The natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells

The natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells Manuela Cipolletti, Claudia Bellucci, Marco Fiocchetti, Matic Pavlin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6706598/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. Metastatic breast cancer (MBC) remains a major clinical challenge, particularly in estrogen receptor α (ERα)-positive patients who develop resistance to endocrine therapy (ET). While hotspot mutations such as Y537S in the ligand-binding domain (LBD) are well-characterized drivers of resistance, other ERα variants remain poorly studied. Understanding the molecular mechanisms underlying resistance in these variants is crucial for identifying novel therapeutic strategies. Here, we investigated the functional role of the L370F and E471D ERα variants, which are spatially close in the ERα structure. Methods. Stable overexpressing HEK293 cells and CRISPR/CAS9 engineered MCF-7 cells were generated and treated with 17β-estradiol (E2), fulvestrant (Ful) and all-trans retinoic acid (ATRA) to measure ERα stability, transcriptional activity and gene expression analyses using different cellular assays and RNASeq techniques. Direct in vitro measurement of ligand binding affinity to ERα were performed using the purified full-length wild type (wt) as well as L370F and Y537S ERα. In silico structural simulations were also performed to predict the structure of the mutated L370F ERα. Senescent analyses of MCF-7 and Y537S MCF-7 cells were performed using direct measurement β-galactosidase activity in vitro and in cell lines. Results The L370F variant conferred resistance to Ful in terms of in vitro ERα binding, ERα transcriptional activity, receptor degradation and cell proliferation by modifying the folding of the receptor structure. Furthermore, L370F-expressing cells exhibited a hyperactive response to low doses of E2 and basally upregulated late estrogen responsive genes. Additionally, we found that both L370F and Y537S ERα variants displayed increased RARα expression, rendering them highly sensitive to ATRA. Notably, ATRA killed L370F-expressing cells and induced senescence in Y537S-expressing cells, highlighting mutation-specific responses. Conclusions Our findings expand the understanding of ERα mutations beyond known hotspots, identifying L370F as a novel mutation contributing to ET resistance and further indicate the necessity to characterize all the less-studied ERα variants found in MBC. Furthermore, we demonstrate that ATRA selectively targets MBC cells harboring L370F and Y537S mutations, suggesting its potential as a mutation-specific therapeutic agent. These results support further investigation of ATRA in clinical settings to improve treatment strategies for ERα-mutant MBC. Cancer Biology Breast cancer estrogen receptor α endocrine resistance ERα mutations fulvestrant all-trans retinoic acid targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Breast cancer (BC) is the deadliest tumor type for women. Approximately 70% of BC cases express the estrogen receptor α (ERα), and patients with this form of the disease are effectively treated with endocrine therapy (ET) drugs (e.g., aromatase inhibitors - AIs, selective ER modulators – SERMs, and selective ER down-modulators – SERDs). However, 20–40% of these patients experience relapse and develop metastatic BC (MBC), which is resistant to the classic ET drugs (e.g., AIs; 4OH-tamoxifen – Tam) and is largely incurable [ 1 ]. BC cells can acquire resistance to ET drugs through multiple molecular mechanisms. Interestingly, in approximately 50% of ERα-expressing MBCs, ERα point mutations emerge and lead to a hyperactive ERα (e.g., Y537S), conferring specific proliferative advantages and mediating resistance to ET drugs [ 1 ]. The actual clinical strategies for MBC currently involve treating patients either with increasing doses of the same ET drugs (e.g., Tam), with the second-line therapy drug fulvestrant (Ful), a SERD inducing ERα degradation, or with targeted agents (e.g., the CDK4/6 inhibitors palbociclib, ribociclib, or abemaciclib), which can be administered in combination with ET drugs [ 1 ]. Recently, novel antiestrogen drugs that bind either non-covalently or covalently to the Y537S ERα mutation, induce its degradation and block its hyperactive phenotype (e.g., the selective ER down-modulators – SERDs such as AZD9833, camizestrant; RAD-1901, elacestrant; GDC-9545, giredestrant; LY3484356, Imlunestrant; GDC-0927; the selective ERα covalent antagonists – SERCAs, H3B-5942; proteolysis targeting chimerics – PROTACs, such as ARV-471; complete estrogen receptor antagonists – CERANs, such as OP-1250), are currently undergoing clinical trials or have been recently approved (i.e., RAD-1901, elacestrant) [ 1 – 9 ]. In addition, we have demonstrated that ‘antiestrogen-like’ activities can be found in Food and Drug Administration (FDA) approved drugs that do not directly bind to ERα but determine its degradation. In particular, we have reported that cardiac glycosides, antivirals, and Chk1 inhibitors induce the degradation of the Y537S ERα variant, block its hyperactive phenotype, and prevent the proliferation of MBC cells expressing it when administered alone or in combination with either ET drugs or with CDK4/6 inhibitors [ 10 – 13 ]. Thus, a broad range of drugs could be used for the clinical treatment of MBC expressing the Y537S ERα [ 1 ]. To date, ~ 50 ERα point mutations have been identified in MBC patients and annotated in the free on line Cosmic and cBioPortal databases [ 14 ], all of which reduce their survival rates [ 14 ]. The most frequent and best-characterized variants map in one hotspot region within the ligand-binding domain (LBD) (e.g., Y537S) [ 14 ]. However, many ERα variants fall outside this hotspot region and have been so far neglected. Recently, the initial characterization of some of them (V422del, G442R, F461V, S463P, L469V [ 15 ], and F404L/I/V [ 16 ]) revealed another hotspot within in the LBD [ 15 ] and indicated that distinct mechanisms underlying uncontrolled proliferation [ 15 ] or resistance to ET drugs [ 16 ] can be operative in MBC. In this study, we investigated the L370F and E471D ERα variants because we observed that, among the receptor point mutations found in MBC located within the LBD and annotated in the COSMIC and cBioPortal databases [ 14 ], the side chains of residues L370, laying on helix 4 (H4) and E471, laying within helix H10 [ 17 ] are facing each other in the LDB structure (Fig. 1 A, yellow residues). Due to this spatial configuration, we hypothesized that they could represent a novel 3D-hotspot and, in turn, their single mutations in MBC could provide specific selective advantages to tumor cells (the double L370F/E471D mutation has not been identified in MBC patients). Consistently, specific ERα cellular assays demonstrated that the L370F ERα variant is resistant to high doses of Ful and leads to a hyperactive response to low doses of 17β-estradiol (E2). Additionally, both the L370F and Y537S ERα variants exhibit high expression levels of retinoic acid receptor α (RARα) and are sensitive to the antiproliferative effects of all-trans retinoic acid (ATRA). Interestingly, ATRA induces cell death in the presence of the L370F ERα mutant and senescence in the presence of the Y537S ERα mutant. These data establish that the L370F ERα variants confer distinct properties to MBC cells, highlighting ATRA as a potential ERα-variant-specific therapeutic agent for MBC driven by ERα function. Material and Methods Cell Culture and Reagents HEK293 and MCF-7 cell lines were obtained from ATCC (USA), and all other cell lines used in this study were derived from these parental lines. Cells were maintained following the manufacturer's recommendations. The following reagents and antibodies were employed: 17β-estradiol (E2), DMEM (with or without phenol red), fetal calf serum, and charcoal-stripped fetal calf serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bradford protein quantification kit and HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) were obtained from Bio-Rad (Hercules, CA, USA). Primary antibodies against ERα (F-10, mouse), RARα (C-1, mouse), ASCL1 (D-7, mouse), CALCR (CT-R, 2F7, mouse), FABP5 (E-FABP, C-20, rabbit), and Caveolin-1 (N-20, rabbit) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Additional antibodies included anti-phospho ERα (Ser118, mouse), anti-phospho H2AX (rabbit), anti-PARP (rabbit), and anti-FASN (rabbit) from Cell Signaling Technology (USA). Anti-vinculin (mouse) and anti-tubulin (mouse) antibodies were obtained from Sigma-Aldrich. Western blot chemiluminescence detection reagents were purchased from Bio-Rad. The following compounds were used in selected experiments: all-trans retinoic acid (ATRA) and digoxin (Digo) from Sigma-Aldrich; Fulvestrant (Ful) and 4-hydroxy-Tamoxifen (Tam) from Selleck Chemicals (USA). The ERα Green Competitor Assay Kit (PolarScreen™, A15882) was obtained from Thermo Scientific. All other reagents were from Sigma-Aldrich and used without further purification, unless otherwise specified. Cell line authentication was confirmed by STR profiling performed by BMR Genomics (Italy). Cell Manipulation for Western Blot Analyses Cell manipulation, Western blotting Image acquisition and consequent manipulations including band quantitations have been described in detail in [ 18 ]. Generation of CRISPR/CAS9 MCF-7 cells expressing the L370F ERα The generation of MCF-7 L370F cells using CRISPR/Cas9 technology was outsourced to Cogentech in 2021. A clone (clone 3–78) was obtained with two correctly modified alleles (HDR alleles carrying the G/C mutation, which converts the TTG (L) codon into TTC (F), along with three synonymous mutations) and one allele with a single-nucleotide insertion, resulting in a frameshift and a premature stop codon immediately after the insertion. Details of the procedure are available upon request. Small Interference RNA For the small interference RNA (siRNA) experiments, cells were transfected with esiRNA [obtained from Sigma-Aldrich (St. Louis, MO, USA)] targeting the specific proteins of interest. The transfection procedure was conducted using Lipofectamine RNAi Max (Thermo Fisher), following established protocols described in [ 19 ]. Cell Proliferation Assays The xCELLigence DP system (ACEA Biosciences, Inc., San Diego, CA) Multi-E-Plate station was utilized to measure the time-dependent response to the specified drugs by real-time cell analysis (RTCA), following previously reported protocols [ 18 ]. Synergy studies were conducted using Crystal Violet staining, as described in [ 18 ]. The synergy was subsequently calculated using Combenefit freeware software [ 13 ]. Measurement of the Cellular Area Following the outlined stimulation, phase-contrast images of MCF-7, L370F, and Y537S cells were randomly acquired using a ZEISS Axio Vert.A1 FL-LED fluorescence microscope (20× objective, 1× zoom) (Zeiss, Oberkochen, Germany). For cell area analysis, images were loaded into the FIJI distribution of the ImageJ program in their native format. Prior to analysis, brightness and contrast were adjusted using the “Brightness/Contrast” tool in FIJI to ensure optimal definition of the cell profile. Cellular contours were manually traced using the “Freehand Selection” tool to define the Region of Interest (ROI). Multiple ROIs were then selected from the ROI Manager, and the “Measure” tool under the “Analyze” menu was applied with the measurement parameter set to “Area”, representing the surface area of the selected ROIs. The obtained values were reported as indicative of cell area in µm². Generation of Stable Cell lines and Measurement of ERα and RARα Transcriptional Activity The generation of HEK293 cells stably transfected with a reporter gene containing an estrogen response element (ERE) controlling the expression of nanoluciferase (NLuc)-PEST was done using the selection methods and the reagents described in [ 20 , 21 ]. These cell lines were then transfected with the pcDNA His HA wild type (wt), L370F and Y537S ERα and selected using neomycin. To generate the pcDNA His HA wt, and Y537S ERα, the pcDNA-HA-ERα [ 22 ] and the pcDNA-HA-Y537S ERα [ 22 ] were purchased from Addgene (USA) and digested with BamHI and ApaI to excide the receptor ORFs. The fragments were ligated into pcDNA His vector sites. To generate the pcDNA His HA L370F ERα, site-directed mutagenesis was performed on the pcDNA His HA ERα using the following forward 5’-gtgccaggctttgtggattttaccctccatgatcaggtccaccttc-3’ and reverse 5’-cacggtccgaaacacctaaaatgggaggtactagtccaggtggaag-3’ primers and using the QuickChange Lightning kit from Agilent (Santa Clara, CA, USA). All the resulting plasmids were sequence verified. The generation of L370F MCF-7 cells stably transfected with a reporter gene containing the ERE sequence controlling the expression of NLuc-PEST was done using the selection methods and the reagents described in [ 20 , 21 ]. This cell line together with the corresponding parental and Y537S MCF-7 ERE-NLuc cells were used to measure the ERα transcriptional activity as previously reported [ 20 , 21 ]. To generate the parental, L370F and Y537S MCF-7 cells stably expressing a reporter gene containing an retinoic acid response element (RARE) controlling the expression of NLuc-PEST, the plasmid pGL2Basic_Neo_RARE-NLuc-PEST was transfected and the cell lines were selected as previously reported [ 20 , 21 ]. To generate the plasmid pGL2Basic_Neo_RARE-NLuc-PEST, the RARE cassette was KpnI and HindIII excised from the pGL3-RARE-luciferase [ 23 ] purchased from Addgene (USA) and ligated into the corresponding restriction sites in the pGL2Basic_Neo_NLuc-PEST [ 20 , 21 ]. Gene Arrays and RNASeq Analyses Gene arrays analyses were conducted as described in [ 18 ]. RNASeq and the relative initial data analyses were performed by Novogene (Cambridge, UK) as an outsourced service. Briefly, three replicates of sub-confluent growing parental, L370F and Y537S MCF-7 cells were pelleted, and sent in dry ice for analysis to Novogene, which performed RNA extraction, quality control, mRNA library preparation (polyA enrichment) and sequencing using the NovaSeq X Plus Series (PE150) (6G raw data per sample) following by standard analysis (i.e., data quality control and data filtering, mapping to reference genome, gene expression quantitation and correlation analysis, differential expression analysis, enrichment analysis of differential expressed genes, GSEA enrichment analysis of expressed genes, protein-protein interaction analysis of differential expressed genes, oncogene functional annotation of differential expressed genes, alternative splicing quantification and differential analysis, SNP/InDel analysis and fusion gene analysis). Full-Length ERα Purification from Expi293F cells To generate the plasmid pcDNA 3.1_ ERα_TEV_TWIN STREP TAG, the TEV_TWIN STREP TAG was PCR amplified from the pcDNA3.1 GIL-11 myc TEV TwinStrep [ 24 ] purchased from Addgene (USA) using the following forward 5’-cggggtaccccgagcgcgtggagccatccgcagt-3’ and reverse 5’-cgcggatccgcgtttttcaaactgcggatggct-3’ primers. The fragment was EcoRI and XhoI digested and subcloned in pcDNA 3.1 to obtain the pcDNA 3.1_TEV_TWIN STREP TAG. Wild type (wt), L370F and Y537S ERα were PCR amplified using pcDNA His HA wt, L370F and Y537S ERα plasmids as templates and the following forward 5’-cgcggatccgcgatgaccatgaccctccacaccaaagcatct-3’ and reverse 5’-ccggaattccgggaccgtggcagggaaaccctc-3’ primers. The resulting fragments were BamHI and EcoRI digested and subcloned into the corresponding restriction sites in the pcDNA 3.1_TEV_TWIN STREP TAG. The plasmids were then sequence-verified. Transfection of the pcDNA 3.1_ ERα_TEV_TWIN STREP TAG encoding for the wt, L370 and Y537S receptor fused in frame with the TWIN STREP TAG (Molecular weight = 70,7 KDa, corresponding to 66,0 KDa of the ERα and 4,7 KDa of the TWIN STREP TAG) was conducted in Expi293F cells and performed by the National Facilities at the Human Technopole (Milan, Italy) as a service. Access to the Biomass Production Unit service @ the Human Technopole (Milan, Italy) was granted following a competitive selection procedure (Project ID 1771263). Expi293F cells were transfected at a density of 2–3 x 10 6 cells/ml using a modified PEI (polyethylenimine) protocol, with 1.1 mg of plasmid DNA and 3 mg of PEI per each liter of cell culture. A total of 10 liters of Expi293F cells for each construct was transfected. After transfection cells have been grown in a Multitron shaker (Infors HT, Bottmingen, Switzerland) for additional 72 hours. The cell suspension was divided into aliquots of 500 ml and then centrifuged at 1000 rcf, at 4°C, for 10 minutes. The 500 ml cell pellets were then washed with PBS, the centrifugation was repeated and the supernatant discarded. The resulting pellets were stored at -80°C until protein purification (see below). A pellet of 500 ml of Expi293F cells was resuspended in 50 ml of the following lysis buffer 100 mM TrisHCl pH 8, 150 mM NaCl, 2% Triton X100, 10% Glycerol, 1mM EDTA, 10 mM MgCl 2 , 1 mM ATP, 2 mM DTT, 10 µM Mg-132, 1mM PMSF and protease inhibitor cocktail and left on ice for 10 minutes. The volume was then centrifuged in 2 ml eppendorf tubes for 5 minutes at 4°C at 10,000 rpm. The supernatant was loaded three times on a 3 ml gravity column Strep-Tactin®XT 4Flow® 50% suspension [IBA-Lifescience (Gottingen, Germany)] packed o.n. at 4°C. After washing the column with > 50 ml of wash buffer (100 mM TrisHCl pH 8, 150 mM NaCl, 1 mM EDTA) 14 fractions of 1 ml were eluted with 100 mM TrisHCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 mM biotin. The eluate was then concentrated in 50 KDa cut-off Amicon® Ultra [obtained from Sigma-Aldrich (St. Louis, MO, USA)] up to 500–800 µl. The purity of the purified ERα was evaluated by staining an SDS-PAGE gel with InstaBlue Protein Stain Solution (CliniScience, Italy). Receptor amount and purity was calculated in reference to a standard curve of bovine serum albumin (Supplementary Fig. 1A). The final yield was in the µM range per each receptor. In Vitro ERα Binding Assay The in vitro ERα binding assay employed a fluorescence polarization (FP) method to assess the binding affinity of 17β-estradiol (E2), and fulvestrant (Ful) with commercially available recombinant ERα and the purified full length wild type (wt), L370F and Y537S ERα-TWIN STREP TAG purified from Expi293F cells as described above. The FP assay was done as reported in [ 18 ]. Briefly, measurement has been performed using different doses of the test compounds in a final assay reaction that contained the above described ERα (50 nM - Supplementary Fig. 1B) and fluomone ES2 (4.5 nM) in ERα binding buffer, Thermo Scientific). Each sample was measured in quadruplicate in black 384 multiwell plates and the experiment was repeated twice. The assay was incubated for two hours in the dark at room temperature before reading on a Tecan Spark Elisa reader. The calculation of the K i was obtained from the apparent IC 50 of the compound toward each receptor by performing the calculation as described in [ 25 ]. Briefly, we applied a modified Cheng-Prusoff equation [ 26 ] [Ki = (apparent IC 50 * K dFluomoneES2 )/[Fluomone ES2] * ([Fluomone ES2] * K dFluomoneES2 )] to take into consideration the K d of the fluomone ES2 toward the receptors (i.e., 18 nM) and the concentration of the flouomone ES2 used in the assay. Classical Molecular Dynamics Simulations Three different ERα systems were prepared, namely the wild type (wt), L370F, and E471D ERα with fulvestrant inside the binding cavity. The L370F and E471D model systems were prepared by mutating corresponding residues in the wt variant. ERα structures were prepared based on the wt ERα-fulvestrant complex from the study by Pavlin et al. 2018 [ 17 ]. 500 ns-long simulations were performed for each system using Amber20 code [ 27 ]. FF19SB force field was used for the description of protein [ 28 ], which was placed in the cubic simulation box solvated with up to ~ 37,000 TIP3P water molecules, making sure that the distance between solute and edge of the box was at least 12 Å. For the description of fulvestrant same parameters as in ref. [ 17 ] were used. Each system was energy minimized by 20,000 steps using the steepest descent algorithm, followed by 30,000 steps of conjugate gradient. This was followed by the canonical NVT equilibration performed in 4 runs of 10,000 steps, with the constraints on the solute gradually releasing (from 100 kcal mol -1 Å -2 in first run to 60 kcal mol -1 Å -2 and 30 kcal mol -1 Å -2 in the second and third run, respectively, while the fourth run was performed without constraints) and the system was gradually heated to 293 K. Subsequently, NPT equilibration at 1 bar was carried out in two successive runs of 100,000 steps each. During the first run, the solute was restrained with a force constant of 20 kcal mol -1 Å -2 and in the second run the constraint was released. Production runs were performed with periodic boundary conditions and electrostatic interaction were considered using Particle Mesh Ewald method [ 29 ] at 293 K and 1 bar by coupling to the Langevin thermostat [ 30 ] and Berendsen barostat [ 31 ]. In all simulations all bond lengths involving hydrogen atoms were constrained using SHAKE algorithm [ 32 ] to achieve a time step of 2 fs. Analyses (namely RMSD, hydrogen bond (H-bond) analysis, clustering and cross correlation anaylsis) on the equilibrated part of trajectories, from 200 to 500 ns, were performed using AmberTools22 [ 33 ]. β-Galactosidase activity β-Galactosidase activity was assessed using the Beta-Glo® assay system (Promega, Madison, MA, USA) following the manufacturer's guidelines. In brief, 2000 cells were seeded in 96-well plates and treated at the designated time point as specified. Luminescence was then read at Tecan Spark Elisa reader. Each condition was tested in triplicate. Replica plates were also used to normalize the experiment for cell number by using Crystal Violet staining, as described above. Detection of activity at pH 6, a known characteristic of senescent cells was performed the Senescence β-galactosidase staining kit (Cell Signaling, Technology Danvers, MA, USA) according to manufacturer’s instructions. The experiment was performed twice triplicate. Statistical Analysis Statistical analysis was conducted as detailed in been described in detail in [ 18 ]. Results Evaluation of the sensitivity to Ful and E2 of HEK293 cells expressing L370F and E471D ERα variants. Since the ERα variants found in MBC are selected following administration of first-line ET drugs such as AI and/or Tam, MBC patients are typically treated with the second-line ET drug Ful [ 1 ]. To investigate whether the L370F and E471D ERα variants are resistant to this antiestrogen, we initially generated isogenic pooled stable HEK293 cell lines (ERα-negative cells) [ 34 ] co-expressing a reporter construct containing the estrogen response element (ERE) regulating a nanoluciferase gene (ERE-NLuc). This system allows evaluation of ERα transcriptional activity in living cells [ 20 , 21 , 35 ]. Additionally, we introduced expression vectors encoding the L370F and E471D ERα variants fused with an N -terminal double tag (HA and His tag). As controls, we generated isogenic pooled stable HEK293 cell lines expressing wild type (wt) ERα and the hyperactive Y537S ERα mutant [ 36 ]. Dose-response analyses were conducted to evaluate the effectiveness of Ful in decreasing basal ERα transcriptional activity. Cells were treated with varying doses of Ful (10 − 13 M to 10 − 5 M) for 24 hours, and the relative inhibitory concentration 50 (IC 50 ) was calculated. To