Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Abstract

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.

Extended Data Fig. 1. Characterization of cells cultured in 5-methyl THF.

(A-B) Histogram (A) or box plot (B) of log₂ MTR mRNA levels for cells in the Cancer Cell Line Encyclopedia (CCLE) (n = 1019/1457 cell lines with detectable MTR). Median (box center line), interquartile range (IQR) (box), 1.5*IQR (whiskers), and outliers (points) are plotted; dashed line represents the overall median. (C) Proliferation rate of T.T cells in media containing no folate source (−), folic acid (+FA), or 5-methyl THF (+5-MeTHF) before (0 days, n = 3 independent samples) or after (3 days, n = 6 independent samples) of culture with no folic acid (“prestarved”). (D) LC/MS measurement of folic acid and 5-methyl THF concentrations in dialyzed fetal bovine serum. (E-H) LC/MS measurement of intracellular methionine, serine, lysine (E), SAM (F), AMP, ADP, ATP, GMP, GDP, GTP, GAR, AICAR, IMP (G), dUMP and dTMP (H) in A549 and T.T cells cultured for up to 3 days in medium lacking folates (n = 3 independent samples). Data are normalized to protein concentration and an internal standard. (I) Proliferation rate of A549 or T.T cells cultured in medium lacking folates for up to 3 days (n = 4 independent samples). (J-L) Correlation between the proliferation rate in 5-methyl THF and folic acid medium (J; dashed line: y=x), log₂ MTR mRNA level and log₂ relative viability in 5-methyl THF versus folic acid medium (K), or log₂ MTR mRNA level and proliferation rate in 5-methyl THF / folic acid (L) for 489 barcoded cell lines (Pearson’s product moment correlation coefficient / p-value for J: R = 0.255, p = 2e-07; K: R = −0.041 p = 0.4; L: 5-methyl THF R = 0.08, p = 0.11 and folic acid R = 0.1, p = 0.05). Purple line is a linear regression fit, shading represents a 95% confidence interval around the mean. (M) Western blot analysis of MTR expression in A549 or T.T cells cultured in medium with the indicated folate source (representative of 1 experiment). (A-M) Mean +/− SD error bars are displayed.

Extended Data Fig. 2. MTR knockout affects levels of intracellular folate species and proliferation in different folate sources.

(A) Proliferation rates of A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA) or 96% 5-methyl THF, 4% folic acid (phys ratio) for 4 days after a folate pre-starvation period (n = 3 independent samples). (B) LC/MS measurement of intracellular folate, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, and 5-formyl THF levels in A549 and T.T MTR knockout cells +EV or +MTR cultured up to 3 days in medium lacking folates (n = 1 independent sample for each time point). Data are normalized to protein concentration and an internal standard. (C) Proliferation rate of A549 or T.T MTR knockout cells +EV or +MTR cultured in medium lacking folates (prestarved) or with folic acid for 3 days (n = 3 independent samples; A549: p = 0.011; T.T: p = 0.046). p values indicated are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05). (A,C) Mean +/− SD error bars are displayed.

Extended Data Fig. 3. MTR knockout results in folate insufficiency and impaired nucleotide synthesis in 5-methyl THF.

(A-D) LC/MS measurement of intracellular AMP, ADP, ATP (A), GMP, GDP, GTP (B), GAR, AICAR, IMP (C), and dTMP (D) levels in T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in folic acid (+FA) or 5-methyl THF (+5-MeTHF) after a prestarvation period (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: AMP: 0.014, ADP: 0.002, ATP: 0.014, GMP: 0.26, GDP: 0.008, GTP: 0.017, GAR: 0.004, AICAR: 0.005, IMP: 0.014, dTMP: 0.01; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: AMP: 0.012, ADP: 0.0097, ATP: 0.007, GMP: 0.0006, GDP: 0.008, GTP: 0.012, GAR: 0.002, AICAR: 0.003, IMP: 0.022, dTMP: 2.51*10⁻⁵; +EV +FA vs +EV +5-methyl THF p-values: AMP: 0.007, ADP: 0.006, ATP: 5.26*10⁻⁵, GMP: 0.0004, GDP: 0.006, GTP: 2.94*10⁻⁵, GAR: 0.002, AICAR: 0.003, IMP: 0.008, dTMP: 0.005). Data are normalized to protein concentration and to an internal standard. Mean +/− SEM error bars are displayed. (E) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured as in A-D in the indicated folate with or without the addition of 100 μM each of the indicated nucleotide precursors hypoxanthine (H), uridine (U), and thymidine (T) (n = 3 independent samples; except +MTR +H,U,T n = 6). (F) Proliferation rates of A549 MTR knockout cells +EV or +MTR passaged in the indicated folate with the addition of nucleotide precursors for up to 16 days (n = 3 independent samples). (G) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured in the indicated folate as in A-D, with or without 1 mM serine or formate (n = 3 independent samples). (E-G) Mean +/− SD error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Extended Data Fig. 4. Neither SAM nor methionine levels are limiting for proliferation in MTR knockout cells in 5-methyl THF.

