Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 16:11:e72512.
doi: 10.7554/eLife.72512.

Stereospecific lasofoxifene derivatives reveal the interplay between estrogen receptor alpha stability and antagonistic activity in ESR1 mutant breast cancer cells

David J Hosfield  1 , Sandra Weber  2 , Nan-Sheng Li  1 , Madline Sauvage  2 , Carstyn F Joiner  3 , Govinda R Hancock  3 , Emily A Sullivan  3 , Estelle Ndukwe  1 , Ross Han  1 , Sydney Cush  1 , Muriel Lainé  1 , Sylvie C Mader  2 , Geoffrey L Greene  1 , Sean W Fanning  3
Affiliations

Stereospecific lasofoxifene derivatives reveal the interplay between estrogen receptor alpha stability and antagonistic activity in ESR1 mutant breast cancer cells

David J Hosfield et al. Elife. .

Abstract

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1. Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles.

Keywords: D538G ESR1 Mutation; E. coli; Y537S ESR1 Mutation; antiestrogen; biochemistry; breast cancer; cancer biology; chemical biology; drug resistance; estrogen receptor degradation; hormone resistance; human.

PubMed Disclaimer

Conflict of interest statement

DH, SW, NL, MS, CJ, GH, ES, EN, RH, SC, ML, SM, GG No competing interests declared, SF In the interest of transparency, Dr. Fanning's laboratory receives sponsored research funds from Olema Oncology Inc. Olema was not involved in this study. This work has no impact on the company

