EZH2 suppresses endogenous retroviruses and an interferon response in cancers

Abstract

Ewing sarcoma is an aggressive cancer of bone and soft tissue in children. It is characterized by the chromosomal translocation between EWS and an Ets family transcription factor, most commonly FLI1. We recently reported that Ewing sarcoma depends on the autocrine signaling mediated by a cytokine, NELL2. NELL2 signaling stimulates the transcriptional output of EWS-FLI1 through the BAF chromatin remodeling complexes. While studying the impact of NELL2 silencing on Ewing sarcoma, we found that suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons, an interferon response, and growth arrest. We determined that a histone methyltransferase, EZH2, is the critical downstream target of NELL2 signaling in suppressing ERVs, LINE-1, an interferon response, and growth arrest. We show that EZH2 inhibitors induce ERVs, LINE-1, and an interferon response in a variety of cancer types. These results uncover the role for NELL2-EZH2 signaling in suppressing endogenous virus-like agents and an antiviral response, and suggest the potential utility of EZH2 inhibitors in enhancing anti-tumor immunity.

Keywords: EZH2; Ewing sarcoma; NELL2; endogenous retroviruses; interferon response.

Figure 1. Suppression of NELL2 signaling induces an interferon response in Ewing sarcoma.

(A–E) siRNA-mediated silencing of NELL2 induces an interferon response in Ewing sarcoma cells. Ewing sarcoma cells were transfected with NELL2 siRNA pool or control siRNA pool and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by quantitative RT-PCR (left) and immunoblotting (right). ^*p < 0.05 compared with control siRNA transfected cells. (A) A673 cells, (B) EW8 cells, (C) TC32 cells, (D) TC71 cells, and (E) SK-N-MC cells. (F) shRNA-mediated silencing of NELL2 induces an interferon response in A673 cells. NELL2 was silenced by lentiviral expression of shRNA in A673 cells and the expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. ^*p < 0.05 compared with control shRNA expressing cells. (G) Extracellular NELL2 signals to suppress an interferon response in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of IFNB1, Mx1, OAS1, p21, and NELL2 was analyzed by qRT-PCR. ^*p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. ^**p < 0.05 compared with cells transfected with NELL2 siRNA and left untreated or treated with recombinant NELL2.

Figure 2. The CD133^low population displays an interferon response, which can be suppressed by exogenous CD133.

A673 cells (A) and NCH-EWS-1 cells (B) were incubated with anti-CD133 (AC133) antibody and were sorted into the CD133^high and CD133^low populations. Part of the CD133^low population was infected with CD133-expressing lentivirus and selected with puromycin. The expression of IFNB1, Mx1, OAS1, p21, NELL2, and CD133 was analyzed by qRT-PCR (left) and immunoblotting (right). ^*p < 0.05 compared with the CD133^high population and with the CD133^low population expressing exogenous CD133.

Figure 3. Suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons in Ewing sarcoma.

(A) Extracellular NELL2 signals to suppress ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of the indicated genes was analyzed by qRT-PCR. NELL2 silencing induced the expression of ERVs and LINE-1, which was suppressed by recombinant NELL2. ^*p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. ^**p < 0.05 compared with cells transfected with NELL2 siRNA and left untreated or treated with recombinant NELL2. (B) The CD133^low population expresses high levels of ERVs and LINE-1, which can be suppressed by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of the indicated genes by qRT-PCR. ^*p < 0.05 compared with the CD133^high population and with the CD133^low population expressing exogenous CD133.

Figure 4. EZH2 suppresses ERVs, LINE-1, and an interferon response downstream of NELL2 signaling.

(A) Extracellular NELL2 signals to maintain histone H3K27me3 modification in ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. Histone H3K27me3 modification in ERVs and LINE-1 was analyzed by chromatin immunoprecipitation. ^*p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (B) EZH2 expression is regulated by NELL2 signaling in Ewing sarcoma. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of EZH2 was analyzed by qRT-PCR. NELL2 silencing resulted in reduced EZH2 expression, which was restored by recombinant NELL2. ^*p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (C) The CD133^low population displays low EZH2 expression, which was rescued by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of EZH2 by qRT-PCR. ^*p < 0.05 compared with the CD133^high population and with the CD133^low population expressing exogenous CD133. (D) NELL2 signaling maintains EZH2 binding to ERVs and LINE-1. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. The binding of EZH2 to ERVs and LINE-1 was assessed by chromatin immunoprecipitation. NELL2 silencing reduced EZH2 binding to ERVs and LINE-1, which was restored by recombinant NELL2. ^*p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (E) EZH2 suppresses an interferon response induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The expression of the indicated genes was analyzed by qRT-PCR. ^*p < 0.05 compared with cells transfected with control siRNA and empty vector and with cells transfected with EZH2. (F) EZH2 suppresses growth arrest induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. Cell proliferation was assessed using the IncuCyte live-cell imaging system. (G) EZH2 expression does not affect siRNA-mediated NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The protein levels of NELL2, IFNB1, Mx1, OAS1, and p21 were assessed by immunoblotting. Tubulin serves as a loading control.

Figure 5. EPZ005687 induces ERVs, LINE-1, and an interferon response in transformed cells.

A673, RD, RMS13, SK-N-BE(2), Y79, WERI-Rb-1, 293T, HCT116, IMR-90, human umbilical vein endothelial cells (HUVEC), and ARPE-19 cells were treated with the indicated concentration of EPZ005687 for 72 hours and the expression of the indicated genes was analyzed by qRT-PCR. ^*p < 0.05 compared with untreated cells.

Figure 6. GSK343 induces ERVs, LINE-1, and an interferon response in transformed cells.

A673, RD, RMS13, SK-N-BE(2), Y79, WERI-Rb-1, 293T, HCT116, IMR-90, human umbilical vein endothelial cells (HUVEC), and ARPE-19 cells were treated with the indicated concentration of GSK343 for 72 hours and the expression of the indicated genes was analyzed by qRT-PCR. ^*p < 0.05 compared with untreated cells.