ERα-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer

Abstract

Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER(+) xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.

Keywords: CDK4; Estrogen receptor; aromatase inhibitor; breast; resistance.

Fig. 1

ER is required for acquired hormone-independent breast cancer cell growth. A) Lysates from cells treated with 10% DCC-FBS ± fulvestrant or 4-OH-T × 24 h were analyzed by immunoblotting using ER and actin antibodies. B) Cells were treated as in (A) ± E2, 4-OH-T, or fulvestrant. Media and drugs were replenished every 2–3 days. Adherent cells were counted after 5–10 days. Data are presented as % parental control, mean of triplicates ± SD. C) Cells transfected with siRNA targeting ER or non-silencing control (siCtl) were treated as in (B), and adherent cells were counted after 6–9 days. Data are presented as % parental siCtl/no hormone, mean of triplicates ± SD. D) Immunoblots of lysates from the same batches of cells used in (C). E) Parental cells were treated as in (B). When control wells reached 30–50% confluence (18–60 days), cells were stained with crystal violet. Representative images are shown. Quantification of staining intensity is noted at bottom as mean of triplicates ± SD (% control). *p<0.05 by Bonferroni post-hoc test compared to control [or siCtl/no hormone in (C)] of each cell line.

Fig. 2

Gene expression and genomic profiling reveal estrogen-independent ER transcriptional activity. A) RNA from MCF-7/LTED and HCC-1428/LTED cells treated ± fulvestrant × 48 h was analyzed using gene expression microarrays. We derived a signature of 141 genes commonly up- or down-regulated by E2 stimulation in MCF-7 cells. Using LTED cell gene expression data, we hierarchically clustered these 141 genes (x-axis). Fulvestrant induced expression changes diametrically opposed to those induced by E2. B) We determined probe sets up- or down-regulated by fulvestrant in each LTED line (≥1.5-fold, p≤0.05), and used Venn diagrams to identify commonly deregulated probe sets (yielded 1,434 probe sets for 718 down- and 285 up-regulated genes). C) ChIP-seq analysis to identify ER genomic binding regions. To focus on functional ER binding sites, we determined which sites were within 60 kb of the transcription start site of a fulvestrant-deregulated gene. D) Forty-eight ER binding regions identified in (C) were tested by ChIP-qPCR. Data presented as heatmap of fold-enrichment in ER-ChIP over IgG-ChIP control, or fold-enrichment score from ChIP-seq. ND- not detected. E2-regulated genes deregulated by fulvestrant in (A), presence of a proximal estrogen-response-element (ERE) within 500 bp of a peak, and overlap of peaks with published ER-ChIP datasets are noted below heatmap. E) ER-bound regions verified in (D) were subcloned into a luciferase reporter vector. Cells transfected with reporter plasmids were treated with 10% DCC-FBS ± fulvestrant or E2. Luciferase activities were measured after 24 or 48 h. ERE- contains two consensus EREs. RLU- relative light units (firefly/Renilla). Data are presented as log₁₀(ratio vs. each untreated control), mean of triplicates ± SD. *p<0.05 by Bonferroni post-hoc test compared to each control. F) ChIP-qPCR as in (D) using human ER+ breast tumor samples acquired from patients following 10–21 days of neoadjuvant letrozole. Data presented as fold-enrichment in ER-ChIP over IgG-ChIP control.

