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. 2020 Jan 11;22(1):46-57.
doi: 10.1093/neuonc/noz136.

NFAT2-HDAC1 signaling contributes to the malignant phenotype of glioblastoma

Yifu Song  1 , Yang Jiang  1   2 , Dongxia Tao  3 , Zixun Wang  1 , Run Wang  1 , Minghao Wang  1 , Sheng Han  1
Affiliations

NFAT2-HDAC1 signaling contributes to the malignant phenotype of glioblastoma

Yifu Song et al. Neuro Oncol. .

Abstract

Background: Deregulation of the nuclear factor of activated T cell (NFAT) pathway has been reported in several human cancers. Particularly, NFAT2 is involved in the malignant transformation of tumor cells and is identified as an oncogene. However, the role of NFAT2 in glioblastoma (GBM) is largely unknown.

Methods: The expression and prognostic value of NFAT2 were examined in the databases of the Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas (TCGA) and clinical samples. The functional effects of silencing or overexpression of NFAT2 were evaluated in glioma stem cell (GSC) viability, invasion, and self-renewal in vitro and in tumorigenicity in vivo. The downstream target of NFAT2 was investigated.

Results: High NFAT2 expression was significantly associated with mesenchymal (MES) subtype and recurrent GBM and predicted poor survival. NFAT2 silencing inhibited the invasion and clonogenicity of MES GSC-enriched spheres in vitro and in vivo. NFAT2 overexpression promoted tumor growth and MES differentiation of GSCs. A TCGA database search showed that histone deacetylase 1 (HDAC1) expression was significantly correlated with that of NFAT2. NFAT2 regulates the transcriptional activity of HDAC1. Rescue of HDAC1 in NFAT2-knockdown GSCs partially restored tumor growth and MES phenotype. Loss of NFAT2 and HDAC1 expression resulted in hyperacetylation of nuclear factor-kappaB (NF-κB), which inhibits NF-κB-dependent transcriptional activity.

Conclusion: Our findings suggest that the NFAT2-HDAC1 pathway might play an important role in the maintenance of the malignant phenotype and promote MES transition in GSCs, which provide potential molecular targets for the treatment of GBMs.

Keywords: HDAC1; NF-κB; NFAT2; glioblastoma; mesenchymal.

