Abstract P6-04-08: FOXA1 expression: regulated by EZH2 and associated with favorable outcome to tamoxifen in advanced breast cancer
Authors: Reijm EA , Helmijr JCA , Soler CMJ , Beekman R , Gerritse FL , Prager-van der Smissen WJC , Ruigrok-Ritstier K , van IJcken WFJ , Stevens MG , Smid M , Look MP , Meijer ME , Sieuwerts AM , Sleijfer S , Foekens JA , Berns EMJJ , Jansen MPHM 
Published: August 2014

Background: Enhancer of Zeste Homolog 2 (EZH2) is a member of the polycomb complex 2 and serves as a histone methyltransferase. Through trimethylation of histone 3 of lysine 27, EZH2 regulates ‘genome wide’ gene transcription silencing.

Downregulation of EZH2 is associated with increased expression of the estrogen receptor (ER) and favorable outcome to tamoxifen in metastatic breast cancer. The binding of ER to its target genes is enhanced by Forkhead Box A1 (FOXA1), a pioneer factor that binds to and opens chromatized DNA. The aim of this study is to investigate the potential association between EZH2 and FOXA1 and their relation with treatment outcome in patients with advanced breast cancer.

Experimental design: EZH2 and FOXA1 mRNA levels were analysed using available gene expression profile-data of 344 primary breast cancer specimens of lymph-node-negative (LNN) patients and in 109 ER-positive (ER+) tumors of patients with metastatic disease treated with first-line tamoxifen. To functionally analyze EZH2, cell lines were generated with different knockdown-constructs for EZH2, followed by evaluation of mRNA and protein expression levels and chromatin immunoprecipitation (ChIP) experiments combined with qPCR and/or next generation sequencing (NGS; ChIPseq).

Results: In the 344 LNN breast tumors, EZH2 was highly expressed in breast tumors of the basal subtype. In contrast, FOXA1 was hardly expressed in this subtype, but highly in the luminal A and B subtypes. FOXA1 was related to a prolonged time to progression (TTP) after tamoxifen (HR = 0.51 [0.35–0.74], P &lt; 0.001) in 59 LNN patients with ER+ primary tumors and advanced disease. This positive association between FOXA1 and TTP could be confirmed in an independent set of 109 ER+ primary tumors of patients with metastatic disease treated with tamoxifen (HR = 0.56 [0.37–0.85] p = 0.006).

We obtained 50–70% EZH2 knockdown in the breast cancer cell lines MCF7 (ER+/PR+/FOXA1+), SUM52PE (ER+/PR−/FOXA1+), and MM231 (ER−/PR−/FOXA1−). ChIP followed by qPCR demonstrated higher levels of H3K27 trimethylation at the ER-locus in the parental cell lines compared to the EZH2-knockdowns for MM231 and at the PR-locus for SUM52PE and MM231. ChIPseq evaluation in the EZH2 knockdown of MM231 identified 200 loci with a significant decrease in read numbers for H3K27me3 compared to the parental. Interestingly, amongst these 200 loci the FOXA1-locus was identified. These results suggest FOXA1 as an EZH2-target since its expression was not seen in the parental MM231.

Conclusion: High EZH2 and low FOXA1 mRNA levels are associated with poor clinical outcome for tamoxifen therapy of advanced breast cancer. Functional studies indicate that EZH2 might regulate the expression of FOXA1. These results suggest that EZH2 and/or FOXA1 could be used as a predictive marker to identify patients at risk for tamoxifen therapy failure and possibly as a target for therapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-08.