(A-B) Proliferation rates of A549 and T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA), 5-methyl THF (5-MeTHF), or no folate (−) for 4 days after a prestarvation period, with or without 900 uM added methionine (A) or T.T MTR knockout cells +EV or +MTR cultured in the indicated folate or 100 uM each nucleotide precursor (hypoxanthine, uridine, and thymidine) at varied extracellular methionine concentrations (B). (C-D) LC/MS measurement of intracellular methionine, serine, lysine (C), SAM, and SAH levels (D) in T.T MTR knockout cells +EV or +MTR cultured in the indicated folate source as in A (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: methionine: 0.002, serine: 0.57, lysine: 9.94*10⁻⁶, SAM: 0.007, SAH: 0.024; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: methionine: 0.01, serine: 2.71*10⁻⁵, lysine: 0.0001, SAM: 0.003, SAH: 0.47; +EV +FA vs +EV +5-methyl THF p-values: methionine: 0.011, serine: 5.57*10⁻⁵, lysine: 0.0001, SAM: 0.24, SAH: 0.27). Data are normalized to protein concentration and an internal standard. (E) Western blots to assess phosphorylation of mTORC1 targets and levels of mono- or dimethyl lysine-containing proteins in T.T MTR knockout cells +EV or +MTR cultured in the indicated folate as in A. The ratio of phospho-protein to total protein signal from 3 independent replicates is shown. (F) Proliferation rates of T.T MTR knockout cells +EV or +MTR cultured in the indicated folate for 16 hours, with or without the addition of 1 mM SAM (n = 3 independent samples for +EV, n = 6 for +MTR; +EV −folate vs +EV +SAM p = 1.6*10⁻⁶, +EV +SAM vs +MTR +SAM p = 0.11). (A,B,F) Mean +/− SD error bars are displayed. (C-E) Mean +/− SEM error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Extended Data Fig. 5. 5-methyl THF medium blunts methotrexate efficacy but not import.

(A-B) Proliferation rates of A549 and T.T cells (A) or T.T MTR knockout cells without (+EV) or with MTR expression (+MTR) (B) cultured in folic acid (+FA) or 5-methyl THF (+5-MeTHF) for 4 days across a range of methotrexate doses (n = 3 independent samples). (C) Log₂ fold change in relative viability of 489 cell lines cultured in 5-methyl THF versus folic acid as in A-B (log2 fc 5-MeTHF/FA) at the indicated methotrexate doses, grouped by tissue of origin (note that the 0 dose is the same data plotted in Fig. 1G). Median (box center line), interquartile range (IQR) (box), and 1.5*IQR (whiskers) are plotted. (D) Mean barcode read counts for the total pool of cancer cell lines, before (day 0) or after (day 4) culture in the indicate folate/methotrexate doses as in A-B. Tukey HSD p-value was calculated by correcting a two-way ANOVA test for multiple comparisons between the dose response curves in the two folates (n = 489 cell lines; folic acid vs. 5-methyl THF dose response p = 0.049). (E-G) LC/MS measurement of intracellular AMP, ADP, ATP, GMP, GDP, GTP, GAR, AICAR, IMP, dUMP, dTMP, dTTP (E), methionine, serine, lysine (F), and intracellular methotrexate (Mtx) (G) levels in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate across a range of doses of methotrexate as in A-B (n = 3 independent samples). Data are normalized to cell number and an internal standard. (A-G) Mean +/− SD error bars are displayed, except in D where mean +/− SEM is displayed.

Figure 1.. Cells can be cultured in medium with 5-methyl THF as the folate source.

