Targeting the enhancer of zeste homologue 2 in medulloblastoma

Abstract

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.

Figure 1

EZH2 gene expression microarray data in medulloblastoma. A. Expression of EZH2 mRNA in normal cerebellum specimens (CB), classic medulloblastoma (MED) patient samples (n = 21), and large cell anaplastic medulloblastoma (LCA MED)(n = 7). B. EZH2 mRNA expression in medulloblastoma samples sorted by genomic categories. C. Quantification of genomic gain of chromosome 7 in medulloblastoma samples. D. Expression of EZH2 mRNA in common medulloblastoma cell lines is increased consistent with patient tumor samples.

Figure 2

Effect of EZH2 depletion on cell viability and clonogenic index. A. Daoy and ONS-76 cells were transfected with plasmid vector control pSIF and shEZH2 plasmid. Cell viability was evaluated by Guava EasyCyte Plus flow cytometer using ViaCount reagent. Viability was normalized to untransfected cells. EZH2 knockdown significantly impaired cell viability in vitro in both cell lines (p < 0.001). B. Colony focus assay was done in both Daoy and ONS-76 cells lines after plasmid transfection. Colony numbers were significantly reduced after transfection with shEZH2 in both cell lines (p < 0.001 in Daoy cells and p < 0.01 in ONS-76 cells). Colony counts were done in triplicate of 3 independents experiments.

Figure 3

Functional relevance of EZH2 in tumorigenesis and signaling pathways. A. Inhibition of EZH2 activity reduces the tumor sphere forming ability of medulloblastoma cells. B. The number of spheres is significantly decreased in cells with shEZH2 knockdown compared to controls (p < 0.008). In addition the sphere diameter (C) was significantly smaller in cells with shEZH2 knockdown compared to controls (p < 0.0003). D. EZH2 knockdown by shRNA leads to significantly decreased E2F1, Myc, and Sox2 activity (p < 0.001, p < 0.005 and p <0.007 respectively). Luciferase activity was measured by the Dual Luciferase Assay system (Promega). E. Inhibition of EZH2 leads to significantly decreased expression of progenitor cell markers Nestin, Nanog, Sox2 and Myc in Daoy tumor spheres.

Figure 4

A. Effect of DZNep treatment on cell proliferation. Pharmacologic depletion of EZH2 reduces medulloblastoma cell proliferation and altered the growth rate of cells. Daoy and ONS-76 medulloblastoma cells were treated with 0.5, 2.5, and 5 μM of DZNep. Both cell lines showed a dose-dependent growth inhibition with DZNep treatment. B. Colony formation assay. DZNep efficiently decreased the clonogenic potential at concentrations starting from 0.5 μM (p < 0.005). The numbers of colonies decreased with increasing DZNep dose. At the highest concentration of DZNep (5 μM), colony formation was reduced up to 30% in ONS-76 cells and over 70% in Daoy cells compared to untreated controls. C. Effect of combination treatment with shEZH2 and DZNep on H3K27me3 levels. Daoy and ONS-76 cell lines were first treated with shEZH2 followed by treatment with either 0.5 μM or 5 μM DZNep. For comparison, these cells were treated with either shEZH2 or DZNep alone. Western blot analysis was performed with 50 μg total cell lysates from each sample, using antibodies directed against H3K27me3 (Cell Signaling. 1:1000) and actin (Santa Cruz, 1:1000). Cells treated with either shEZH2 or DZNep (5 μM) alone showed a decrease in H3K27me3 levels. Interestingly, treatment with both knocked down H3K27me3 levels even further.

Figure.5

Knockdown of EZH2 induces apoptosis. Daoy cells were treated with control plasmid (pSIF), shEZH2 plasmid, or DZNep (0.5 μM). Following treatment, cells were stained for Annexin, a cell surface marker of apoptosis. Annexin-positive cells were measured by Guava Nexin Assay. DZNep significantly induced apoptosis in non-transfected and shEZH2-transfected Daoy cells (n=4, p < 0.0005).

Figure 6

Anchorage-independent colony formation in soft agar. C17.2 cells were transfected with REST or a combination of REST and shEZH2, and the ability of cells to form colonies in soft agar measured. Unmodified C17.2 cells do not grow colonies while REST transformed C17.2 cells exhibit robust colony formation in soft agar. Inhibition of EZH2 reduced the ability of REST to transform cells (p < 0.01). Inhibition with shRNA and 0.5μM DZNep was most potent in inhibiting transformation of C17.2 cells (p < 0.001).