Coagulation Factor X Regulated by CASC2c Recruited Macrophages and Induced M2 Polarization in Glioblastoma Multiforme

Yan Zhang,Qiang Liu,Yingnan Sun,Jianbo Feng,Qing Liu,Zhibin Yu,Haijuan Fu,Guiyuan Li,Tao Liu,Yang Liu,Changhong Liu,Chunhua Zhao,Xiaoling She,Minghua Wu,Peiyao Li

doi:10.3389/fimmu.2018.01557

Abstract

Tumor-associated macrophages (TAMs) constitute a major component of inflammatory cells in the glioblastoma multiforme (GBM) tumor microenvironment. TAMs have been implicated in GBM angiogenesis, invasion, local tumor recurrence, and immunosuppression. Coagulation factor X (FX) is a vitamin K-dependent plasma protein that plays a role in the regulation of blood coagulation. In this study, we first found that FX was highly expressed and positively correlated with TAM density in human GBM. FX exhibited a potent chemotactic capacity to recruit macrophages and promoted macrophages toward M2 subtype polarization, accelerating GBM growth. FX bound to extracellular signal-related kinase (ERK)1/2 and inhibited p-ERK1/2 in GBM cells. FX was secreted in the tumor microenvironment and increased the phosphorylation and activation of ERK1/2 and AKT in macrophages, which may have been responsible for the M2 subtype macrophage polarization. Moreover, although the lncRNA CASC2c has been verified to function as a miR-101 competing endogenous RNA (ceRNA) to promote miR-101 target genes in GBM cells, we first confirmed that CASC2c did not function as a miR-338-3p ceRNA to promote FX expression, and that FX was a target gene of miR-338-3p. CASC2c interacted with and reciprocally repressed miR-338-3p. Both CASC2c and miR-388-3p bound to FX and commonly inhibited its expression and secretion. CASC2c repressed M2 subtype macrophage polarization. Taken together, our findings revealed a novel mechanism highlighting CASC2c and FX as potential therapeutic targets to improve GBM patients by altering the GBM microenvironment.

Highlights

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor in adults and is associated with poor prognosis [1, 2]
We further investigated whether factor X (FX) mediate macrophage polarization through ERK1/2 and AKT. p-extracellular signal-related kinase (ERK) and p-AKT were decreased when THP-1 cells were treated with cell culture supernatants from G1124 cells in which FX was knocked down, while when THP-1 cells were treated with cell culture supernatants from U251 cells which FX was overexpressed, p-ERK1/2 and p-AKT were increased (Figure 5G). p-ERK1/2 and p-AKT increased when THP-1 cells were treated with recombinant FX (rFX) protein in a concentration-dependent manner (Figure 5H)
Flow cytometry showed that the proportion of cluster of differentiation molecule 11b (CD11b)+Cd80+ M1 macrophages increased and CD11b+CD206+ M2 macrophages decreased when THP-1 treated with supernatants from G1124 cells transfected with CASC2c compared with control vector (Figures 7L,M)

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor in adults and is associated with poor prognosis [1, 2]. FX Recruited M2 TAMs in GBM grades and poor prognosis in many cancers, including GBM [4]. Abundant macrophages infiltrate GBM and promote tumor progression in multiple aspects. TAMs secrete cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interferon-γ, which have been shown to promote tumor cell growth [5]. TAMs could facilitate angiogenesis by releasing vascular endothelial growth factor-α and associating with adjacent endothelial tip cells, facilitating vascular anastomosis [6]. TAMs secrete various cytokines and chemokines that suppress CD4+ and CD8+ T cell effector function directly or indirectly by recruiting regulatory T cells to the tumor microenvironment [8]

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Results

Conclusion