EZH2 mRNA transcript and protein levels are elevated in breast cancer. (a) Quantitative SYBR green RT-PCR of EZH2 transcript in laser-capture microdissected normal and breast cancer epithelia. Each sample was performed in duplicate, and a ratio was calculated relative to the housekeeping gene hydroxymethylbilane synthase (HMBS). (b) Immunoblot analysis of EZH2 and EED in breast tissue extracts. Metastatic (Met) prostate cancer was used as a positive control. β-Tubulin was included as a loading control. (c) Representative breast tissue sections stained with an antibody to EZH2. (Left) Normal breast epithelia (open triangle) and adjacent intravascular breast cancer emboli (filled triangle). (Center) An invasive breast cancer expressing high levels of EZH2. (Right) An invasive breast cancer expressing low levels of EZH2. (d) Tissue microarray analysis of EZH2 expression in breast cancer progression. Tumor specimens were stratified into high EZH2 expressors (filled bars, scored 3 or 4) and low EZH2 expressors (open bars, scored 1 or 2). The y axis represents the percentage of patients in each category.

High EZH2 levels are associated with aggressive breast cancer. (a) Kaplan–Meier analysis of metastasis-free survival according to EZH2 mRNA transcript levels as measured using DNA microarrays by van't Veer et al. (30). Kaplan–Meier analysis of disease-specific (b) and overall (c) survival according to EZH2 protein levels as assessed by immunohistochemical analysis. Patients grouped on the basis of high (+) or low (–) EZH2 expression levels. P values were calculated by using the log-rank test.

Anchorage-independent growth mediated by EZH2. (a) Immunoblot analysis of breast cell line H16N2 infected with adenovirus encoding EZH2 or EZH2 Δ SET mutant. (b) Ectopic overexpression of EZH2 does not significantly enhance growth of breast epithelial cells in culture. H16N2 cells were infected with EZH2 adenovirus and controls, and cells were counted at indicated time points. LacZ adenovirus and vector adenovirus were used as controls. (c) EZH2 expression enhances anchorage-independent growth in vitro. H16N2 cells were infected with EZH2, EZH2 Δ SET, or vector adenoviruses. Anchorage-independent growth was determined by assaying colony formation in soft agar as described in Methods. After 25 days, the plates were stained and photographed. (d) Quantitation of soft agar colonies from experiments described in c. Colonies from three wells were quantitated for each condition. (e) EZH2 induces HDAC activity in breast epithelial cells. HDAC activity was measured in extracts from H16N2 cells infected with indicated viruses ± treatment with TSA (1.0 μM). As indicated by the manufacturer (Biomol), nuclear extracts from HeLa cells were used as positive controls. Extract 2 had 2-fold more HDAC activity than Extract 1. AFU, arbitrary fluorescence units.

EZH2 orchestrates cell invasion both in vitro and in vivo.(a) A reconstituted basement membrane invasion chamber assay (Chemicon) was used to assess breast epithelial cell lines infected with EZH2 and control adenoviruses. Representative fields of invaded and stained cells are shown. (b) The numbers of invaded cells were counted in six fields, and the mean values were determined. Quantitation by colorimetry (absorbance at 560 nm) is shown in Inset.(c) EZH2-mediated invasion of SU-ECM. H16N2 cells were infected with EZH2, EZH2 Δ SET, or control adenoviruses. (d) EZH2 overexpression mediates invasion of breast epithelial cells in a CAM assay. (Upper) CAM tissues stained with hematoxylin/eosin. Arrows indicate the cells that have invaded the CAM. Because cells were labeled with Fluoresbrite carboxylated polystyrene nanospheres, they could also be visualized by fluorescence (Lower).