depth studies of transcriptional and growth-promoting properties endowed by ESR1 LBD
point mutations, the role of such mutations in driving cell invasion and tumor metastasis is underexplored.
A scratch wound assay was performed on Y537S and D538G mutant expressing MCF7 cells to examine cell
motility which showed enhanced cell migration under hormone-deprived conditions driven by these ESR1
mutants [54,61] . A recent study sheds light on ER mutant-driven metastatic biology, showing a remarkable
[64]
enrichment of metastasis-associated gene sets in ESR1 mutant cells . Consequently, Y537S and D538G
mutant expressing MCF7 cells developed metastases after survival surgery to remove primary tumors in
xenograft models. The Y537S mutant greatly potentiated both tumor growth and metastasis compared to
[64]
D538G mutant .

Mechanisms and therapeutic vulnerabilities of breast cancers harboring ESR1 point mutations
Structural analysis has revealed that the formation of hydrogen bonds between S537 or G538 and D351
[53]
located within helix 12 of ESR1 LBD confers an agonist conformation to ESR1 mutant proteins . In wild-
type ER, the binding of ligand alters the position of helix 12 into an open pocket, favoring recruitment of
transcriptional coactivators such as p160 family members that include SRC-3, and histone acetylases CBP
and p300. In contrast, tamoxifen results in disposition of helix 12 that hinders coactivators binding and
[65]
results in recruitment of corepressors such as N-CoR/SMRT . The substitution of D538 to glycine mimics
[54]
the active conformation of wild-type ER bound by estrogen .
To better understand the consequences of coactivator recruitment to mutant ER proteins, a proteomic
profiling approach was used and revealed enhanced recruitment of transcriptional coactivators, histone
H3 lysine 4 (H3K4) methyltransferase KMT2D/2C complex, as well as steroid receptor coactivators (SRCs),
[66]
to ERE-bound ESR1-Y537S and ESR1-D538G mutants compared to ERE-bound wild-type ER . Genetic
inhibition of SRC-3 in HeLa cells expressing ESR1-Y537S and ESR1-D538G significantly suppressed activity
of an ERE-luciferase reporter. Pharmacological inhibition using a pan-SRC inhibitor, SI-1, also suppressed
transcriptional activation in ESR1 mutant expressing HeLa cell lines and blocked cell proliferation in
ER+ breast cancer cells stably expressing ESR1-Y537S and ESR1-D538G. Using a PDX naturally harboring
the ESR1-Y537S mutation (WHIM20), treatment with an improved pan-SRC inhibitor, SI-2, suppressed
growth in vivo. Suppression of WHIM20 tumor growth was even greater when SI-2 was administered in
combination with an oral SERD, AZD9496, compared to either single agent alone, suggesting that targeting
coactivator recruitment in combination with endocrine therapy could be a promising therapeutic strategy
[66]
for breast tumors harboring ESR1 LBD mutants such as Y537S and D538G . Another study identified that
[62]
the transcription factor TFIIH was also recruited by the ESR1-Y537S mutant . Phosphorylation of Ser118
was found to be mediated by TFIIH kinase, cyclin-dependent kinase (CDK) 7 and subsequent ESR1-Y537S
[62]
driven cell proliferation was suppressed by a CDK7 inhibitor, THZ1 . These results suggest that CDK7 may
represent another target that is associated with ESR1 mutant proteins for therapeutic intervention.

Targeting non-genomic signaling pathways activated by ESR1 mutants has also been investigated. As
discussed above, interactions between ER with RTKs such as EGFR, HER2, and IGF1-R can activate
downstream kinases. This results in phosphorylation of multiple transcriptional factors, including ER, and
[67]
coregulators leading to changes in gene expression in a hormone-independent manner . A recent study
[61]
demonstrated that IGF1 signaling was the most activated pathway in ESR1 mutant MCF7 cells . IGF1
stimulation lead to increased phosphorylation of both IGF1-Rβ and insulin receptor substrate-1 (pIRS-1).
Treatment with an IGF1-Rβ inhibitor (GSK1838705A) monotherapy was able to block Y537S-driven cell
motility and combinatorial treatment with tamoxifen abrogated transcriptional activity and cell growth
[61]
driven by Y537S, Y537N, and D538G mutants . These results suggest that targeting non-genomic signaling
pathways activated by ESR1 mutants may be an additional therapeutic strategy to block ESR1 mutant driven
breast tumors.

Fulvestrant is used to treat metastatic ER+ breast cancer patients who have developed resistance to AI and
tamoxifen. In preclinical models, transcriptional activity and cell proliferation of ESR1 LBD mutant cells