Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells

Abstract

Introduction: Overexpression of the Polycomb repressive complex 2 (PRC2) subunit Enhancer of Zeste 2 (EZH2) occurs in several malignancies, including prostate cancer, breast cancer, medulloblastoma, and glioblastoma multiforme. Recent evidence suggests that EZH2 may also have a role in rhabdoid tumors. Atypical teratoid/rhabdoid tumor (ATRT) is a rare, high-grade embryonal brain tumor that occurs most commonly in young children and carries a very poor prognosis. ATRTs are characterized by absence of the chromatin remodeling protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes, we investigated the role of EZH2 in ATRT.

Methods: Microarray analysis was used to evaluate expression of EZH2 in ATRT tumor samples. We used shRNA and a chemical inhibitor of EZH2 to examine the impact of EZH2 inhibition on cell growth, proliferation, and tumor cell self-renewal.

Results: Here, we show that targeted disruption of EZH2 by RNAi or pharmacologic inhibition strongly impairs ATRT cell growth, suppresses tumor cell self-renewal, induces apoptosis, and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity, we found the cyclin D1-E2F axis to be repressed after EZH2 depletion in ATRT cells.

Conclusions: Our observations provide evidence that EZH2 disruption alters cell cycle progression and may be an important new therapeutic target, particularly in combination with radiation, in ATRT.

Fig. 1.

EZH2 expression in ATRT. (A) Scatter Plot of EZH2 expression in 18 ATRT patient tissues, compared with normal brain (cortex and cerebellum). (B) GSEA of EZH2 responsive genes in ATRT, compared with normal brain. Gene sets from three individual studies were evaluated. The enhancement score was significantly negative in all 3 cases. (C) Heat map of the top 25 most suppressed genes in ATRT, compared with normal brain. (D) Expression of EZH2 protein and SMARCB1 protein in normal brain, control GBM cell line U251G, and ATRT cell lines (BT12, BT16, and UPN 737).

Fig. 2.

EZH2 is required for ATRT cell growth. (A) RNAi-mediated inhibition of EZH2 suppresses ATRT cell proliferation. (B and C) EZH2 inhibition suppresses ATRT cell clonogenic survival. (D) RNAi-mediated inhibition of EZH2 results in decreased EZH2 protein compared to PSIF vector control. (E and F) RNAi-mediated inhibition of EZH2 results in senescence of ATRT cells. Senescent cells shown by red arrows.

Fig. 3.

EZH2-regulated signaling pathways in ATRT. (A) Activity of 45 transcription factors as measured by promoter luciferase activity as a measure of signaling activity in response to RNAi inhibition of EZH2. (B and C) EZH2 inhibition suppresses activity of E2F and c-Myc. (D) Western blot analysis of c-Myc–cyclin D–RB pathway.

Fig. 4.

Chemical inhibition of EZH2 in ATRT. (A) DZNep inhibits clonogenic potential of ATRT cells. Quantification of colony formation with representative wells in control and DZNep-treated cells (*P < .01, **P < .001). (B) DZNep induces a G2/M arrest in BT12 cells. Quantification of cell cycle fractions with representative flow plots. (C) Inhibition of EZH2 induces apoptosis in BT12 ATRT cells. Representative flow plots of Annexin V expression and quantification of apoptosis in control and DZNep-treated cells.

Fig. 5.

DZNep induces radiation sensitivity in ATRT cells. Line graph plotting the surviving fraction of (A) BT16 cells or (B) UPN 737 cells given different radiation doses after 24 h of treatment with DZNep or the vehicle control. Blue line represents control treated cells. Red line represents cells treated with 0.5 µM DZNep. Bar graphs show the quantification of the surviving fraction following 2 Gy irradiation.

Fig. 6.

DZNep suppresses the tumor sphere formation of ATRT cells. (A) Representative images of UPN 737 cells grown as tumor spheres in control or DZNep-treated conditions. (B) Quantification of tumor sphere number after 5 days of DZNep treatment (*P < .01). (C) Quantification of tumor sphere size in DZNep-treated cells compared to control (*P < .01). (D) Representative images of secondary tumor spheres in DZNep-treated UPN 737 cells. DZNep-treated cells did not form any spheres >50 cells. Sphere size of DZNep-treated cells was significantly smaller (*P < .01, **P < .001).