High miR-26a and low CDC2 levels associate with decreased EZH2 expression and with favorable outcome on tamoxifen in metastatic breast cancer

Abstract

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.

Fig. 1

Kaplan–Meier curves of TTP as a function of miR-26a, EZH2, CCNE1, and CDC2 expression levels. Patients were evenly divided into three groups according to their expression levels. Curves were generated as function of low, intermediate, and high miR-26a, EZH2, CCNE1, and CDC2 expression levels. Patients at risk at different time points are indicated

Fig. 2

Global testing approach result of the cyclins and cell cycle regulation pathway. This pathway was overlapping between miR-26a- and EZH2-related pathways. Red bars illustrate high expression levels of the pathway gene in samples with high miR-26a or EZH2 levels, whereas green bars indicate high expression levels in samples with low miR-26a or EZH2 levels. The number of vertical markers in a bar indicates the significance and the height of a bar the contribution of a gene to the pathway. The continuous line shows the threshold for significance; bars with more than two lines above this border are significantly (P < 0.05) differentially expressed genes within the pathway, which are also indicated with an asterisk. Only CCNE1 and CDC2 showed significant associations with both miR-26a and EZH2

Fig. 3

The regulatory network of EZH2. A model for the modulation of the expression and activity of EZH2 based on our results and available data in the literature. Binding of miR-101 and miR-26a to the 3′-UTR blocks transcription of EZH2 [10, 11]. Our data linked expression levels of miR-26a and EZH2 by the GTA of pathways to the cyclins and cell cycle regulation pathway with two significant genes [CCNE1 and CDC2 (CDK1)]. CDC2 (CDK1) and CDK2 activate EZH2 through the phosphorylation of its Thr350 residue [–36]. Our study shows that, in breast cancer, miR-26a and CDC2 might be involved in the regulation EZH2 expression and activity, respectively, and as a result associate with response to tamoxifen