Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability

•A variety of cancer cells rely on cytosolic 1C flux in physiological folate medium•Intracellular levels determine the versus mitochondrial flux•SLC19A1 regulates compartmentalized by modulating intracellular levels•SHMT1 is essential for growth low RFC-expressing tumors vitro and vivo Folate metabolism supplies one-carbon (1C) units biosynthesis methylation has long been a target chemotherapy. Mitochondrial serine catabolism considered sole contributor folate-mediated proliferating cells. Here, we show that under cell environment, serine-hydroxymethyltransferase (SHMT1) predominant source cancers, while overly repressed. Tumor-specific reliance associated with poor capacity to retain folates, which determined expression SLC19A1, encodes reduced carrier (RFC). We silencing SHMT1 RFC impairs pyrimidine tumor vivo. Overall, our findings reveal major diversity utilization cycle across SLC19A1 as marker increased SHMT1. 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Food 2007; 51: 267-292Crossref (301) Still, understanding mostly relies experiments performed standard tissue culture conditions, total concentration order magnitude higher than (150–450 nM [Fazili 2008Fazili Pfeiffer Zhang R.B. Koontz Influence 5,10-methylenetetrahydrofolate reductase polymorphism whole-blood concentrations measured LC-MS/MS, microbiologic assay, bio-rad radioassay.Clin. Chem. 2008; 54: 197-201Crossref (52) Wishart 2018Wishart D.S. Feunang Y.D. Marcu Guo A.C. Liang Vázquez-Fresno Sajed Johnson Karu al.HMDB 4.0: metabolome database 2018.Nucleic Acids 46: D608-D617Crossref (1519) Scholar] 2.2 ?M 9 RPMI DMEM, respectively). analyzing series challenge current view find ~50% lines. shown depend controlling levels. To examine relative contribution levels, stable isotope panel Cells fed [2,3,3-2H]-serine incorporation deuterium labeling synthesized thymidine triphosphate (dTTP) monitored. Thymidine atoms (M+2) via through SHMT1, one (M+1) SHMT2; 5,10-methylene-THF oxidized formate, transported incorporated into THF, eventually back 5,10-methylene-THF, synthesizing dTTP (Figure 1A). lowering media level (from 200 nM, growing them at least 2 weeks ensure steady state) ~10-fold increase average different lines 1B). Under folate, became origins (dTTP M+2/M+1 > 1; Figure S1A), including T glioblastoma, non-small-cell lung carcinoma. Notably, had no rate, suggesting sufficient meet demands conditions S1B). producing drop whole-cell flux, marked decrease specifically SHMT2 flux. Reduced evident ~5-fold consumption CCRF-CEM grown high (p < 0.001; 1C) consistent products, formate. While excreted media, substantial uptake observed folates (~0.8 mM/h; uptake). Increased cellular extracellular novo production further significant M+1 feeding 1D). Formate rate showed ~7-fold reduction 1C). condition NADP+ MTHFD2. Feeding significantly lowered fractional proline 1E), d-1-pyrroline-5-carboxylate synthase (ALDH18A1), oxidizing transferring hydrogen precursor L-glutamate-5-semialdehyde Jurkat A549 switched relying S2). These results suggest appears some Nilsson it may not be ubiquitous all bias interpretation based exchange effects, where near chemical equilibrium simultaneously catalyze forward backward directions (Fan 2013aFan Kamphorst J.J. Shlomi Fatty glutamine hypoxia explained without net reductive isocitrate (IDH) flux.J. Biol. 288: 31363-31369Abstract (46) overcome quantitatively infer fluxes, employed variant analysis (MFA), modeling fluxes experiments. Our approach searches likely whole-cell-level-measured isotopic matches convolution simulated these metabolites (STAR Methods; S3; Tables S1, S2, S3, S4). CCRF- CEM 1F). Exploring how regulate response availability, 2A) or global mRNA pattern S4A). Metabolomics using liquid chromatography-mass spectrometry (LC-MS) revealed >50-fold purine intermediates 5?-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) 5?-phosphoribosyl-glycinamide (GAR) (Figures 2B S4B), indicating pools Indeed, THF 10-formyl-THF 80%–90% 2C 2D). comparison, MCF-7 HCT116, showing smaller 0.05; Figures substrate-level downregulation depletion folates. Accordingly, resulted induction decreased ~16% 1 h after media; p 2E). explore mechanism underlying line-specific analyzed genes studied (utilizing transcriptomics data Cell Line Encyclopedia Collection; CCLE) (Klijn 2015Klijn Durinck Stawiski E.W. Haverty P.M. Jiang Degenhardt Mayba Gnad al.A comprehensive transcriptional portrait lines.Nat. Biotechnol. 2015; 33: 306-312Crossref (348) (RFC), lower 3A). validated qRT-PCR have 3B), affected 3C). anion antiporter utilizes phosphate gradient achieve uphill transport ubiquitously expressed tissues (Hou Matherly, 2014Hou Matherly L.H. Biology facilitative transporters SLC46A1.Curr. Top. Membr. 73: 175-204Crossref (65) Note other SLC46A1 coding proton-coupled (PCFT) receptors (Fra-FOLR1, Frb-FOLR2, Frg-FOLR3) correlated test whether overexpressed 3D). overexpression induced preventing shift unaffected overexpress SLC19A1; 3E). Induced upon 3F). 3G), also evidenced AICAR GAR overexpressing control S5A S5B). Similarly, CEM-7A (Rothem 2002Rothem Ifergan Kaufman Priest D.G. Jansen Assaraf Y.G. Resistance multiple novel antifolates mediated drug resulting clustered mutations leukaemia lines.Biochem. 2002; 367: 741-750Crossref Scholar) compared their parental S5C–S5G). Analogously, depleting HCT116 utilize 3H) repression 0.01; 3I). knockdown 3J). Finally, 4K), increasing S5H S5I). Taken together, modulates availability. Considering expression, sought knockout will hinder growth. SHMT1-deficient 4A) ~65% 0.001), larger 4B). (KO) S6A). KO partially rescued impaired growth, cannot demand proliferation ~27% fraction phase 4C) dUMP ratio 4D), insufficient thymidylate (TS) replication. contrast S6B S6C) S6D), did lead dUMP/dTTP pool size S6E). evaluate vivo, implanted onto hind flanks immunocompromised NOD/SCID mice. As expected, grew slower, volume 8 4E, 4F, S7A, S7B). xenograft phenotype vitro; TS inhibited tumors, ~100-fold UMP dTMP 4G). Furthermore, ~15-fold intratumor 4H), inhibiting biosynthesis, accumulation phosphoribosyl-N-formylglycineamide (FGAR; S7C). Consistently, AMP GMP dropped 10- 6-fold respectively 0.01) 4H). confirm identification characterization specific design effective stratified treatments supplying 2020Li Seoane Xiao Melemenidis Zhou Vanharanta Graves E.E. al.Metabolic profiling reveals dependency metastatic breast unit metabolism.Mol. 2020; 18: 599-611Crossref (22) formyl-methionine (Minton 2018Minton Nam McLaughlin D.J. Shin Bayraktar E.C. Alvarez S.W. Sviderskiy V.O. Papagiannakopoulos Birsoy Possemato proper initiation maintenance formylmethionyl-tRNAs.Mol. 69: 610-621.e5Abstract (69) S1A) confirmed inhibits S8A–S8D). Exploiting small-molecule enzyme, dual inhibitors, active investigation García-Cañaveras 2020García-Cañaveras J.C. Lancho Gh