Down Regulation of EZH2 Is Associated with ESR1 Upregulation and Response to Endocrine Therapy in Breast Cancer.
Authors: Reijm E. , Ruigrok-Ritstier K. , van Staveren I. , Sieuwerts A. , Look M. , Meijer-vanGelder M. , Sleijfer S. , Foekens J. , Berns P. , Jansen M. 
Published: December 2010

Background: We have previously identified a gene signature for resistance to first-line tamoxifen therapy in advanced breast cancer. One of these genes is Enhancer of Zeste Homolog 1 (EZH1), a member of the EZH family of which EZH2 has been identified as being prognostic. Both genes are involved in transcriptional control and epigenetic memory maintenance and act as polycomb repressors. The aim of this study is to investigate these genes for their predictive value and, if associated with clinical outcome, to evaluate the functional role in treatment response in vitro.Experimental design: Expression levels of EZH1 and EZH2 expression were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. Functional studies were done in MCF7, a human estrogen sensitive breast cancer cell line. Expression levels of EZH2 were downregulated with siRNAs, and changes in cell numbers were determined treating with (the anti-estrogen) ICI164.384. In addition, alterations in expression levels of putative downstream genes, including the estrogen receptor (ESR1), were measured.Results: When analysed as continuous variable in univariate analysis, only EZH2 was significantly associated with therapy resistance and a shorter Progression Free Survival (PFS) in 278 patients with advanced disease treated with first-line tamoxifen monotherapy. In univariate analysis the tertile with highest EZH2 levels was associated with clinical benefit (OR=0.48, 95%CI: 0.26-0.89; P=0.02) and with PFS (HR=1.80, 95%CI: 1.32-2.46;P&lt;0.001). In multivariate analysis including traditional predictive factors, highest EZH2 levels were independently related with a shorter PFS (HR=1.58, 95%CI: 1.10-2.26; P=0.01). Despite, EZH2 mRNA levels were not significantly correlated with metastasis free survival (HR=1.14, 95%CI: 0.98-1.32; P=0.10) in 688 lymph node negative patients who did not receive adjuvant systemic therapy.EZH2 silencing in MCF7 caused a significant decrease in cell numbers (38%, range 17-53%, N=3) whereas ICI164.384 treatment resulted in decrease of 25% (range 12-30%, N=3) compared to controls. Combining EZH2 silencing with ICI-treatment reduced cell numbers with 67% (range 54-75%, N=3, P&lt;0.001). Interestingly, downregulation of EZH2 was associated with an almost 2-fold upregulation of ESR1 (N=6, P=0.001). This inverse relation has been confirmed in additional “in silico” analyses.Conclusion: High levels of EZH2 are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. EZH2 silencing in MCF7 cells inhibits cell proliferation, it upregulates ESR1 levels and increases sensitivity to ICI164.384. Further validation is needed to confirm that silencing of EZH2 leads to an upregulation of the ESR1 and as a consequence to a better response to anti-estrogens. This finding sheds new light on the involvement of EZH2 in anti-estrogen treatment offering possibilities for novel management strategies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5131.