evaluate the sensitivity of each cell line (wt, L370F, E471D, and Y537S ERα-expressing HEK293 cells) to Ful, the IC 50 values were mathematically transformed to -Log 2 and plotted. As shown in Fig. 1 B, HEK293 cells expressing the Y537S ERα mutant exhibited significantly reduced sensitivity to Ful, as expected [ 36 ]. Interestingly, while HEK293 cells expressing the E471D ERα mutant showed similar sensitivity to Ful as the wt ERα-expressing cells (Fig. 1 D), the L370F ERα mutant cells displayed reduced sensitivity to Ful’s inhibitory effect on receptor transcriptional activity compared to wt ERα cells (Fig. 1 C). These data suggest that the L370F, but not the E471D ERα mutation, impairs Ful’s ability to inhibit receptor transcriptional activity. Therefore, the E471D ERα mutant was excluded from further analysis. Next, we evaluated Ful’s capacity to induce ERα degradation in HEK293 cells expressing wt, L370F, and Y537S ERα mutants. Western blot (WB) analyses were conducted after 24-hour treatments with varying doses of Ful (10 − 13 M to 10 − 5 M) and Ful induced receptor degradation in all cell lines tested. As previously reported [ 36 ], the Y537S mutation reduced Ful-induced receptor degradation compared to wt ERα cells (Fig. 1 E and 1 E’). Interestingly, in HEK293 cells expressing the L370F mutant, higher doses of Ful (10 − 8 M to 10 − 5 M) were less effective in inducing receptor degradation as compared to wt ERα containing cells (Fig. 1 E and 1 E’’). These results indicate that the L370F ERα mutation hinders Ful's ability to induce receptor degradation. Similarly to the Y537S ERα mutant, many other point mutations located within the LBD lead to constitutively hyperactive ERα by causing structural changes that mimic the E2-activated conformation [ 37 ]. We then investigated the effect of E2 on the transcriptional activity of both wild-type and L370F ERα. HEK293 cells were treated with varying doses of E2 (10 − 14 M to 10 − 8 M) for 24 hours, and transcriptional activity was measured. No significant differences in basal transcriptional activity were detected between wt and L370F receptors, and E2 induced a dose-dependent increase in transcriptional activity in both cell types (Fig. 1 F). Nevertheless, at low doses of E2 (from 10 − 14 M to 10 − 10 M), the L370F ERα exhibited enhanced transcriptional activity as compared to its wt counterpart (Fig. 1 F). These findings suggest that the L370F ERα mutation enhances the responsiveness of ERα to low doses of E2, increasing receptor transcriptional activity. Evaluation of Ful sensitivity in parental, L370F and Y573S CRISPR/CAS9-engineered MCF-7 cells. Previous results suggest that the L370F variant found in MBC may confer resistance to Ful treatment and promote hyperactivity in response to E2 in cells expressing this mutation. Consequently, cells expressing this receptor point mutation may evade the effects of ET drugs while continuing to proliferate under the influence of E2. To test this hypothesis, we generated a CRISPR/CAS9 knock-in L370F-mutated MCF-7 cell line that exclusively expresses the mutated receptor (please see Material and Method section) and studied them together with parental MCF-7 and Y537S MCF-7 cells [ 36 ]. Initial experiments were conducted to validate the findings from stable HEK293 cells. The ability of Ful to induce ERα degradation was assessed through WB analysis in parental, L370F, and Y537S MCF-7 cells treated for 24 hours with varying doses of Ful (10 − 14 M to 10 − 5 M). As shown in Fig. 2 A and 2 A’, Ful induced dose-dependent ERα degradation in all cell lines tested. As expected [ 36 ], the ability of Ful to degrade ERα was reduced in Y537S MCF-7 cells (Fig. 2 A and 2 A’). Notably, in L370F MCF-7 cells, high doses of Ful (10 − 7 M to 10 − 6 M) were less effective at inducing receptor degradation as compared to parental MCF-7 cells (Fig. 2 A and 2 A’). Next, we stably transfected the nanoluciferase reporter construct (ERE-NLuc) into parental, L370F, and Y537S MCF-7 cells to create cell lines for studying wt and ERα variant transcriptional activity (i.e., MCF-7 NLuc cells), as previously described [ 38 ]. In these cell lines, dose-response analyses of Ful’s ability to reduce basal ERα transcriptional activity were performed by administering varying doses of Ful (10 − 13 M to 10 − 5 M) for 24 hours. As shown in Fig. 2 B, the inhibitory effect of Ful on receptor transcriptional activity was significantly reduced in L370F and Y537S MCF-7 NLuc cells compared to parental MCF-7 NLuc cells. Growth curve analyses were performed in parental, L370F, and Y537S MCF-7 cells to evaluate the antiproliferative effects of Ful. Each cell line was treated with different doses of Ful (10 − 11 M to 10 − 5 M), and the resulting dose-dependent effect was measured at the time point when the control for each cell line reached maximum growth. Figure 2 C shows that the antiproliferative effect induced by Ful in parental MCF-7 cells was significantly diminished in L370F MCF-7 cells, particularly at high Ful doses, and was almost entirely abolished in Y537S MCF-7 cells, as previously reported [ 36 ]. Overall, these data corroborate the results obtained from stable HEK293 cells and demonstrate that the L370F mutation in ERα confers resistance to Ful-induced ERα degradation and reduces the inhibitory effect of this ET drug on receptor transcriptional activity and cell proliferation. Evaluation of the proliferation rate of parental, L370F and Y573S MCF-7 cells in the presence and absence of E2. Next, we studied the proliferation rate of parental, L370F, and Y537S MCF-7 cells under normal growth conditions (10% FBS) and in the presence of E2-deprived serum (10% CS-FBS) using the xCELLigence apparatus. Under normal growth conditions, parental MCF-7 cells reached maximal growth later than both the L370F and Y537S MCF-7 cells (Fig. 3 A), with Y537S MCF-7 cells exhibiting the fastest growth rate, as previously reported [ 36 , 39 ]. Accordingly, L370F and Y537S MCF-7 cells displayed a significantly shorter doubling time compared to parental MCF-7 cells, with Y537S MCF-7 cells having the shortest doubling time among the tested lines (Fig. 3 B). When growth curve analyses were performed in the presence of E2-deprived serum (10% CS-FBS), we confirmed that the growth of Y537S MCF-7 cells was E2-independent [ 36 , 39 ] (Fig. 3 C). Moreover, in contrast to parental MCF-7 cells, L370F MCF-7 cells did not exhibit reduced growth during prolonged E2 deprivation (Fig. 3 C). Next, we evaluated the proliferative effect of E2 in parental, L370F, and Y537S MCF-7 cells. Growth curve analyses revealed that administration of various doses of E2 (10 − 15 M to 10 − 10 M) induced a dose-dependent increase in the proliferation of parental MCF-7 cells, both under normal growth conditions (10% FBS) (Fig. 3 D) and in the presence of E2-deprived serum (10% CS-FBS) (Fig. 3 H). As expected, the dose-dependent proliferative effect of E2 was more pronounced in the presence of E2-deprived serum (10% CS-FBS), reaching a maximum at 10 − 12 M E2 in both conditions (Fig. 3 G and 3 K). Consistent with the E2-independent growth of Y537S MCF-7 cells, the effect of E2 at different doses was negligible in this cell line under both normal growth conditions (10% FBS) (Fig. 3 F and 3 G) and in E2-deprived serum (10% CS-FBS) (Fig. 3 J and 3 K). Interestingly, administration of increasing doses of E2 to L370F MCF-7 cells under normal growth conditions (10% FBS) induced a modest, yet significant increase in cell proliferation (Fig. 3 E). Dose-response analysis showed that the E2-induced increase in proliferation was maximal at low hormone concentrations (10 − 15 M) (Fig. 3 G). Conversely, in the presence of E2-deprived serum (10% CS-FBS), E2 induced a dose-dependent increase in L370F MCF-7 cell proliferation (Fig. 3 I). The E2-dependent effect was greater in E2-deprived conditions than in normal growth conditions, peaking at 10 − 12 M E2 and becoming significant even at lower doses (10 − 15 M) (Fig. 3 K). These data indicate that the proliferation of MCF-7 cells expressing the L370F ERα variant is partially E2-independent and can be stimulated by low E2 doses. Analysis of E2-dependent regulation of ERα transcriptional functions in parental, and L370F MCF-7 cells. The parental and L370F MCF-7 NLuc cells were next used to evaluate the ability of E2 to modulate ERα transcriptional activity. A 24-hour administration of different doses of E2 (10 − 14 M to 10 − 8 M) revealed that the hormone induces a dose-dependent increase in ERα transcriptional activity in both cell lines (Fig. 4 A). Notably, low-dose hormone treatment (10 − 13 M, 10 − 12 M) triggered a greater E2-dependent increase in receptor transcriptional activity in L370F MCF-7 NLuc cells compared to the parental MCF-7 NLuc line (Fig. 4 A). Phosphorylation of ERα at serine 118 (S118) is essential for its transcriptional activation in response to E2 [ 40 ]. E2-triggered ERα S118 phosphorylation occurs rapidly, reaching a maximum after 30 min of hormone administration in MCF-7 cells [ 40 ]. ERα S118 phosphorylation was evaluated in parental and L370F MCF-7 cells treated with different doses of E2 (10 − 14 M to 10 − 8 M) for 30 minutes. As shown in Fig. 4 B and 4 B’, E2 dose-dependently increased the ERα S118 phosphorylation in both cell lines. However, the E2-induced receptor S118 phosphorylation was increased at low doses of the hormone (10 − 13 M to 10 − 10 M) in L370F MCF-7 cells with respect to the parental MCF-7 cells. To confirm that the inhibitory effect of Ful on ERα transcriptional activity is reduced, and that E2-induced receptor transcriptional activity is increased at low hormone doses in L370F MCF-7 cells, we co-administered different doses of E2 and Ful for 24 hours in both parental and L370F MCF-7 NLuc cells. The data revealed that antagonism between E2 and Ful was observed in both cell lines. However, the antagonistic interaction between E2 and Ful in L370F MCF-7 NLuc cells was significantly weaker as compared to parental MCF-7 NLuc cells (Fig. 4 C). This indicates that Ful exhibits reduced antagonistic activity against E2 in regulating ERα transcriptional activity in L370F MCF-7 cells as compared to parental cells. Since ERα regulates the expression of various genes, with or without the presence of ERE sequences in their promoter regions [ 41 ], we next assessed the E2 ability to modulate gene expression in parental and L370F MCF-7 cells. Based on previous data suggesting that the effect of E2 is enhanced in L370F MCF-7 cells treated with low E2 doses as compared to parental cells, we used an RT-qPCR-based array targeting 89 E2-sensitive genes [ 38 ]. We hybridized cDNA samples generated from total RNA extracted from parental MCF-7 cells treated with E2 at 10 − 9 M and L370F MCF-7 cells treated with E2 at 10 − 12 M. Interestingly, despite the difference in concentration, E2 modulated most of the genes included in the array similarly in both parental and L370F MCF-7 cells (Fig. 4 D and Supplementary Table 1), with a large overlap of genes regulated by E2 in both cell lines (Fig. 4 E and Supplementary Table 1). Notably, for most of the genes commonly regulated by E2 at different doses, the E2 effect was larger in L370F MCF-7 cells than in parental cells (Fig. 4 F and Supplementary Table 1). Overall, these data demonstrate that the L370F mutation renders ERα hypersensitive to low doses of E2, enhancing ERα transcriptional activity and E2-modulated gene expression. Global gene expression profiling of ERα mutants reveals an increase in late E2 response gene in L370F MCF-7 cells. The data reported previously demonstrate that culturing the cells in E2-depleted medium allows the continuous growth of Y537S MCF-7 cells and causes a growth arrest in the L370F MCF-7 cells that is not followed by subsequent cell death, as it occurs in parental MCF-7 cells (Fig. 3 A- 3 C). Thus, to further evaluate the impact of the L370F ERα mutation in the regulation of E2 gene expression we performed RNA-seq analysis in parental, L370F and Y537S MCF-7 cells that had been maintained in E2-containing medium (i.e., normal growing condition – 10%-FBS) to identify changes in basal gene expression independent of the potential interferences caused by cell cycle-dependent regulation of gene expression. We conducted RNA-seq analysis using three biological replicates for each cell line to estimate gene expression levels, measured as FPKM (Fragments Per Kilobase of transcript sequence per Million mapped reads) [ 42 , 43 ]. Principal component analysis (PCA) of gene expression levels (FPKM) revealed strong clustering of biological replicates within each cell line, while both mutant lines (L370F and Y537S) showed clear separation from the parental MCF-7 cells (Supplementary Fig. 2A). Furthermore, we calculated the correlation coefficients between samples across groups. A heatmap of these coefficients revealed high correlation within the biological replicates of each cell line (i.e., parental, L370F, and Y537S MCF-7 cells), indicating robust experimental consistency. Notably, parental and Y537S MCF-7 cells exhibited the lowest intergroup correlation coefficients, while the L370F MCF-7 cells showed an intermediate correlation, bridging both parental and Y537S profiles (Supplementary Fig. 2B). The DESeq2 analysis (|log2(FoldChange)| >= 1 and padj < = 0.05) was performed to evaluate differential gene expression between L370F and parental MCF-7 cells, as well as Y537S and parental MCF-7 cells. This was followed by gene enrichment analysis using GSEA, which generated a ranked gene list in L370F (Fig. 5 A) and Y537S (Fig. 5 B) MCF-7 cells. Initially, we analyzed the gene lists and observed that a substantial number of modulated genes were shared between L370F and Y537S MCF-7 cells (Fig. 5 C and Supplementary Table 2). Subsequently, we assessed whether the genes modulated in L370F and Y537S MCF-7 cells (relative to parental MCF-7 cells) with a signal-to-noise ratio > 0.5 or < 0.5 included both early and late E2-regulated genes. Identification of these genes was guided by the corresponding gene sets applied in the GSEA (i.e., hallmark estrogen response early and hallmark estrogen response late) (Fig. 5 D, 5 E, and Supplementary Table 2). Notably, the majority of early and late E2-regulated genes (referred to as E2 Responsive Genes in Figs. 5 D and 5 E) were also modulated in L370F and Y537S MCF-7 cells. Specifically, 213 out of 299 E2-regulated genes (71.2%) were modulated in L370F MCF-7 cells (Fig. 5 D and Supplementary Table 2), while 248 out of 299 genes (82.9%) were modulated in Y537S MCF-7 cells (Fig. 5 E and Supplementary Table 2). Interestingly, the proportion of upregulated and downregulated E2-responsive genes was comparable between L370F (Fig. 5 D, smaller pie chart and Supplementary Table 2) and Y537S (Fig. 5 E, smaller pie chart and Supplementary Table 2) MCF-7 cells. Altogether, these data indicate that the basal regulation of E2-responsive genes in L370F MCF-7 cells closely resembles that observed in Y537S MCF-7 cells. This suggests that the introduction of the L370F mutation in ERα may enhance the activity of the mutant receptor compared to the wt ERα, particularly with respect to E2-sensitive genes. To further investigate this hypothesis, we analyzed the lists of E2 upregulated genes in L370F and Y537S MCF-7 cells (as shown in the smaller pie charts in Fig. 5 D, 5 E and Supplementary Table 2). The Venn diagram in Fig. 5 F and the Supplementary Table 2 highlights 65 genes that were commonly upregulated in both L370F and Y537S MCF-7 cells, while 46 genes were specifically upregulated in L370F cells and 50 genes in Y537S cells. Consistent with this, basal levels of RARα, and FASN were upregulated in both L370F and Y537S MCF-7 cells. Conversely, expression levels of Cav-1 and FABP5 were specifically upregulated in Y537S cells, while ASCL1 and CALCR were specifically upregulated in L370F cells (Fig. 5 F’ and right panels). Finally, we assessed the proportion of genes upregulated by E2 that were classified as belonging to early, late, or both early and late E2 responses. As shown in Fig. 5 G, the 65 genes commonly upregulated in both L370F and Y537S cells (Fig. 5 F and Supplementary Table 2) were distributed among the three categories with a similar pattern. Notably, the 50 genes uniquely upregulated in Y537S MCF-7 cells were uniformly distributed among the three categories (Fig. 5 H, right pie chart and Supplementary Table 2). In contrast, the 46 genes specifically upregulated in L370F MCF-7 cells predominantly belonged to the late E2-responsive gene category (Fig. 5 H, left pie chart and Supplementary Table 2). These findings demonstrate that while the Y537S receptor mutation upregulates both early and late E2-responsive genes equally, the L370F ERα variant preferentially enhances the expression of late E2-responsive genes. The impact of the L370F mutation on ERα ligand binding and on receptor structure. The biological effects of the L370F ERα mutation may be due to a reduced or impaired binding of agonist (E2) or antagonist (Ful) to ERα. To assess the binding affinities of E2 and Ful to the L370F ERα, we transiently expressed full-length wild-type (wt), L370F, and Y537S ERα with a twin-strep tag in Epi293F cells and purified them using affinity chromatography. The resulting recombinant full-length wt, L370F, and Y537S ERα were then included in fluorescence polarization competitive binding assays, as previously described [ 13 ]. As additional control, we incorporated a commercially available wt ERα (Thermo Scientific), purified from insect cells, into our experimental plan. Both E2 and Ful were able to displace the fluorescent E2 tracer across all tested receptors (Table 1). Notably, no significant differences were observed in the K i values for E2 and Ful between the commercially available wt ERα and the recombinant ERα purified from Epi293F cells (relative binding affinity for E2 (RBA E2 ) = 0.78 ± 0.42; RBA Ful = 0.93 ± 0.45). However, as expected [ 37 ], the K i of the Y537S mutant ERα for both E2 and Ful was significantly higher than that of the wt ERα (Table 1) (RBA E2 = 0.072 ± 0.036; RBA Ful = 0.20 ± 0.09). Interestingly, while the K i of the L370F mutant ERα for E2 slightly increased with respect to that of the wt ERα, its absolute value remained within the same low nanomolar range (Table 1) (RBA E2 = 0.48 ± 0.30). In contrast, the K i of the L370F mutant ERα for Ful was significantly higher than that of the wt ERα (Table 1) (RBA Ful = 0.31 ± 0.14), and no significant differences were detected between the K i values of the L370F and Y537S mutants for Ful (Table 1) (RBA Ful = 0.71 ± 0.30). These findings confirm that the Y537S mutation reduces the receptor's affinity for both E2 and Ful [ 37 ]. Notably they also reveal that while L370F mutation has only a minor impact on E2 binding to ERα, it instead significantly reduces ERα’s affinity for Ful. Because we observed a reduction in Ful binding affinity towards the L370F receptor, we next performed classical molecular dynamics simulations to understand if the introduction of this mutation could alter the receptor structure, as observed for the Y537S ERα mutant. As shown in Fig. 6 A- 6 F, the L370F and to lower extend also E471D mutations result in a remodeling of helix H3 as compared to the wt ERα and to an ERα crystal structure in the antagonist conformation (pdb id:3ert [ 44 ]). In addition, the loop connecting H11 and H12 undergoes remodeling and moves closer to H3, leading to a partial disruption of H11’s secondary structure. Consistently, with the above findings this behavior is more pronounced in the L370F mutant. Aiming to inspect how the mutation affect the internal dynamical coupling of the receptor thus making it less sensitive to Ful, we then calculated the cross correlation coupling among the different ERα structural element, as done in previous studies [ 45 ] (Supplementary Fig. 3). Remarkably, the sum of correlation coefficients of H12 with H5 is higher than 4 only in the L370F mutant (Fig. 6 G). This was previously considered as the threshold for the ERα activation in the previous study [ 17 ]. While the hydrogen bond network does not change significantly in any of the simulations, Ful shows less persistent hydrogen bonds with ERα in the mutant receptors than in the wt (Supplementary Tables 3–5). Altogether these data indicate that the L370F mutation remodels ERα, reducing its affinity to Ful. Evaluation of the ATRA antiproliferative effects in parental, L370F and Y537S MCF-7 cells. Previous data showed that L370F and Y537S MCF-7 cells express higher levels of RARα compared to parental MCF-7 cells (Fig. 5 F’). Given that ATRA is a known antiproliferative agent for BC cells [ 46 ] and no information is available regarding its effects on cell lines expressing ERα point mutations found in MBC, we investigated the impact of this FDA-approved drug on these cell lines. Growth curve analyses were performed on parental, L370F, and Y537S MCF-7 cells treated with varying doses of ATRA (10 − 10 M to 10 − 4 M). As expected [ 46 ], a 7-day ATRA treatment reduced the proliferation rate in all tested cell lines in a dose-dependent manner (Fig. 7 A-C). Interestingly, during the first 3–4 days of treatment, the normalized cell index (CI) measured using the xCELLigence system showed a transient increase in all cell lines, which was most pronounced in L370F MCF-7 cells (Fig. 7 A-C). This increase was not due to enhanced proliferation but was instead attributable to ATRA-induced enlargement of the cellular surface, a feature accurately detected by the xCELLigence apparatus (Supplementary Fig. 4A and 4A’). IC 50 calculations revealed notable differences among the cell lines: L370F MCF-7 cells displayed the lowest IC 50 value (15.3 ± 0.62 nM), parental MCF-7 cells showed an intermediate value (9.1 ± 0.32 nM), and Y537S MCF-7 cells exhibited the highest IC 50 (5.2 ± 3.9 µM) (Fig. 7 D). Furthermore, the antiproliferative effect of ATRA was significantly greater in L370F MCF-7 cells compared to both parental and Y537S MCF-7 cells (Fig. 7 D). Detailed analysis of growth curve profiles (Fig. 7 A-C) showed that ATRA rapidly reduced the proliferation rate in L370F MCF-7 cells within 4–6 days, whereas the reduction in parental and Y537S MCF-7 cells over the same timeframe was slower. These findings suggest that ATRA exerts distinct antiproliferative effects across the different cell lines. To explore this hypothesis, we extended the growth curve analyses to 12 days, monitoring proliferation in each cell line treated with the indicated doses of ATRA. Consistent with previous results, ATRA exhibited the strongest antiproliferative effect in L370F MCF-7 cells, the weakest in Y537S MCF-7 cells, and an intermediate effect in parental MCF-7 cells (Fig. 7 E-G). Interestingly, L370F MCF-7 cells failed to proliferate entirely in the presence of ATRA (Fig. 7 F). In contrast, parental and Y537S MCF-7 cells maintained their control-level proliferation rates for the first 2–3 days of ATRA treatment but then plateaued, sustaining an almost constant cell number over the remaining 9–10 days of the assay (Fig. 7 E, G). These findings confirm the antiproliferative effect of ATRA on BC cells and demonstrate that its magnitude varies depending on the ERα variant expressed. Moreover, the results suggest that ATRA induce distinct cellular responses: cell death in L370F MCF-7 cells and a senescent-like phenotype in parental and Y537S MCF-7 cells. To assess cell death, we next examined the ability of ATRA to induce PARP cleavage. Parental, L370F, and Y537S MCF-7 cells were treated with different doses of ATRA (10 − 7 M to 10 − 5 M) for 3 days. Western blotting analysis revealed that ATRA induced a