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Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migratio

Issuing time:2022-05-27 11:44

Abstract

Metastasis is the most common cause of death in cancer patients. Canonical drugs target mainly the proliferative capacity of cancer cells, which leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions are still poorly understood. Here we show that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism during cytosolic 1C metabolism impairment. Interestingly, antifolate dependent growth-arrest does not correlate with decreased migration capacity. Therefore, using methotrexate as a tool compound allows us to disentangle proliferation and migration to profile the metabolic phenotype of migrating cells. We observe that increased serine de novo synthesis (SSP) supports mitochondrial serine catabolism and inhibition of SSP using the competitive PHGDH-inhibitor BI-4916 reduces cancer cell migration. Furthermore, we show that sole inhibition of mitochondrial serine catabolism does not affect primary breast tumor growth but strongly inhibits pulmonary metastasis. We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

Introduction

The survival rates of patients harboring primary tumors steadily increase due to targeted treatment schemes. However, in case of tumor relapse and metastatic disease, effective therapies are mostly lacking with the consequence that secondary tumors account for the majority of cancer deaths1. Classical chemotherapeutic approaches to counteract cancer growth aim to target biomass production by interfering with the synthesis of proteins, lipids, and nucleotides and thereby directly interfere with central pathways of cancer cell metabolism2,3. Prospectively, therapeutic approaches targeting metabolic pathways that also support the invasive and migratory properties of cancer cells would help to prevent metastatic progression of the initial disease. However, profound knowledge of specific metabolic dependencies that support growth-independent processes during the metastatic cascade, are only starting to be understood4.

Nucleotide synthesis is an essential requirement for cancer cell proliferation. Consequently, antifolates such as Methotrexate (MTX) and related compounds have been proven as successful chemotherapeutics for several decades. Although differences between the various antifolates exist, a shared mode of action is the inhibition of one-carbon (1C) metabolism which provides essential building blocks for thymidylate and purine synthesis. Additionally, 1C metabolism has implications in supporting methylation reactions, glutathione, heme, sphingolipid and protein synthesis5. 1C metabolism describes a metabolic cycle that is spread across mitochondrion and cytoplasm and it is mainly fueled by the non-essential amino acid serine. De-novo serine synthesis is catalyzed by the rate limiting enzyme phosphoglycerate dehydrogenase (PHGDH) which is amplified in different tumors6,7,8. Hence, targeting serine synthesis, serine catabolism or serine availability by starvation or inhibition of PHDGH has been proven successful to inhibit cancer progression9,10,11,12,13,14,15,16,17. To support the various anabolic programs, cytosolic 1C metabolism is of particular importance, while mitochondrial 1C metabolism is dispensable due to the reversibility of the cytosolic 1C metabolism16. In fact, depending on intracellular folate levels, cytosolic 1C metabolism can be the dominant route to provide one-carbon units for nucleotide synthesis17. In that light, reasons for conserved mitochondrial 1C metabolism are still not fully understood. Previous evidence suggests implications of mitochondrial 1C metabolism for mitochondrial translation18,19. One additional explanation resides in the observation that mitochondrial derived formate lowers the pool of free cytosolic folates by increasing the 10-CHO-THF pool thereby protecting it from degradation. Upon MTX, degradation is facilitated as folates accumulate in the oxidized DHF form, impeding the 10-CHO-THF synthetase activity of MTHFD120. However, if mitochondrial folate-dependent 1C metabolism and formate production remains functional under conditions of cytosolic 1C metabolism impairment and whether mitochondrial 1C metabolism itself confers additional functions that contribute to tumorigenesis remains unknown.

We have previously shown that a reduction of biomass demand can result in increased formate release from cancer cells to their surrounding microenvironment21 and that the rate of serine catabolism to formate is significantly increased in adenomas of the small intestine and breast tumors compared to normal tissue22. Importantly, enhanced formate release rates and increased extracellular formate concentrations have been shown to increase the invasiveness of glioblastoma cells in vitro22.