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Figures

Fig. 1
Fig. 1
Correlation between NFAT2 expression and the clinic-pathologic features of gliomas. (A) NFAT2 mRNA expression is shown according to the histopathologic grades of REMBRANDT and TCGA gliomas. (B) Messenger RNA expression of NFAT2 is shown according to the molecular subtypes of REMBRANDT and TCGA GBMs. (C) NFAT2 mRNA expression is compared between primary and recurrent TCGA GBMs. (D) Prognostic significance of NFAT2 in REMBRANDT gliomas. (E–G) The prognostic significance of NFAT2 in MES GBMs was tested using the REMBRANDT database (E), Lee Y dataset (F), and Freije dataset (G). *P < 0.05, ***P < 0.001.
Fig. 2
Fig. 2
NFAT2 expression in primary GSCs. (A) Representative IHC images of NFAT2 expression in WHO grade IV, grade III, and grade II glioma clinical samples. Intense immunostaining was observed in the nuclei of grade IV glioma tissues. Scale bar = 50 μm. (B) Representative WB image of NFAT2 protein expression in WHO grade IV, grade III, and grade II glioma clinical samples. (C) Western blotting shows the expression of NFAT2, YKL40, CD44, and Olig2 in the indicated GSCs. (D) Immunofluorescence of NFAT2 in the primary GSCs. Scale bar = 25 μm. (E, F) The expression of NFAT2 in CD44high and CD44low GSCs was examined by real-time PCR (E) and WB (F). Results are presented as mean ± SD of triplicate samples from 3 independent experiments. *P< 0.05 and **P< 0.01.
Fig. 3
Fig. 3
NFAT2 silencing inhibits MES GSC-enriched spheres clonogenicity in vitro. (A) Western blotting of NFAT2, CD44, and YKL40 in GSCs transfected with shRNA targeting NFAT2 (shNFAT2-1 or shNFAT2-2) or a control shRNA (shC). (B) Cell viability assay shows that NFAT2 knockdown markedly decreases the proliferation of G08 and G10 GSCs. (C) Targeting of NFAT2 via specific shRNAs significantly increases the apoptosis of G08 and G10 GSCs, as assessed with an assay by TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling). Scale bar = 50 μm. (D) Representative microphotographs showing the invasion of G08 and G10 GSCs in the presence of NFAT2-shRNA or control-shRNA using the Matrigel assay. Cells were allowed to invade the Matrigel-coated filters toward the lower compartment for 20 h. Scale bar = 50 μm. Histogram showing the quantification of invasive cells. (E) The 3D spheroid invasion assay demonstrates that NFAT2 knockdown significantly decreased the invasion of G08 and G10 neurospheres. Scale bar: 100 μm. (F) Representative images of G10 and G08 neurospheres transfected with shRNA targeting NFAT2. Histogram showing the quantification of neurosphere size. Scale bar = 50 μm. (G) Limiting dilution neurosphere formation assays of effect of NFAT2 silencing on GSC renewal. Results are presented as mean ± SD of triplicate samples from 3 independent experiments. *P < 0.05 and **P < 0.01.
Fig. 4
Fig. 4
NFAT2 regulates GSC growth in vivo. (A) H&E-stained brain sections of mice after intracranial transplantation of G08 GSCs transfected with shC, shNFAT2-1, or shNFAT2-2. Brains were harvested at 15 days after transplantation. NFAT2 silencing significantly inhibited tumor growth in vivo. Scale bars = 1 mm. (B) Kaplan–Meier survival curves of mice injected with G08 GSCs transfected with shC, shNFAT2-1, or shNFAT2-2. (C) IHC staining of NFAT2 and CD44 in intracranial tumors derived from G08 GSCs transfected with shC, shNFAT2-1, or shNFAT2-2. Scale bar = 25 μm. (D) H&E-stained brain sections of mice after intracranial transplantation of G12 GSCs transfected with the NFAT2 overexpression plasmids or empty vector. Brains were harvested at 15 days after transplantation. NFAT2 overexpression markedly enhanced tumor growth in vivo. Scale bars = 1 mm. (E) Kaplan–Meier survival curves of mice injected with G12 GSCs transfected with the NFAT2 overexpression plasmids or empty vector. (F) IHC staining of NFAT2 and CD44 in intracranial tumors derived from G12 GSCs transfected with the NFAT2 overexpression plasmids or empty vector. Scale bar = 25 μm. *P < 0.05, **P < 0.01.
Fig. 5
Fig. 5
NFAT2 regulates the expression of HDAC1 in GSCs. (A) The expression of HDAC1 was significantly correlated with that of NFAT2 in TCGA gliomas. (B) The mRNA expression of HDAC1 is shown according to molecular subtypes of TCGA GBMs. (C) The prognostic significance of HDAC1 is examined in TCGA gliomas. (D, E) The expression of HDAC1 in CD44high and CD44low GSCs was examined by real-time PCR (D) and WB (E). (F) The effect of NFAT2 knockdown on the expression of HDAC1 was examined by western blotting in G08 and G10 GSCs. (G) The effect of NFAT2 overexpression on the expression of HDAC1 was examined by western blotting in G12 GSCs. (H, I) Double-labeled immunofluorescence staining demonstrates that NFAT2-knockdown is accompanied by reduced HDAC1 expression in G08 (H), while NFAT2 overexpression is accompanied by increased HDAC1 expression in G12 (I). Scale bar = 25 μm. (J, K) Effect of NFAT2 on HDAC1 promoter activities. Knockdown of NFAT2 in G08 significantly suppresses the luciferase activity driven by the wildtype HDAC1 promoters compared with control-shRNA. Mutating NFAT2 binding sites markedly decreases promoter activity compared with the wildtype promoter (J). NFAT2 overexpression in G12 enhances luciferase promoter activities (K). (L) Binding of NFAT2 to HDAC1 promoters. Binding of NFAT2 is suppressed when NFAT2 is knocked down in G08, while binding is enhanced when NFAT2 is overexpressed in G12. Results are presented as mean ± SD of triplicate samples from 3 independent experiments. *P < 0.05, ***P < 0.001.
Fig. 6
Fig. 6
Rescue of HDAC1 in NFAT2-silenced GSCs partially restores clonogenicity in vitro and in vivo. (A) Rescuing HDAC1 in NFAT2-silenced G08 restores the expression of CD44 and YKL40 in vitro. (B) Rescue of HDAC1 partially restores proliferation compared with an empty vector control in NFAT2-silenced G08. (C) Reexpression of HDAC1 rescues neurosphere growth compared with an empty vector control in NFAT2-silenced G08. Scale bar = 25 μm. (D) Neurosphere formation after HDAC1 is rescued in NFAT2-silenced G08. Neurosphere formation capacity is significantly increased after HDAC1 rescue. (E) NFAT2 silencing in G08 inhibits the tumor growth in vivo. Rescuing HDAC1 in NFAT2-silenced G08 reverts tumor growth in vivo. (F) NFAT2 silencing in G08 prolongs the survival of tumor-bearing mice. Reexpression of HDAC1 in NFAT2-silenced G08 reverts the survival time. (G) Rescuing HDAC1 in NFAT2-silenced G08 reverts the expression of CD44 in vivo. Scale bar = 25 μm. (H) Gene set enrichment analysis plots of NF-κB pathway signatures in high NFAT2/HDAC1 expression versus low NFAT2/HDAC1 expression TCGA gliomas. Normalized enrichment score (NES) and false discovery rate (FDR) are shown in the plot. (I–K) HDAC1 reexpression in NFAT2-silences G08 inhibits hyperacetylation of p65 (I), restores the ability of p65 to bind κB-DNA (J), and recovers NF-κB–dependent transcriptional activity (K). (L) A working model of mesenchymal transition mediated by NFAT2/HDAC1/NF-κB pathway in gliomas. Results are presented as mean ± SD of triplicate samples from 3 independent experiments. *P < 0.05, **P < 0.01.
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