BRPF1 is an essential gene in AE-sensitive and -resistant ER + BC cells

Genome-wide ‘drop-out’ screenings led to the identification of several genetic dependencies of human diseases, focused analyses are now required to highlight cancer-specific molecular signatures involved in the deregulation of essential pathways in cancer progression and response to therapy. Considering the relevance of ERα as therapeutic target in hormone-dependent BC, and the biological key role of the components of its associated multiprotein complexes for cancer development [3, 4], we focused our investigation to ER-interacting partners essential for LBC subtype, uncovering novel exploitable therapeutic targets to prevent and/or overcome ET-resistance (Fig. 1A). We compared the subset of ER + BC cells fitness genes, essential for BC cell growth and proliferation, obtained from a focused investigation of ERα co-essentiality from DepMap and ProjectScore [5], with a subgroup of proteins retrieved from databases of ERα interacting partners [3, 10, 11], identifying a subset of 36 estrogen receptor-associated essential proteins whose corresponding mRNAs result highly expressed in BCs (Additional File 1: Fig. S1A), specifically in Luminal A and B subtypes (Fig. 1B), when compared to normal mammary gland, in TCGA BC datasets. These proteins, which are by functional enrichment analysis mainly involved in histone modifications and chromatin organization (Fig. 1C), include ERα (ESR1) and previously characterized ER partners such as BAZ1B, DOT1L, KMT2D, LBD1, MEN1 and the pioneering factor FOXA1 [3, 4, 12, 13]. To validate our in silico results, we performed a small scale siRNA screening in MCF-7 cells by using two different siRNAs against each gene target and assessing the consequences of gene kd on key cellular functions, such as cell proliferation and death, and on ERα-mediated activation of estrogen signaling (Fig. 1D). Results demonstrate that the candidate genes selected for this study are involved in BC cell proliferation, apoptotic cell death and efficient receptor-mediated transactivation of target genes (in this case a reporter gene: ERE-luc) already after 72 h of siRNA transfection and without showing significant toxicity under these experimental conditions (Additional File 1: Fig. S1B). Of note, these analyses, together with the above-mentioned known receptor coregulators reveled new and previously uncharacterized BC cell factors, including in particular BRPF1 (Bromodomain and PHD Finger Containing 1). This gene caught our attention as it scored fifth in the essential gene list according to fitness score and its inhibition exerted a strong effect on all the three cellular parameters analyzed, comparable to those elicited by ESR1 kd (Additional File 1: Fig. S1C-E). Moreover, a protein complex assembly analysis, performed to gather information on possible complex formation among our candidates, revealed the MOZ-complex, of which BRPF1 is a functional component, as the highest enriched (p-value = 0.0316) in our dataset of estrogen receptor-associated essential proteins (Additional File 1: Fig. S1F). Given the ability of this epigenetic factor to influence cellular behaviors through transcriptional control events mediated by its chromatin remodeling activity, we further characterized functional role of BRPF1 in ER + BC cells. BRPF1-ERα interaction was experimentally validated by co-IP in nuclear lysates from MCF-7 cells (Additional File 1: Fig. S1G). Analyzing TCGA datasets, we observed that both genes are often co-expressed in the same ER + tumors (Fig. 1E left), with a positive correlation (R = 0.53, p-value = 0,) between each other (Additional File 1: Fig. S1H). Interrogation of CPTAC BC dataset revealed that BRPF1 is expressed at a higher level in luminal-like BC compared to normal mammary gland (Additional File 1: Fig. S1I), and this associates to worse overall survival in BC patients (Fig. 1E right). When considering the mutational status of the BRPF1 in BCs from TCGA, this gene results mutated in only 2% and 0.7% of ER + LBCs. All these results suggest that, within the gene set identified here, BRPF1 might represent a novel prognostic biomarker and actionable target against ER-expressing BCs. To this end, after assessing BRPF1 protein expression level in a panel of ER + and ER- BC cells (Supp. Fig. S1J), we selected exponentially growing MCF-7 for further analyses. Cells were subjected to transient BRPF1 kd with two siRNAs, a reduction of up to 80% of mRNA level (Additional File 1: