Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

Abstract

Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

Figure 1. Acetate rescues hypoxia-induced reduction of histone acetylation.

(a) ¹H-NMR spectra of acetate concentration in fresh medium, medium of cancer cells cultured under normoxia or hypoxia. The results represented mean±s.d. of triplicate experiments. (^**P<0.01; *P<0.05; by two-tailed unpaired Student's t-test). (b) Acetate rescues hypoxia-reduced H3K9, H3K27 and H3K56 acetylation levels. HepG2 cells were treated with or without 2.5 mM acetate under hypoxia (1% O₂) for 4 h. The histone acetylation levels were determined by western blot. Total H3 served as a loading control. (c) Cancer cells are more sensitive to acetate under hypoxia. HepG2 cells were treated with indicated concentrations of acetate under normoxia or hypoxia (1% O₂) for 4 h. The histone acetylation levels were determined by western blot. Total H3 served as a loading control. (d) Acetate increases H3K9, H3K27 and H3K56 acetylation levels in a time-dependent manner under hypoxia. HepG2 cells were treated with or without 5 mM acetate for 0.5, 1, 2 and 4 h under hypoxia (1% O₂), respectively. The global histone acetylation levels were determined by western blot. Total histone H3 served as a loading control. (e) Acetate increases H3K9, H3K27 and H3K56 acetylation levels in a dose-dependent manner under hypoxia. HepG2 cells were treated with the indicated concentrations of acetate for 4 h under hypoxia (1% O₂). The histone acetylation levels were determined by western blot. Total H3 served as a loading control.

Figure 2. Acetate predominately activates lipid synthesis pathway through epigenetic regulation under hypoxia.

(a) Scatter plot of mRNA expression data of 139 metabolic genes in HepG2 cells, comparing cells treated with hypoxia (y axis) to normoxia (x axis). The mRNA expression value of triplicate experiments was shown on a log 2 scale. Grey lines indicated two-fold differences in the mRNA expression levels between two groups. Upregulated genes (>2-fold change, P<0.05) were shown in magenta. Downregulated expressed genes (>2-fold change, P<0.05) were shown in blue. Two-tailed unpaired Student's t-test was used. (b) Scatter plot of mRNA expression data of 139 metabolic genes in HepG2 cells under normoxia, comparing cells treated with 2.5 mM acetate (y axis) to acetate-free (x axis). The mRNA expression value of triplicate experiments was shown on a log2 scale. Grey lines indicated two-fold differences in the mRNA expression levels between two groups. Downregulated genes (>2-fold change, P<0.05) were shown in blue. Two-tailed unpaired Student's t-test was used. (c) Scatter plot of mRNA expression data of 139 metabolic genes in HepG2 cells under hypoxia, comparing cells treated with 2.5 mM acetate (y axis) with acetate-free (x axis). The mRNA expression value of triplicate experiments was shown on a log 2 scale. Grey lines indicated two-fold differences in the mRNA expression levels between two groups. Upregulated genes (>2-fold change, P< 0.05) were shown in magenta. Two-tailed unpaired Student's t-test was used. (d) Fold-change analysis of the mRNA expression of 139 genes in HepG2 cells treated with or without 2.5 mM acetate under hypoxia. (e,f) FASN (e) and ACACA (f) mRNA levels in HepG2 cells treated with indicated concentrations of acetate for 12 h under normoxia or hypoxia were quantified by qPCR. The results were presented as mean±s.d. of triplicate experiments. (g,h) ChIP-qPCR assays showing H3K9, H3K27 and H3K56 acetylation enrichment at FASN (g) and ACACA (h) promoter regions in HepG2 cells treated with indicated concentrations of acetate under normoxia or hypoxia for 4 h. Rabbit IgG was included as a negative control. Each histogram was presented as mean±s.d. of triplicate experiments (*P<0.05; ^**P<0.01; NS, not significant; by two-tailed unpaired Student's t-test).

Figure 3. Acetate epigenetically activates lipogenic genes without reflecting cellular lipid demands.