significant and dose-dependent increase in PARP cleavage in L370F MCF-7 cells, whereas the effect was only marginal in parental and Y537S MCF-7 cells (Fig. 7 H, upper panels and Fig. 7 H’). Since it has been reported that ATRA could induce DNA damage in BC [ 47 ], which is closely linked to the initiation of cell death, we further evaluated its effect on the phosphorylation of histone H2AX (γH2AX), a well-established marker of DNA double-strand breaks [ 48 ]. As shown in Fig. 7 H and 7 H’’, ATRA administration for 3 days caused a robust and significant increase in γH2AX levels in L370F MCF-7 cells, while only a minor and not significant effect was observed in parental and Y537S MCF-7 cells. To investigate the senescence-inducing potential of ATRA [ 47 ], parental and Y537S MCF-7 cells were treated with the indicated doses of ATRA for 12 days, after which the activation of the senescence associated β-galactosidase (SA-βGal) activity, a classical marker of senescence [ 49 ], was evaluated. As a positive control, etoposide was used to induce senescence in parental MCF-7 cells [ 50 ]. Figures 7 I and 7 J demonstrate that ATRA induced SA-βGal enzymatic activity in a dose-dependent manner in both cell lines, further confirming the onset of a senescent phenotype. These findings collectively demonstrate that ATRA exerts differential effects on MCF-7 cell lines depending on the ERα variant expressed. Specifically, ATRA triggers cell death in L370F MCF-7 cells, while it induces senescence in both parental and Y537S MCF-7 cells. Evaluation of the RARα and ERα crosstalk in parental, L370F and Y537S MCF-7 cells. Since previous studies have demonstrated that ATRA and E2 signaling transcriptionally antagonize each other in BC cells [ 51 ], we hypothesized that the effects of ATRA in promoting cell death in L370F MCF-7 cells may be attributed to differences in the reciprocal regulation of RARα and ERα expression across parental, L370F, and Y537S MCF-7 cell lines. To test this hypothesis, we assessed whether siRNA-induced depletion of either RARα or ERα differentially influenced the expression of ERα and RARα, respectively. Seventy-two hours after transfecting cells with siRNAs targeting RARα or ERα, the expression levels of both receptors were measured in parental, L370F, and Y537S MCF-7 cells. As expected, RARα-targeting siRNA reduced RARα expression, and ERα-targeting siRNA reduced ERα expression across all cell lines (Supplementary Fig. 5A, 5A’ and 5A’’). Notably, RARα depletion similarly affected ERα expression in all cell lines, and ERα depletion reciprocally reduced RARα expression to a comparable extent in parental, L370F, and Y537S MCF-7 cells (Supplementary Fig. 5A, 5A’ and 5A’’). These findings confirm a regulatory crosstalk between RARα and ERα in BC cells and demonstrate that the introduction of the L370F and Y537S mutations in the ERα does not alter the mutual influence these receptors exert on each other's expression. Next, we investigated the effects of ATRA on the expression levels of both ERα and RARα in parental, L370F, and Y537S MCF-7 cells. Cells were treated with increasing concentrations of ATRA (10 − 7 M to 10 − 5 M) for 72 hours, and receptor levels were assessed via WB analysis. As shown in Fig. 8 A and 8 A’, ATRA induced a dose-dependent reduction in ERα expression in all three cell lines, with the effect being most pronounced in Y537S MCF-7 cells. In contrast, ATRA induced a dose-dependent decrease in RARα expression in Y537S MCF-7 cells, showing the greatest reduction among the three cell lines. However, only the highest dose (10 − 5 M) resulted in a significant decrease in RARα levels in parental and L370F MCF-7 cells (Fig. 8 B and 8 B’). Interestingly, baseline RARα expression levels were elevated in both L370F and Y537S MCF-7 cells compared to parental cells (control samples). However, ATRA treatment reduced RARα levels in Y537S MCF-7 cells below those of parental cells, while RARα levels in L370F MCF-7 cells remained higher than in parental cells (Fig. 8 B and 8 B’’). Prompted by these observations, we hypothesized that ATRA-induced RARα activity might differ among parental, L370F, and Y537S MCF-7 cells. To test this, we generated stable cell lines expressing a reporter construct containing the retinoic acid receptor response element (RARE) fused to the nanoluciferase gene (RARE-NLuc). These cells were treated with ATRA at varying concentrations (10 − 11 M to 10 − 6 M) for 24 hours to evaluate RARα transcriptional activity. ATRA dose-dependently increased RARα transcriptional activity in all cell lines (Fig. 8 C). However, the response was highest in L370F MCF-7 RARE-NLuc cells and lowest in Y537S MCF-7 RARE-NLuc cells compared to parental cells. Because ATRA reduced ERα expression in these cell lines and ERα degradation is intrinsically linked to the activation of ERα transcriptional activity [ 52 , 53 ], we further assessed ATRA's impact on ERα transcriptional activity using parental, L370F, and Y537S MCF-7 ERE-NLuc cells. ATRA administration for 24 hours caused a dose-dependent reduction in ERα transcriptional activity in all three cell lines, with the strongest effect observed in Y537S MCF-7 ERE-NLuc cells (Fig. 8 D). These findings indicate that ATRA-induced RARα transcriptional activity differs significantly in L370F and Y537S MCF-7 cells compared to parental cells. Notably, the ATRA-induced effects on RARα transcriptional activity (Fig. 8 C) closely parallel the drug’s antiproliferative effects in these cell lines (Fig. 7 D), suggesting that the enhanced antiproliferative effect of ATRA in L370F MCF-7 cells may result from hyperactivation of RARα transcriptional activity. Evaluation of the effect of digoxin on ATRA-induced senescence in parental, and Y537S MCF-7 cells. The induction of a senescent phenotype in cancer cells by therapeutic agents provides an opportunity to explore novel anticancer strategies, such as using compounds that selectively eliminate senescent cells (i.e., senolytics) [ 54 ]. Therefore, we investigated the potential of digoxin (Digo), an FDA-approved cardiac glycoside with known senolytic activity [ 55 ], to target ATRA-induced senescent parental and Y537S MCF-7 cells. To this end, we performed growth curve analyses on parental and Y537S MCF-7 cells treated with various concentrations of Digo (10 − 8 M to 10 − 6 M) both in the absence and presence of ATRA (10 − 6 M). As expected, Digo induced a dose-dependent reduction in the proliferation rate of both parental and Y537S MCF-7 cells. Similarly, ATRA (10 − 6 M) consistently reduced the proliferation of these cell lines over time (Fig. 9 A and 9 B). Following 10 days of continuous ATRA administration, when senescence was previously confirmed (Fig. 7 ), we introduced different doses of Digo (10 − 8 M to 10 − 6 M) to the parental and Y537S MCF-7 cells and monitored their proliferation rates over an additional 5-day period. Under these conditions, Digo again caused a dose-dependent decrease in the proliferation rate of both cell lines (Fig. 9 A and 9 B). To quantitatively assess the effect of Digo, we calculated its IC 50 values at different time points after administration, both in the absence and presence of ATRA. The IC 50 values were mathematically transformed (i.e., -Log 2 ) to evaluate the sensitivity of both cell lines to Digo. The overall sensitivity to Digo was similar in both cell lines both in the absence and presence of ATRA (Fig. 9 A’ and 9B’). However, while the parental MCF-7 cells exhibited no significant differences in Digo sensitivity over time, regardless of ATRA treatment (Fig. 9 A’), Y537S MCF-7 cells showed enhanced sensitivity to Digo in the presence of ATRA compared to its absence (Fig. 9 B’). These findings demonstrate that the antiproliferative effect of Digo during prolonged ATRA treatment manifests more rapidly in Y537S MCF-7 cells compared to parental MCF-7 cells. This observation strongly suggests that Digo could function as a senolytic agent specifically in ATRA-induced senescent Y537S MCF-7 cells. Finally, we investigated the combined effects of ATRA and 4OH-tamoxifen (Tam), a cornerstone therapy for patients with ERα-expressing primary BC [ 1 ], by treating parental MCF-7 cells with varying doses of both drugs. The results demonstrated a synergistic interaction between ATRA and Tam in parental MCF-7 cells (Fig. 9 C and 9 C’). These findings suggest the potential use of ATRA in combination with Tam for the treatment of primary BC. Discussion The mainstay treatment for ERα-expressing primary breast cancer (BC) involves endocrine therapy (ET) with drugs such as aromatase inhibitors (AIs) and 4OH-tamoxifen (Tam). This treatment is typically administered for 5 to 10 years following diagnosis and has significantly reduced BC mortality rates. However, the extended duration of therapy often leads to the development of resistance to ET drugs in a substantial subset of patients, resulting in relapse and progression to metastatic breast cancer (MBC), which is frequently fatal [ 1 ]. Resistance to ET drugs arises through multiple mechanisms, including the selection of ERα point mutations in MBC cells that drive uncontrolled proliferation. Most of these ERα variants occur within the receptor's ligand-binding domain (LBD) and induce structural rearrangements that mimic the three-dimensional conformation of the wt receptor bound to E2. This constitutively active agonist conformation not only promotes continuous proliferative signaling but also renders the mutated receptor insensitive to anti-estrogen therapies, such as Ful [ 14 , 37 ]. Targeting ERα point mutations in MBC is therefore crucial for developing strategies to manage the disease. Various therapeutic approaches, including SERDs, SERCAs, PROTACs, CERANs, and other targeted drugs [ 10 – 13 ], have been explored to inhibit the metastatic potential of mutant ERα variants. Promisingly, clinical trials have demonstrated the efficacy of some of these agents, such as elacestrant [ 6 ] and camizestrant [ 5 , 7 ], in combating receptor mutations, leading to the approval of elacestrant for clinical use [ 2 , 6 ]. Despite these advancements, preclinical and clinical studies have predominantly focused on the most frequent ERα mutations, such as Y537S, while neglecting the functional impact of less common ERα variants. These lesser-studied mutations are equally associated with reduced patient survival and contribute to therapy resistance via variant-specific mechanisms [ 14 ]. For example, Irani et al. [ 15 ] reported that mutations in the ERα dimerization domain lead to constitutive transcriptional activation, promoting cell proliferation. This study also proposed that disrupting receptor dimerization could serve as a novel therapeutic strategy for MBC [ 15 ]. These findings not only highlight the urgent need to characterize all ERα variants expressed in MBC to identify new therapeutic targets but also underscore the role of three-dimensional structural determinants in deregulating specific receptor functions (e.g., such as ERα dimerization, and nuclear localization), thus opening the possibility that specific neglected ERα variant could be treated with specific drugs. In this context, we investigated two clinically observed ERα mutations, L370F and E471D, which are annotated in the COSMIC and cBioPortal databases. These mutations affect residues positioned across from each other on helices H4 and H10 within the ERα ligand-binding domain (LBD), pointing to a potential new structural hotspot. The characterization of these mutations demonstrates that they differently affect sensitivity to Ful. Specifically, the L370F variant reduces sensitivity to Ful, whereas the E471D variant does not. Further characterization of the L370F mutation revealed that it confers several distinct properties to BC cells such as i) reduced sensitivity to Ful in terms of ERα transcriptional activity, receptor stability, and cell proliferation; ii) partially E2-independent growth and hyperactive proliferation in response to low E2 doses; and iii) basal upregulation of late E2 response genes. Our data show that the recombinant purified L370F receptor exhibits reduced binding affinity for Ful compared to the wt receptor. All atoms’ simulations further confirmed this finding elucidating that the lower binding of Ful to this ERα variant is due to a structural reorganization of the H4-H5-H12 region in the ERα antagonist conformation. Altogether in vitro and in silico findings provide evidence that the natural mutation of ERα at residue L370 alters the receptor's structure, which impairs Ful binding, similarly to what observed for the Y537S ERα variant [ 37 ]. Notably, simulation analyses performed on the E471D mutant ERα revealed that this mutation has a minor effect on the receptor structure and Ful binding. On one hand, this result supports the lack of Ful effect on ERα transcriptional activity; on the other hand, it suggests that the different point mutations found in MBC patients could also affect three-dimensional structural clusters. This further opens the possibility that distant mutations (e.g., in domains other than the LBD) could influence the structure-function of the LBD, and in turn, antagonist binding and receptor cellular functions. The L370F mutant structural remodeling and reduced binding affinity to Ful contribute to the diminished effect of Ful on the transcriptional activity, receptor degradation, and cell proliferation of the L370F ERα variant. In both stable HEK293 cells overexpressing the L370F ERα variant and L370F MCF-7 cells, we observed that this mutation attenuates the effect of Ful, particularly at high drug doses. These results demonstrate that the L370F ERα point mutation confers resistance to the ET drug Ful. Notably, this resistance is significant in the context of high-dose Ful administration (e.g., 500mg/Kg), which has been shown to enhance anti-tumor effects in patients [ 1 ]. Growth curve analysis under 10% serum conditions revealed that cells expressing the L370F ERα variant have a reduced doubling time compared to wt ERα-expressing cells, and their proliferation rate is similar to that of Y537S MCF-7 cells. Interestingly, the growth behavior of L370F-expressing cells mirrors that of Y537S MCF-7 cells. Conversely, when grown in an E2-deprived medium, the Y537S MCF-7 cells continue to proliferate, while parental MCF-7 cells stop growing and eventually die. In contrast, L370F MCF-7 cells cease growth but survive in the absence of E2. Upon E2 administration, parental MCF-7 cells remain sensitive to the hormone, while Y537S MCF-7 cells are unresponsive. However, L370F MCF-7 cells respond to E2 in terms of cell proliferation only when grown in an E2-deprived medium. In a medium containing E2, L370F MCF-7 cells exhibit only a minimal response to E2. Notably, at low E2 doses, the L370F ERα variant displays hyperactivity in terms of E2-dependent cell proliferation compared to the wt receptor. This effect is also evident in terms of receptor transcriptional activity. Although the specific mechanisms underlying this effect were not investigated, our in silico simulations of the L370F ERα suggest a structural reorganization of the receptor. This, combined with the similar binding affinity for E2 as observed in the wt ERα, could facilitate the response to E2. Alternatively, the differential sensitivity to E2 may result from the recruitment of distinct co-activators by the L370F mutant receptor compared to the wt ERα. Interestingly, the ability of the L370F ERα variant to respond to low E2 doses may support metastatic cell proliferation. Indeed, in menopausal women or those undergoing ovarian function inhibition, plasma E2 levels are in the picomolar range [ 56 ]. In this context, the L370F mutation activates key functions of the mutant receptor, suggesting that receptor activation at low E2 doses may represent a novel mechanism by which BC cells adapt to the hostile environment created by ET drugs (e.g., AI). One hallmark of the Y537S mutation is the receptor’s ability to constitutively modulate E2-regulated genes, independent of E2 binding [ 37 ]. Our transcriptomic analysis revealed that the Y537S and L370F ERα mutants regulate a similar number of genes, with several E2-sensitive genes being upregulated in both mutant cell lines. Interestingly, the extent of upregulation differed between the two cell lines, with some genes specifically upregulated in one line or the other. Moreover, our data showed that in Y537S MCF-7 cells, genes from both early and late E2 responses were upregulated, whereas in L370F MCF-7 cells, genes from the late E2 response class were more prominently upregulated. Thus, the overall E2 response differs between the two cell lines and may be due to the ability of the L370F ERα to associate with distinct chromatin regions compared to the Y537S variant. Future ChIP-Seq analyses are necessary to further elucidate these differences. However, the selective upregulation of late E2 response genes by the L370F ERα variant highlights its role in promoting cell cycle progression and supporting the development of resistance to ET drugs [ 57 ]. It is important to clarify the potential clinical significance of the L370F ERα variant. Among annotated ERα mutations, some are frequently observed in patients, while others are rarer [ 58 ]. Regardless of their prevalence, these mutations generally exhibit poor responsiveness to existing therapies and negatively impact patient survival [ 58 ]. Despite their rarity, our findings demonstrate that the L370F ERα mutation provides BC cells with selective advantages for metastatic growth. These findings highlight the need to investigate the biological impact of rare ERα mutations in MBC. Moreover, it is possible that some ERα mutations are classified as rare simply because they are overlooked in current research and diagnostic efforts. Currently, next-generation sequencing or droplet digital PCR experiments (ddPCR) are used to detect ERα mutations [ 59 ]. ddPCR is used to identify only known and most frequent ERα mutations (e.g., exon 5 to exon 8, i.e., ERα LBD) while other mutations and/or those located outside the LBD are not actively searched in patient samples, unless whole exome sequencing, an expensive and time consuming method [ 58 ], is performed. For example, no screening protocols exist for mutations located in the ERα A/B domain, and the L370F mutation located in exon 5 cannot be identified as the standard oligonucleotides used for ddPCR-based detection of that region contain the L370 wt codon [ 59 ]. This diagnostic bias neglects mutations that may significantly contribute to disease progression, as exemplified by the L370F ERα variant described herein, and others previously reported [ 15 , 16 ]. This limitation is critical because no specific treatment protocols currently exist for MBC patients harboring these neglected ERα mutations, leaving these individuals without access to personalized therapy. Interestingly, we observed that both L370F and Y537S MCF-7 cells express elevated levels of RARα, prompting us to investigate the antiproliferative effects of ATRA on these cell lines [ 46 ]. Our results show that L370F ERα-expressing cells are more sensitive to ATRA than parental or Y537S MCF-7 cells. Importantly, ATRA triggers substantial DNA damage and pronounced cell death selectively in L370F cells. Initially, we hypothesized that this effect might arise from an interaction between RARα and ERα [ 51 ]. However, the presence of the L370F and Y537S mutations did not alter the reciprocal regulation of receptor levels or the ability of ATRA to activate RARα transcriptional activity and to inhibit ERα transcriptional activity. Furthermore, ATRA did not bind in vitro to wt, L370F and Y537S ERα (data not shown). Nevertheless, we found that ATRA-induced activation of RARα transcriptional activity is significantly enhanced in L370F cells. Given previous findings that R-loops generated during the E2 transcriptional response contribute to DNA damage and genomic instability in BC cells [ 60 , 61 ], we speculate that ATRA-dependent hyperactivation of RARα transcription leads to transcriptional stress (e.g., R-loops), DNA damage, and subsequent cell death, thereby explaining the heightened sensitivity of L370F cells to ATRA. Growth curve analyses and cellular assays revealed that ATRA induces senescence in parental and Y537S MCF-7 cells. While ATRA-induced senescence in BC cells has been previously reported [ 47 ], this is, to our knowledge, the first evidence of a drug inducing senescence in a BC cell line expressing the Y537S ERα variant. Exploiting senescence as a therapeutic strategy is increasingly recognized for its potential to overcome treatment resistance, as it creates vulnerabilities that can be targeted with senolytic agents in a ‘one-two punch’ approach [ 54 ]. Supporting this, we found that digoxin, an FDA-approved cardiac glycoside with known senolytic activity [ 55 ], selectively targets ATRA-induced senescence in Y537S MCF-7 cells but not in parental cells. The clinical potential of ATRA has been demonstrated by its success in treating acute promyelocytic leukemia, inspiring efforts to explore its use in solid tumors [ 62 , 63 ]. Although studies consistently report ATRA’s inhibitory effects on BC cell growth, this knowledge has led to only a limited number of clinical trials investigating ATRA as an anti-BC agent [ 64 ]. Moreover, no data currently address the potential use of ATRA in treating MBC expressing ERα mutations. However, our findings highlight three potential applications of ATRA in BC clinical practice. First, we observed that co-administration of ATRA with Tam produces a synergistic antiproliferative effect in parental MCF-7 cells, indicating its potential use in managing primary ERα expressing BC that are suitable for ET. Additionally, ATRA could be implemented as a chemotherapeutic agent for treating MBC expressing the L370F ERα variant. Finally, ATRA could be employed to induce cellular senescence in MBC expressing the Y537S ERα variant, creating an opportunity for subsequent senolytic therapy in a ‘one-two punch’ treatment strategy. Conclusions This study identifies the L370F ERα mutation as a novel natural variant associated with MBC and elucidates the mechanisms through which it contributes to ET resistance and metastatic growth. Our findings highlight the importance of characterizing all ERα mutations, including those currently neglected, to better understand their roles in disease progression and therapy resistance. Furthermore, we provide evidence that different ERα variants exhibit unique sensitivities to specific drugs, such as ATRA, which can elicit distinct therapeutic effects depending on the mutation. Overall, this work demonstrates that tailoring treatment based on the specific ERα variant expressed in MBC can pave the way for more effective and personalized BC therapies. Abbreviations AI: aromatase inhibitors ASCL1 : Achaete-Scute Family BHLH Transcription Factor 1 ATRA: all-trans retinoic acid BC: breast cancer CALCR : Calcitonin Receptor CDK4: cyclin-dependent kinase 4 CDK6: cyclin-dependent kinase 6 CERAN: complete estrogen receptor antagonists CS-FBS : Charcoal Stripped Fetal bovine serum ddPCR : droplet digital PCR Digo : Digoxin E2: 17β-estradiol ERE: estrogen responsive element ER α : estrogen receptor α ET: Endocrine therapy FABP5 : fatty acid binding protein 5 FASN : fatty acid synthase FBS : Fetal bovine serum FDA: Food and Drug Administration FPKN : Fragments Per Kilobase of transcript Ful: fulvestrant HER2: Human Epidermal Growth Factor Receptor 2 LBD: ligand binding domain MBC: metastatic breast cancer MBC: metastatic breast cancer NLuc : Nanoluciferase PCA : Principal component analysis PROTAC: proteolysis targeting chimerics RARE : retinoic acid response element RAR α : retinoic acid receptor α SERCA: selective estrogen receptor covalent antagonists SERD: selective estrogen receptor downmodulators SERM: selective estrogen receptor modulators Tam: 4OH-tamoxifen WB : Western Blot wt : wild type γ H2AX: phosphorylated H2A Histone Family Member X Declarations Ethics approval and consent to participate: 'Not applicable' Consent for publication: 'Not applicable' Availability of data and materials Densitometric analyses of each WB, as well as original data for growth curves and synergy proliferation experiments, are available from the corresponding author on reasonable request. All the original Western blots are available in Supplementary Materials. Competing interests: The authors declare that they have no competing interests. Funding. The research leading to these results has received funding from AIRC under IG 2018 - ID. 21325 project – P.I. Acconcia Filippo. The Grant of Excellence Departments 2023-2027, MIUR (ARTICOLO 1, COMMI 314 – 337 LEGGE 232/2016) to the Department of Science, University Roma TRE is also gratefully acknowledged. This study was also supported by grants from Ministero della Salute RF‐2021‐12372851; CUPF83C22002620001 to Acconcia Filippo. Author’s Contributions. M.C. performed most of the experimental work. C.B. performed transcriptional studies. M.F. performed measurement of cellular surface. M.P. and A.M. performed in silico studies. F.A. conceptualized the research, formally analyzed the data, wrote, reviewed, and edited the manuscript. All authors reviewed the manuscript. Acknowledgements We thank A. Scardua and J. Weber (Biomass Production Unit, National Facility for Structural Biology, Human Technopole) for support and service provision. Access to National Facility was granted to Project ID 1771263. The authors are grateful to Prof. Simak Ali, University of London Imperial College for the gift of the MCF-7 Y537S cells. The anti-FASN antibody was a generous gift of Prof. Andrea Morandi, Dipartimento di Scienze Biomediche Sperimentali e Cliniche ‘Mario Serio’, Firenze, Italy. References Will M, Liang J, Metcalfe C and Chandarlapaty S 2023 Therapeutic resistance to anti-oestrogen therapy in breast cancer. 