Fig. S2A, left panel), followed by gene expression profiling by RNA-Seq. As shown in Fig. 1F, BRPF1 silencing had a significant effect on MCF-7 cells transcriptome (196 up- and 957 down-regulated genes, Additional File 2: Table S1), affecting signal transduction pathways known to control key cellular processes involved in cancer, such as cell cycle checkpoints, glucose metabolism, VEGF signaling and above all, those controlled by ERα in BC (Fig. 1F). Indeed, BRPF1 silencing significantly downregulates well-known receptor target and pioneering genes, like TFF1, GATA3 and FOXA1, probably because of a reduction of ERα mRNA (Fig. 1G left panel and Additional File 1: Fig S2A right panel) and protein (Fig. 1G right panel) levels in the cell, confirming that the activity of this protein is essential to allow efficient estrogen signaling. This evidence suggested that this enzyme could also represent a candidate target for silencing ER-mediated pathway in AE-resistant breast tumors, such as Tamoxifen-resistant (TAM-R) ones, that maintain hormone responsiveness and express mutated and/or constitutively active receptors, where a significantly higher BRPF1 expression was found when compared to AE-sensitive ones (Fig. 1H). Thus, we validated the association between ERα and BRPF1 also in TAM-R MCF-7 cells (Fig. 1I). The effects of BRPF1 kd were assessed on cell proliferation, death and the transcriptome of TAM-R MCF-7 cell lines (Additional File 1: Fig. S2B left panel). Consistently with the results obtained in AE-sensitive cells, BRPF1 kd in TAM-R cells resulted in reduction of cell proliferation (Fig. 1J), increased apoptosis (Fig. 1K), and deregulation of receptor signaling (Fig. 1L; Additional File 3: Table S2), primarily driven by downregulation of ERα mRNA and protein levels (Fig. 1M and Additional File 1, Fig. S2B right panel). Comparable results on cell proliferation and death were detected in Fulvestrant/ICI-resistant (ICI-R) BC cells (Additional File 1: Fig. S2C). Also here, RNA-seq showed a deregulation of receptor signaling (Additional File 1: Fig. S2D; Additional File 4: Table S3), downregulation of ERα mRNA and protein levels (Additional File 1: Fig. 2E). A significant inhibition of cell proliferation and ER downregulation following BRPF1 kd was observed also in T47D and ZR75.1 ERα + BC cells, further supporting the direct involvement of BRPF1 in ER mediated mitogenic signaling (Additional File 1: Fig S2F-G).

Fig. 1

BRPF1 gene essentiality in AE-sensitive and -resistant BC cells. (A) Venn diagram showing the overlap between luminal-like BC fitness genes and ERα interacting partners. (B) Box plots showing the mRNA expression levels of the 36 ERα essential interactors in BC subtypes obtained from datasets deposited in The Cancer Genome Atlas (TCGA) and analyzed with GEPIA2. (C) Graphical display showing functional enrichment analysis of statistically significant molecular function encoded in the 36 essential gene encoding ERα interactors. (D) Heatmaps showing the fitness (essentiality) score and impact of siRNA mediate kd of the 36 ERα essential interactors on cell proliferation, caspase, and ERE-Luc reporter gene activity. Data related to fitness score represent the median of essentiality value of each molecule in BC cell analyzed [5]. Data related to cell proliferation, caspase, and ERE-Luc activity are analyzed with respect to the scramble siRNA (CTRL). Data are presented as the mean ± SD of determinations from a representative experiment performed with 2 siRNAs (#1 and #2) in six independent replicates after 72 h of silencing. Triangle forms indicate the orientation of each scale in terms of decrease (decr) or increase (incr) of the value respect to the CTRL. (E) Histogram (left panel) showing BRPF1 and ERα mRNA co-expression from two additional luminal-like BC patient datasets from TCGA and Kaplan-Meier curves (right panel) showing the probability of overall survival, according to BRPF1 mRNA expression levels, of luminal-like BCs contained in TCGA database analyzed by GEPIA2. (F) Graphical display summarizing functional enrichment analysis of statistically significant modulated pathways by RNA-seq following BRPF1 kd for 72 h in MCF-7 cells. (G) Left panel: Bar chart showing mRNA expression levels of ESR1 (ERα) and its genomic partners FOXA1 and GATA3 and downstream target TFF1 following BRPF1 kd for 72 h in MCF-7 cells. Data from RNA-seq analysis were analyzed with respect to a scramble siRNA (CTRL); asterisks indicate statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005) respect