Abstract
Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP–NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin’s lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN–PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.
Highlights
Oncogenic de novo lipogenesis, which is catalyzed by the overexpressed fatty acid synthase (FASN), is an important metabolic phenotype observed in many cancers [1]
We investigated the impact of FASN inhibition on de novo nucleotide biosynthesis by the following experiments: pulse labeling and isotope tracing of carbon flow from C13-glucose into nucleotides showed a reduction in the proportion of C13-labeled C5 fractions associated with nucleotide monophosphates (AMP, CMP, GMP, and IMP) while increasing the amounts of 6PG-labeled fractions, confirming that phospho-gluconate dehydrogenase (PGDH) and nucleotide biosynthesis are suppressed by FASN inhibition with cerulenin (Figures 4A, B)
The results from FASN inhibition with cerulenin treatment show the transcriptomic downregulation of nucleotide metabolism and cell cycle processes (Supplementary Figure S2) to be correlated with reduced nucleotide biosynthesis, nucleic acid metabolism, and cell cycle impairment (Figure 4)
Summary
Oncogenic de novo lipogenesis, which is catalyzed by the overexpressed fatty acid synthase (FASN), is an important metabolic phenotype observed in many cancers [1]. FASN activity is entirely dependent on cytosolic glucose metabolism for the substrates acetyl-CoA and malonyl-CoA, both derived through glycolysis/citric acid cycle, and coenzyme NADPH, derived from the pentose phosphate pathway (PPP) [2], as indicated in the following reaction equation. Palmitic acid, which is the end-product in FASN enzyme activity, is a key intermediate for lipid metabolism. Several palmitic acid-derived lipids act as second messengers and are involved in the regulation of growth and immunerelated signaling pathways, including, PI3K, MAPK, and NFkB [2, 4,5,6]. FASN upregulation mediated by oncogenic signals (including HER2, EGFR, MAPK, and PI3K) through sterol response element binding proteins is surmised as lipogenic in nature [2]
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