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Cancer Treat Rev 40:739-49. doi: 10.1016/j.ctrv.2014.01.001 Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA and Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753-8. doi: 10.1038/39645 Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files OriginalWB.pptx Table1.jpg Table1 SupplementaryFigureCaptions.docx SupplementaryFigures.pptx SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTables35.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6706598","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459255564,"identity":"32349172-ee62-4518-a9c6-d064b3e42e22","order_by":0,"name":"Manuela Cipolletti","email":"","orcid":"","institution":"1Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy","correspondingAuthor":false,"prefix":"","firstName":"Manuela","middleName":"","lastName":"Cipolletti","suffix":""},{"id":459256032,"identity":"889b194c-2acf-4589-9bac-195aa7d25311","order_by":1,"name":"Claudia Bellucci","email":"","orcid":"","institution":"1Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Bellucci","suffix":""},{"id":459256033,"identity":"f9050fb3-1a36-4620-9933-ab39367645b0","order_by":2,"name":"Marco Fiocchetti","email":"","orcid":"","institution":"1Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Fiocchetti","suffix":""},{"id":459256034,"identity":"e12b63b6-82f8-4b75-9ab5-fe12deb624f3","order_by":3,"name":"Matic Pavlin","email":"","orcid":"","institution":"2Department of Chemical Reaction Engineering, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia","correspondingAuthor":false,"prefix":"","firstName":"Matic","middleName":"","lastName":"Pavlin","suffix":""},{"id":459256035,"identity":"fd42c086-542e-480c-8e00-229d888069b3","order_by":4,"name":"Alessandra Magistrato","email":"","orcid":"","institution":"National Research Council-Insitute of Material Foundry (CNR-IOM) at International School for Advanced Studies (SISSA/ISAS) via Bonomea 265 34136 Trieste Italy.","correspondingAuthor":false,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Magistrato","suffix":""},{"id":459256036,"identity":"636ccdb2-a411-4d75-9a4a-876883a28165","order_by":5,"name":"Filippo Acconcia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBAC9mbmhgNIfAkGfiAJFpFnYGzApoXnMCOaFskGqBbDBsZGbHp4DqAbZQA34QB2a3jYGRsPF+6wYeBvP/zwcUGNhbzxjdyDhysqtskzNjC3P8CmhZmx4fDMM2kMEmfSjI1nHJMw3HYjL+HgmTO3DdsZsDvMHqSFt+0wA8MNHjZp3gYJxm03cgwONrbdBqrH4ReYFnmoFvvNMyBa7BsOENBiANWSuEECoiURrxagX3gMQX7hOSaRPOPMG4ODDWduJ29sZmycgU0L/+HDn4EhJid3HBhiPDV1tv3tOcYfGypu285nb3/wAYsWEABaxMCDTRyHeqiWUTAKRsEoGAW4AQDAcWoaJ2rqJAAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Sciences, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy","correspondingAuthor":true,"prefix":"","firstName":"Filippo","middleName":"","lastName":"Acconcia","suffix":""}],"badges":[],"createdAt":"2025-05-20 10:15:02","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6706598/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6706598/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83294612,"identity":"5d20c120-6f0a-4d23-aa9c-476387a91ae8","added_by":"auto","created_at":"2025-05-22 13:38:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the fulvestrant effect in stable L370F and E471D ERα variants expressing HEK293 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Structure of the wild type (wt) ERα ligand binding domain (pdb code: 1A52 [65]) where the L370 and E471 residues are highlighted in yellow. (B-D) Dose-response of ERα transcriptional activity have been performed in HEK293 cells stably expressing both the estrogen responsive element nanoluciferase (ERE-NLuc) reporter construct and the expression vectors encoding for the indicated ERα variants. Each dot represents the -Log\u003csub\u003e2\u003c/sub\u003e transformation of the inhibitory concentration 50 (IC\u003csub\u003e50\u003c/sub\u003e) of the Ful effect in each cell line. Dotted lines indicate the two Ful IC\u003csub\u003e50\u003c/sub\u003e obtained in the same experiment in which each condition was tested in triplicate. * indicates the significant differences calculated using the Student’s t-test (p \u0026lt; 0.05). (E) Western blot and (E’ and E’’) relative densitometric analyses of ERα levels in HEK293 cells stably expressing both the ERE-NLuc reporter construct and the expression vectors encoding for the indicated ERα variants treated for 24 hours with the indicated doses of fulvestrant (Ful). Each experiment was performed at least in triplicate and significant differences in the effect of Ful between the wt and mutant ERα expressing HEK293 cells were calculated using the Student’s t-test (* indicates p \u0026lt; 0.05). (F) Dose-response of ERα transcriptional activity in HEK293 cells stably expressing both the ERE-NLuc reporter construct and the expression vectors encoding for the wt and L370F ERα treated with the indicated doses of 17b-estradiol (E2). Experiments were performed three times and each condition was tested in triplicate.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/9e2b9fda894960331be4535f.jpg"},{"id":83296236,"identity":"410f9cfb-75d3-4b10-ba8e-69a2acd22410","added_by":"auto","created_at":"2025-05-22 13:54:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the fulvestrant effect in CRISPR/CAS9 engineered L370F MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot and (A’) relative densitometric analyses of ERα levels in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 24 hours with the indicated doses of fulvestrant (Ful). Each experiment was performed at least in triplicate and significant differences in the effect of Ful between the parental and L370F ERα expressing MCF-7 cells (* indicates p \u0026lt; 0.05) and the Y537S MCF-7 cells (° indicates p \u0026lt; 0.05) were calculated using the Student’s t-test. (B) Dose-response of ERα transcriptional activity in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells stably expressing the ERE-NLuc reporter construct (NLuc) treated with the indicated doses of Ful. Experiments were performed three times, and each condition was tested in triplicate. Significant differences in the effect of Ful between the parental and L370F ERα expressing MCF-7 cells (* indicates p \u0026lt; 0.05) and the Y537S MCF-7 cells (° indicates p \u0026lt; 0.05) were calculated using the Student’s t-test. (C) Growth curve analyses in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated with the indicated doses Ful. Experiments were performed using the xCelligence RTCA system. Dose response curves were calculated at the maximum proliferation of each cell line. The compound effect is the difference between the normalized cell index (NCI) value in the treated samples and the NCI value in the untreated samples at the time point when cells reach the maximal growth (i.e., confluency). Significant differences in the effect of Ful between the parental and L370F ERα expressing MCF-7 cells (* indicates p \u0026lt; 0.05) and the Y537S MCF-7 cells (° indicates p \u0026lt; 0.05) were calculated using the Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/23474e467c8a37523a4f16a8.jpg"},{"id":83295624,"identity":"c3c2f5e2-d746-4255-9627-d50c0a69ebb7","added_by":"auto","created_at":"2025-05-22 13:46:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the proliferation rate of the CRISPR/CAS9 engineered L370F MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Growth curve analyses in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells plated in growing conditions (i.e., 10% FBS). Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation the untreated sample in each cell line. (B) Doubling time of the parental and the CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells. Significant differences between the parental and the L370F or Y537S ERα expressing MCF-7 cells (**** indicates p \u0026lt; 0.001) and between the L370F and the Y537S MCF-7 cells (°°°° indicates p \u0026lt; 0.001) were calculated using the Student’s t-test. Each dot indicates an experimental replicate. (C) Growth curve analyses in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells plated in the presence of charcoal stripped fetal calf serum (i.e., 10% CS-FBS). Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation of the CRISPR/CAS9 engineered Y537S MCF-7 cells. Growth curve analyses in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells plated in growing conditions (i.e., 10% FBS) (D-F) or in the presence of charcoal stripped fetal calf serum (i.e., 10% CS-FBS) (H-J) and treated with the indicated doses of 17β-estradiol (E2). Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation of the untreated sample in each cell line. (G, K) Dose response curves of E2 effect were calculated at the maximum proliferation of the most effective E2 treated sample in each cell line. The effect of E2 is the difference between the normalized cell index (NCI) value in the treated samples and the NCI value in the untreated samples at the time point when cells reach the maximal growth (i.e., confluency). Significant differences in the effect of E2 between the parental and L370F ERα expressing MCF-7 cells (** indicates p \u0026lt; 0.01) were calculated using the Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/8b7ccf5e9d41536f157a0f05.jpg"},{"id":83295626,"identity":"f5748b51-c9cd-48f8-ba93-3f39082cb88d","added_by":"auto","created_at":"2025-05-22 13:46:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the E2-dependent regulation of ERα transcriptional activity in CRISPR/CAS9 engineered L370F MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dose-response of ERα transcriptional activity in parental and CRISPR/CAS9 engineered L370F MCF-7 cells plated in growing conditions (i.e., 10% FBS) treated with the indicated doses of 17b-estradiol (E2). Experiments were performed three times, and each condition was tested in triplicate. Significant differences in the effect of E2 between the parental and L370F MCF-7 cells were calculated using the Student’s t-test (**** indicates p \u0026lt; 0.0001 and ** indicates p \u0026lt; 0.01). (B) Western blot and (B’) relative densitometric analyses of S118 phosphorylated and total ERα levels in parental and CRISPR/CAS9 engineered L370F MCF-7 cells treated for 30 minutes with the indicated doses of 17b-estradiol (E2). The experiment was performed in triplicate and significant differences in the effect of E2 between the parental and L370F ERα expressing MCF-7 cells were calculated using the Student’s t-test (* indicates p \u0026lt; 0.05). (C) Antagonism (red) and synergy (blue) surfaces of ERα transcriptional activity in parental and CRISPR/CAS9 engineered L370F MCF-7 cells plated in growing conditions (i.e., 10% FBS) in the presence or in the absence of the co-treatment with 17b-estradiol (E2) (10\u003csup\u003e-14\u003c/sup\u003e-10\u003csup\u003e-8\u003c/sup\u003eM) and fulvestrant (Ful) (10\u003csup\u003e-11\u003c/sup\u003e-10\u003csup\u003e-5\u003c/sup\u003eM). Experiments were performed in duplicate. (D) Pie diagrams illustrating the percentages of modulated and un-modulated array genes in parental MCF-7 cells treated with 17b-estradiol (E2) (10\u003csup\u003e-9\u003c/sup\u003eM) for 24 hours and in CRISPR/CAS9 engineered L370F MCF-7 cells treated with E2 (10\u003csup\u003e-12\u003c/sup\u003eM) for 24 hours. Percentages and categories of genes are indicated. (E) Pie diagrams illustrating the similarity in the E2-modulated genes between the parental MCF-7 cells treated with E2 (10\u003csup\u003e-9\u003c/sup\u003eM) for 24 hours and the CRISPR/CAS9 engineered L370F MCF-7 cells treated with E2 (10\u003csup\u003e-12\u003c/sup\u003eM) for 24 hours. (E) Pie diagrams illustrating the percentages of the E2 effect on the common modulated genes in the array both in parental MCF-7 cells and in CRISPR/CAS9 engineered L370F MCF-7 cells.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/83595ad531450c4244f2698c.jpg"},{"id":83295628,"identity":"7a1567b5-dfa9-49ca-a5b3-bf9ccdbf3ae6","added_by":"auto","created_at":"2025-05-22 13:46:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal gene expression analysis in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRanked gene list correlation profile of CRISPR/CAS9 engineered L370F (A) and of Y537S (B) MCF-7 cells with respect to parental MCF-7 cells. (C) Venn diagram showing the number of modulated genes in CRISPR/CAS9 engineered L370F and of Y537S MCF-7 cells with respect to parental MCF-7 cells with a signal-to-noise ratio \u0026gt;0.5 or \u0026lt;0.5. Pie diagrams showing the percentage of 17β-estradiol (E2) regulated genes in the gene sets in GSEA (i.e., hallmark estrogen response early and hallmark estrogen response late) of CRISPR/CAS9 engineered L370F (D) and of Y537S (E) MCF-7 cells with respect to parental MCF-7 cells. The smaller pie diagrams indicate the percentage of the E2 responsive genes either up regulated or down regulated in the same cell lines (D, and E). (F) Venn diagram showing the number of the E2 genes upregulated RISPR/CAS9 engineered L370F or Y537S MCF-7 cells with respect to parental MCF-7 cells (F’) Western blot and relative densitometric analyses (right panels) of RARα, FASN, FABP5, Cav1, ASCL1, and CALCR levels in growing parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells. Each dot indicates an experimental replicate. Significant differences with respect to the parental MCF-7 cells were calculated using the Student’s t-test. * indicates p\u0026lt;0.05; ** indicates p\u0026lt;0.01; *** indicates p\u0026lt;0.001 and **** indicates p\u0026lt; 0.0001.\u003cstrong\u003e \u003c/strong\u003e(G) Pie diagram showing the percentage of E2 commonly up regulated genes [from (F)] in CRISPR/CAS9 engineered L370F and in Y537S (G) MCF-7 cells or up regulated in CRISPR/CAS9 engineered L370F (H, left panel) and in Y537S (H, right panel) with respect to parental MCF-7 cells and divided as genes modulated by E2 in early and late response (grey), genes modulated by E2 in early response (blue) and in genes modulated by E2 in late response (purple).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/3ac6209c8ed1477b282667e4.jpg"},{"id":83294621,"identity":"d6c81d2a-7024-49dc-90e2-902818d4d2c7","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural profile of wt and ERα L370F variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHighest populated clusters of ERα (wt – gray, L370F – red, E471D – blue) from the equilibrated part of the trajectory superimposed on the 3ert crystal structure (green) (pdb id:3ert [44]), wt side view (A), wt top view (B), L370F side view (C), L370F top view (D), E471D side view (E), and E471D top view (F). Residues 370 and 471 are shown in balls and stick representation in the corresponding colour scheme of the protein and fulvestrant is depicted in violet. Helices H3, H11, and H12 are marked. (G) Sum of cross-correlation coefficients for interactions of residues with H12, monomer A is shown on the left and monomer B on the right side of the plot.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/8e558ea99a76932aec759eec.jpg"},{"id":83295633,"identity":"1abda666-dffe-4009-8f33-cbbc5adc3be3","added_by":"auto","created_at":"2025-05-22 13:46:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":148009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of ATRA in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth curve analyses in parental (A), CRISPR/CAS9 engineered L370F (B) and Y537S (C) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) for the indicated time. Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation the untreated sample in each cell line. (D) Dose response curves of ATRA effect were calculated at the maximum proliferation of the control sample (CTR). The effect of ATRA is the difference between the normalized cell index (NCI) value in the untreated samples and the NCI value in the treated samples at the time point when cells reach the maximal growth (i.e., confluency). Growth curve analyses in parental (E), CRISPR/CAS9 engineered L370F (F) and Y537S (G) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) for the indicated time. Arrows indicate the time point where the medium was changed and ATRA administration was repeated. Experiments were performed using the xCelligence RTCA system. Curves were calculated at the maximum proliferation of the untreated sample in each cell line. (H) Western blot and (H’ and H’’) relative densitometric analyses of PARP and phosphorylated gH2AX levels in parental, and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 3 days with the indicated doses of ATRA. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. ** (p \u0026lt; 0.01) and *** (p \u0026lt; 0.001) indicates differences with respect the untreated (0) sample in each cell line while °° (p \u0026lt; 0.01) indicates differences between the corresponding sample in parental MCF-7 cells. (I) Measurement of β-galactosidase activity (βGal) in parental and in CRISPR/CAS9 engineered Y537S MCF-7 cells treated with the indicated doses of ATRA for 12 days. As positive control MCF-7 cells were treated with etoposide (Eto 12.5 µM) and allowed to recover for 4 days. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. **** (p \u0026lt; 0.0001) indicates differences with respect to the untreated (0) sample in each cell line. (J) β-Galactosidase staining at pH 6.0 on parental and on CRISPR/CAS9 engineered Y537S MCF-7 cells treated with ATRA (10\u003csup\u003e-7\u003c/sup\u003eM for MCF-7 cells and 10\u003csup\u003e-6\u003c/sup\u003eM for Y537S MCF-7 cells) for 12 days. Scale bar = 100 µm\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/f56bcda7f06ced8dd61544d1.jpg"},{"id":83294625,"identity":"073b54d9-06cc-4dd7-b304-8c30b6096614","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":127734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATRA-dependent regulation of RARα and ERα expression and transcriptional activity in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot and (A’) relative densitometric analyses of ERα levels in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 72 hours with the indicated doses of all-trans retinoic acid (ATRA). Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. *** and **** indicates p \u0026lt; 0.001 and p \u0026lt; 0.0001 with respect to control (-) samples. ° and °°°° indicates p \u0026lt; 0.05 and p \u0026lt; 0.0001 with respect to the corresponding samples in parental MCF-7 cells. (B) Western blot and (B’ and B’’) relative densitometric analyses of RARα levels in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells treated for 72 hours with the indicated doses of ATRA. Each dot indicates an experimental replicate. Significant differences in the effect of ATRA were calculated using the Student’s t-test. **** indicates p \u0026lt; 0.0001 with respect to control (-) samples. ° and °°°° indicates p \u0026lt; 0.05 and p \u0026lt; 0.0001 with respect to the corresponding samples in parental MCF-7 cells. Dose-response of RARα transcriptional activity (C) and ERα transcriptional activity (D) in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells stably expressing the RARE-NLuc reporter construct (NLuc) or the ERE-NLuc reporter construct (NLuc), respectively, treated with the indicated doses of ATRA. Experiments were performed three times, and each condition was tested in triplicate.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/4cf4f7efc9a9864b91e9ca58.jpg"},{"id":83295631,"identity":"77b7525d-1a1c-4313-967b-877fb3a1393f","added_by":"auto","created_at":"2025-05-22 13:46:06","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":126453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe senolytic activity of digoxin in parental and CRISPR/CAS9 engineered L370F and Y537S MCF-7 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth curve analyses in parental (A), and in CRISPR/CAS9 engineered Y537S (B) MCF-7 cells plated in growing conditions (i.e., 10% FBS) and treated with the indicated doses of all-trans retinoic acid (ATRA) or digoxin (Digo) for the indicated time. Black arrows indicate the time point in which media was changed and ATRA was re-administered together with the indicated doses of Digo and the proliferation profile was followed up to 15 days. Experiments were performed twice in duplicate using the xCelligence RTCA system. Curves were calculated at the maximum proliferation the untreated sample in each cell line. (A) Time dependent inhibitory concentration 50 (IC\u003csub\u003e50\u003c/sub\u003e) -Log\u003csub\u003e2\u003c/sub\u003e transformed to indicate the sensitivity of parental (A’) and CRISPR/CAS9 engineered Y537S (B’) MCF-7 cells to Digo both in the absence (CTR) and in the presence of the indicated dose of ATRA.\u003c/p\u003e\n\u003cp\u003e(C) Synergy map of 12-day-treated parental MCF-7 cells with different doses of 4OH-Tamoxifen (Tam) and ATRA the MELK inhibitor MELK-8a (MELKin). (C’) Growth curves in parental MCF-7 cells showing the synergistic effect of each combination of compounds with selected doses. Significant differences were calculated using the ANOVA test. **** (p-value \u0026lt; 0.0001) indicates significant differences with respect to the untreated (i.e., -,-) sample. ° (p-value \u0026lt; 0.05) indicates significant differences with respect to Tam treated sample. ^ (p-value \u0026lt; 0.05) indicates significant differences with respect to ATRA treated sample.