Volume 24, Issue 2, Pages 311-323 (August 2016) Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation  Cyril Corbet, Adán Pinto, Ruben Martherus, João Pedro Santiago de Jesus, Florence Polet, Olivier Feron  Cell Metabolism  Volume 24, Issue 2, Pages 311-323 (August 2016) DOI: 10.1016/j.cmet.2016.07.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Chronic Acidosis Triggers Mitochondrial Protein Hyperacetylation (A) Schematic of antibody-based peptide enrichment combined with tandem mass spectrometry for quantitative profiling of acetylation in parental and acidic pH-adapted cells. (B) Subcellular localization of proteins containing acetyl-sites downregulated (white bars) and upregulated (black bars) in acidic pH-adapted SiHa cells. (C) Representative immunoblotting for acetylated proteins, GCN5L1, and SIRT3 in isolated mitochondria extracts of parental and acidic pH-adapted tumor cells. ATP5O was used as a loading control. (D) Acetyl-CoA levels in isolated mitochondria extracts of parental and acidic pH-adapted tumor cells. (E) Protein acetylation in isolated mitochondria (from SiHa/7.4) incubated in vitro with increasing concentrations of acetyl-CoA. (F) Immunoblotting of acetylated respiratory chain-associated proteins in acetyl-CoA-treated mitochondria (from SiHa/7.4). For (D), the data are represented as mean ± SEM. ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1 and Tables S1 and S2. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Mitochondrial Protein Acetylation Is Driven by Acetyl-CoA from Fatty Acid Oxidation (A) Extent of 14C incorporation into mitochondrial proteins collected from anti-acetylated lysine IP (eluates) and IP supernatants (outputs) following SiHa cell exposure for 24 hr to 14C-labeled glucose, glutamine, or palmitate. (B and C) Representative immunoblotting (B) and corresponding quantification (C) of protein acetylation in isolated mitochondria extracts from parental and acidic pH-adapted SiHa cells treated for 24 hr with 10 mM 2-DG, 20 nM CB-839, or 30 μM etomoxir. (D) Representative immunoblotting for acetylated proteins in isolated mitochondria extracts from parental and acidic pH-adapted SiHa cells transfected with ACSL1- or CPT1A-targeting siRNA. (E) Acetyl-CoA concentration in isolated mitochondria extracts of parental and acidic pH-adapted SiHa cells treated as in (B). (F) [U-14C]palmitate 10 min uptake measured in parental and acidic pH-adapted SiHa cells. For (A), (C), (E), and (F), the data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figure S2. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Acidosis-Induced Mitochondrial Hyperacetylation Regulates Complex I Activity and ROS Production (A and B) Palmitate-dependent OCR (A) and mitochondrial membrane potential (B) in parental and acidic pH-adapted SiHa cells. (C and D) State 3 respiration of mitochondria from SiHa/6.5 pre-challenged with DMSO (named as “acetylation +”) or with 30 μM etomoxir (named as “acetylation -”) for 24 hr before isolation. Representative results are shown (C), and oxygen consumption rate by each complex was calculated (D). (E) Immunoprecipitation experiments depicting the extent of complex I and NDUF subunit acetylation on lysine residues in parental and acidic pH-adapted SiHa cells pre-challenged or not with etomoxir. NDUFA9 and IgG heavy chains were used as loading controls. (F) Complex I activity in parental and acidic pH-adapted SiHa cells transfected with wild-type or indicated NDUFA9 mutants; transfection (overexpression) was validated by immunoblotting. (G) Membrane potential in isolated mitochondria, from acidic pH-adapted SiHa cells, measured in the presence of pyruvate and malate with or without succinate. (H) Measurement of H2O2 production using Amplex Red in isolated mitochondria from acidic pH-adapted SiHa cells exposed to pyruvate and malate. For (A)–(D) and (F)–(H), data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. See also Figure S3. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Reductive Carboxylation Is the Primary Route of Glutamine to Lipids under Chronic Acidosis (A) Schematic of carbon atom (circles) transitions and [5-13C]glutamine (blue) tracer used to detect contribution of reductive glutamine metabolism to palmitate synthesis. Molecular symmetry is shown for oxidative metabolism. ACLY, ATP citrate lyase; ME, malic enzyme; PDH, pyruvate dehydrogenase. (B and C) Absolute intracellular abundance of citrate and alpha-ketoglutarate (B) and calculated alpha-ketoglutarate to citrate ratio (C) in parental and acidic pH-adapted SiHa cells. (D) Relative abundance of palmitate mass isotopomers in parental and acidic pH-adapted SiHa cells cultured for 72 hr with [5-13C]glutamine. (E and F) Relative abundance of TCA cycle intermediates (E) and malic enzyme-derived metabolites (F) in parental and acidic pH-adapted SiHa cells cultured for 24 hr with [5-13C]glutamine. (G and H) Absolute rate (G) and specific contribution of 14C-tracers (H) for lipid synthesis in parental and acidic pH-adapted SiHa cells. (I) Representative immunoblotting for SLC25A1 in parental and acidic pH-adapted tumor cells; immunoblot exposure was optimized to avoid signal saturation. Longer exposure also shows a SLC25A1 band in parental cells. (J) SiHa cell growth (72 hr) following transfection with SLC25A1-targeting siRNA. For (B)–(H) and (J), the data are represented as mean ± SEM. ∗p < 0.05; ∗∗∗p < 0.001; ns, not significant. See also Figure S4. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Histone Deacetylation in ACACB Promoter Facilitates the Simultaneity of FAS and FAO in Acidic pH-Adapted Tumor Cells (A and B) Representative immunoblotting (A) and quantification (B) for ACC1 and ACC2 expression in parental and acidic pH-adapted tumor cells. (C) Relative mRNA expression for ACACB in parental and acidic pH-adapted tumor cells. (D and E) Representative immunoblotting (D) and quantification (E) for acetylation of histones H3 (K9) and H4 (K8) from parental and acidic pH-adapted cells. (F and G) Representative immunoblotting (F) and quantification (G) for histone acetylation in cells transfected with SIRT1- and SIRT6-targeting siRNA for 72 hr. (H) Representative immunoblotting for ACC2 in parental and acidic pH-adapted SiHa cells transfected with SIRT1- and SIRT6-targeting siRNA for 72 hr. (I) Level of promoter-associated acetylated histones H3 and H4 in the ACACB promoter. For (B), (C), (E), (G), and (I), the data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S5. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Inhibition of Fatty Acid Metabolism Delays the Growth of Tumor Cells Preadapted to Acidic pH (A–E) Representative immunoblotting (A) and quantification (B) for ACC1 and ACC2 protein expression, palmitate uptake (C), palmitate-dependent OCR (D), and cell growth (E) in SiHa/7.4 and SiHa/6.5 upon expression of recombinant wild-type ACC2. (F) SiHa cell growth (72 hr) following transfection with ACSL1- or CPT1A-targeting siRNA. (G) SiHa cell growth (72 hr) after etomoxir treatment. (H) Tumor growth of SiHa/7.4 and SiHa/6.5 xenografts in nude mice treated daily or not with BPTES (10 mg/kg), etomoxir (40 mg/kg) alone, or in combination. For (B)–(H), the data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S6. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Model of Tumor Metabolic Rewiring under Acidosis Extracellular acidic pH leads to a shift in the source of mitochondrial acetyl-CoA from glucose-derived pyruvate to FA oxidation (FAO). Acetyl-CoA fuels the TCA cycle together with oxidative metabolism of glutamine (not shown) and supports oxidative phosphorylation (OXPHOS). In parallel, reductive metabolism of glutamine-derived α-ketoglutarate ensures concomitant cytosolic FA synthesis (FAS). This profound reprogramming of FA metabolism is under the regulation of two acetylation-related modifications: non-enzymatic mitochondrial protein hyperacetylation restrains the activity of complex I activity, thereby limiting mitochondria overfeeding (including ROS generation), and SIRT1/6-mediated nuclear histone deacetylation leads to downregulation of ACC2, a mitochondrion-anchored enzyme that normally prevents the degradation of neo-synthesized FA. Cell Metabolism 2016 24, 311-323DOI: (10.1016/j.cmet.2016.07.003) Copyright © 2016 Elsevier Inc. Terms and Conditions