Figures

Figure 1.
Figure 1.. Estrogen receptor alpha ligands evaluated in this study including estradiol, selective estrogen receptor modulators (SERMs), and selective estrogen receptor degraders/downregulators (SERDs).
Figure 2.
Figure 2.. Impact of ligand and mutation on Halo-estrogen receptor alpha (ERα) lifetime in T47D breast cancer cells.
(A–C) Halo-618 fluorescence measured every 4 hr in T47D cells expressing WT halo-ERα (A), Y537S (B), and D538G (C) treated over 100 hr with vehicle (Veh), 1 μM estradiol (E2), fulvestrant (ICI), or GDC0927 following induction of expression. (D–F) Same conditions as in (A–C), except that cells were treated with Veh, 4-hydroxytamoxifen (4OHT) or RU39411. Data are normalized to cell count in each well and are shown as the mean of two biological replicates ± SD (G–I) TMR signal in T47D breast cancer WT (G), Y537S (H), or D538G (I) ERα treated for 24 hr with between 2.5 pm and 1 μM E2, 4OHT, ICI, lasofoxifene (Laso), or GDC0927. All data are normalized to vehicle and are shown as the mean of two biological replicates ± SD.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Doxycycline induction of Halo-ERα after 24 hr.
Data are representative of the mean of three replicates ± standard error.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. IC50 of fulvestrant (ICI) in normal T47D cells.
Data are representative of three replicates per concentration ± SD. All data were normalized to actin control in the individual treatments.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. In-cell western of T47D breast cancer cells treated for 24 hr with 4-hydroxytamoxifen (4OHT) or fulvestrant (ICI).
Data are shown as the mean of 3 replicates ± SD.
Figure 3.
Figure 3.. Stereospecific methyl additions onto the pyrrolidine of lasofoxifene (Laso) impact estrogen receptor alpha (ERα) levels in T47D breast cancer cells.
(A) Chemical structures of Laso and the synthesized stereospecific methyl derivatives. (B) Dose-response curves of hormone (E2) alongside LASOLaso and derivatives after 24 hr treatment for WT halo-ERα. (C) Dose-response curves of chirally purified LA3 and LA5 alongside E2 and 4-hydroxytamoxifen (4OHT) for WT halo-ERα. LA3/5-1 and LA3/5-2 represent the first and second major peaks separated by chiral affinity chromatography. (D/E) Dose-response curves of LA-Deg and LA-Stab for Y537S and D537G halo-ERα after 24 hr compared to E2, Laso, and 4OHT. (F–H) TMR fluorescence measured every 4 hr in T47D breast cancer cells with WT halo-ERα (F), Y537S (G), and D538G (H) treated over 100 hr at 1 μM LA-Stab or LA-Deg following induction of expression. Data are shown as the mean of two biological replicates ± SD.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Chiral separation of 3 R (LA-Deg) and 2S-methylpyrrolidine (LA-Stab) lasofoxifene derivatives.
(A) Chromatogram of LA-Deg chiral separation. (B) Purification of the LA-Deg peak 1. (C) Purification of LA-Deg peak 2. (D) Chromatogram of LA-Stab chiral separation. (E) Purification of LA-Stab peak 1. (F) Purification of LA-Stab peak 2.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. In-cell western of T47D breast cancer cells treated with lasofoxifene (Laso).
(A), LA-Stab (B), or LA-Deg (C) for 24 hours. Data shown are the mean of 3 replicates ± SD.
Figure 4.
Figure 4.. Impact of Y537S and D538G mutation on ligand-induced estrogen receptor alpha (ERα) SUMOylation and SRC1 coactivator binding.
SUMOylation of WT (A), Y537S (B), or D538G (C) ERα in the presence of vehicle, fulvestrant, GDC0927, raloxifene, AZD9496, 4-hydroxytamoxifen (4OHT), lasofoxifene-degrader (LA-Deg), or lasofoxifene-stabilizer (LA-Stab). Data are shown as the mean ± SEM n=3–5 biological replicates. Association of WT (D), Y537S (E), and D538G (F) ERα and the receptor-interacting domain of SRC1. Data are shown as the mean ± SEM, n=3 biologic replicates.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Fulvestrant-induced SUMOylation WT.
(A), Y537S (B), and D538G (C) estrogen receptor alpha in the presence and absence of 5 nM estradiol (E2). Data are mean of three biological replicates ± SD.
Figure 5.
Figure 5.. Transcriptional reporter gene assays in MCF7 cells with WT, WT/Y537S, and WT/D538G ESR1.
Selective estrogen receptor modulators (SERMs) in (A) WT, (B) WT/Y537S, and (C) WT/D538G MCF7 cells. SERM/selective estrogen receptor degraders/downregulators (SERDs) in (D) WT, (E) WT/Y537S, (F) WT/D538G MCF7 cells. SERDs in (G) WT, (H) WT/Y537S, and (I) WT/D538G MCF7 cells. (J) Summary of IC50 and normalized fluorescence at 5 μM compound after 24 hr for each compound. Data are ordered from left to right based on normalized fluorescence at highest dose in Y573S MCF7. Poor IC50 fits were omitted. Data are shown as the mean of two biologic replicates ± SD. All data are normalized to cell count in their respective wells.
Figure 6.
Figure 6.. LA-Stab induces uterotrophic activity.
