EZH2 suppression in glioblastoma shifts microglia toward M1 phenotype in tumor microenvironment

Yatao Yin,Yun Xu,Xiangpen Li,Ying Peng,Bo Huang,Shuwei Qiu

doi:10.1186/s12974-017-0993-4

Abstract

BackgroundGlioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis. Enhancer of zeste homolog 2 (EZH2) is abundant in GBM samples and cell lines and is involved in GBM proliferation, cell cycle, and invasion, whereas its association with innate immune response is not yet reported. Herein, the aim of this study was to investigate the role of EZH2 in GBM immune.MethodsCo-culturing models of human/murine GBM cells with PBMC-derived macrophages/primary microglia were employed. EZH2 mRNAs and function were suppressed by siEZH2 and DZNep. Real-time PCR and flow cytometry were used to determine levels of microglia/macrophages markers. The fluorescence-labeled latex beads and flow cytometry were utilized to evaluate phagocytic abilities of microglia. CCK8 assay was performed to assess microglia proliferation.ResultsEZH2 inhibition led to significant reduction of TGFβ1-3 and IL10 and elevation of IL1β and IL6 in human and murine GBM cells. More importantly, EZH2 suppression in GBM cells resulted in significant increase of M1 markers (TNFα and iNOS) and decrease of a pool of M2 markers in murine microglia. The proportion of CD206+ cells was decreased in PBMC-derived macrophages as co-incubated with EZH2-inhibited GBM cells. Functional researches showed that phagocytic capacities of microglia were significantly ameliorated after EZH2 inhibition in co-culturing GBM cells and microglia proliferation was declined after addition of TGFβ2 antibodies to co-incubated GBM cells with EZH2 inhibition. Besides, we found that EZH2 suppression in GBM cells enhanced co-culturing microglia engulfment through activation of iNOS.ConclusionsOur data demonstrates that EZH2 participates in GBM-induced immune deficient and EZH2 suppression in GBM can remodel microglia immune functions, which is beneficial for understanding GBM pathogenesis and suggests potential targets for therapeutic approaches.

Highlights

Glioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis
Enhancer of zeste homolog 2 (EZH2) inhibition in GBM cells is associated with immune responses Initially, global gene expression pattern related with EZH2 inhibition by siRNAs in GBM U87 cell line was investigated using gene microarray assay
We found that EZH2-related genes were enriched in KEGG TNF signaling pathway and biological process immune response, which suggested that EZH2-associated genes played roles in immune response in GBM cells

Summary

Introduction

Glioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis. TAMs facilitate tumor proliferation, survival, and migration, acting as an anti-immunological and protumorigenic role in the tumor microenvironment. The classically activated microglia/macrophages, M1 phenotype, stimulate anti-tumor immune response through secretion of pro-inflammatory cytokines, such as IFNgamma, IL1β, iNOS, etc., whereas the alternatively activated or M2 phenotypes promote tumor survival via producing anti-inflammatory cytokines like IL4, TGFβ, IL10, etc. TAMs are forced forwards M2 phenotypes by GBM cells via secreting a wide variety of factors such as IL10, IL4, IL6, M-CSF, macrophage inhibitory factor (MIF), TGFβ, and prostaglandin E2 (PGE2), and subsequently support tumor growth and invasion [4]. The pathogenesis of elevation of these anti-immune factors and reduction of pro-immune factors in GBM remains elucidated

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