Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells

Abstract

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKβ establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKβ signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Keywords: breast cancer; crystallography; cytokines; drug resistance; estrogen receptor; inflammation; tamoxifen.

Figure 1. Inflammatory cytokines regulate E2-dependent target genes through ERα

(A) Diagram of the hormone activation conformer of ERα where both AF1 and AF2 contribute to gene activation. (B) Diagram of TOT-bound ERα actively repressing transcription via recruitment of corepressors to multiple domains of ERα. (C) Diagram of TOT- bound ERα activating gene expression through coactivator recruitment to AF1. (D) Heat map for mRNA-Seq expression of the 801 transcripts regulated in MCF7 cells treated with E2, IL1β, or TNFα. The mRNA expression is shown for MCF7 cells treated with Veh, E2, E2 + ICI, IL1β, IL1β + ICI, TNFα, and TNFα + ICI for 3 hours. (E) Gene ontology analysis for genes that are regulated by E2, IL1β, and TNFα. (F) Cancer cell extravasation assay using the CAM assay. MCF7 WT and MCF7 ERα null cells were treated with Veh, IL1β or TNFα. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) compared to MCF7 WT ERα cells. (G) QPCR analysis for MYC mRNA in MCF7 cells treated with siRNA for control (CTL) or ERα (ESR1) and stimulation with vehicle (Veh), E2, IL1β or TNFα. Values are expressed as mean ± SEM.*p<0.05 compared to siCtl, E2. **p<0.05 compared to siCtl, IL1β. ***p<0.05 compared to siCtl, TNFα. (H) MCF-7 cells were transferred to steroid free media and treated with a dose curve of TOT and assayed for proliferation after 5 days. Mean +/−SD (N=4). (I) MCF-7 cells were treated with a dose curve of IL1β + TOT and assayed for proliferation after 5 days. Mean +/− SD (N=4).

Figure 2. Inflammatory cytokines activate the ERα cistrome

(A) Heat map for 15,213 ERα ChIP-Seq peaks in MCF-7 cells treated with vehicle, E2, IL1β, TNFα. (B) Venn diagram of ERα cistrome in MCF-7 cells treated with E2, IL1β, and TNFα. (C) Boxplot of the GRO-Seq signal at distal top 10% of E2-ERα binding sites in MCF-7 cells treated with Veh, E2, TNFα, and IL1β. (D) Boxplot for DNase hypersensitivity (GSE33216) at E2 preferential ERα peaks and E2+Cytokine ERα peaks. (E) Boxplot for H3K4me2 (top, GSE33216) or H3K27ac (bottom, GSE45822) at E2 preferential ERα peaks and E2+Cytokine ERα peaks. (F) Venn diagram for the ERα and p65 cistromes in MCF-7 cells treated with E2 or IL1β. (G) Top, De novo motif analysis for the E2 preferential cistome (n=8,296) identified in figure 2A, or bottom, for ERα binding sites identified in the presence of E2 and either TNFα, IL1β, or both TNFα and IL1β treatments (n=5,442).

Figure 3. Inflammatory cytokine treatments increase S305 phosphorylation on ERα

(A) Western blot analysis for ERα-S118-P, ERα-S305P, and ERα in MCF-7 cells treated with Veh, E2, IL1β or TNFα. (B) Western blot analysis for ERα and Histone H3 in MCF-7 cells (ERα+), CRISPR-cas9 knock out ERα MCF7 cells (ER-), and CRISPR-cas9 ESR1 MCF7 cells transfected with WT or S305A. (C) QPCR analysis for the indicated mRNAs in MCF7 cells expressing WT or S305A estrogen receptors treated with Veh, E2, IL1β or TNFα. Values are expressed as mean ± SEM.*p<0.05, (Student’s t-test) compared to MCF7 WT ERα cells. (D) ChIP-PCR of ERα-S305p recruitment in MCF-7 cells to the indicated genomic locus in the presence of Veh, E2 or IL1β. Values are expressed as mean ± SEM.*p<0.05, (Student’s t-test) compared to Veh sample.

Figure 4. IKKβ phosphorylation of S305 ERα is required for cytokine-dependent ERα activation

(A) Western blot analysis for ERα-S305-P and ERα in MCF-7 cells treated with Veh, or IL1β in the absence or presence of inhibitors for PKA, PAK1and IKKα/β. (B) Quantification of two independent western blot analysis as performed in figure 4A. Values are expressed as mean ± SEM.*p<0.05, (Student’s t-test) compared to IL1β sample. (C) Western blot analysis for ERα-S305-P and total ERα in MCF-7 cells treated with siRNA for Ctl, IKKα, or IKKβ and treated with Veh or IL1β. (D) Histogram of ERα ChIP-Seq signal in the presence of Veh, IL1β, IKK7, or a combination ofIL1β andIKK7. (E) ChIP-PCR of ERα recruitment to the MYC genomic locus in the presence of Veh, IL1β, or a combination of IL1β and IKK7. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) IL1β + IKK7 compared to IL1β sample. (F) Quantitative real-time PCR data for MYC mRNA in MCF-7 cells treated with Veh, IL1β, or a combination of IL1β and IKK7. Values are expressed as mean ± SEM.*p<0.05, (Student’s t-test) IL1β +IKK7 compared to IL1β sample. (G) Same as F, but cells were also treated with TOT or TNFα, as indicated.

