Metabolic reprogramming in response to chronic acidosis regulates tumor cell protein acetylation

Corbet, Cyril [UCL]

INTRODUCTION: Tumor cells must adapt to survive under acidosis. Recently, we reported a metabolic shift toward glutamine metabolism in tumor cells acclimated to pH 6.5. This effect was largely mediated by HIF-2 deacetylation by SIRT1, a protein deacetylase activated by the increased pool of NAD+ in acidic pH-adapted cells. The goal of the current study was to determine whether other signaling or metabolism-related proteins were also differentially acetylated under acidosis. METHODS: The AcetylScan technology was used to identify and quantify global differences in acetylation between native and acidic pH-adapted tumor cells. This technology utilizes antibodies with high affinity to acetylated-lysine peptides for enrichment of acetylated peptides from protease-digested cell samples, followed by LC-MS/MS analysis for quantitative profiles of non-redundant acetylated sequences. Immunoblotting was performed to evaluate the expression of major mitochondrial acetyltransferase and deacylases. AcetylCoA measurements and supplementation experiments were used to evaluate non-enzymatic acetylation process, and the CPT1 inhibitor etomoxir was used to determine the contribution of fatty acids to the mitochondrial pool of acetylCoA. RESULTS AND DISCUSSION: The analysis of the acetylome profiles in various cancer cells led us to identify 9,750 redundant peptide assignments containing acetyl-lysines. Among these sites, 773 acetylated lysines from 529 non-redundant proteins had a cut-off > 2.5-fold. Clustering analysis identified 277, 331 and 201 acetyl-sites in cytoplasm, nucleus and mitochondria, respectively. The most dramatic change was a net increase in global protein acetylation in the mitochondria of acidic pH-adapted tumor cells with more than 90% of hyperacetylated proteins. This process was not associated with changes in the expression of mitochondrial acetyltransferase GCN5L1 and sirtuin deacylases SIRT3, SIRT4 and SIRT5. Instead, we documented that non-enzymatic acetylation resulting from the larger pool of mitochondrial acetylCoA accounted for the hyperacetylation observed under acidosis. This was further supported by a significant increase in fatty acid beta-oxidation in acidic pH-adapted cells. CONCLUSION: Chronic acidosis strongly affects tumor cell metabolism by increasing glutamine and fatty acid use while decreasing glycolysis. This metabolic shift is associated with a global change in the acetylation of mitochondrial proteins that might be critical to support the adaptation of tumor cell metabolism under acidosis.