(a) FASN expression in HepG2 cells treated with indicated concentrations of acetate for 12 h under normoxia or hypoxia were quantified by qPCR. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; NS, not significant; by Student's t-test). (b) ChIP-qPCR assays showing histone acetylation enrichment at FASN promoter region in HepG2 cells treated with indicated concentrations of acetate under normoxia or hypoxia for 4 h. Rabbit IgG was included as a negative control. Each histogram was presented as mean±s.d. of triplicate experiments (^**P<0.01; NS, not significant; by Student's t-test). (c) ACACA expression in HepG2 cells treated as in panel (a) were quantified by qPCR. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; NS, not significant; by Student's t-test). (d) ChIP-qPCR assays showing histone acetylation enrichment at ACACA promoter region in HepG2 cells treated as in b. Each histogram was presented as mean±s.d. of triplicate experiments (^**P<0.01; NS, not significant; by Student's t-test). (e) Quantification of FASN expression in HepG2 cells treated with or without 2.5 mM acetate in media plus 10% FBS or 10% LPDS for 12 h under hypoxia by qPCR. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; by Student's t-test). (f) ChIP-qPCR assays showing histone acetylation levels at FASN promoter region in HepG2 cells treated as in e for 4 h under hypoxia. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; by Student's t-test). (g) Quantification of FASN expression in HepG2 cells treated with 10% LPDS plus the indicated concentration of palmitate (PA) for 12 h under normoxia or hypoxia by qPCR. The results were presented as mean±s.d. of triplicate experiments (NS, not significant; by Student's t-test). (h) ChIP-qPCR assays showing histone acetylation levels enrichment at FASN promoter region in HepG2 cells treated as in g for 4 h under normoxia or hypoxia. The results were presented as mean±s.d. of triplicate experiments (NS, not significant; by Student's t-test).

Figure 4. Both ACSS1 and ACSS2 are involved in acetate-induced epigenetic regulation of de novo lipogenesis.

(a) Stably concurrent knockdown of ACSS1 and ACSS2 totally blocks acetate-induced increase of histone acetylation levels. Histone acetylation levels in shscr or shACSS1/2 HepG2 cells treated with or without 2.5 mM acetate under normoxia or hypoxia were analysed by western blot. (b) ¹³C₂-labelled H3K9ac and H3K27ac levels are decreased by ACSS1/2 knockdown. Quantification of ¹³C₂-labelled histone H3 acetylation levels in shscr or shACSS1/2 HepG2 stable cell line treated with [U-¹³C₂]-acetate for 4 h under hypoxia was analysed by LC-MRM MS. The percentage indicates the ratio of H3K[¹³C₂-ac]/total H3Kac in each site. The results were presented as mean±s.d. of triplicate experiments (*P<0.05; ^***P<0.001; by Student's t-test). (c,d) ACSS1/2 knockdown totally blocks acetate-induced histone acetylation association with FASN and ACACA promoter. ChIP-qPCR assays were performed to determine H3K9, H3K27 and H3K56 acetylation enrichment at FASN (c) and ACACA (d) promoter region in shscr or shACSS1/2 HepG2 stable cell lines treated with or without acetate under hypoxia for 4 h. IgG was included as a negative control. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; ^***P<0.001; NS, not significant; by Student's t-test). (e) ChIP-qPCR assays were performed to determine H3K9, H3K27 and H3K56 acetylation enrichment at ACLY promoter region in shscr or shACSS1/2 HepG2 cells treated as in c. IgG was included as a negative control. The results were presented as mean±s.d. of triplicate experiments (NS, not significant; by Student's t-test).

Figure 5. Epigenetic regulation of de novo lipogenesis by acetate is vital for cell survival under hypoxia.

