ERRγ target genes are poor prognostic factors in Tamoxifen-treated breast cancer

Abstract

Background: One-third of estrogen (ER+) and/or progesterone receptor-positive (PGR+) breast tumors treated with Tamoxifen (TAM) do not respond to initial treatment, and the remaining 70% are at risk to relapse in the future. Estrogen-related receptor gamma (ESRRG, ERRγ) is an orphan nuclear receptor with broad, structural similarities to classical ER that is widely implicated in the transcriptional regulation of energy homeostasis. We have previously demonstrated that ERRγ induces resistance to TAM in ER+ breast cancer models, and that the receptor's transcriptional activity is modified by activation of the ERK/MAPK pathway. We hypothesize that hyper-activation or over-expression of ERRγ induces a pro-survival transcriptional program that impairs the ability of TAM to inhibit the growth of ER+ breast cancer. The goal of the present study is to determine whether ERRγ target genes are associated with reduced distant metastasis-free survival (DMFS) in ER+ breast cancer treated with TAM.

Methods: Raw gene expression data was obtained from 3 publicly available breast cancer clinical studies of women with ER+ breast cancer who received TAM as their sole endocrine therapy. ERRγ target genes were selected from 2 studies that published validated chromatin immunoprecipitation (ChIP) analyses of ERRγ promoter occupancy. Kaplan-Meier estimation was used to determine the association of ERRγ target genes with DMFS, and selected genes were validated in ER+, MCF7 breast cancer cells that express exogenous ERRγ.

Results: Thirty-seven validated receptor target genes were statistically significantly altered in women who experienced a DM within 5 years, and could classify several independent studies into poor vs. good DMFS. Two genes (EEF1A2 and PPIF) could similarly separate ER+, TAM-treated breast tumors by DMFS, and their protein levels were measured in an ER+ breast cancer cell line model with exogenous ERRγ. Finally, expression of ERRγ and these two target genes are elevated in models of ER+ breast cancer with hyperactivation of ERK/MAPK.

Conclusions: ERRγ signaling is associated with poor DMFS in ER+, TAM-treated breast cancer, and ESRRG, EEF1A2, and PPIF comprise a 3-gene signaling node that may contribute to TAM resistance in the context of an active ERK/MAPK pathway.

Figure 1

Selected clinical studies have similar proportions of distant metastasis-free survival (DMFS). Survival data were plotted using the Kaplan-Meier estimator, which show non-significant differences in DMFS data amongst the 3 studies. Log-rank p = 0.09.

Figure 2

ERRγ and its target genes predict poor DMFS in ER+, but not ER-, breast cancer. Gene symbols for 35 of the 37 ERR target DEGs (2 were not annotated) were entered into KM Plotter and used to classify DMFS data from women with ER+, TAM-treated breast cancer (A, n = 504, HR = 1.75, log-rank p = 0.006) or ER-, chemotherapy-treated breast cancer (B, n = 53, HR = 0.35, log-rank p = 0.024).

Figure 3

Predictive value and expression of ERRγ target genes EEF1A2 and PPIF in ER+ breast cancer. A, The 3-gene signature of ESRRG, EEF1A2, and PPIF predicts poor DMFS in ER+, TAM-treated breast cancer using KM Plotter (n = 504, HR = 1.57, log-rank p = 0.022). B, Expression of EEF1A2 and PPIF protein in MCF7 cells transiently expressing exogenous hemagglutinin (HA-) tagged ERRγ. β-actin serves as the loading control. Relative expression of EEF1A2 (1.57) and PPIF (1.07) in ERRγ-transfected cells vs. empty vector control (1.0) was analyzed using NIH Image J.

Figure 4

Expression of ESRRG, EEF1A2, and PPIF correlates with ERK/MAPK activation status in ER+ breast cancer cells. Gene expression data from Rae et al. obtained from ONCOMINE were analyzed for ESRRG (A), EEF1A2 (B), and PPIF (C) in n = 3 replicates per cell line. Mann Whitney rank-sum p < 0.05 (*).