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/2d0dadce6d30cef1df68cf7d.jpg"},{"id":83297127,"identity":"41345473-aab4-4980-bef8-38496f6b7c6c","added_by":"auto","created_at":"2025-05-22 14:10:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2864456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/cbb6c312-0d45-424e-b396-aebe771d30c5.pdf"},{"id":83294630,"identity":"50f0503f-4937-4009-afc4-ffc50db66686","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3760710,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWB.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/8aff393735b01daf60be478e.pptx"},{"id":83296524,"identity":"beda1cae-1c78-4d4d-a434-e03072c9d875","added_by":"auto","created_at":"2025-05-22 14:02:06","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":385235,"visible":true,"origin":"","legend":"\u003cp\u003eTable1\u003c/p\u003e","description":"","filename":"Table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/f7c785b6957e31934bad53c9.jpg"},{"id":83296237,"identity":"f27bdb98-26fb-4ebc-bcdd-2eb47da14912","added_by":"auto","created_at":"2025-05-22 13:54:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16900,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureCaptions.docx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/e855a84911c2d0f9ed7ac240.docx"},{"id":83294633,"identity":"ac114a57-860b-45b7-9152-c4fae0f233a4","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10813514,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/e5682dfac367ecc10c36bd81.pptx"},{"id":83294626,"identity":"a712a1e1-23bb-46c6-88f9-5ea32721d718","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12215,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/40a1f64c94a6d334abae7d3c.xlsx"},{"id":83294628,"identity":"70c50337-11be-44c6-854d-6168776d5b5c","added_by":"auto","created_at":"2025-05-22 13:38:06","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":588554,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/1725464b13ce9af32ba4b3fe.xlsx"},{"id":83294617,"identity":"e8876eb5-ad7f-40d2-9d15-631aca192cd5","added_by":"auto","created_at":"2025-05-22 13:38:05","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":18355,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables35.docx","url":"https://assets-eu.researchsquare.com/files/rs-6706598/v1/0e18be457391739d9384ecb3.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eBreast cancer (BC) is the deadliest tumor type for women. Approximately 70% of BC cases express the estrogen receptor α (ERα), and patients with this form of the disease are effectively treated with endocrine therapy (ET) drugs (e.g., aromatase inhibitors - AIs, selective ER modulators \u0026ndash; SERMs, and selective ER down-modulators \u0026ndash; SERDs). However, 20\u0026ndash;40% of these patients experience relapse and develop metastatic BC (MBC), which is resistant to the classic ET drugs (e.g., AIs; 4OH-tamoxifen \u0026ndash; Tam) and is largely incurable [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. BC cells can acquire resistance to ET drugs through multiple molecular mechanisms. Interestingly, in approximately 50% of ERα-expressing MBCs, ERα point mutations emerge and lead to a hyperactive ERα (e.g., Y537S), conferring specific proliferative advantages and mediating resistance to ET drugs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe actual clinical strategies for MBC currently involve treating patients either with increasing doses of the same ET drugs (e.g., Tam), with the second-line therapy drug fulvestrant (Ful), a SERD inducing ERα degradation, or with targeted agents (e.g., the CDK4/6 inhibitors palbociclib, ribociclib, or abemaciclib), which can be administered in combination with ET drugs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, novel antiestrogen drugs that bind either non-covalently or covalently to the Y537S ERα mutation, induce its degradation and block its hyperactive phenotype (e.g., the selective ER down-modulators \u0026ndash; SERDs such as AZD9833, camizestrant; RAD-1901, elacestrant; GDC-9545, giredestrant; LY3484356, Imlunestrant; GDC-0927; the selective ERα covalent antagonists \u0026ndash; SERCAs, H3B-5942; proteolysis targeting chimerics \u0026ndash; PROTACs, such as ARV-471; complete estrogen receptor antagonists \u0026ndash; CERANs, such as OP-1250), are currently undergoing clinical trials or have been recently approved (i.e., RAD-1901, elacestrant) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, we have demonstrated that \u0026lsquo;antiestrogen-like\u0026rsquo; activities can be found in Food and Drug Administration (FDA) approved drugs that do not directly bind to ERα but determine its degradation. In particular, we have reported that cardiac glycosides, antivirals, and Chk1 inhibitors induce the degradation of the Y537S ERα variant, block its hyperactive phenotype, and prevent the proliferation of MBC cells expressing it when administered alone or in combination with either ET drugs or with CDK4/6 inhibitors [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, a broad range of drugs could be used for the clinical treatment of MBC expressing the Y537S ERα [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, ~\u0026thinsp;50 ERα point mutations have been identified in MBC patients and annotated in the free on line Cosmic and cBioPortal databases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], all of which reduce their survival rates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The most frequent and best-characterized variants map in one hotspot region within the ligand-binding domain (LBD) (e.g., Y537S) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, many ERα variants fall outside this hotspot region and have been so far neglected. Recently, the initial characterization of some of them (V422del, G442R, F461V, S463P, L469V [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and F404L/I/V [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]) revealed another hotspot within in the LBD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and indicated that distinct mechanisms underlying uncontrolled proliferation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] or resistance to ET drugs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] can be operative in MBC.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the L370F and E471D ERα variants because we observed that, among the receptor point mutations found in MBC located within the LBD and annotated in the COSMIC and cBioPortal databases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the side chains of residues L370, laying on helix 4 (H4) and E471, laying within helix H10 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] are facing each other in the LDB structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, yellow residues). Due to this spatial configuration, we hypothesized that they could represent a novel 3D-hotspot and, in turn, their single mutations in MBC could provide specific selective advantages to tumor cells (the double L370F/E471D mutation has not been identified in MBC patients).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistently, specific ERα cellular assays demonstrated that the L370F ERα variant is resistant to high doses of Ful and leads to a hyperactive response to low doses of 17β-estradiol (E2). Additionally, both the L370F and Y537S ERα variants exhibit high expression levels of retinoic acid receptor α (RARα) and are sensitive to the antiproliferative effects of all-trans retinoic acid (ATRA). Interestingly, ATRA induces cell death in the presence of the L370F ERα mutant and senescence in the presence of the Y537S ERα mutant.\u003c/p\u003e \u003cp\u003eThese data establish that the L370F ERα variants confer distinct properties to MBC cells, highlighting ATRA as a potential ERα-variant-specific therapeutic agent for MBC driven by ERα function.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture and Reagents\u003c/h2\u003e \u003cp\u003eHEK293 and MCF-7 cell lines were obtained from ATCC (USA), and all other cell lines used in this study were derived from these parental lines. Cells were maintained following the manufacturer's recommendations. The following reagents and antibodies were employed: 17β-estradiol (E2), DMEM (with or without phenol red), fetal calf serum, and charcoal-stripped fetal calf serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bradford protein quantification kit and HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) were obtained from Bio-Rad (Hercules, CA, USA). Primary antibodies against ERα (F-10, mouse), RARα (C-1, mouse), ASCL1 (D-7, mouse), CALCR (CT-R, 2F7, mouse), FABP5 (E-FABP, C-20, rabbit), and Caveolin-1 (N-20, rabbit) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Additional antibodies included anti-phospho ERα (Ser118, mouse), anti-phospho H2AX (rabbit), anti-PARP (rabbit), and anti-FASN (rabbit) from Cell Signaling Technology (USA). Anti-vinculin (mouse) and anti-tubulin (mouse) antibodies were obtained from Sigma-Aldrich. Western blot chemiluminescence detection reagents were purchased from Bio-Rad. The following compounds were used in selected experiments: all-trans retinoic acid (ATRA) and digoxin (Digo) from Sigma-Aldrich; Fulvestrant (Ful) and 4-hydroxy-Tamoxifen (Tam) from Selleck Chemicals (USA). The ERα Green Competitor Assay Kit (PolarScreen\u0026trade;, A15882) was obtained from Thermo Scientific. All other reagents were from Sigma-Aldrich and used without further purification, unless otherwise specified. Cell line authentication was confirmed by STR profiling performed by BMR Genomics (Italy).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Manipulation for Western Blot Analyses\u003c/h3\u003e\n\u003cp\u003eCell manipulation, Western blotting Image acquisition and consequent manipulations including band quantitations have been described in detail in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGeneration of CRISPR/CAS9 MCF-7 cells expressing the L370F ERα\u003c/h3\u003e\n\u003cp\u003eThe generation of MCF-7 L370F cells using CRISPR/Cas9 technology was outsourced to Cogentech in 2021. A clone (clone 3\u0026ndash;78) was obtained with two correctly modified alleles (HDR alleles carrying the G/C mutation, which converts the TTG (L) codon into TTC (F), along with three synonymous mutations) and one allele with a single-nucleotide insertion, resulting in a frameshift and a premature stop codon immediately after the insertion. Details of the procedure are available upon request.\u003c/p\u003e\n\u003ch3\u003eSmall Interference RNA\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the small interference RNA (siRNA) experiments, cells were transfected with esiRNA [obtained from Sigma-Aldrich (St. Louis, MO, USA)] targeting the specific proteins of interest. The transfection procedure was conducted using Lipofectamine RNAi Max (Thermo Fisher), following established protocols described in [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell Proliferation Assays\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe xCELLigence DP system (ACEA Biosciences, Inc., San Diego, CA) Multi-E-Plate station was utilized to measure the time-dependent response to the specified drugs by real-time cell analysis (RTCA), following previously reported protocols [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Synergy studies were conducted using Crystal Violet staining, as described in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The synergy was subsequently calculated using Combenefit freeware software [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of the Cellular Area\u003c/h2\u003e \u003cp\u003eFollowing the outlined stimulation, phase-contrast images of MCF-7, L370F, and Y537S cells were randomly acquired using a ZEISS Axio Vert.A1 FL-LED fluorescence microscope (20\u0026times; objective, 1\u0026times; zoom) (Zeiss, Oberkochen, Germany). For cell area analysis, images were loaded into the FIJI distribution of the ImageJ program in their native format. Prior to analysis, brightness and contrast were adjusted using the \u0026ldquo;Brightness/Contrast\u0026rdquo; tool in FIJI to ensure optimal definition of the cell profile. Cellular contours were manually traced using the \u0026ldquo;Freehand Selection\u0026rdquo; tool to define the Region of Interest (ROI). Multiple ROIs were then selected from the ROI Manager, and the \u0026ldquo;Measure\u0026rdquo; tool under the \u0026ldquo;Analyze\u0026rdquo; menu was applied with the measurement parameter set to \u0026ldquo;Area\u0026rdquo;, representing the surface area of the selected ROIs. The obtained values were reported as indicative of cell area in \u0026micro;m\u0026sup2;.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of Stable Cell lines and Measurement of ERα and RARα Transcriptional Activity\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe generation of HEK293 cells stably transfected with a reporter gene containing an estrogen response element (ERE) controlling the expression of nanoluciferase (NLuc)-PEST was done using the selection methods and the reagents described in [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These cell lines were then transfected with the pcDNA His HA wild type (wt), L370F and Y537S ERα and selected using neomycin. To generate the pcDNA His HA wt, and Y537S ERα, the pcDNA-HA-ERα [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and the pcDNA-HA-Y537S ERα [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] were purchased from Addgene (USA) and digested with BamHI and ApaI to excide the receptor ORFs. The fragments were ligated into pcDNA His vector sites. To generate the pcDNA His HA L370F ERα, site-directed mutagenesis was performed on the pcDNA His HA ERα using the following forward 5\u0026rsquo;-gtgccaggctttgtggattttaccctccatgatcaggtccaccttc-3\u0026rsquo; and reverse 5\u0026rsquo;-cacggtccgaaacacctaaaatgggaggtactagtccaggtggaag-3\u0026rsquo; primers and using the QuickChange Lightning kit from Agilent (Santa Clara, CA, USA). All the resulting plasmids were sequence verified.\u003c/p\u003e \u003cp\u003eThe generation of L370F MCF-7 cells stably transfected with a reporter gene containing the ERE sequence controlling the expression of NLuc-PEST was done using the selection methods and the reagents described in [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This cell line together with the corresponding parental and Y537S MCF-7 ERE-NLuc cells were used to measure the ERα transcriptional activity as previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo generate the parental, L370F and Y537S MCF-7 cells stably expressing a reporter gene containing an retinoic acid response element (RARE) controlling the expression of NLuc-PEST, the plasmid pGL2Basic_Neo_RARE-NLuc-PEST was transfected and the cell lines were selected as previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To generate the plasmid pGL2Basic_Neo_RARE-NLuc-PEST, the RARE cassette was KpnI and HindIII excised from the pGL3-RARE-luciferase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] purchased from Addgene (USA) and ligated into the corresponding restriction sites in the pGL2Basic_Neo_NLuc-PEST [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eGene Arrays and RNASeq Analyses\u003c/h3\u003e\n\u003cp\u003eGene arrays analyses were conducted as described in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. RNASeq and the relative initial data analyses were performed by Novogene (Cambridge, UK) as an outsourced service. Briefly, three replicates of sub-confluent growing parental, L370F and Y537S MCF-7 cells were pelleted, and sent in dry ice for analysis to Novogene, which performed RNA extraction, quality control, mRNA library preparation (polyA enrichment) and sequencing using the NovaSeq X Plus Series (PE150) (6G raw data per sample) following by standard analysis (i.e., data quality control and data filtering, mapping to reference genome, gene expression quantitation and correlation analysis, differential expression analysis, enrichment analysis of differential expressed genes, GSEA enrichment analysis of expressed genes, protein-protein interaction analysis of differential expressed genes, oncogene functional annotation of differential expressed genes, alternative splicing quantification and differential analysis, SNP/InDel analysis and fusion gene analysis).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFull-Length ERα Purification from Expi293F cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo generate the plasmid pcDNA 3.1_ ERα_TEV_TWIN STREP TAG, the TEV_TWIN STREP TAG was PCR amplified from the pcDNA3.1 GIL-11 myc TEV TwinStrep [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] purchased from Addgene (USA) using the following forward 5\u0026rsquo;-cggggtaccccgagcgcgtggagccatccgcagt-3\u0026rsquo; and reverse 5\u0026rsquo;-cgcggatccgcgtttttcaaactgcggatggct-3\u0026rsquo; primers. The fragment was EcoRI and XhoI digested and subcloned in pcDNA 3.1 to obtain the pcDNA 3.1_TEV_TWIN STREP TAG. Wild type (wt), L370F and Y537S ERα were PCR amplified using pcDNA His HA wt, L370F and Y537S ERα plasmids as templates and the following forward 5\u0026rsquo;-cgcggatccgcgatgaccatgaccctccacaccaaagcatct-3\u0026rsquo; and reverse 5\u0026rsquo;-ccggaattccgggaccgtggcagggaaaccctc-3\u0026rsquo; primers. The resulting fragments were BamHI and EcoRI digested and subcloned into the corresponding restriction sites in the pcDNA 3.1_TEV_TWIN STREP TAG. The plasmids were then sequence-verified.\u003c/p\u003e \u003cp\u003eTransfection of the pcDNA 3.1_ ERα_TEV_TWIN STREP TAG encoding for the wt, L370 and Y537S receptor fused in frame with the TWIN STREP TAG (Molecular weight\u0026thinsp;=\u0026thinsp;70,7 KDa, corresponding to 66,0 KDa of the ERα and 4,7 KDa of the TWIN STREP TAG) was conducted in Expi293F cells and performed by the National Facilities at the Human Technopole (Milan, Italy) as a service. Access to the Biomass Production Unit service @ the Human Technopole (Milan, Italy) was granted following a competitive selection procedure (Project ID 1771263). Expi293F cells were transfected at a density of 2\u0026ndash;3 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml using a modified PEI (polyethylenimine) protocol, with 1.1 mg of plasmid DNA and 3 mg of PEI per each liter of cell culture. A total of 10 liters of Expi293F cells for each construct was transfected. After transfection cells have been grown in a Multitron shaker (Infors HT, Bottmingen, Switzerland) for additional 72 hours. The cell suspension was divided into aliquots of 500 ml and then centrifuged at 1000 rcf, at 4\u0026deg;C, for 10 minutes. The 500 ml cell pellets were then washed with PBS, the centrifugation was repeated and the supernatant discarded. The resulting pellets were stored at -80\u0026deg;C until protein purification (see below).\u003c/p\u003e \u003cp\u003eA pellet of 500 ml of Expi293F cells was resuspended in 50 ml of the following lysis buffer 100 mM TrisHCl pH 8, 150 mM NaCl, 2% Triton X100, 10% Glycerol, 1mM EDTA, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM ATP, 2 mM DTT, 10 \u0026micro;M Mg-132, 1mM PMSF and protease inhibitor cocktail and left on ice for 10 minutes. The volume was then centrifuged in 2 ml eppendorf tubes for 5 minutes at 4\u0026deg;C at 10,000 rpm. The supernatant was loaded three times on a 3 ml gravity column Strep-Tactin\u0026reg;XT 4Flow\u0026reg; 50% suspension [IBA-Lifescience (Gottingen, Germany)] packed o.n. at 4\u0026deg;C. After washing the column with \u0026gt;\u0026thinsp;50 ml of wash buffer (100 mM TrisHCl pH 8, 150 mM NaCl, 1 mM EDTA) 14 fractions of 1 ml were eluted with 100 mM TrisHCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 mM biotin. The eluate was then concentrated in 50 KDa cut-off Amicon\u0026reg; Ultra [obtained from Sigma-Aldrich (St. Louis, MO, USA)] up to 500\u0026ndash;800 \u0026micro;l. The purity of the purified ERα was evaluated by staining an SDS-PAGE gel with InstaBlue Protein Stain Solution (CliniScience, Italy). Receptor amount and purity was calculated in reference to a standard curve of bovine serum albumin (Supplementary Fig.\u0026nbsp;1A). The final yield was in the \u0026micro;M range per each receptor.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn Vitro ERα Binding Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe in vitro ERα binding assay employed a fluorescence polarization (FP) method to assess the binding affinity of 17β-estradiol (E2), and fulvestrant (Ful) with commercially available recombinant ERα and the purified full length wild type (wt), L370F and Y537S ERα-TWIN STREP TAG purified from Expi293F cells as described above. The FP assay was done as reported in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, measurement has been performed using different doses of the test compounds in a final assay reaction that contained the above described ERα (50 nM - Supplementary Fig.\u0026nbsp;1B) and fluomone ES2 (4.5 nM) in ERα binding buffer, Thermo Scientific). Each sample was measured in quadruplicate in black 384 multiwell plates and the experiment was repeated twice. The assay was incubated for two hours in the dark at room temperature before reading on a Tecan Spark Elisa reader. The calculation of the K\u003csub\u003ei\u003c/sub\u003e was obtained from the apparent IC\u003csub\u003e50\u003c/sub\u003e of the compound toward each receptor by performing the calculation as described in [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, we applied a modified Cheng-Prusoff equation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] [Ki = (apparent IC\u003csub\u003e50\u003c/sub\u003e * K\u003csub\u003edFluomoneES2\u003c/sub\u003e )/[Fluomone ES2] * ([Fluomone ES2] * K\u003csub\u003edFluomoneES2\u003c/sub\u003e)] to take into consideration the K\u003csub\u003ed\u003c/sub\u003e of the fluomone ES2 toward the receptors (i.e., 18 nM) and the concentration of the flouomone ES2 used in the assay.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eClassical Molecular Dynamics Simulations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThree different ERα systems were prepared, namely the wild type (wt), L370F, and E471D ERα with fulvestrant inside the binding cavity. The L370F and E471D model systems were prepared by mutating corresponding residues in the wt variant. ERα structures were prepared based on the wt ERα-fulvestrant complex from the study by Pavlin et al. 2018 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e500 ns-long simulations were performed for each system using Amber20 code [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. FF19SB force field was used for the description of protein [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which was placed in the cubic simulation box solvated with up to ~\u0026thinsp;37,000 TIP3P water molecules, making sure that the distance between solute and edge of the box was at least 12 \u0026Aring;. For the description of fulvestrant same parameters as in ref. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were used. Each system was energy minimized by 20,000 steps using the steepest descent algorithm, followed by 30,000 steps of conjugate gradient. This was followed by the canonical NVT equilibration performed in 4 runs of 10,000 steps, with the constraints on the solute gradually releasing (from 100 kcal mol\u003csup\u003e-1\u003c/sup\u003e \u0026Aring;\u003csup\u003e-2\u003c/sup\u003e in first run to 60 kcal mol\u003csup\u003e-1\u003c/sup\u003e \u0026Aring;\u003csup\u003e-2\u003c/sup\u003e and 30 kcal mol\u003csup\u003e-1\u003c/sup\u003e \u0026Aring;\u003csup\u003e-2\u003c/sup\u003e in the second and third run, respectively, while the fourth run was performed without constraints) and the system was gradually heated to 293 K. Subsequently, NPT equilibration at 1 bar was carried out in two successive runs of 100,000 steps each. During the first run, the solute was restrained with a force constant of 20 kcal mol\u003csup\u003e-1\u003c/sup\u003e \u0026Aring;\u003csup\u003e-2\u003c/sup\u003e and in the second run the constraint was released. Production runs were performed with periodic boundary conditions and electrostatic interaction were considered using Particle Mesh Ewald method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] at 293 K and 1 bar by coupling to the Langevin thermostat [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and Berendsen barostat [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In all simulations all bond lengths involving hydrogen atoms were constrained using SHAKE algorithm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] to achieve a time step of 2 fs.\u003c/p\u003e \u003cp\u003eAnalyses (namely RMSD, hydrogen bond (H-bond) analysis, clustering and cross correlation anaylsis) on the equilibrated part of trajectories, from 200 to 500 ns, were performed using AmberTools22 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eβ-Galactosidase activity\u003c/h2\u003e \u003cp\u003eβ-Galactosidase activity was assessed using the Beta-Glo\u0026reg; assay system (Promega, Madison, MA, USA) following the manufacturer's guidelines. In brief, 2000 cells were seeded in 96-well plates and treated at the designated time point as specified. Luminescence was then read at Tecan Spark Elisa reader. Each condition was tested in triplicate. Replica plates were also used to normalize the experiment for cell number by using Crystal Violet staining, as described above. Detection of activity at pH 6, a known characteristic of senescent cells was performed the Senescence β-galactosidase staining kit (Cell Signaling, Technology Danvers, MA, USA) according to manufacturer\u0026rsquo;s instructions. The experiment was performed twice triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted as detailed in been described in detail in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEvaluation of the sensitivity to Ful and E2 of HEK293 cells expressing L370F and E471D ERα variants.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince the ERα variants found in MBC are selected following administration of first-line ET drugs such as AI and/or Tam, MBC patients are typically treated with the second-line ET drug Ful [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo investigate whether the L370F and E471D ERα variants are resistant to this antiestrogen, we initially generated isogenic pooled stable HEK293 cell lines (ERα-negative cells) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] co-expressing a reporter construct containing the estrogen response element (ERE) regulating a nanoluciferase gene (ERE-NLuc). This system allows evaluation of ERα transcriptional activity in living cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, we introduced expression vectors encoding the L370F and E471D ERα variants fused with an \u003cem\u003eN\u003c/em\u003e-terminal double tag (HA and His tag). As controls, we generated isogenic pooled stable HEK293 cell lines expressing wild type (wt) ERα and the hyperactive Y537S ERα mutant [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDose-response analyses were conducted to evaluate the effectiveness of Ful in decreasing basal ERα transcriptional activity. Cells were treated with varying doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) for 24 hours, and the relative inhibitory concentration 50 (IC\u003csub\u003e50\u003c/sub\u003e) was calculated. To evaluate the sensitivity of each cell line (wt, L370F, E471D, and Y537S ERα-expressing HEK293 cells) to Ful, the IC\u003csub\u003e50\u003c/sub\u003e values were mathematically transformed to -Log\u003csub\u003e2\u003c/sub\u003e and plotted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, HEK293 cells expressing the Y537S ERα mutant exhibited significantly reduced sensitivity to Ful, as expected [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, while HEK293 cells expressing the E471D ERα mutant showed similar sensitivity to Ful as the wt ERα-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), the L370F ERα mutant cells displayed reduced sensitivity to Ful\u0026rsquo;s inhibitory effect on receptor transcriptional activity compared to wt ERα cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These data suggest that the L370F, but not the E471D ERα mutation, impairs Ful\u0026rsquo;s ability to inhibit receptor transcriptional activity. Therefore, the E471D ERα mutant was excluded from further analysis.\u003c/p\u003e \u003cp\u003eNext, we evaluated Ful\u0026rsquo;s capacity to induce ERα degradation in HEK293 cells expressing wt, L370F, and Y537S ERα mutants. Western blot (WB) analyses were conducted after 24-hour treatments with varying doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) and Ful induced receptor degradation in all cell lines tested. As previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the Y537S mutation reduced Ful-induced receptor degradation compared to wt ERα cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026rsquo;). Interestingly, in HEK293 cells expressing the L370F mutant, higher doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) were less effective in inducing receptor degradation as compared to wt ERα containing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026rsquo;\u0026rsquo;). These results indicate that the L370F ERα mutation hinders Ful's ability to induce receptor degradation.\u003c/p\u003e \u003cp\u003eSimilarly to the Y537S ERα mutant, many other point mutations located within the LBD lead to constitutively hyperactive ERα by causing structural changes that mimic the E2-activated conformation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We then investigated the effect of E2 on the transcriptional activity of both wild-type and L370F ERα. HEK293 cells were treated with varying doses of E2 (10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM) for 24 hours, and transcriptional activity was measured. No significant differences in basal transcriptional activity were detected between wt and L370F receptors, and E2 induced a dose-dependent increase in transcriptional activity in both cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Nevertheless, at low doses of E2 (from 10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003eM), the L370F ERα exhibited enhanced transcriptional activity as compared to its wt counterpart (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings suggest that the L370F ERα mutation enhances the responsiveness of ERα to low doses of E2, increasing receptor transcriptional activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of Ful sensitivity in parental, L370F and Y573S CRISPR/CAS9-engineered MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious results suggest that the L370F variant found in MBC may confer resistance to Ful treatment and promote hyperactivity in response to E2 in cells expressing this mutation. Consequently, cells expressing this receptor point mutation may evade the effects of ET drugs while continuing to proliferate under the influence of E2.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we generated a CRISPR/CAS9 knock-in L370F-mutated MCF-7 cell line that exclusively expresses the mutated receptor (please see Material and Method section) and studied them together with parental MCF-7 and Y537S MCF-7 cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInitial experiments were conducted to validate the findings from stable HEK293 cells. The ability of Ful to induce ERα degradation was assessed through WB analysis in parental, L370F, and Y537S MCF-7 cells treated for 24 hours with varying doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026rsquo;, Ful induced dose-dependent ERα degradation in all cell lines tested. As expected [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the ability of Ful to degrade ERα was reduced in Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026rsquo;). Notably, in L370F MCF-7 cells, high doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM) were less effective at inducing receptor degradation as compared to parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026rsquo;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we stably transfected the nanoluciferase reporter construct (ERE-NLuc) into parental, L370F, and Y537S MCF-7 cells to create cell lines for studying wt and ERα variant transcriptional activity (i.e., MCF-7 NLuc cells), as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In these cell lines, dose-response analyses of Ful\u0026rsquo;s ability to reduce basal ERα transcriptional activity were performed by administering varying doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) for 24 hours. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the inhibitory effect of Ful on receptor transcriptional activity was significantly reduced in L370F and Y537S MCF-7 NLuc cells compared to parental MCF-7 NLuc cells.\u003c/p\u003e \u003cp\u003eGrowth curve analyses were performed in parental, L370F, and Y537S MCF-7 cells to evaluate the antiproliferative effects of Ful. Each cell line was treated with different doses of Ful (10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM), and the resulting dose-dependent effect was measured at the time point when the control for each cell line reached maximum growth. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC shows that the antiproliferative effect induced by Ful in parental MCF-7 cells was significantly diminished in L370F MCF-7 cells, particularly at high Ful doses, and was almost entirely abolished in Y537S MCF-7 cells, as previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, these data corroborate the results obtained from stable HEK293 cells and demonstrate that the L370F mutation in ERα confers resistance to Ful-induced ERα degradation and reduces the inhibitory effect of this ET drug on receptor transcriptional activity and cell proliferation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the proliferation rate of parental, L370F and Y573S MCF-7 cells in the presence and absence of E2.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we studied the proliferation rate of parental, L370F, and Y537S MCF-7 cells under normal growth conditions (10% FBS) and in the presence of E2-deprived serum (10% CS-FBS) using the xCELLigence apparatus.\u003c/p\u003e \u003cp\u003eUnder normal growth conditions, parental MCF-7 cells reached maximal growth later than both the L370F and Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with Y537S MCF-7 cells exhibiting the fastest growth rate, as previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Accordingly, L370F and Y537S MCF-7 cells displayed a significantly shorter doubling time compared to parental MCF-7 cells, with Y537S MCF-7 cells having the shortest doubling time among the tested lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When growth curve analyses were performed in the presence of E2-deprived serum (10% CS-FBS), we confirmed that the growth of Y537S MCF-7 cells was E2-independent [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, in contrast to parental MCF-7 cells, L370F MCF-7 cells did not exhibit reduced growth during prolonged E2 deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we evaluated the proliferative effect of E2 in parental, L370F, and Y537S MCF-7 cells. Growth curve analyses revealed that administration of various doses of E2 (10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003eM) induced a dose-dependent increase in the proliferation of parental MCF-7 cells, both under normal growth conditions (10% FBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and in the presence of E2-deprived serum (10% CS-FBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). As expected, the dose-dependent proliferative effect of E2 was more pronounced in the presence of E2-deprived serum (10% CS-FBS), reaching a maximum at 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003eM E2 in both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Consistent with the E2-independent growth of Y537S MCF-7 cells, the effect of E2 at different doses was negligible in this cell line under both normal growth conditions (10% FBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) and in E2-deprived serum (10% CS-FBS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eInterestingly, administration of increasing doses of E2 to L370F MCF-7 cells under normal growth conditions (10% FBS) induced a modest, yet significant increase in cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Dose-response analysis showed that the E2-induced increase in proliferation was maximal at low hormone concentrations (10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003eM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Conversely, in the presence of E2-deprived serum (10% CS-FBS), E2 induced a dose-dependent increase in L370F MCF-7 cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). The E2-dependent effect was greater in E2-deprived conditions than in normal growth conditions, peaking at 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003eM E2 and becoming significant even at lower doses (10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003eM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eThese data indicate that the proliferation of MCF-7 cells expressing the L370F ERα variant is partially E2-independent and can be stimulated by low E2 doses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of E2-dependent regulation of ERα transcriptional functions in parental, and L370F MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe parental and L370F MCF-7 NLuc cells were next used to evaluate the ability of E2 to modulate ERα transcriptional activity. A 24-hour administration of different doses of E2 (10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM) revealed that the hormone induces a dose-dependent increase in ERα transcriptional activity in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, low-dose hormone treatment (10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003eM, 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003eM) triggered a greater E2-dependent increase in receptor transcriptional activity in L370F MCF-7 NLuc cells compared to the parental MCF-7 NLuc line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhosphorylation of ERα at serine 118 (S118) is essential for its transcriptional activation in response to E2 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. E2-triggered ERα S118 phosphorylation occurs rapidly, reaching a maximum after 30 min of hormone administration in MCF-7 cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. ERα S118 phosphorylation was evaluated in parental and L370F MCF-7 cells treated with different doses of E2 (10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM) for 30 minutes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026rsquo;, E2 dose-dependently increased the ERα S118 phosphorylation in both cell lines. However, the E2-induced receptor S118 phosphorylation was increased at low doses of the hormone (10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003eM) in L370F MCF-7 cells with respect to the parental MCF-7 cells.\u003c/p\u003e \u003cp\u003eTo confirm that the inhibitory effect of Ful on ERα transcriptional activity is reduced, and that E2-induced receptor transcriptional activity is increased at low hormone doses in L370F MCF-7 cells, we co-administered different doses of E2 and Ful for 24 hours in both parental and L370F MCF-7 NLuc cells. The data revealed that antagonism between E2 and Ful was observed in both cell lines. However, the antagonistic interaction between E2 and Ful in L370F MCF-7 NLuc cells was significantly weaker as compared to parental MCF-7 NLuc cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This indicates that Ful exhibits reduced antagonistic activity against E2 in regulating ERα transcriptional activity in L370F MCF-7 cells as compared to parental cells.\u003c/p\u003e \u003cp\u003eSince ERα regulates the expression of various genes, with or without the presence of ERE sequences in their promoter regions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], we next assessed the E2 ability to modulate gene expression in parental and L370F MCF-7 cells. Based on previous data suggesting that the effect of E2 is enhanced in L370F MCF-7 cells treated with low E2 doses as compared to parental cells, we used an RT-qPCR-based array targeting 89 E2-sensitive genes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We hybridized cDNA samples generated from total RNA extracted from parental MCF-7 cells treated with E2 at 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003eM and L370F MCF-7 cells treated with E2 at 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003eM. Interestingly, despite the difference in concentration, E2 modulated most of the genes included in the array similarly in both parental and L370F MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and Supplementary Table\u0026nbsp;1), with a large overlap of genes regulated by E2 in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary Table\u0026nbsp;1). Notably, for most of the genes commonly regulated by E2 at different doses, the E2 effect was larger in L370F MCF-7 cells than in parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eOverall, these data demonstrate that the L370F mutation renders ERα hypersensitive to low doses of E2, enhancing ERα transcriptional activity and E2-modulated gene expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlobal gene expression profiling of ERα mutants reveals an increase in late E2 response gene in L370F MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe data reported previously demonstrate that culturing the cells in E2-depleted medium allows the continuous growth of Y537S MCF-7 cells and causes a growth arrest in the L370F MCF-7 cells that is not followed by subsequent cell death, as it occurs in parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Thus, to further evaluate the impact of the L370F ERα mutation in the regulation of E2 gene expression we performed RNA-seq analysis in parental, L370F and Y537S MCF-7 cells that had been maintained in E2-containing medium (i.e., normal growing condition \u0026ndash; 10%-FBS) to identify changes in basal gene expression independent of the potential interferences caused by cell cycle-dependent regulation of gene expression.\u003c/p\u003e \u003cp\u003eWe conducted RNA-seq analysis using three biological replicates for each cell line to estimate gene expression levels, measured as FPKM (Fragments Per Kilobase of transcript sequence per Million mapped reads) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Principal component analysis (PCA) of gene expression levels (FPKM) revealed strong clustering of biological replicates within each cell line, while both mutant lines (L370F and Y537S) showed clear separation from the parental MCF-7 cells (Supplementary Fig.\u0026nbsp;2A). Furthermore, we calculated the correlation coefficients between samples across groups. A heatmap of these coefficients revealed high correlation within the biological replicates of each cell line (i.e., parental, L370F, and Y537S MCF-7 cells), indicating robust experimental consistency. Notably, parental and Y537S MCF-7 cells exhibited the lowest intergroup correlation coefficients, while the L370F MCF-7 cells showed an intermediate correlation, bridging both parental and Y537S profiles (Supplementary Fig.\u0026nbsp;2B).\u003c/p\u003e \u003cp\u003eThe DESeq2 analysis (|log2(FoldChange)| \u0026gt;= 1 and padj\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05) was performed to evaluate differential gene expression between L370F and parental MCF-7 cells, as well as Y537S and parental MCF-7 cells. This was followed by gene enrichment analysis using GSEA, which generated a ranked gene list in L370F (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and Y537S (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) MCF-7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInitially, we analyzed the gene lists and observed that a substantial number of modulated genes were shared between L370F and Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Table\u0026nbsp;2). Subsequently, we assessed whether the genes modulated in L370F and Y537S MCF-7 cells (relative to parental MCF-7 cells) with a signal-to-noise ratio\u0026thinsp;\u0026gt;\u0026thinsp;0.5 or \u0026lt;\u0026thinsp;0.5 included both early and late E2-regulated genes. Identification of these genes was guided by the corresponding gene sets applied in the GSEA (i.e., hallmark estrogen response early and hallmark estrogen response late) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, and Supplementary Table\u0026nbsp;2). Notably, the majority of early and late E2-regulated genes (referred to as E2 Responsive Genes in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) were also modulated in L370F and Y537S MCF-7 cells. Specifically, 213 out of 299 E2-regulated genes (71.2%) were modulated in L370F MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Table\u0026nbsp;2), while 248 out of 299 genes (82.9%) were modulated in Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Supplementary Table\u0026nbsp;2). Interestingly, the proportion of upregulated and downregulated E2-responsive genes was comparable between L370F (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, smaller pie chart and Supplementary Table\u0026nbsp;2) and Y537S (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, smaller pie chart and Supplementary Table\u0026nbsp;2) MCF-7 cells.\u003c/p\u003e \u003cp\u003eAltogether, these data indicate that the basal regulation of E2-responsive genes in L370F MCF-7 cells closely resembles that observed in Y537S MCF-7 cells. This suggests that the introduction of the L370F mutation in ERα may enhance the activity of the mutant receptor compared to the wt ERα, particularly with respect to E2-sensitive genes.\u003c/p\u003e \u003cp\u003eTo further investigate this hypothesis, we analyzed the lists of E2 upregulated genes in L370F and Y537S MCF-7 cells (as shown in the smaller pie charts in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Supplementary Table\u0026nbsp;2). The Venn diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and the Supplementary Table\u0026nbsp;2 highlights 65 genes that were commonly upregulated in both L370F and Y537S MCF-7 cells, while 46 genes were specifically upregulated in L370F cells and 50 genes in Y537S cells. Consistent with this, basal levels of RARα, and FASN were upregulated in both L370F and Y537S MCF-7 cells. Conversely, expression levels of Cav-1 and FABP5 were specifically upregulated in Y537S cells, while ASCL1 and CALCR were specifically upregulated in L370F cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026rsquo; and right panels).