(A) Schematic showing reactions involved in folate and methionine metabolism. THF: tetrahydrofolate. SAM: S-adenosyl methionine. SAH: S-adenosyl homocysteine. dTMP: deoxythymidine monophosphate. dUMP: deoxyuridine monophosphate. MTR: methionine synthase. DHFR: dihydrofolate reductase. SHMT: serine hydroxymethyl transferase. MTHFR: methylenetetrahydrofolate reductase. MTHFD: methylenetetrahydrofolate dehydrogenase. MTHFS: methylenetetrahydrofolate synthetase. AHCY: adenosylhomocysteinase. MAT: methionine adenosyltransferase. TYMS: thymidylate synthase. AICARFT: aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (AICAR). GARFT: phosphoribosylglycinamide (GAR) formyltransferase. (B) LC/MS measurement of 5-methyl THF (5-MeTHF) and folic acid levels in plasma from NSG mice (n = 10 mice; p = 4*10⁻⁷). p-value is derived from a two-tailed, unpaired Welch’s t test. (C) Proliferation rate of A549 cells in medium containing no folate (−), folic acid (+FA), or 5-methyl THF (+5-MeTHF) before (0 days, n = 3 independent samples) or after (3 days, n = 6) of culture with no folate (“prestarved”). (D) LC/MS measurement of intracellular folic acid (folate), THF, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, and 5-formyl THF levels in A549 or T.T cells cultured for the indicated number of days in medium lacking folates (n = 3 independent samples). Data are normalized to cell number and an internal standard. (E) Proliferation rate of A549 cells when switched to medium with the indicated folate for 4 days, after 3 weeks of continuous passaging in folic acid or 5-methyl THF (n = 3 independent samples). (F) Proliferation rate of A549 cells in medium containing the indicated amounts of either folic acid or 5-methyl THF (n = 3 independent samples). (G) Log₂ fold change in relative viability for 489 barcoded cell lines grouped by tissue of origin when cultured 5-methyl THF versus folic acid (log2 fc 5-MeTHF/FA). Median (box center line), interquartile range (IQR) (box), and 1.5*IQR (whiskers) are plotted; dashed line represents the population median. (A-G) Mean +/− SD error bars are displayed.

Figure 2.. Methionine synthase is only essential for proliferation when 5-methyl THF is the folate source.

(A) Western blot analysis of MTR expression in parental A549 and T.T cells as well as MTR knockout cells expressing an empty vector (+EV) or MTR (+MTR), cultured in RPMI (representative of 2 independent experiments). (B) Proliferation rates of A549 and T.T MTR knockout cells +EV or +MTR after culture in folic acid (+FA) or 5-methyl THF (+5-MeTHF) for 4 days after a folate pre-starvation period (n = 3 independent samples). Mean +/− SD error bars are displayed. (C) LC/MS measurement of intracellular folate, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, and 5-formyl THF levels in A549 and T.T MTR knockout cells +EV or +MTR after culturing cells in the indicated folate as in B. Data are normalized to cell number and an internal standard. Mean +/− SEM error bars are displayed.

Figure 3.. MTR is required for nucleotide synthesis in 5-methyl THF medium.

(A-D) LC/MS measurement of intracellular AMP, ADP, ATP (A), GMP, GDP, GTP (B), GAR, AICAR, IMP (C), and dTMP (D) levels in A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in folic acid (+FA) or 5-methyl THF (5-MeTHF) after a folate prestarvation period (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: AMP: 0.006, ADP: 0.003, ATP: 0.015, GMP: 0.004, GDP: 0.007, GTP: 0.011, GAR: 0.002, AICAR: 0.003, IMP: 0.007, dTMP: 0.79; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: AMP: 0.01, ADP: 0.022, ATP: 0.005, GMP: 0.019, GDP: 0.015, GTP: 0.009, GAR: 0.003, AICAR: 0.005, IMP: 0.025, dTMP: 0.001; +EV +FA vs +EV +5-methyl THF p-values: AMP: 0.0002, ADP: 0.008, ATP: 0.0006, GMP: 0.54, GDP: 0.0004, GTP: 0.0007, GAR: 0.01, AICAR: 0.07, IMP: 0.016, dTMP: 0.019). Data are normalized to protein concentration and an internal standard. Mean +/− SEM error bars are displayed. (E-F) Proliferation rates of A549 MTR knockout cells without +EV or +MTR cultured as in A-D in the indicated folate, with or without the addition of 100 μM each of the indicated nucleotide precursors hypoxanthine (H), uridine (U), thymidine (T) (n = 3 independent samples; except +MTR +H,U,T n = 6) or the addition of 1 mM serine or formate (n = 3 independent samples). Mean +/− SD error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 4.. MTR knockout reduces SAM but not methionine levels.