to CTRL. Right panel: Representative western blot and relative densitometry showing BRPF1 and ERα protein levels following BRPF1 kd in MCF-7 cells for 72 h. β-actin (ACTB) was used as loading control and images were processed with ImageJ software (https://imagej.Net) for densitometry readings. (H) Graphical display of BRPF1 mRNA expression in TAM-sensitive and -resistant BC samples from TCGA datasets. (I) Representative western blot showing BRPF1 and ERα co-immunoprecipitation in TAM- resistant MCF-7 (MCF7 TAM-R) nuclear extracts. IgG was used as negative control. (J) MCF7 TAM-R cell proliferation rate measured by MTT assay following BRPF1 silencing in MCF-7 cells. K) Caspase 3/7 activity assay following BRPF1 silencing in MCF-7 cells. Data are presented as the mean ± SD of determinations from a representative experiment performed in six independent replicates after 72 h of silencing All data are analyzed with respect to the scramble siRNA (CTRL). Asterisks indicate statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005) to CTRL. L) Graphical display summarizing functional enrichment analysis of statistically significant modulated pathways by RNA-seq following BRPF1 kd for 72 in MCF-7 TMA-R BC cells. (M) Representative western blot and relative densitometry showing BRPF1 and ERα protein levels following BRPF1 silencing or treatment with ICI (100 nM) in MCF7 TAM-R BC cells. β-actin (ACTB) was used as loading control and images were processed with ImageJ software (https://imagej.Net) for densitometry readings

Fig. 2

Impact of BRPF1 pharmacological inactivation on cell functions in AE-sensitive and -resistant BC cells and PDOs. (A) Western blot analysis and (B) cell proliferation rate in MCF-7 and MCF-7-flag cells before and after BRPF1 silencing or treatment with ICI (100 nM). For western blot analysis β-actin (ACTB) was used as loading control. For MTT assays all data are analyzed with respect to scramble siRNA (CTRL) and presented as the mean ± SD of determinations from a representative experiment performed in six independent replicates. Both experiments were performed after 72 h of silencing. Asterisks indicate statistically significant differences (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005) to CTRL. (C) Heatmap showing the effects of ERα silencing on ERα-BRPF1 shared binding sites in MCF-7 genome. (D) Effect of BRPF1 pharmacological blockade on AE-sensitive (MCF-7) BC cells following increasing concentrations of GSK, TAM (100nM) and ICI (100 nM) after 3, 6, 9 and 12 days. DMSO (vehicle) was used as control. Data are presented as mean ± SD from six independent replicates. (E) Cell cycle phase distribution (Percentages of G1, S, and G2/M) in hormone-deprived MCF-7 cell cultures before (-) or after treatment with E2 alone (24 h) or in combination with GSK or ICI at the indicated times and concentrations determined by flow cytometry after PI staining. (F) Cell cycle sub-G phase analyzed as described above. Results shown represent the means ± SD of multiple determinations from a representative experiment performed at least in triplicate. (G) Heatmap shoving ATAC-seq (normalized) signals at sites of increasing (“opening”, gained: red) and decreasing (“closing”, lost: blue) chromatin accessibility following GSK treatment compared to vehicle (DMSO, V) in MCF-7 cells. (H) Correlation graph between RNA expression and accessibility changes following GSK treatment in MCF-7 cells showing that the transcriptome changes identified positively correlate with changes in chromatin accessibility of the corresponding transcription units. l) RT-qPCR analysis of ESR1 (ERα) and TFF1 mRNA levels following treatment with GSK9311 (negative control inhibitor of GSK: C) or GSK in MCF-7 cells. Data are analyzed with respect to vehicle (DMSO: CTRL) and presented as the mean ± SD of triplicate determinations from a representative experiment. J) Representative western blot and relative densitometry showing BRPF1 and ERα protein levels following treatment with vehicle (DMSO: V), GSK9311 (negative control inhibitor of GSK: C) or GSK in MCF-7 cells. β-actin (ACTB) was used as loading control and images were processed with ImageJ software (https://imagej.Net) for densitometry readings. K) Effect of BRPF1 pharmacological blockade on AE-resistant (MCF-7 TAM-R) BC cells after 3, 6, 9 and 12 days of treatment with the indicated concentrations of GSK, TAM or ICI. DMSO (vehicle) was used as control. Data are presented as mean ± SD from six independent replicates. L) RT-qPCR