(a) ACSS1/2 mediates acetate-induced palmitate and stearate production. Fractional abundance of palmitate (left) and stearate (right) isotopologues in ACSS1/2 stably double-knockdown HepG2 cells (designated as magenta) compared with that of control cells (designated as black) after 24 h of culture under hypoxia with [U-¹³C₂]-acetate. Each histogram was presented as mean±s.d. of triplicate experiments. The inset shows the percentage of lipogenic acetyl-CoA derived from acetate carbon (^**P<0.01; by Student's t-test). (b) Fractional abundance of palmitate (left panel) and stearate (middle panel) isotopologues in scramble (siscr) and siFASN HepG2 cells treat as in a. Each histogram was presented as mean±s.d. of triplicate experiments. The inset shows the percentage of lipogenic acetyl-CoA derived from acetate carbon. The knockdown efficiency was detected by PCR with reverse transcription (right panel) (^**P<0.01; NS, not significant; by Student's t-test). (c)Acetate promotes cell survival under hypoxia. Viability of HepG2 cells treated with indicated concentrations of acetate under normoxia or hypoxia was determined via CCK8 assay. The results were presented as mean±s.d. of triplicate experiments (^**P<0.01; ^***P<0.001; NS, not significant; by Student's t-test). (d) Viability of control or siFSAN or siACACA HepG2 cells treated with or without 2.5 mM acetate under hypoxia was determined via CCK8 assay. The results were presented as mean±s.d. of triplicate experiments (^***P<0.001; by Student's t-test). (e) ACSS1/2 mediates the promotion of cell survival by acetate. Viability of shscr or shACSS1/2 HepG2 stable cell lines treated as indicated was determined via CCK8 assay. The results were presented as mean±s.d. of triplicate experiments (*P<0.05; NS, not significant; by Student's t-test). (f) ACSS1/2 mediates cells growth advantage by acetate in Matrigel. Shscr or shACSS1/2 HepG2 cells were plated into the Matrigel bed and cultured with DMEM with 10% FBS, and 2% Matrigel for 12 days before the images were taken (left). Scale bar, 0.25 mm. The quantitative data were presented as mean±s.d. (n>3) (right) (^***P<0.001; NS, not significant; by Student's t-test).

Figure 6. ACSS1/2 positively correlates with histone acetylation and FASN expression in human hepatocellular carcinoma.

(a) FASN, ACSS1 and ACSS2 protein levels (upper panels, normalized against β-actin) and H3K9, H3K14, H3K18, H3K23, H3K27 and H3K56 acetylation levels (lower panels, normalized against histone H3) in 53 pairs of HCC (each paired with cancerous tissue (designated as C) and adjacent normal tissue (designated as N)) were analysed by western blot. Two pairs of representative samples were shown. For the other 51 pairs of samples, please refer to Supplementary Fig. 6b. (b) Heatmap of protein expression (ACSS1, ACSS2 and FASN) and histone acetylation levels (H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac and H3K56ac) in all 53 pairs of HCC. Data were presented as Z-score of relative protein expression or histone acetylation. Tumours with 1.5-fold higher expression of ACSS1 or ACSS2 or both than that of adjacent normal control tissue are grouped into ACSS-high tumours (tumour/normal ≥1.5, n=26), while ACSS-low tumours (tumor/normal<1.5, n=27) express both ACSS proteins at 1.5-fold lower than its normal control. (c) H3K9ac (P=0.0220), H3K14ac (P=0.0289), H3K27ac (P=0.0034), and H3K56ac (P=0.0410), are significantly stronger in ACSS-high tumours than that in ACSS-low tumours. Statistical analyses were performed with a two-tailed unpaired Student's t-test (*P<0.05; ^**P<0.01; NS, not significant). (d) FASN protein level is significantly upregulated in ACSS-high tumours (P=0.002). Statistical analyses were performed with a two-tailed unpaired Student's t-test (^**P<0.01). (e) Representative immunohistochemical staining results for ACSS1, ACSS2, FASN and H3K9/K27/K56 acetylation in adjacent normal tissue (N) and hepatocellular carcinoma (C). Scale bar, 100 μm.

Figure 7. The model for acetate-induced epigenetic regulation of de novo lipogenesis.

In addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, acetate also functions as an epigenetic metabolite to induce H3 acetylations in both dose- and time-dependent manners under hypoxia, enhancing H3K9, H3K27 and H3K56 acetylation levels at FASN and ACACA promoter regions, which upregulates FASN and ACACA expression and increases de novo lipid synthesis to promote tumour cell survival.