\u003c/p\u003e \u003cp\u003eFinally, we assessed the proportion of genes upregulated by E2 that were classified as belonging to early, late, or both early and late E2 responses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, the 65 genes commonly upregulated in both L370F and Y537S cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and Supplementary Table\u0026nbsp;2) were distributed among the three categories with a similar pattern. Notably, the 50 genes uniquely upregulated in Y537S MCF-7 cells were uniformly distributed among the three categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, right pie chart and Supplementary Table\u0026nbsp;2). In contrast, the 46 genes specifically upregulated in L370F MCF-7 cells predominantly belonged to the late E2-responsive gene category (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, left pie chart and Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that while the Y537S receptor mutation upregulates both early and late E2-responsive genes equally, the L370F ERα variant preferentially enhances the expression of late E2-responsive genes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe impact of the L370F mutation on ERα ligand binding and on receptor structure.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe biological effects of the L370F ERα mutation may be due to a reduced or impaired binding of agonist (E2) or antagonist (Ful) to ERα. To assess the binding affinities of E2 and Ful to the L370F ERα, we transiently expressed full-length wild-type (wt), L370F, and Y537S ERα with a twin-strep tag in Epi293F cells and purified them using affinity chromatography. The resulting recombinant full-length wt, L370F, and Y537S ERα were then included in fluorescence polarization competitive binding assays, as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As additional control, we incorporated a commercially available wt ERα (Thermo Scientific), purified from insect cells, into our experimental plan.\u003c/p\u003e \u003cp\u003eBoth E2 and Ful were able to displace the fluorescent E2 tracer across all tested receptors (Table\u0026nbsp;1). Notably, no significant differences were observed in the K\u003csub\u003ei\u003c/sub\u003e values for E2 and Ful between the commercially available wt ERα and the recombinant ERα purified from Epi293F cells (relative binding affinity for E2 (RBA\u003csub\u003eE2\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42; RBA\u003csub\u003eFul\u003c/sub\u003e = 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45). However, as expected [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the K\u003csub\u003ei\u003c/sub\u003e of the Y537S mutant ERα for both E2 and Ful was significantly higher than that of the wt ERα (Table\u0026nbsp;1) (RBA\u003csub\u003eE2\u003c/sub\u003e = 0.072\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036; RBA\u003csub\u003eFul\u003c/sub\u003e = 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09).\u003c/p\u003e \u003cp\u003eInterestingly, while the K\u003csub\u003ei\u003c/sub\u003e of the L370F mutant ERα for E2 slightly increased with respect to that of the wt ERα, its absolute value remained within the same low nanomolar range (Table\u0026nbsp;1) (RBA\u003csub\u003eE2\u003c/sub\u003e = 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30). In contrast, the K\u003csub\u003ei\u003c/sub\u003e of the L370F mutant ERα for Ful was significantly higher than that of the wt ERα (Table\u0026nbsp;1) (RBA\u003csub\u003eFul\u003c/sub\u003e = 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14), and no significant differences were detected between the K\u003csub\u003ei\u003c/sub\u003e values of the L370F and Y537S mutants for Ful (Table\u0026nbsp;1) (RBA\u003csub\u003eFul\u003c/sub\u003e = 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30).\u003c/p\u003e \u003cp\u003eThese findings confirm that the Y537S mutation reduces the receptor's affinity for both E2 and Ful [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably they also reveal that while L370F mutation has only a minor impact on E2 binding to ERα, it instead significantly reduces ERα\u0026rsquo;s affinity for Ful.\u003c/p\u003e \u003cp\u003eBecause we observed a reduction in Ful binding affinity towards the L370F receptor, we next performed classical molecular dynamics simulations to understand if the introduction of this mutation could alter the receptor structure, as observed for the Y537S ERα mutant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, the L370F and to lower extend also E471D mutations result in a remodeling of helix H3 as compared to the wt ERα and to an ERα crystal structure in the antagonist conformation (pdb id:3ert [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]). In addition, the loop connecting H11 and H12 undergoes remodeling and moves closer to H3, leading to a partial disruption of H11\u0026rsquo;s secondary structure. Consistently, with the above findings this behavior is more pronounced in the L370F mutant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAiming to inspect how the mutation affect the internal dynamical coupling of the receptor thus making it less sensitive to Ful, we then calculated the cross correlation coupling among the different ERα structural element, as done in previous studies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;3). Remarkably, the sum of correlation coefficients of H12 with H5 is higher than 4 only in the L370F mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This was previously considered as the threshold for the ERα activation in the previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While the hydrogen bond network does not change significantly in any of the simulations, Ful shows less persistent hydrogen bonds with ERα in the mutant receptors than in the wt (Supplementary Tables\u0026nbsp;3\u0026ndash;5).\u003c/p\u003e \u003cp\u003eAltogether these data indicate that the L370F mutation remodels ERα, reducing its affinity to Ful.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the ATRA antiproliferative effects in parental, L370F and Y537S MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious data showed that L370F and Y537S MCF-7 cells express higher levels of RARα compared to parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026rsquo;). Given that ATRA is a known antiproliferative agent for BC cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and no information is available regarding its effects on cell lines expressing ERα point mutations found in MBC, we investigated the impact of this FDA-approved drug on these cell lines.\u003c/p\u003e \u003cp\u003eGrowth curve analyses were performed on parental, L370F, and Y537S MCF-7 cells treated with varying doses of ATRA (10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003eM). As expected [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], a 7-day ATRA treatment reduced the proliferation rate in all tested cell lines in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, during the first 3\u0026ndash;4 days of treatment, the normalized cell index (CI) measured using the xCELLigence system showed a transient increase in all cell lines, which was most pronounced in L370F MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). This increase was not due to enhanced proliferation but was instead attributable to ATRA-induced enlargement of the cellular surface, a feature accurately detected by the xCELLigence apparatus (Supplementary Fig.\u0026nbsp;4A and 4A\u0026rsquo;).\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e calculations revealed notable differences among the cell lines: L370F MCF-7 cells displayed the lowest IC\u003csub\u003e50\u003c/sub\u003e value (15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 nM), parental MCF-7 cells showed an intermediate value (9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 nM), and Y537S MCF-7 cells exhibited the highest IC\u003csub\u003e50\u003c/sub\u003e (5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, the antiproliferative effect of ATRA was significantly greater in L370F MCF-7 cells compared to both parental and Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eDetailed analysis of growth curve profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C) showed that ATRA rapidly reduced the proliferation rate in L370F MCF-7 cells within 4\u0026ndash;6 days, whereas the reduction in parental and Y537S MCF-7 cells over the same timeframe was slower. These findings suggest that ATRA exerts distinct antiproliferative effects across the different cell lines.\u003c/p\u003e \u003cp\u003eTo explore this hypothesis, we extended the growth curve analyses to 12 days, monitoring proliferation in each cell line treated with the indicated doses of ATRA. Consistent with previous results, ATRA exhibited the strongest antiproliferative effect in L370F MCF-7 cells, the weakest in Y537S MCF-7 cells, and an intermediate effect in parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). Interestingly, L370F MCF-7 cells failed to proliferate entirely in the presence of ATRA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). In contrast, parental and Y537S MCF-7 cells maintained their control-level proliferation rates for the first 2\u0026ndash;3 days of ATRA treatment but then plateaued, sustaining an almost constant cell number over the remaining 9\u0026ndash;10 days of the assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, G).\u003c/p\u003e \u003cp\u003eThese findings confirm the antiproliferative effect of ATRA on BC cells and demonstrate that its magnitude varies depending on the ERα variant expressed. Moreover, the results suggest that ATRA induce distinct cellular responses: cell death in L370F MCF-7 cells and a senescent-like phenotype in parental and Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003eTo assess cell death, we next examined the ability of ATRA to induce PARP cleavage. Parental, L370F, and Y537S MCF-7 cells were treated with different doses of ATRA (10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) for 3 days. Western blotting analysis revealed that ATRA induced a significant and dose-dependent increase in PARP cleavage in L370F MCF-7 cells, whereas the effect was only marginal in parental and Y537S MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, upper panels and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH\u0026rsquo;). Since it has been reported that ATRA could induce DNA damage in BC [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which is closely linked to the initiation of cell death, we further evaluated its effect on the phosphorylation of histone H2AX (γH2AX), a well-established marker of DNA double-strand breaks [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH\u0026rsquo;\u0026rsquo;, ATRA administration for 3 days caused a robust and significant increase in γH2AX levels in L370F MCF-7 cells, while only a minor and not significant effect was observed in parental and Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003eTo investigate the senescence-inducing potential of ATRA [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], parental and Y537S MCF-7 cells were treated with the indicated doses of ATRA for 12 days, after which the activation of the senescence associated β-galactosidase (SA-βGal) activity, a classical marker of senescence [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], was evaluated. As a positive control, etoposide was used to induce senescence in parental MCF-7 cells [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ demonstrate that ATRA induced SA-βGal enzymatic activity in a dose-dependent manner in both cell lines, further confirming the onset of a senescent phenotype.\u003c/p\u003e \u003cp\u003eThese findings collectively demonstrate that ATRA exerts differential effects on MCF-7 cell lines depending on the ERα variant expressed. Specifically, ATRA triggers cell death in L370F MCF-7 cells, while it induces senescence in both parental and Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the RARα and ERα crosstalk in parental, L370F and Y537S MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince previous studies have demonstrated that ATRA and E2 signaling transcriptionally antagonize each other in BC cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], we hypothesized that the effects of ATRA in promoting cell death in L370F MCF-7 cells may be attributed to differences in the reciprocal regulation of RARα and ERα expression across parental, L370F, and Y537S MCF-7 cell lines.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we assessed whether siRNA-induced depletion of either RARα or ERα differentially influenced the expression of ERα and RARα, respectively. Seventy-two hours after transfecting cells with siRNAs targeting RARα or ERα, the expression levels of both receptors were measured in parental, L370F, and Y537S MCF-7 cells. As expected, RARα-targeting siRNA reduced RARα expression, and ERα-targeting siRNA reduced ERα expression across all cell lines (Supplementary Fig.\u0026nbsp;5A, 5A\u0026rsquo; and 5A\u0026rsquo;\u0026rsquo;). Notably, RARα depletion similarly affected ERα expression in all cell lines, and ERα depletion reciprocally reduced RARα expression to a comparable extent in parental, L370F, and Y537S MCF-7 cells (Supplementary Fig.\u0026nbsp;5A, 5A\u0026rsquo; and 5A\u0026rsquo;\u0026rsquo;). These findings confirm a regulatory crosstalk between RARα and ERα in BC cells and demonstrate that the introduction of the L370F and Y537S mutations in the ERα does not alter the mutual influence these receptors exert on each other's expression.\u003c/p\u003e \u003cp\u003eNext, we investigated the effects of ATRA on the expression levels of both ERα and RARα in parental, L370F, and Y537S MCF-7 cells. Cells were treated with increasing concentrations of ATRA (10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) for 72 hours, and receptor levels were assessed via WB analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026rsquo;, ATRA induced a dose-dependent reduction in ERα expression in all three cell lines, with the effect being most pronounced in Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, ATRA induced a dose-dependent decrease in RARα expression in Y537S MCF-7 cells, showing the greatest reduction among the three cell lines. However, only the highest dose (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM) resulted in a significant decrease in RARα levels in parental and L370F MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026rsquo;). Interestingly, baseline RARα expression levels were elevated in both L370F and Y537S MCF-7 cells compared to parental cells (control samples). However, ATRA treatment reduced RARα levels in Y537S MCF-7 cells below those of parental cells, while RARα levels in L370F MCF-7 cells remained higher than in parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026rsquo;\u0026rsquo;).\u003c/p\u003e \u003cp\u003ePrompted by these observations, we hypothesized that ATRA-induced RARα activity might differ among parental, L370F, and Y537S MCF-7 cells. To test this, we generated stable cell lines expressing a reporter construct containing the retinoic acid receptor response element (RARE) fused to the nanoluciferase gene (RARE-NLuc). These cells were treated with ATRA at varying concentrations (10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM) for 24 hours to evaluate RARα transcriptional activity. ATRA dose-dependently increased RARα transcriptional activity in all cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). However, the response was highest in L370F MCF-7 RARE-NLuc cells and lowest in Y537S MCF-7 RARE-NLuc cells compared to parental cells.\u003c/p\u003e \u003cp\u003eBecause ATRA reduced ERα expression in these cell lines and ERα degradation is intrinsically linked to the activation of ERα transcriptional activity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], we further assessed ATRA's impact on ERα transcriptional activity using parental, L370F, and Y537S MCF-7 ERE-NLuc cells. ATRA administration for 24 hours caused a dose-dependent reduction in ERα transcriptional activity in all three cell lines, with the strongest effect observed in Y537S MCF-7 ERE-NLuc cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese findings indicate that ATRA-induced RARα transcriptional activity differs significantly in L370F and Y537S MCF-7 cells compared to parental cells. Notably, the ATRA-induced effects on RARα transcriptional activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) closely parallel the drug\u0026rsquo;s antiproliferative effects in these cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), suggesting that the enhanced antiproliferative effect of ATRA in L370F MCF-7 cells may result from hyperactivation of RARα transcriptional activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the effect of digoxin on ATRA-induced senescence in parental, and Y537S MCF-7 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe induction of a senescent phenotype in cancer cells by therapeutic agents provides an opportunity to explore novel anticancer strategies, such as using compounds that selectively eliminate senescent cells (i.e., senolytics) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Therefore, we investigated the potential of digoxin (Digo), an FDA-approved cardiac glycoside with known senolytic activity [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], to target ATRA-induced senescent parental and Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003eTo this end, we performed growth curve analyses on parental and Y537S MCF-7 cells treated with various concentrations of Digo (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM) both in the absence and presence of ATRA (10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM). As expected, Digo induced a dose-dependent reduction in the proliferation rate of both parental and Y537S MCF-7 cells. Similarly, ATRA (10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM) consistently reduced the proliferation of these cell lines over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing 10 days of continuous ATRA administration, when senescence was previously confirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), we introduced different doses of Digo (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003eM to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003eM) to the parental and Y537S MCF-7 cells and monitored their proliferation rates over an additional 5-day period. Under these conditions, Digo again caused a dose-dependent decrease in the proliferation rate of both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo quantitatively assess the effect of Digo, we calculated its IC\u003csub\u003e50\u003c/sub\u003e values at different time points after administration, both in the absence and presence of ATRA. The IC\u003csub\u003e50\u003c/sub\u003e values were mathematically transformed (i.e., -Log\u003csub\u003e2\u003c/sub\u003e) to evaluate the sensitivity of both cell lines to Digo. The overall sensitivity to Digo was similar in both cell lines both in the absence and presence of ATRA (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026rsquo; and 9B\u0026rsquo;). However, while the parental MCF-7 cells exhibited no significant differences in Digo sensitivity over time, regardless of ATRA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026rsquo;), Y537S MCF-7 cells showed enhanced sensitivity to Digo in the presence of ATRA compared to its absence (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB\u0026rsquo;).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that the antiproliferative effect of Digo during prolonged ATRA treatment manifests more rapidly in Y537S MCF-7 cells compared to parental MCF-7 cells. This observation strongly suggests that Digo could function as a senolytic agent specifically in ATRA-induced senescent Y537S MCF-7 cells.\u003c/p\u003e \u003cp\u003eFinally, we investigated the combined effects of ATRA and 4OH-tamoxifen (Tam), a cornerstone therapy for patients with ERα-expressing primary BC [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], by treating parental MCF-7 cells with varying doses of both drugs. The results demonstrated a synergistic interaction between ATRA and Tam in parental MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC\u0026rsquo;). These findings suggest the potential use of ATRA in combination with Tam for the treatment of primary BC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe mainstay treatment for ERα-expressing primary breast cancer (BC) involves endocrine therapy (ET) with drugs such as aromatase inhibitors (AIs) and 4OH-tamoxifen (Tam). This treatment is typically administered for 5 to 10 years following diagnosis and has significantly reduced BC mortality rates. However, the extended duration of therapy often leads to the development of resistance to ET drugs in a substantial subset of patients, resulting in relapse and progression to metastatic breast cancer (MBC), which is frequently fatal [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResistance to ET drugs arises through multiple mechanisms, including the selection of ERα point mutations in MBC cells that drive uncontrolled proliferation. Most of these ERα variants occur within the receptor's ligand-binding domain (LBD) and induce structural rearrangements that mimic the three-dimensional conformation of the wt receptor bound to E2. This constitutively active agonist conformation not only promotes continuous proliferative signaling but also renders the mutated receptor insensitive to anti-estrogen therapies, such as Ful [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Targeting ERα point mutations in MBC is therefore crucial for developing strategies to manage the disease. Various therapeutic approaches, including SERDs, SERCAs, PROTACs, CERANs, and other targeted drugs [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], have been explored to inhibit the metastatic potential of mutant ERα variants. Promisingly, clinical trials have demonstrated the efficacy of some of these agents, such as elacestrant [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and camizestrant [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], in combating receptor mutations, leading to the approval of elacestrant for clinical use [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these advancements, preclinical and clinical studies have predominantly focused on the most frequent ERα mutations, such as Y537S, while neglecting the functional impact of less common ERα variants. These lesser-studied mutations are equally associated with reduced patient survival and contribute to therapy resistance via variant-specific mechanisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, Irani et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] reported that mutations in the ERα dimerization domain lead to constitutive transcriptional activation, promoting cell proliferation. This study also proposed that disrupting receptor dimerization could serve as a novel therapeutic strategy for MBC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings not only highlight the urgent need to characterize all ERα variants expressed in MBC to identify new therapeutic targets but also underscore the role of three-dimensional structural determinants in deregulating specific receptor functions (e.g., such as ERα dimerization, and nuclear localization), thus opening the possibility that specific neglected ERα variant could be treated with specific drugs.\u003c/p\u003e \u003cp\u003eIn this context, we investigated two clinically observed ERα mutations, L370F and E471D, which are annotated in the COSMIC and cBioPortal databases. These mutations affect residues positioned across from each other on helices H4 and H10 within the ERα ligand-binding domain (LBD), pointing to a potential new structural hotspot. The characterization of these mutations demonstrates that they differently affect sensitivity to Ful. Specifically, the L370F variant reduces sensitivity to Ful, whereas the E471D variant does not. Further characterization of the L370F mutation revealed that it confers several distinct properties to BC cells such as i) reduced sensitivity to Ful in terms of ERα transcriptional activity, receptor stability, and cell proliferation; ii) partially E2-independent growth and hyperactive proliferation in response to low E2 doses; and iii) basal upregulation of late E2 response genes.