analysis of ESR1 (ERα) and TFF1 mRNA levels following treatment with GSK9311 (control inhibitor, C) or GSK in MCF-7 TAM-R cells. Data are analyzed with respect to vehicle (DMSO: CTRL) and presented as the mean ± SD of triplicate determinations from a representative experiment. M) Representative western blot and relative densitometry showing BRPF1 and ERα protein levels following treatment with vehicle (DMSO, V), GSK9311 (control inhibitor, C) or GSK in MCF-7 cells treatment in MCF-7 TAM-R cells. β-actin (ACTB) was used as loading control and images were processed with ImageJ software (https://imagej.Net) for densitometry readings. N) Representative microscope photographs of ERɑ immuno-staining on organoids from n = 3 ER + BCs (see also Additional file 1: Table 1). O) Box plot showing proliferation rate in the 3 PDOs following treatment with vehicle (DMSO: V) and the indicated concentrations of GSK or with 100 nM ICI for 10 days. Results shown represent the means ± SD of multiple determinations of biological and technical replicates obtained from independent experiments performed on each PDO. P) Caspase activity assay in 2 PDOs following treatment with vehicle (DMSO, V) and the indicated concentrations of GSK or with 100 nM ICI for 10 days. Results shown represent the means ± SD of multiple determinations of biological replicates obtained from independent experiments performed on each PDO. Q) Graphical display of functional enrichment analysis of statistically significant deregulated pathways following BRPF1 pharmacological blockade in BC PDOs analyzed by RNA-seq. R) Heatmap showing differentially expressed genes identified by RNA-seq following pharmacological blockade of BRPF1 with GSK compared to vehicle (DMSO, V) in BC PDOs

Pharmacological inhibition of BRPF1 induces deregulation of chromatin accessibility, causing growth arrest and cell death, via inhibition of ERα-mediated signaling, in both AE-sensitive and -resistant BC cells and in preclinical BC models

Given the results described above, and the response of ET-sensitive and -resistant cells to BRPF1 kd, we sought then to elucidate the involvement of this enzyme on ER-cistrome and ERα-mediated transcriptional program. To characterize the mechanistic and functional interplay of the BRPF1-ERα nuclear complex, we used first an MCF-7 cell line stably expressing full-length-3xFlag-ESR1 (ERα-flag) [4]. These clones carry double ERα expression, from the endogenous and the transfected genes, the first under the control of the natural promoter and the second from the exogenous one. The effects of BRPF1 silencing on cell proliferation and ERα protein expression were assessed in this complemented (ERα + ERα-flag) cellular model, compared to the same in wt MCF-7 cells (ERα only). Results reported in Fig. 2A and B confirmed a role of BRPF1 on ERα gene, as expression of exogenous ERα and proliferation rate of cells expressing it were not affected by BRPF1 kd, that caused instead downregulation of endogenous ER, demonstrating that the endogenous ER gene is the target of this epigenetic factor. Subsequently, we mapped BRPF1 binding to MCF-7 cell genome by Chromatin Immunoprecipitation coupled to sequencing (ChIP-seq). As shown in Supp. Fig. S3A, ChIP-western blotting showed an association of the two proteins on MCF-7 cell chromatin and their colocalization [3] in a sizable number of chromatin sites (6332 binding sites). In addition, analysis of the BRPF1 cistrome (20049 binding sites) revealed, among others, enriched binding motifs for HOX13 and GFLI factors (Additional File 1: Fig. S3B), while co-occupied ER + BRPF1 binding sites showed a prevalence of ERE (estrogen response element) or ERE-like sequences (Additional File 1: Fig. S3C). ER + BRPF1 binding sites resulted distributed within heterochromatin, enhancers, and promoters (Additional File 1: Fig. S3D left panel) and with a significant enrichment in these regulatory regions when considering the observed/expected distribution, also in comparison to ERα binding sites (Additional File 1: Fig. S3D right panel). Considering that BRPF1 does not bind DNA directly, our results suggest that the association with ERα targets this bromodomain protein to specific chromatin locations, where the two proteins act together to regulate gene activity. In fact, when performing BRPF1 ChIP-seq following ER kd, we observed that this factor lost the ability to bind to the subset of binding sites shared with ERα but not to those independent form the presence of the receptor, confirming