Fig. 3

A gene expression signature of E2F activation correlates with poor tumor response to aromatase inhibitors in patients. A) The set of 1,003 genes commonly deregulated by fulvestrant was used to query TRANSFAC database. Top 20 Genesets are shown, 12 of which contain E2F motifs (highlighted). The number of fulvestrant-deregulated genes as a percentage of the total genes in each set is shown. We derived a set of 24 genes containing E2F motifs but lacking a Gene Ontology cell cycle annotation. B–E) Expression values of these 24 genes were used to generate E2F activation scores for ER+ primary breast tumors from patients treated with an AI using pre- and post-treatment gene expression data. E2F scores were standardized to generate Z-scores. Patients were treated with neoadjuvant anastrozole for 14 days (B–C), or with letrozole for 10–14 days (D–E). The associations of the pre-anastrozole (B), post-anastrozole (C), pre-letrozole (D), and post-letrozole (E) E2F Z-scores with log₂-transformed post-AI Ki67 score were analyzed by linear regression. Pearson correlation coefficients (r) and ANOVA p values were calculated. F) In a third cohort, protein levels of FANCD2 and CDK1 were measured by RPPA in ER+ primary breast tumors following 10–21 days of neoadjuvant letrozole. Log₂-transformed signal values were standardized into Z-scores, which were compared to post-letrozole Ki67 score. G) The protein (Z-score) and mRNA levels (from post-treatment microarrays) of FANCD2 and CDK1 (CDC2) were compared to Ki67 score in all 3 datasets using linear regression. adj = adjusted p-value from multiple regression analysis.

Fig. 4

CDK4 is required for the hormone-independent growth of ER+ breast cancer cells. A) MCF-7 and MCF-7/LTED cells transiently transfected with a siRNA library targeting 779 kinases were reseeded in 10% DCC-FBS. Cell viability was measured 4–5 days later. Cell growth for each kinase siRNA relative to non-silencing controls (siCtl) was transformed to a Z-score, and the median Z-score across 3–4 independent experiments was calculated. B) Cells transfected with an independent siRNA targeting CDK4 or siCtl were treated with 10% DCC-FBS ± 1 μM fulvestrant. Media and drugs were replenished every 2–3 days. Adherent cells were counted after 6–9 days. Data are presented as % parental siCtl/no hormone, mean of triplicates ± SD. We were unable to achieve CDK4 knockdown in HCC-1428 lines. C) Cells were treated with 10% DCC-FBS ± fulvestrant or 0.2 μM PD-0332991. Adherent cells were counted after 6–16 days. Data are presented as % parental control, mean of triplicates ± SD. *p<0.05 by Bonferroni post-hoc test compared to control [or siCtl/no hormone in (B)] for each cell line. D) Immunoblots of lysates from cell treated as in (C) × 24 h. E) Mice bearing established MCF-7 xenografts were randomized to the indicated treatments. Mean tumor volumes ± SEM are shown. *p<0.05 by general linear model compared to vehicle control at the indicated time point. F) Immunoblot analysis of lysates from tumors from (E) using the indicated antibodies. G) IHC scoring for phospho-Histone H3Ser10 and cleaved caspase-3/7 was performed on tumors from (E). *p<0.05 by Bonferroni post-hoc test. ns-not significant.

Fig. 5

Combined downregulation of ER and inhibition of PI3K induces regression of ER+ xenografts. A) Cells were treated with 10% DCC-FBS ± fulvestrant, BKM120, or both. Combination index (CI) values were calculated using the Median Effect method at the IC₅₀, IC₇₅, and IC₉₀ for both drugs. CI<1 is indicative of synergy. B) Mice bearing MCF-7 tumors were randomized to the indicated treatments. Mean tumor volumes ± SEM are shown. *p<0.05 by general linear model compared to vehicle control at the indicated time point.#p<0.05 compared to both single-agent treatment arms. C) Tumor-bearing mice were imaged before and after nine days of treatment by [¹⁸F]FDG-PET. Representative images show [¹⁸F]FDG uptake pre- and post-BKM120 (T- tumor). Quantified in Fig. S12. D) Immunoblot analysis of lysates of tumors from (B) using the indicated antibodies. E) H&E staining and IHC for Ki67 and cleaved caspase-3/7 of tumors from (B). Quantification is shown below. *p<0.001 by Bonferroni post-hoc test. ns- not significant. F) Model depicting the synergistic effects of fulvestrant and BKM120.