Essential gene screening identifies the bromodomain-containing protein BRPF1 as a new actionable target for endocrine therapy-resistant breast cancers

Abstract

Identifying master epigenetic factors controlling proliferation and survival of cancer cells allows to discover new molecular targets exploitable to overcome resistance to current pharmacological regimens. In breast cancer (BC), resistance to endocrine therapy (ET) arises from aberrant Estrogen Receptor alpha (ERα) signaling caused by genetic and epigenetic events still mainly unknown. Targeting key upstream components of the ERα pathway provides a way to interfere with estrogen signaling in cancer cells independently from any other downstream event. By combining computational analysis of genome-wide 'drop-out' screenings with siRNA-mediated gene knock-down (kd), we identified a set of essential genes in luminal-like, ERα + BC that includes BRPF1, encoding a bromodomain-containing protein belonging to a family of epigenetic readers that act as chromatin remodelers to control gene transcription. To gather mechanistic insights into the role of BRPF1 in BC and ERα signaling, we applied chromatin and transcriptome profiling, gene ablation and targeted pharmacological inhibition coupled to cellular and functional assays. Results indicate that BRPF1 associates with ERα onto BC cell chromatin and its blockade inhibits cell cycle progression, reduces cell proliferation and mediates transcriptome changes through the modulation of chromatin accessibility. This effect is elicited by a widespread inhibition of estrogen signaling, consequent to ERα gene silencing, in antiestrogen (AE) -sensitive and -resistant BC cells and pre-clinical patient-derived models (PDOs). Characterization of the functional interplay of BRPF1 with ERα reveals a new regulator of estrogen-responsive BC cell survival and suggests that this epigenetic factor is a potential new target for treatment of these tumors.

Keywords: BRPF1; Breast cancer; Endocrine therapy resistance; Estrogen signaling.

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