Apart from our previous findings on formate overflow and its potential implications for cancer cell invasion21,22, other reports substantiate a role of serine metabolism in the context of metastasis7,14,23,24,25. However, such studies were so far focused on the relevance of serine de novo synthesis via PHGDH or extracellular serine availability for metastatic outgrowth (proliferative aspect) in the secondary tissue7,14,23,24,25. In contrast, whether mitochondrial serine catabolism also has a growth-independent role in promoting cancer cell motility that potentially increases the rate of cancer cell escape from the primary tumor has not been addressed so far. Being able to identify and target metabolic programs that promote the early steps of the metastatic cascade would help to contain the tumor, a clinical phenotype with more favorable therapeutic outcome compared to advanced metastatic stages.

In the present study, we observed that antifolates, albeit being strongly cytostatic, did not decrease the cell’s motility potential. Using MTX as a tool compound to selectively inhibit cytosolic 1C metabolism, we show that mitochondrial 1C metabolism runs as an autarkic, compartmentalized pathway that is important to drive cancer cell motility. In contrast, the cytosolic part of 1C metabolism, which is essential for nucleotide synthesis during proliferation, is dispensable for the cellular motility potential. Hence, we provide mechanistic evidence that one selective advantage of mitochondrial 1C metabolism is to support cell motility. We corroborate this hypothesis in an orthotopic breast cancer model. Here, we demonstrate that ablating mitochondrial 1C metabolism effectively blocks mitochondrial serine catabolism but does not affect primary tumor growth. Yet, it strongly inhibits pulmonary metastasis formation.

Results

Inhibition of anabolic synthesis routes differentially impacts the motility potential of cancer cells

To assess the relative importance of different anabolic pathways for cancer cell migration, we employed a panel of metabolic perturbations targeting glycolysis, mitochondrial electron transport chain (ETC) as well as lipid, protein, and nucleotide synthesis (Fig. 1a). As expected, the chosen metabolic interventions inhibited proliferation and affected cell cycle progression of MDA-MB-468 breast cancer cells with no to mild toxic effects after 48 h treatment (Fig. 1b–c, S1A). Inhibition of glycolysis, ETC, lipid, and protein synthesis significantly reduced migration of MDA-MB-468 cells (Fig. 1d). Surprisingly and in contrast to all other growth-inhibiting conditions, inhibition of nucleotide synthesis with various drugs did not diminish migration of MDA-MB-468 cells (Fig. 1e). Calculation of the area under curve (AUC) allowed us to quantitatively compare wound closure over time (Fig. 1d, e) and confirmed that wound closure was significantly reduced upon treatment with rotenone (Rot), galactose (Gal), simvastatin (SIM) and sirolimus (rapamycin, SIR). However, treatment with the antifolates methotrexate (MTX) and pemetrexed (PEM), as well as with hydroxyurea (HU) and clofarabine (CLO) did not affect or did even significantly increase wound closure in scratch assays in comparison to fully proliferative, untreated control cells. While cell migration correlates with reduced cell growth for all other metabolic perturbations, nucleotide synthesis inhibition does not reduce cell migration in correlation to growth inhibition (Fig. 1f, g). Sustained migration in response to nucleotide synthesis inhibition was verified in LN229 glioblastoma and 4T1 breast cancer cells (Fig. S1B–D). Of note, using dialyzed or normal FBS did not change the result (Fig. S1E). This observation was also confirmed in trans-well migration assays using Boyden chambers (Fig. 1h, S1F, G). Furthermore, using ECM-collagen coating in trans-well assay, we found that nucleotide synthesis inhibition also had no impact on the invasive capacity of MDA-MB-468 cells (Fig. 1h), LN229 and 4T1 cells (Fig. S1F, G). To validate if MTX resistance results in a pro-migratory effect, we generated MTX-resistant MDA-MB-468 cells by long-term cultivation in 50 nM MTX for 2 months. The resulting MTX-resistant MDA-MB-468 cells proliferate in the presence of increasing concentrations of MTX (Fig. 1i) and display enhanced migratory capacity compared to the parental MDA-MB-468 cell line (Fig. 1j).