(A) Proliferation rates of A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA) or 5-methyl THF (5-MeTHF) or in 100 uM each nucleotide precursor (hypoxanthine, uridine, and thymidine) for 4 days after a folate prestarvation period, at varied extracellular methionine concentrations (n = 3 independent samples, except n = 6 for +FA 100 uM methionine +EV/+MTR). (B-C) LC/MS measurement of intracellular methionine, serine, lysine (B), SAM, and SAH levels (C) in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate as in A (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: methionine: 0.01, serine: 0.0001, lysine: 0.936, SAM: 0.0009, SAH: 0.012; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: methionine: 0.003, serine: 0.007, lysine: 0.0001, SAM: 0.012, SAH: 0.152; +EV +FA vs +EV +5-methyl THF p-values: methionine: 0.001, serine: 0.01, lysine: 9.1*10⁻⁶, SAM: 0.47, SAH: 0.1). Data are normalized to protein concentration and an internal standard. (D) Western blots to assess phosphorylation of mTORC1 targets and levels of mono- or dimethyl lysine-containing proteins in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate for 16 hours. The ratio of phospho-protein to total protein signal from 3 independent replicates is shown. (E) Proliferation rates of A549 MTR knockout cells +EV or +MTR cultured in the indicated folate as in A, with or without the addition of 1 mM SAM (n = 3 independent samples for +EV, n = 6 for +MTR; +EV −folate vs +EV +SAM p = 0.084, +EV +SAM vs +MTR +SAM p = 0.001). (A,E) Mean +/− SD error bars are displayed. (B-D) Mean +/− SEM error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 5.. Physiological folates prevent growth of MTR knockout tumors and cause methotrexate resistance.

(A) Tumor weight of subcutaneous xenografts generated by injection of 100,000 A549 MTR knockout cells without (+EV) or with (+MTR) MTR expression into the flanks of NSG mice. Tumors were harvested after 4 months (n=3 mice per condition; n.d.: not detected). (B-C) Plasma concentration of folic acid (B) and 5-methyl THF (C) in NSG mice provided with unlimited water (control water) or water containing 0.1 g/L folic acid (+folic acid water) for 3 weeks (n=10 mice per condition; folic acid p = 0.1; 5-methyl THF p = 0.053). (D) Tumor weight of subcutaneous xenografts formed by injecting 1,000,000 A549 MTR knockout cells +EV or +MTR into the flanks of NSG mice provided with control or folic acid water as in B-C since the day of injection. Tumors were harvested after 3 months (n=5 mice per condition; +MTR control vs. folic acid water p = 0.1, +EV vs +MTR folic acid water p = 0.5; n.d.: not detected). (E) Proliferation rates of A549 MTR knockout cells +EV or +MTR cultured in folic acid (+FA) or 5-methyl THF (5-MeTHF) for 4 days across a range of methotrexate doses (n = 3 independent samples). (F) Overall population log₂ fold change in relative viability of 489 cell lines cultured in 5-methyl THF versus folic acid as in E at the indicated methotrexate doses. Median (box center line), interquartile range (IQR) (box), and 1.5*IQR (whiskers) are plotted (n = 489 cell lines). (G) Overall pooled 489 cell line population bulk proliferation rate in doublings per day (left panel, n = 4 measurements of 1 pooled sample; overall folic acid vs. 5-methyl THF dose-response p-value = 0.013, pairwise comparison adjusted p-values: 0 nM p = 1, 7.8 nM p = 0.233, 31.3 nM p = 0.001, 1000 nM p = 0.28) and averaged proliferation rates across all cell lines (right panel, n = 489 cell lines; overall folic acid vs. 5-methyl THF dose-response p-value = 3.5*10⁻⁵, pairwise comparison adjusted p-values: 0 nM p = 0.85, 7.8 nM p = 5.2*10⁻⁵, 31.3 nM p ~ 0, 1000 nM p ~ 0) cultured in 5-methyl THF or folic acid as in E at the indicated methotrexate doses. Tukey HSD and pairwise comparison p-values at each dose were calculated by correcting a two-way ANOVA test for multiple comparisons between the dose response curves in the two folate sources. (H) LC/MS measurement of intracellular folate, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, THF, and DHF levels in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate as in E across a range of methotrexate doses (n = 3 independent samples). Data are normalized to protein concentration and an internal standard. (I) Proliferation rates of A549 MTR knockout cells +MTR cultured in the indicated folate as in E with or without the addition of 100 μM each of the indicated nucleotide precursors hypoxanthine (H), uridine (U), and thymidine (T), as well as methotrexate or vehicle (DMSO) (n = 3 independent samples except n=6 for samples +FA − vehicle, − 4 uM, and +H,U,T +4 uM and +5-MeTHF − vehicle). For the +H and +U,T conditions, only the lower dose (500 nM) of methotrexate was assessed. (A-I) Mean +/− SD error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test except as otherwise indicated in G (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).