Ishikawa cells treated with representative selective estrogen receptor modulators (SERM) or selective estrogen receptor degraders/downregulators (SERD) for 3 days in the absence of E2 and assayed for AP activity. Data shown are the mean ± SD. (n=3 independent replicates, 9 technical replicates total).
Figure 7.
Figure 7.. Anti-proliferative activities of selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders/downregulators (SERDs) in MCF7 cells with heterozygous WT/Y537S ESR1.
(A) 4-hydroxytamoxifen (4OHT). (B) Fulvestrant (ICI). (C) Lasofoxifene (Laso). (D) Lasofoxifene-Stabilizer (LA-Stab). (E) Lasofoxifene-Degrader (LA-Deg). (F) Normalized cell count after 84 hr. All antagonist treatments are in the presence of 1 nM estradiol (E2). All data are normalized to initial cell count of the vehicle wells in their respective plates. All data are mean ± SEM for three biological replicates and a total of nine technical replicates.
Figure 8.
Figure 8.. Enforcing helix 12 AF-2 cleft burial enhances Y537S estrogen receptor alpha (ERα) transcriptional inhibition.
(A) Superposition of each monomer in the asymmetric unit of WT (green) or Y537S (cyan) ERα LBD in complex with RAL. (B) 2mFo-DFc difference map (yellow mesh) of the electron density around E380 (magenta) and H12 (cyan) of the Y537S-RAL structure contoured to 1.0 σ. (C) Hydrogen bond formed between E380 and S537 in Chain A of Y537S-RAL. (D) Superposition of each monomer in the asymmetric unit of WT (green) or Y537S (cyan) ERα in complex with 4OHT. (E) 2mFo-DFc difference map (yellow mesh) of the electron density around E380 (magenta) and H12 (cyan) of the Y537S-4OHT structure contoured to 1.0 σ. (F) Position of S537 relative to E380 in the Y537S-4OHT structure. Raloxifene PDBs: 7KBS and 7UJC and 4OHT PDBs: 5 W9C and 7UJ8.
Figure 8—figure supplement 1.
Figure 8—figure supplement 1.. Representative crystal contact formed between D538 and R436 of a symmetry mate that pulls 537 S out of hydrogen bonding distance to E380.
Figure 8—figure supplement 2.
Figure 8—figure supplement 2.. Selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders/downregulators (SERDs) with increased transcriptional antagonistic efficacies show improved helix 12 (H12) packing in the AF-2 cleft.
Average main chain B-factors are shown at each H12 position normalized to average B-factor for each Y537S estrogen receptor alpha LBD structure. Lower B-factor indicates less mobility within the crystal.
Figure 8—figure supplement 3.
Figure 8—figure supplement 3.. 2mFo-DFc difference maps for antiestrogens in complex with WT and Y537S estrogen receptor alpha (ERα) LBD contoured to 1.5σ.
(A) Y537S-BZA. (B) WT-RAL. (C) Y537S-RAL. (D) Y537S-4OHT. (E) WT-LA-Deg. (F) Y537S-LA-Deg. (G) WT-LA-Stab. (H) Y537S-LA-Stab. (I) WT-RU39411. (J) Y537S-RU39411. (K) WT-Clomiphene. (L) Y537S-LSZ102. All ligands are shown as sticks, all maps are shown as blue mesh, and all protein are shown as light grey ribbons.
Figure 9.
Figure 9.. Experimental for the synthesis of lasofoxifene analogues (9a-9d).
Author response image 1.
Author response image 1.
Author response image 2.
Author response image 2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andreano KJ, Baker JG, Park S, Safi R, Artham S, Oesterreich S, Jeselsohn R, Brown M, Sammons S, Wardell SE, Chang CY, Norris JD, McDonnell DP. The Dysregulated Pharmacology of Clinically Relevant ESR1 Mutants is Normalized by Ligand-activated WT Receptor. Molecular Cancer Therapeutics. 2020;19:1395–1405. doi: 10.1158/1535-7163.MCT-19-1148. - DOI - PMC - PubMed
    1. Anurag M, Ellis MJ, Haricharan S. DNA damage repair defects as a new class of endocrine treatment resistance driver. Oncotarget. 2018;9:36252–36253. doi: 10.18632/oncotarget.26363. - DOI - PMC - PubMed
    1. Bardia A, Linden HM, Ulaner GA, Chandarlapaty S, Gosselin A, Doroumian S, Celanovic M, Campone M. Dose-escalation study of SAR439859, an oral selective estrogen receptor (ER) degrader (SERD), in postmenopausal women with ER+/HER2- metastatic breast cancer (mBC) Journal of Clinical Oncology. 2019;37:e1054. doi: 10.1200/JCO.2019.37.15_suppl.1054. - DOI
    1. Burstein HJ, Lacchetti C, Anderson H, Buchholz TA, Davidson NE, Gelmon KA, Giordano SH, Hudis CA, Solky AJ, Stearns V, Winer EP, Griggs JJ. Adjuvant Endocrine Therapy for Women With Hormone Receptor-Positive Breast Cancer: ASCO Clinical Practice Guideline Focused Update. Journal of Clinical Oncology. 2019;37:423–438. doi: 10.1200/JCO.18.01160. - DOI - PubMed
    1. Chandarlapaty S, Chen D, He W, Sung P, Samoila A, You D, Bhatt T, Patel P, Voi M, Gnant M, Hortobagyi G, Baselga J, Moynahan ME. Prevalence of ESR1 Mutations in Cell-Free DNA and Outcomes in Metastatic Breast Cancer: A Secondary Analysis of the BOLERO-2 Clinical Trial. JAMA Oncology. 2016;2:1310–1315. doi: 10.1001/jamaoncol.2016.1279. - DOI - PMC - PubMed

Publication types

Grants and funding