EZH2 Phosphorylation Promotes Self-Renewal of Glioma Stem-Like Cells Through NF-κB Methylation

Abstract

Cancer stem-like cells (CSCs) is a cell population in glioma with capacity of self-renewal and is critical in glioma tumorigenesis. Parallels between CSCs and normal stem cells suggest that CSCs give rise to tumors. Oncogenic roles of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) have been reported to play a crucial role in glioma tumorigenesis. Herein, we focus on mechanistic contributions of downstream molecules to maintaining stemness of glioma stem-like cells (GSCs). Transcriptional factor, NF-κB, co-locates with MELK/EZH2 complex. Clinically, we observe that the proportion of MELK/EZH2/NF-κB complex is elevated in high-grade gliomas, which is associated with poor prognosis in patients and correlates negatively with survival. We describe the interaction between these three proteins. Specifically, MELK induces EZH2 phosphorylation, which subsequently binds to and methylates NF-κB, leading to tumor proliferation and persistence of stemness. Furthermore, the interaction between MELK/EZH2 complex and NF-κB preferentially occurs in GSCs compared with non-stem-like tumor cells. Conversely, loss of this signaling dramatically suppresses the self-renewal capability of GSCs. In conclusion, our findings suggest that the GSCs depend on EZH2 phosphorylation to maintain the immature status and promote self-proliferation through NF-κB methylation, and represent a novel therapeutic target in this difficult to treat malignancy.

Keywords: EZH2 phosphorylation; NF-κB methylation; cancer stem-like cells; glioma; self-renewal.

Figure 1

MELK, EZH2, and NF-κB are highly expressed in high-grade glioma. (A) Representative IHC panels across four grades of glioma and adjacent normal tissues (200×) showing the expression of MELK, EZH2, and NF-κB. (B) Pearson analysis showing the association of strong MELK/EZH2/NF-κB expression with high Ki-67 index (Pearson r = 0.89, 0.84, and 0.83, respectively). (C) Up, immunoblotting analysis showing protein levels of MELK, EZH2, and NF-κB. Down, qPCR analysis displaying the mRNA level of melk, ezh2, and nf-κb in surgical glioma samples. The experiments were repeated three times. (D–F) The postsurgical OS and PFS curves evaluated by Kaplan-Meier methods among GBM patients showing poor survival was associated with high expression of MELK, EZH2, or NF-κB (low, the IHC score of MELK, EZH2, or NF-κB was <1.2, 1.5, and 1.2, respectively; high, the IHC score was more than 1.3, 1.6, and 1.3, respectively). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

MELK, EZH2, and NF-κB are enriched in GSCs. (A) Frozen sections from human GBM samples immunostained for MELK, EZH2, and Nestin (400×) displaying the co-location of MELK/EZH2 and Nestin. (B) Immunofluorescence staining against NF-κB and CD44 in human GBM (400×) showing the co-location of CD44 and NF-κB. The white arrow represented one cell which was double positive for NF-κB and CD44. (C) Cells sorted from the fresh human GBM by FACS utilizing CD133/CD44 surface markers were immunostained for MELK, EZH2, and NF-κB. (D) qPCR analysis showing the high mRNA levels of melk, ezh2, and nf-κb in GSCs. The experiments were repeated three times. **p < 0.01, ***p < 0.001.

Figure 3

EZH2 is phosphorylated by MELK in GSCs. (A) Immunoblotting analysis showing higher expression of MELK, EZH2, and NF-κB proteins in GSCs compared to that in NSCs. (B) Immunoblotting analysis showing endogenous MELK bound to EZH2 in GSCs (9,982 and 10,012) following co-IP with MELK antibody. Western blot showing the binding of endogenous EZH2 to MELK in GSCs following co-IP with EZH2 antibody. (C) Western blot showing phosphorylation of EZH2 after IP of the lysates with EZH2 antibody in GSCs (9,982) cultured with or without serum. Actin was used as the loading control. (D) IP and western blot showing the impaired p-EZH2 activity based on the low expression of H3K27me in GSCs (9,982) infected with shMELK. (E) IP and western blot showing impaired p-EZH2 activity based on the low expression of H3K27me in GSCs (9,467) after treatment of OTSSP167. The experiments were repeated three times.

Figure 4

NF-κB is methylated by EZH2 in GSCs. (A) Western blot analysis showing binding of endogenous EZH2 to NF-κB in GSCs (9,982) following co-IP with EZH2 antibody. Western blot analysis showing binding of endogenous NF-κB to EZH2 after co-IP with NF-κB antibody. (B) Immunoblotting analysis showing the expression of Methyl K after IP of the lysates with NF-κB antibody. (C) Immunoblotting assay showing the expression of the Methyl K following IP of the lysates from GSCs (9,982) receiving the shEZH2 or scrambled shRNA infection with NF-κB antibody. (D) Immunoblotting assay showing the expression of Methyl K following IP of the lysates from GSCs (9,467) with NF-κB antibody after GSCs receiving the treatment of DZNep. (E) Responsive luciferase reporter assay showing the inhibited methylation activity of NF-κB in shEZH2 expressing and DZNep treatment group. (F) qPCR analysis determining the decreased mRNA levels of IL-6, relb, and tnf in EZH2 deficient GSCs. The experiments were repeated three times. *p < 0.05, **p < 0.01. shE#2, shEZH2#2; shE#3, shEZH2#3.

Figure 5

Stemness maintenance is required for NF-κB activity. (A) Colony formation assay showing the decreased proliferation of GSCs sorted from U87 cells after EZH2/NF-κB inhibition. (B) qPCR analysis determining the mRNA expression of sox2, olig2, and nestin. (C) The growth curves showing the growth rate of xenografts derived from the primary GSCs expressing shEZH2 and shNF-κB. (D) Immunoblotting assay showing the expression of Methyl K following IP of GSCs lysates with NF-κB antibody in the shEZH2 expressing GSCs derived tumors. (E) IHC staining showing the expression of P65 in shEZH2 and shNF-κB infected GSCs derived tumors (200×). (F) H.E. staining showing the Grade 2 morphology in the EZH2 and NF-κB knockdown group. Immunostaining images showing the expression of Ki-67, Nestin and GFAP in subcutaneous xenograft samples derived from scrambled shRNA, shEZH2, and shNF-κB GSCs. The experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001. shE, shEZH2; shN, shNF-κB.

Figure 6

MELK deficiency suppresses the function of NF-κB. (A) Immunoblotting analysis showing the expression of Methyl K following IP of GSCs lysates with NF-κB antibody after GSCs (10,012) receiving shMELK infection. (B) Immunoblotting analysis presenting the expression of Methyl K following IP of GSCs lysates with NF-κB antibody after GSCs (10,012) receiving the treatment of OTSSP167. (C) Responsive luciferase reporter assay showing the inhibited NF-κB activity in MELK deficiency condition. (D) CHIP-qPCR analysis showing the mRNA levels of IL-6, mcp-1, and Alox5 after MELK inhibition. (E) IHC staining showing the expression of P65 in shMELK infected and OTSSP167 treated GSCs derived tumors (200×). (F) Immunoblotting assay showing the suppressed expression of Methyl K following IP of xenografts lysates with NF-κB antibody in shMELK expressing GSCs derived tumors. The experiments were repeated three times. (G) The schematic model illustrates the mechanism that MELK-mediated EZH2 phosphorylation methylates NF-κB in GSCs (P, phosphorylation; Me, methylation). *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: shM#1, shMELK#1; shM#2, shMELK#2.