\u003c/p\u003e \u003cp\u003eOur data show that the recombinant purified L370F receptor exhibits reduced binding affinity for Ful compared to the wt receptor. All atoms\u0026rsquo; simulations further confirmed this finding elucidating that the lower binding of Ful to this ERα variant is due to a structural reorganization of the H4-H5-H12 region in the ERα antagonist conformation. Altogether in vitro and in silico findings provide evidence that the natural mutation of ERα at residue L370 alters the receptor's structure, which impairs Ful binding, similarly to what observed for the Y537S ERα variant [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, simulation analyses performed on the E471D mutant ERα revealed that this mutation has a minor effect on the receptor structure and Ful binding. On one hand, this result supports the lack of Ful effect on ERα transcriptional activity; on the other hand, it suggests that the different point mutations found in MBC patients could also affect three-dimensional structural clusters. This further opens the possibility that distant mutations (e.g., in domains other than the LBD) could influence the structure-function of the LBD, and in turn, antagonist binding and receptor cellular functions.\u003c/p\u003e \u003cp\u003eThe L370F mutant structural remodeling and reduced binding affinity to Ful contribute to the diminished effect of Ful on the transcriptional activity, receptor degradation, and cell proliferation of the L370F ERα variant. In both stable HEK293 cells overexpressing the L370F ERα variant and L370F MCF-7 cells, we observed that this mutation attenuates the effect of Ful, particularly at high drug doses. These results demonstrate that the L370F ERα point mutation confers resistance to the ET drug Ful. Notably, this resistance is significant in the context of high-dose Ful administration (e.g., 500mg/Kg), which has been shown to enhance anti-tumor effects in patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGrowth curve analysis under 10% serum conditions revealed that cells expressing the L370F ERα variant have a reduced doubling time compared to wt ERα-expressing cells, and their proliferation rate is similar to that of Y537S MCF-7 cells. Interestingly, the growth behavior of L370F-expressing cells mirrors that of Y537S MCF-7 cells. Conversely, when grown in an E2-deprived medium, the Y537S MCF-7 cells continue to proliferate, while parental MCF-7 cells stop growing and eventually die. In contrast, L370F MCF-7 cells cease growth but survive in the absence of E2. Upon E2 administration, parental MCF-7 cells remain sensitive to the hormone, while Y537S MCF-7 cells are unresponsive. However, L370F MCF-7 cells respond to E2 in terms of cell proliferation only when grown in an E2-deprived medium. In a medium containing E2, L370F MCF-7 cells exhibit only a minimal response to E2. Notably, at low E2 doses, the L370F ERα variant displays hyperactivity in terms of E2-dependent cell proliferation compared to the wt receptor. This effect is also evident in terms of receptor transcriptional activity. Although the specific mechanisms underlying this effect were not investigated, our in silico simulations of the L370F ERα suggest a structural reorganization of the receptor. This, combined with the similar binding affinity for E2 as observed in the wt ERα, could facilitate the response to E2. Alternatively, the differential sensitivity to E2 may result from the recruitment of distinct co-activators by the L370F mutant receptor compared to the wt ERα. Interestingly, the ability of the L370F ERα variant to respond to low E2 doses may support metastatic cell proliferation. Indeed, in menopausal women or those undergoing ovarian function inhibition, plasma E2 levels are in the picomolar range [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In this context, the L370F mutation activates key functions of the mutant receptor, suggesting that receptor activation at low E2 doses may represent a novel mechanism by which BC cells adapt to the hostile environment created by ET drugs (e.g., AI).\u003c/p\u003e \u003cp\u003eOne hallmark of the Y537S mutation is the receptor\u0026rsquo;s ability to constitutively modulate E2-regulated genes, independent of E2 binding [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our transcriptomic analysis revealed that the Y537S and L370F ERα mutants regulate a similar number of genes, with several E2-sensitive genes being upregulated in both mutant cell lines. Interestingly, the extent of upregulation differed between the two cell lines, with some genes specifically upregulated in one line or the other. Moreover, our data showed that in Y537S MCF-7 cells, genes from both early and late E2 responses were upregulated, whereas in L370F MCF-7 cells, genes from the late E2 response class were more prominently upregulated. Thus, the overall E2 response differs between the two cell lines and may be due to the ability of the L370F ERα to associate with distinct chromatin regions compared to the Y537S variant. Future ChIP-Seq analyses are necessary to further elucidate these differences. However, the selective upregulation of late E2 response genes by the L370F ERα variant highlights its role in promoting cell cycle progression and supporting the development of resistance to ET drugs [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is important to clarify the potential clinical significance of the L370F ERα variant. Among annotated ERα mutations, some are frequently observed in patients, while others are rarer [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Regardless of their prevalence, these mutations generally exhibit poor responsiveness to existing therapies and negatively impact patient survival [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Despite their rarity, our findings demonstrate that the L370F ERα mutation provides BC cells with selective advantages for metastatic growth. These findings highlight the need to investigate the biological impact of rare ERα mutations in MBC. Moreover, it is possible that some ERα mutations are classified as rare simply because they are overlooked in current research and diagnostic efforts.\u003c/p\u003e \u003cp\u003eCurrently, next-generation sequencing or droplet digital PCR experiments (ddPCR) are used to detect ERα mutations [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. ddPCR is used to identify only known and most frequent ERα mutations (e.g., exon 5 to exon 8, i.e., ERα LBD) while other mutations and/or those located outside the LBD are not actively searched in patient samples, unless whole exome sequencing, an expensive and time consuming method [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], is performed. For example, no screening protocols exist for mutations located in the ERα A/B domain, and the L370F mutation located in exon 5 cannot be identified as the standard oligonucleotides used for ddPCR-based detection of that region contain the L370 wt codon [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This diagnostic bias neglects mutations that may significantly contribute to disease progression, as exemplified by the L370F ERα variant described herein, and others previously reported [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This limitation is critical because no specific treatment protocols currently exist for MBC patients harboring these neglected ERα mutations, leaving these individuals without access to personalized therapy.\u003c/p\u003e \u003cp\u003eInterestingly, we observed that both L370F and Y537S MCF-7 cells express elevated levels of RARα, prompting us to investigate the antiproliferative effects of ATRA on these cell lines [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our results show that L370F ERα-expressing cells are more sensitive to ATRA than parental or Y537S MCF-7 cells. Importantly, ATRA triggers substantial DNA damage and pronounced cell death selectively in L370F cells. Initially, we hypothesized that this effect might arise from an interaction between RARα and ERα [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, the presence of the L370F and Y537S mutations did not alter the reciprocal regulation of receptor levels or the ability of ATRA to activate RARα transcriptional activity and to inhibit ERα transcriptional activity. Furthermore, ATRA did not bind in vitro to wt, L370F and Y537S ERα (data not shown). Nevertheless, we found that ATRA-induced activation of RARα transcriptional activity is significantly enhanced in L370F cells. Given previous findings that R-loops generated during the E2 transcriptional response contribute to DNA damage and genomic instability in BC cells [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], we speculate that ATRA-dependent hyperactivation of RARα transcription leads to transcriptional stress (e.g., R-loops), DNA damage, and subsequent cell death, thereby explaining the heightened sensitivity of L370F cells to ATRA.\u003c/p\u003e \u003cp\u003eGrowth curve analyses and cellular assays revealed that ATRA induces senescence in parental and Y537S MCF-7 cells. While ATRA-induced senescence in BC cells has been previously reported [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], this is, to our knowledge, the first evidence of a drug inducing senescence in a BC cell line expressing the Y537S ERα variant. Exploiting senescence as a therapeutic strategy is increasingly recognized for its potential to overcome treatment resistance, as it creates vulnerabilities that can be targeted with senolytic agents in a \u0026lsquo;one-two punch\u0026rsquo; approach [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Supporting this, we found that digoxin, an FDA-approved cardiac glycoside with known senolytic activity [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], selectively targets ATRA-induced senescence in Y537S MCF-7 cells but not in parental cells.\u003c/p\u003e \u003cp\u003eThe clinical potential of ATRA has been demonstrated by its success in treating acute promyelocytic leukemia, inspiring efforts to explore its use in solid tumors [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Although studies consistently report ATRA\u0026rsquo;s inhibitory effects on BC cell growth, this knowledge has led to only a limited number of clinical trials investigating ATRA as an anti-BC agent [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Moreover, no data currently address the potential use of ATRA in treating MBC expressing ERα mutations. However, our findings highlight three potential applications of ATRA in BC clinical practice. First, we observed that co-administration of ATRA with Tam produces a synergistic antiproliferative effect in parental MCF-7 cells, indicating its potential use in managing primary ERα expressing BC that are suitable for ET. Additionally, ATRA could be implemented as a chemotherapeutic agent for treating MBC expressing the L370F ERα variant. Finally, ATRA could be employed to induce cellular senescence in MBC expressing the Y537S ERα variant, creating an opportunity for subsequent senolytic therapy in a \u0026lsquo;one-two punch\u0026rsquo; treatment strategy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identifies the L370F ERα mutation as a novel natural variant associated with MBC and elucidates the mechanisms through which it contributes to ET resistance and metastatic growth. Our findings highlight the importance of characterizing all ERα mutations, including those currently neglected, to better understand their roles in disease progression and therapy resistance. Furthermore, we provide evidence that different ERα variants exhibit unique sensitivities to specific drugs, such as ATRA, which can elicit distinct therapeutic effects depending on the mutation.\u003c/p\u003e \u003cp\u003eOverall, this work demonstrates that tailoring treatment based on the specific ERα variant expressed in MBC can pave the way for more effective and personalized BC therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAI:\u003c/em\u003e\u003c/strong\u003e aromatase inhibitors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eASCL1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Achaete-Scute Family BHLH Transcription Factor 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eATRA:\u003c/em\u003e\u003c/strong\u003e all-trans retinoic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBC:\u003c/em\u003e\u003c/strong\u003e breast cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCALCR\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Calcitonin Receptor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCDK4:\u003c/em\u003e\u003c/strong\u003e cyclin-dependent kinase 4\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCDK6:\u003c/em\u003e\u003c/strong\u003e cyclin-dependent kinase 6\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCERAN:\u003c/em\u003e\u003c/strong\u003e complete estrogen receptor antagonists\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCS-FBS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Charcoal Stripped Fetal bovine serum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eddPCR\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e droplet digital PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDigo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Digoxin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eE2:\u003c/em\u003e\u003c/strong\u003e 17\u0026beta;-estradiol\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eERE:\u003c/em\u003e\u003c/strong\u003e estrogen responsive element\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eER\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e estrogen receptor \u0026alpha;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eET:\u003c/em\u003e\u003c/strong\u003e Endocrine therapy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFABP5\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e fatty acid binding protein 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFASN\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e fatty acid synthase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFBS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Fetal bovine serum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFDA:\u003c/em\u003e\u003c/strong\u003e Food and Drug Administration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFPKN\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Fragments Per Kilobase of transcript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFul:\u003c/em\u003e\u003c/strong\u003e fulvestrant\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHER2:\u003c/em\u003e\u003c/strong\u003e Human Epidermal Growth Factor Receptor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLBD:\u003c/em\u003e\u003c/strong\u003e ligand binding domain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMBC:\u003c/em\u003e\u003c/strong\u003e metastatic breast cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMBC:\u003c/strong\u003e metastatic breast cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNLuc\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Nanoluciferase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Principal component analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePROTAC:\u003c/em\u003e\u003c/strong\u003e proteolysis targeting chimerics\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRARE\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e retinoic acid response element\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRAR\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e retinoic acid receptor \u0026alpha;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSERCA:\u003c/em\u003e\u003c/strong\u003e selective estrogen receptor covalent antagonists\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSERD:\u003c/em\u003e\u003c/strong\u003e selective estrogen receptor downmodulators\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSERM:\u003c/em\u003e\u003c/strong\u003e selective estrogen receptor modulators\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTam:\u003c/em\u003e\u003c/strong\u003e 4OH-tamoxifen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWB\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e Western Blot\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ewt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e wild type\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026gamma;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eH2AX:\u003c/em\u003e\u003c/strong\u003e phosphorylated H2A Histone Family Member X\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e\u0026apos;Not applicable\u0026apos;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003e\u0026apos;Not applicable\u0026apos;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDensitometric analyses of each WB, as well as original data for growth curves and synergy proliferation experiments, are available from the corresponding author on reasonable request. All the original Western blots are available in Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research leading to these results has received funding from AIRC under IG 2018 - ID. 21325 project \u0026ndash; P.I. Acconcia Filippo. The Grant of Excellence Departments 2023-2027, MIUR (ARTICOLO 1, COMMI 314 \u0026ndash; 337 LEGGE 232/2016) to the Department of Science, University Roma TRE is also gratefully acknowledged. This study was also supported by grants from Ministero della Salute RF‐2021‐12372851; CUPF83C22002620001 to Acconcia Filippo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.C. performed most of the experimental work. C.B. performed transcriptional studies. M.F. performed measurement of cellular surface. M.P. and A.M. performed in silico studies. F.A. conceptualized the research, formally analyzed the data, wrote, reviewed, and edited the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank A. Scardua and J. Weber (Biomass Production Unit, National Facility for Structural Biology, Human Technopole) for support and service provision. Access to National Facility was granted to Project ID 1771263. The authors are grateful to Prof. Simak Ali, University of London Imperial College for the gift of the MCF-7 Y537S cells. The anti-FASN antibody was a generous gift of Prof. Andrea Morandi, Dipartimento di Scienze Biomediche Sperimentali e Cliniche \u0026lsquo;Mario Serio\u0026rsquo;, Firenze, Italy.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWill M, Liang J, Metcalfe C and Chandarlapaty S 2023 Therapeutic resistance to anti-oestrogen therapy in breast cancer. Nat Rev Cancer 23:673-685. doi: 10.1038/s41568-023-00604-3\u003c/li\u003e\n\u003cli\u003eShah M, Lingam H, Gao X, Gittleman H, Fiero MH, Krol D, Biel N, Ricks TK, Fu W, Hamed S, Li F, Sun JJ, Fan J, Schuck R, Grimstein M, Tang L, Kalavar S, Abukhdeir A, Pathak A, Ghosh S, Bulatao I, Tilley A, Pierce WF, Mixter BD, Tang S, Pazdur R, Kluetz P and Amiri-Kordestani L 2024 US Food and Drug Administration Approval Summary: Elacestrant for Estrogen Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative, ESR1-Mutated Advanced or Metastatic Breast Cancer. 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Acconcia Filippo","order_by":0},{"identity":"6e0ad097-7e63-4b3d-bb86-f078d7aeeebb","identifier":"10.13039/501100003407","name":"Ministero dell’Istruzione, dell’Università e della Ricerca","awardNumber":"The Grant of Excellence Departments 2023-2027","order_by":1},{"identity":"baf54849-3eb7-4943-bc23-ee1417fb4267","identifier":"10.13039/501100003196","name":"Ministero della Salute","awardNumber":"RF‐2021‐12372851; CUPF83C22002620001 ","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, estrogen receptor α, endocrine resistance, ERα mutations, fulvestrant, all-trans retinoic acid, targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-6706598/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6706598/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eMetastatic breast cancer (MBC) remains a major clinical challenge, particularly in estrogen receptor α (ERα)-positive patients who develop resistance to endocrine therapy (ET). While hotspot mutations such as Y537S in the ligand-binding domain (LBD) are well-characterized drivers of resistance, other ERα variants remain poorly studied. Understanding the molecular mechanisms underlying resistance in these variants is crucial for identifying novel therapeutic strategies. Here, we investigated the functional role of the L370F and E471D ERα variants, which are spatially close in the ERα structure.\u003c/p\u003e\u003ch2\u003eMethods.\u003c/h2\u003e \u003cp\u003eStable overexpressing HEK293 cells and CRISPR/CAS9 engineered MCF-7 cells were generated and treated with 17β-estradiol (E2), fulvestrant (Ful) and all-trans retinoic acid (ATRA) to measure ERα stability, transcriptional activity and gene expression analyses using different cellular assays and RNASeq techniques. Direct in vitro measurement of ligand binding affinity to ERα were performed using the purified full-length wild type (wt) as well as L370F and Y537S ERα. In silico structural simulations were also performed to predict the structure of the mutated L370F ERα. Senescent analyses of MCF-7 and Y537S MCF-7 cells were performed using direct measurement β-galactosidase activity in vitro and in cell lines.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe L370F variant conferred resistance to Ful in terms of in vitro ERα binding, ERα transcriptional activity, receptor degradation and cell proliferation by modifying the folding of the receptor structure. Furthermore, L370F-expressing cells exhibited a hyperactive response to low doses of E2 and basally upregulated late estrogen responsive genes. Additionally, we found that both L370F and Y537S ERα variants displayed increased RARα expression, rendering them highly sensitive to ATRA. Notably, ATRA killed L370F-expressing cells and induced senescence in Y537S-expressing cells, highlighting mutation-specific responses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings expand the understanding of ERα mutations beyond known hotspots, identifying L370F as a novel mutation contributing to ET resistance and further indicate the necessity to characterize all the less-studied ERα variants found in MBC. Furthermore, we demonstrate that ATRA selectively targets MBC cells harboring L370F and Y537S mutations, suggesting its potential as a mutation-specific therapeutic agent. These results support further investigation of ATRA in clinical settings to improve treatment strategies for ERα-mutant MBC.\u003c/p\u003e","manuscriptTitle":"The natural L370F ERα Variant Confers Endocrine Resistance and Sensitivity to ATRA in Metastatic Breast Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 13:38:01","doi":"10.21203/rs.3.rs-6706598/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e1dc8260-eee8-4d30-9899-1ad560c80ca3","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48786336,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2025-05-22T13:38:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 13:38:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6706598","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6706598","identity":"rs-6706598","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}