the physical/functional cooperation between these two proteins in BC cell chromatin (Fig. 2C). This relationship between the receptor and BRPF1 was then further investigated through pharmacological inhibition of the latter with specific small molecule inhibitors: GSK5959, GSK6853, namely GSK, and its less active analogue, GSK9311, used as negative control [14]. This is of particular interest since bromodomain inhibitors represent a novel class of epigenetic drugs which hold great promise for anti-cancer therapy, although their therapeutic potential in BC remains still unexplored. To this end, the effects of GSK on proliferation, death, and transcriptome changes in a panel of ER + AE-sensitive and -resistant and, as control, in ER- BC cells was evaluated in detail. Considering MCF-7 cells, results showed that these compounds cause a dose-dependent inhibition of cell growth to levels comparable to, or even stronger than, those observed with the AEs (TAM or ICI), both in exponential growth conditions or following treatment of hormone-deprived cells with a mitogenic dose of estrogen (E2) (Fig. 2D and Additional File 1: Fig. S3E). This response resulted absent and comparable with the vehicle (DMSO; V) when considering the negative control inhibitor GSK9311 (Additional File 1: Fig. S3F), inducing us to use the vehicle (DMSO) in other or more complex experiments. The effect observed results from blockade of cell cycle progression in G1 (Fig. 2E and Additional File 1: Fig. S3G left panel), massive induction of apoptotic cell death (Fig. 2F and Additional File 1: Fig. S3G right panel and H) and interference with receptor-mediated transactivating functions resulting in deregulation of ER target genes expression (Additional File 1: Fig. S3I), all comparable to what induced by cell treatment with the pure AE ICI. This confirms the data obtained after BRPF1 kd, and further support the functional link between these two regulatory factors.

BRPF1 is a chromatin remodeler [9], and chromatin accessibility exerts a functional role in gene regulation. We therefore hypothesized that BRPF1 blockade may alter chromatin structure, thereby influencing receptor-mediated BC cell transcriptional regulatory networks, as determined by RNA-seq analyses (Additional File 1: Fig. S3J and Additional File 5: Table S4). To characterize changes in chromatin accessibility upon BRPF1 inhibition, we thus performed chromatin accessibility ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) test. In MCF-7 cells, BRPF1 inhibition resulted in 615 differentially accessible chromatin regions, 80% of which consisting of less accessible chromatin sites following BRPF1 inhibition (Fig. 2G and Additional File 6: Table S5A-B). Of note, most of the chromatin regions detected following BRPF1 blockade were similarly influenced also by ICI, although this compound induced more pronounced chromatin accessibility changes in the same BC cell line (data not shown). Integrating ATAC- and RNA-seq data following GSK treatment, we observed a positive correlation between chromatin remodeling and transcriptome changes (Fig. 2H). Moreover, several deregulated genes associated with modulated chromatin regions are involved in key survival signal transduction pathways, such as TP53 transcription and DNA repair and PIK/AKT signaling (Additional File 1: Fig. S3J), and harbor BRPF1/ERα-BRPF1 binding sites in their regulatory elements. These include genes involved in ER-mediated signaling harboring ERα-BRPF1 binding sites, and of note, the ESR1 gene itself. Moreover, closed (lost, blue) regions resulted statistically significant enriched by ERα/ERα-BRPF1 binding sites (p-value 0,000099; z-score 145.80) when compared to open (gained, red) ones. These data provided mechanistic evidence to explain the effects of the drug on downregulation of estrogen signaling, which was achieved through ESR1 silencing confirmed both at mRNA and protein levels and in comparison, also, to the control (C) inhibitor GSK9311 (Fig. 2I and J). Results, consistent with those described above, were also obtained with the use of an additional BRPF1 specific small molecule inhibitor, GSK5959, in MCF-7 (Additional File 1: Fig. S3K-N), and with both GSK and GSK5959 in T47D BC cells (Additional File 1: Fig. S4A-F). The availability of an already approved drug to block this enzyme, mimicking the effects of gene silencing in AE-sensitive BC cells, led us to translate our findings also in AE-resistant cells. We therefore tested the effects of GSK in TAM-R and ICI-R BC cell