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. 2018 Apr 1;32(7-8):497-511.
doi: 10.1101/gad.311027.117. Epub 2018 Apr 19.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling

Joyce V Lee  1   2 , Corbett T Berry  3   4 , Karla Kim  1   2 , Payel Sen  5 , Taehyong Kim  6 , Alessandro Carrer  1   2 , Sophie Trefely  1   2   7 , Steven Zhao  1   2 , Sully Fernandez  1   2 , Lauren E Barney  8 , Alyssa D Schwartz  8 , Shelly R Peyton  8 , Nathaniel W Snyder  7 , Shelley L Berger  5   9   10 , Bruce D Freedman  3 , Kathryn E Wellen  1   2
Affiliations

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling

Joyce V Lee et al. Genes Dev. .

Abstract

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Keywords: NFAT1; acetyl-CoA; calcium; glioblastoma; histone acetylation; metabolism.

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Figures

Figure 1.
Figure 1.
Acetyl-CoA promotes cell adhesion and migration in GBM cells. (A) Relative mRNA levels of acetyl-CoA-up-regulated genes as determined by RT-qPCR in LN229 cells. (#) P < 0.05; (##) P < 0.01, significance of acetate treated over 1 mM glucose. (**) P < 0.01; (***) P < 0.001, significance of 10 mM glucose treated over 1 mM glucose. (B) Wound healing assay in LN229 cells. (***) P < 0.001; (****) P < 0.0001. (Right panel) Lines indicate the boundary of the scratch. Photos were captured at 0 h and after 24 h. (C) Transwell migration of LN229 across an 8.0-µm polycarbonate membrane. (*) P < 0.05; (**) P < 0.01. (Right panel) Cells on the membrane were stained with Hoechst, and photos were captured 24 h after seeding. (D) Adhesion quantified on biomaterial platform with ECM components of the brain. Cells were pretreated with the indicated conditions for 24 h, and the area covered by cells was measured over time. (*) P < 0.05, significance of acetate conditions over 1 mM glucose conditions determined by Tukey's post hoc test. (E–H) Relative adhesion to 1% fibronectin after 24 h of the indicated treatments in LN229 (E), LN18 (F), U87 (G), and U251 (H) cells. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. (I) LN229 cells were incubated in 1 mM glucose overnight, and then the medium was changed to the indicated conditions. Quantification of adhesion to 1% fibronectin after the indicated hours of treatment. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. All panels show mean ± SEM of triplicates.
Figure 2.
Figure 2.
Glucose-dependent cell migration requires ACLY. (A) Wound healing assay in U251 with stable knockdown of ACLY. (*) P < 0.05. (B) Relative adhesion in U251 cells with knockdown of ACLY. (**) P < 0.01; (****) P < 0.0001. (C) Transwell migration of LN229 cells after treatment with 50 µM ACLY inhibitor (ACLYi; BMS303141) or vehicle in 10 mM glucose. No FBS is a negative control. (**) P < 0.01. (D) Adhesion assay in with U251 cells after treatment with 50 µM ACLYi or vehicle for 24 h. (E) Relative mRNA expression of acetyl-CoA-regulated genes in LN229 cells treated with 50 µM ACLYi. (###) P < 0.001; (####) P < 0.0001; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. (F) Relative adhesion in two different ACLY knockout clones (sg3.6 and sg3.8) on 1% fibronectin. Parental cells were infected with vector expressing Cas9 but without guide RNA. (**) P < 0.01; (****) P < 0.0001. (G) Relative expression of ITGA2B in cells described in F. (*) P < 0.05; (**) P < 0.01. All panels show mean ± SEM.
Figure 3.
Figure 3.
H3K27ac near TSSs is sensitive to acetyl-CoA availability. (A) PDGFRA ChIP-seq tracks with reference normalization (top) and traditional normalization (bottom). (B) H3K27ac ChIP-qPCR analyzing the PDGFRA promoter region. (C) Metagene analysis of H3K27ac in the region −10 to +10 kb around the TSS of acetyl-CoA-up-regulated genes identified in Lee et al. (2014) and for all genes. (Top) Reference genome-normalized data. (Bottom) Traditional normalization. (D) Heat map of H3K27ac at acetyl-CoA-up-regulated genes (reference-normalized).
Figure 4.
Figure 4.
The transcription factor NFAT1 mediates acetyl-CoA-dependent cell adhesion and migration. (A) GSEA of acetyl-CoA-up-regulated genes, comparing 1 mM glucose with “rest” (10 mM glucose and 1 mM glucose + acetate conditions). (B) LN229 cell adhesion onto 1% fibronectin after 24 h of treatment with 10 µM cyclosporin A (CsA) or vehicle control. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C) Relative mRNA expression of acetyl-CoA-up-regulated genes after knockdown of NFAT1 in LN229 cells. Representative genes from the list generated from GSEA in A are shown (see also Supplemental Fig. S5B). (D) Relative adhesion onto 1% fibronectin after shRNA-mediated knockdown of NFAT1 in LN229 cells. (***) P < 0.001. (E) Wound healing assay in LN229 cells after shRNA-mediated knockdown of NFAT1. (*) P < 0.05. All panels show mean ± SEM.
Figure 5.
Figure 5.
NFAT1 nuclear localization is acetyl-CoA-regulated and required for cell adhesion. (A) Immunofluorescent detection of endogenous NFAT1 localization in LN229 cells. Representative images are shown. (B) Quantification of NFAT1 detected in the nucleus, as shown in A, as percentage of cells in each field. At least 170 cells were scored per condition. (**) P < 0.01. (C) Immunofluorescent detection of endogenous NFAT1 localization in ACLY knockout sg3.6 cells with or without murine wild-type ACLY cDNA expression. Representative images are shown. (D) Quantification of endogenous NFAT1 detected in the nucleus, as shown in C, as percentage of cells in each field. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001. (E) Murine HA-tagged NFAT1 was transiently expressed in LN229 cells, cells were treated with the indicated doses of ACLYi, and nuclear and cytosolic fractions were prepared and analyzed by Western blot. (F) Glucose regulation of cell adhesion on 1% fibronectin in LN229 cells expressing empty vector, murine wild-type NFAT1, or constitutively active (CA) NFAT1 (CA-NFAT1). (G) ACLYi regulation of cell adhesion on 1% fibronectin in cells expressing empty vector (plx303), wild-type NFAT1, or CA-NFAT1. (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001.
Figure 6.
Figure 6.
Ca2+ signaling is acetyl-CoA-regulated. (A) Representative Ca2+ traces from the GCaMP6f reporter in the indicated conditions. Each trace represents one cell, and at least 15 cells were analyzed from two different fields in each condition. (B) Quantification of Ca2+ spikes after nutrient add-back. At least 116 cells were scored in each condition in a blinded manner. (*) P < 0.05; (**) P < 0.01. (C) Representative Ca2+ traces in the presence or absence of EGTA are shown. (D) LN229 cell adhesion onto 1% fibronectin after 20 h of incubation in the indicated concentrations of glucose and acetate followed by 4 h of treatment with 0.5 µM ionomycin or vehicle control. (****) P < 0.0001. (E) Relative adhesion after 20 h of treatment with ACLYi followed by 4 h of treatment with 0.5 µM ionomycin or vehicle control in LN229 cells expressing murine wild-type NFAT1. (*) P < 0.05; (**) P < 0.01. (F) Model showing how acetyl-CoA promotes GBM cell adhesion and migration through Ca2+–NFAT signaling.
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