ERK/MAPK regulates ERRγ expression, transcriptional activity and receptor-mediated tamoxifen resistance in ER+ breast cancer

Abstract

Selective estrogen receptor modulators such as tamoxifen (TAM) significantly improve breast cancer-specific survival for women with estrogen receptor-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. The resistant phenotype is usually not driven by loss or mutation of the estrogen receptor; instead, changes in multiple proliferative and/or survival pathways over-ride the inhibitory effects of TAM. Estrogen-related receptor γ (ERRγ) is an orphan member of the nuclear receptor superfamily that promotes TAM resistance in ER+ breast cancer cells. This study sought to clarify the mechanism(s) by which this orphan nuclear receptor is regulated, and hence affects TAM resistance. mRNA and protein expression/phosphorylation were monitored by RT-PCR and western blotting, respectively. Site-directed mutagenesis was used to disrupt consensus extracellular signal-regulated kinase (ERK) target sites. Cell proliferation and cell-cycle progression were measured by flow cytometric methods. ERRγ transcriptional activity was assessed by dual-luciferase promoter-reporter assays. We show that ERRγ protein levels are affected by the activation state of ERK/mitogen-activated protein kinase, and mutation of consensus ERK target sites impairs ERRγ-driven transcriptional activity and TAM resistance. These findings shed new light on the functional significance of ERRγ in ER+ breast cancer, and are the first to demonstrate a role for kinase regulation of this orphan nuclear receptor.

Keywords: ER+ breast cancer; ERK/MAPK; estrogen-related receptor γ; tamoxifen; transcription.

Figure 1. ERRγ expression in ER+ breast tumors and breast cancer cells

A, Expression of ESRRG in ER+, TAM-treated breast tumors is associated with worse overall survival. HR = hazard ratio calculated by [19]. B, Relative expression of ESRRG normalized to RPLP0 in MCF7 and MCF7/RR cells by quantitative RT-PCR. Mean cycle threshold (C_T) values for parental (MCF7) cells are shown. Bars, n=3 replicates from a representative assay performed independently twice. Error, standard deviation (SD). **p≤0.01 for t test. C, Expression of ESRRG and RPLP0 in MCF7 and MCF7/RR cells by non-quantitative RT-PCR. Upper and lower arrowheads identify ESRRG and RPLP0 amplicons, respectively. Plasmid denotes ERRγ ORF cDNA clone. D, Expression of ERRγ protein in MCF7 and MCF7/RR cells by Western blot analysis. *denotes a non-specific band detected by the ERRγ antibody. Purified protein denotes human ERRγ transcript variant 2. β–actin = loading control.

Figure 2. Effect of MEK and ERK on ERRγ protein levels

A, Inhibition of ERK, but not p38 or JNK, reduces exogenous ERRγ expression. MCF7 cells were transiently transfected with the pSG5 empty vector or HA-ERRγ, then treated with DMSO vehicle, 5 μM U0126 (MEK inhibitor), 25 μM SB203580 (p38 inhibitor), or 10 μM SP600125 (JNK inhibitor) for 24 hours prior to lysis and Western blot analysis. Left panels show ERRγ (HA) levels, phosphorylated ERK (pERK), and total ERK from a representative experiment repeated at least twice. Right panels show total and phosphorylated p38 and JNK (p-p38 and pJNK, respectively) from the same experiment. β–actin = loading control. B, Constitutively active, mutant MEK enhances ERRγ protein levels. MCF7 cells were transiently co-transfected with HA-ERRγ and either MEKDD or additional pSG5 empty vector. *denotes the transfected MEKDD construct. β–actin = loading control. C, Exogenous, wild type ERK2 enhances ERRγ protein levels. MCF7 cells were transiently co-transfected with HA-ERRγ and either MEKDD, wild type HA-tagged ERK2, or additional pSG5 empty vector. *denotes the transfected MEKDD construct. The arrowhead and ^ denote transfected HA-ERRγ and HA-ERK2, respectively. β–actin = loading control. D, EGF-mediated enhancement of ERRγ protein levels is reversed by concomitant ERK inhibition. MCF7 cells were transiently transfected with HA-ERRγ, then cultured in low-serum conditions for 20 hours before treatment with DMSO vehicle, 25 ng/ml EGF, or 25 ng/ml EGF plus 5 μM U0126 for 2 hours. β–actin = loading control. E, Inhibition of ERK reduces exogenous ERRγ expression in a second ER+ breast cancer cell line. SUM44 cells were transiently transfected with the pSG5 empty vector or HA-ERRγ, then treated with DMSO vehicle or 5 μM U0126 for 22 hours. β–actin = loading control.

Figure 3. Contribution of serines 57,81, and 219 to ERRγ protein levels

A, Concomitant serine-to-alanine mutation at residues 57, 81, and 219 reduces basal HA-ERRγ levels. B, MEKDD fails to increase protein levels of S_57,81,219A HA-ERRγ. C, Erk inhibition does not reduce S_57,81,219A HA-ERRγ. MCF7 cells were transiently transfected and treated with 5 μM U0126 or DMSO vehicle for 24 hours where indicated (C) prior to lysis and Western blot analysis. β–actin = loading control. Densitometric values for the ratio of HA:β-actin are normalized to the level of wild-type receptor in the absence of treatment (1.0). Data are from representative experiments that were performed independently at least 3 times.

Figure 4. Effect of S_57,81,219A mutation on Tamoxifen response

A, Inhibition of BrdU incorporation by 4HT is reversed by wild type but not S_57,81,219A HA-ERRγ. MCF7 cells were transiently transfected as shown, treated with ethanol vehicle or 1 μM 4HT for 48 hours, then incubated with BrdU for an additional 18–20 hours before fixation and staining for HA and BrdU. Dashed line denotes BrdU incorporation in vehicle-treated cells (set to 1.0). Points, n=3 independent assays. Error, SD. *p<0.05 for post hoc Dunnet’s test following one-way ANOVA for pSG5 vs. wild type HA-ERRγ; n.s. denotes no statistical significance between pSG5 and S_57,81,219A mutant HA-ERRγ. For transfections with wild type or S_57,81,219A ERRγ, data are from HA-positive, FACS-sorted cells only. For transfections with the empty vector pSG5 control, data are from all cells in the population. B, 4HT-mediated induction of cell cycle inhibitors p21 and p27 is reversed by wild type but not S_57,81,219A HA-ERRγ, and the phosphorylation state of Rb is differentially affected by wild type vs. mutant receptor. MCF7 cells were transiently transfected as shown, then treated with ethanol vehicle or 2.5 μM 4HT for 21 hours prior to lysis and Western blot analysis. β–actin = loading control. Densitometric values for the ratio of the indicated proteins to β-actin in 4HT-treated conditions are normalized to the level of their expression in the absence of treatment (1.0) for each transfected construct; for pRb Ser780, the ratio of phosphorylated:total signal (which was then normalized to β-actin) is shown. Data are from a representative experiment that was performed independently 3 times.

Figure 5. Effect of S_57,81,219A mutation on ERRγ transcriptional activity

MCF7 and SUM44 cells were transiently co-transfected with pSG5 empty vector, wild type HA-ERRγ, or S_57,81,219A HA-ERRγ plus the ERRE- (A, D), ERE- (B, E), or ERRE/ERE-driven promoter-reporter luciferase construct (C, F) and the Renilla internal control for 24 (MCF7) or 48 hours (SUM44) prior to lysis and luciferase assay. Bars, luciferase:Renilla ratio of n=3 replicate wells from a representative assay performed 3 times independently. Error, SD. ***p≤0.001 for one-way ANOVA with post hoc Tukey’s tests.