models. As shown in Fig. 2K, GSK administration to MCF-7 TAM-R cells determined a marked growth inhibition not observable with the control (C) inhibitor GSK9311 (Additional File 1: Fig. S4G), coupled with a transcriptome deregulation (Supp. Fig. S4H and Additional File 7: Table S6), via blockade of cell cycle progression accompanied by increased apoptotic BC cell death (Additional File 1: Fig. S4I and J). This effect was observed under both, exponential growth conditions and after E2 stimulation, and it is due to decreased ER mRNA and protein levels in comparison, also, to the control (C) inhibitor GSK9311 (Fig. 2, L and M). MCF-7 TAM-R responded to BRPF1 inhibition similarly to wild-type MCF-7 cells (Fig. 2D-F). Likewise, GSK administration to ICI-R cells inhibited cell growth and ER expression via deregulation of essential BC cell pathways (Additional File 1: Fig. S4 K-N and Additional File 8: Table S7). The antiproliferative effects of GSK were also observed in several other E2-responsive and TAM-R BC cell lines, namely T47D-TAM, ZR-75.1 and ZR-75.1 TAM-R, BT-474 and BT474 TAM-R. At same time, significant responses to GSK or GSK6959 were not detected in ER-negative, E2-unresponsive MDA-MB 231 and HS578T BC cells and in ER- mammary epithelial MCF10 cells (Additional File 1: Fig S5 A to I). The results demonstrate that ERα gene silencing through BRPF1 inhibition, and the downstream effects on estrogen signaling, occur in LBC independently from the cellular background and the molecular mechanism responsible for ET resistance. Given the practical implications of these findings for cancer therapy, we challenged them in preclinical models derived from ER + BC patients (PDOs), since these have been demonstrated to hold molecular characteristics of the original tumors, including tumor heterogeneity [15, 16]. PDOs are also consolidated forecaster models for ex vivo patient’s drug response, mirroring those clinically observed [16]. We therefore tested our findings in organoids derived from three ER-positive breast carcinoma biopsies, from three different patients, all retaining ERα expression to different extents and being estrogen responsive (Fig. 2N and Additional File 1: Table 1). Using GSK at concentrations comparable and also higher than those employed previously in 2D BC cells, we analyzed the effect of BRPF1 pharmacological inhibition on proliferation and death in PDOs. As shown in Fig. 2O and P, GSK was able to dramatically decrease cell growth and induce apoptotic events in a dose-dependent manner in all PDOs tested after 9 days of treatment, at an extent comparable to or higher than that elicited by ICI. To confirm that the observed phenotype would resemble, mechanistically, the results obtained in 2D models, we performed transcriptome analyses on two independent PDOs after BRPF1 pharmacological blockade. Results show that also in these preclinical models, attenuation of BC growth was significantly mediated by cell cycle and ER-mediated signaling, coupled to an enhancement of apoptosis (Fig. 2Q and Additional file 9: Table S8), by downregulation of ESR1 gene and additional key ER pioneering factors and co-activators, such as FOXA1 and NCOA3 and with a consequent impact on expression of ER target genes such as TFF1 (Fig. 2R). Finally, considering that a combinatorial therapeutic approach, using more than one compound targeting different cellular targets, could represent a novel strategy to improve treatment of ER + BCs resistant to actual adopted therapies (AE and CDK4/6 inhibitors) and help maximize the therapeutic efficacy, we examined the effect of combinatory treatment with AE (TAM and ICI) or CDK4/6 inhibitor Palbociclib (Palb) [17] in combination with GSK. Interestingly, the test revealed a synergistic effect of suboptimal concentrations of the pairs of drugs used on growth inhibition of both AE-sensitive and -resistant LBC cells (Additional file 1: Fig. S6 A–B). When combined, the results described above demonstrate a pivotal role of BRPF1 in regulation of ERα activity in E2-responsive BC cells, due both to a functional interplay of the encoded protein with the receptor itself and to enhancement of its gene expression, both resulting in efficient regulation of the estrogen signaling pathway in LBC cells. Interestingly, this occurs also in AE-resistant BC cells, suggesting that this epigenetic factor is a molecular target exploitable to silence ERα signaling in ET-resistant tumors, particularly, in the majority of these that retain responsiveness to estrogen.