Figure 5. Phosphorylation of ERα at S305 is required for cytokine-dependent ERα activation

(A) Cell proliferation assay for MCF7 cells expressing either WT or S305A estrogen receptors. Cells were treated with Veh, E2, E2 + TOT, E2 + TOT + IL1β for 6 days. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) compared to MCF7 WT ERα cells. (B) Heat map for mRNA-Seq expression of the 202 transcripts regulated in MCF7 cells treated with E2 that are sensitive to TOT. The mRNA expression is shown for MCF7 cells treated with Veh, E2, E2 + TOT, E2 + TOT + IL1β, and E2 + TOT + TNFα. (C) Pie graphs showing the percentage of E2 regulated genes sensitive to TOT treatment. The genes sensitive to TOT treatments are further stratified based on whether addition of IL1β or TNFα, restores expression mRNA levels to near E2 induced levels. (D) QPCR data for MYC mRNA in MCF-7 cells expressing either ERα or ERα-S305A treated with Veh, E2, E2 + TOT, and E2+ TOT+ IL1β. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) ERα compared to ERα S305 sample. (E) UCSC genome browser image for mRNA-Seq expression at the MYC genomic locus. (F) ChIP-PCR of ERα, Pol II, or SRC3 recruitment to the MYC genomic locus in the presence of Veh, E2, I L1 β, and TOT, or E2 + TOT + IL1β. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) compared to Veh treatment. #p<0.05, (Student’s t-test) compared to E2 treatment.

Figure 6. S305 ERα modulates the AF2 surface

(A) Crystal structure of the PA-E2-bound ERα LBD showing helix-1 (h1) and the PTM cassette of the hinge domain (residues 299-310) in coral, the PTM cassette-interacting region of h10 in yellow, and the coactivator-binding site, AF2 in cyan. (B) A closer view of panel A showing the network of hydrogen bonds (green dashed lines) that reinforces LBD interaction with the PTM cassette of the hinge domain. (C) Structure of the testosterone-bound AR LBD in complex with a BF3-binding compound (PDB 2YLP, PMID: 22047606). The hinge domain-interacting region of the ERα (panel B) corresponds to the BF3-binding site of AR. (D) Hinge domain interaction with the LBD alters the AF2 surface. Structures of the PA-E2-bound ERα LBD with (cyan) or without (gray) an SRC2 peptide (green) were superposed. (E) U2OS cells were transfected with either WT-ERα or ΔAB-ERα, lacking AF-1, and a 3xERE-luc reporter. The next day cells were treated with dose curves of TOT or ICI. Data is mean ± SEM, n=3 (F) Transient transfection in MCF7 ER- cells with WT or ΔAF1 estrogen receptors and 3ERE-Luiciferase, and treated with Veh, E2, IL1β, E2 + TOT, E2 + TOT + IL1β. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) compared to MCF7 WT ERα cells.

Figure 7. S305 ERα controls tamoxifen resistance through inter-domain communication

(A) Surface of the ERα LBD bound to an agonist, with h12 colored blue, and an LxxLL peptide from SRC2 colored yellow. 3UUD.PDB (B) The ERα LBD Δh12 shown as surface bound to a phage display derived corepressor peptide, colored red. (C) Model of S305-P making a salt bridge to R548 was generated from the TOT structure, 3ERT.pdb using VMD, as described in the methods. (D) The phosphate was removed from S305 in the model generated in Figure 7C, and both models were used for molecular dynamics simulations. (E) The S305 and S305-P models were subject to steered molecular dynamics simulations, where an increasing force was applied to h12 to pull it off of the CoRnR groove. (F) Transient transfection in MCF7 ER- cells with WT or F domain mutant estrogen receptors and 3ERE-Luiciferase, and treated with Veh, E2, IL1β, E2+TOT, E2+TOT+IL1β. Values are expressed as mean ± SEM. *p<0.05, (Student’s t-test) compared to Veh treatment (G) Structure of ERα with h12 trapped in the conformer seen with TOT. The CoRnR groove is shown as gray ribbons and h12 is shown as cyan α-carbon trace. Two point mutants are shown. From the PDB_REDO (Joosten et al., 2014) version of 3os8.pdb. (H) HepG2 cells transfected with a 3xERE-driven luciferase reporter and ERα WT or Leu372Ser/Leu536Ser expression plasmids, were stimulated with TOT or ICI. (I) Model of full-length ERα showing how p-305 orients the F domain towards the hinge, DNA – binding domain (DBD), and AF1. (J) Structure of the ERα-LBD showing how the agonist conformer orients the beginning of the F-domain (PDB: 4ZNY).