Fig. 1: Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells.
figure 1

a Synthesis routes of metabolic building blocks (lipids, nucleotides, and proteins) and pharmacologic intervention points of the selected drug panel. b MDA-MB-468 cells were counted after 48 h treatment with 100 nM Sirolimus (SIR) (n = 3), 1 μM Simvastatin (SIM) (n = 3), 50 nM Rotenone (Rot) (n = 5), galactose (Gal) (n = 5) supplementation, 50 nM Methotrexate (MTX) (n = 4), 1 μM Pemetrexed (PEM) (n = 4), 0.5 mM hydroxyurea (HU) (n = 4), and 100 nM Clofarabine (CLO) (n = 4). Mean ± SD; ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Unlabeled comparisons to Ctrl are all significant with p < 0.0001. c MDA-MB-468 cells were treated as in (b). Cell death was assessed by flow cytometry and AnnexinV-FITC/PI-staining; mean ± SD of independent experiments (n = 8 (Ctrl), n = 4 (SIR, SIM, PEM, CLO), n = 3 (Rot, Gal, MTX, HU)). Two-way ANOVA with Dunnett’s multiple comparisons test. d, e Migration of MDA-MB-468 cells upon (d) 50 nM Rot (n = 5), Gal (n = 5), 1 μM SIM (n = 3), and 100 nM SIR (n = 4) or (e) 50 nM MTX (n = 7), 1 μM PEM (n = 3), 0.5 mM HU (n = 4), and 100 nM CLO (n = 4) and respective area under curve (AUC); mean ± SEM of independent experiments; Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. f, g Correlation of cell migration (AUC over 40 h, mean ± SEM, (n = as in (d, e)) and proliferation (fold cell growth after 48 h, mean ± SD (n = as in (b)). MDA-MB-468 cells were treated as in (b) and (d, e). h Migration and invasion of MDA-MB-468 cells treated for 24 h with 50 nM MTX using non-coated (migration) or ECM-Collagen-coated (invasion) Boyden chambers. Mean ± SEM (Migration: n = 3, Invasion: n = 7 (Ctrl), 5 (MTX, 3 (Pem)); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. i Proliferation of MTX-resistant MDA-MB-468 cells upon the indicated concentrations of MTX [nM]; mean ± SEM of independent experiments (n = 4). j Migration of parental and MTX-resistant MDA-MB-468 cells and respective AUC; mean ± SEM of independent experiments (parental n = 15, resistant n = 6); unpaired, two-tailed t-test with Welch’s correction. Time point of 50% wound closure is indicated. Source data are provided as a Source Data file.

In conclusion, the perturbation of any major anabolic route results in growth repression. However, the impact on cell migration highly depends on the targeted metabolic pathway, with nucleotide synthesis being an ineffective target to abrogate cell motility. Furthermore, prolonged nucleotide synthesis inhibition in drug-resistant cells acts as a selection pressure for more motile cancer subpopulations.

Sustained motility upon MTX is supported by a ROS-driven EMT phenotype

As MTX induced the strongest growth repression without increasing cell death (Fig. 1b, c), we used it as a tool compound to disentangle proliferation from migration to investigate the metabolic processes that support cancer cell migration. After testing multiple concentrations (Fig. S2A), we chose a concentration of 50 nM MTX for subsequent experiments. 50 nM MTX is significantly growth- and cell cycle-arresting in MDA-MB-468 cells (Fig. 1b, S1A) and has also been shown to be within the therapeutic window26,27,28,29. As this concentration of 50 nM is significantly lower compared to earlier in vitro studies20,30,31,32,33, we first set out to validate known drug effects of MTX in our system. We observed that 48 h MTX treatment of MDA-MB-468 cells resulted in altered cellular morphology with individual cells visibly increasing in cell size and exhibiting an outstretched cell shape with protruding edges (Fig. 2a). MTX-resistant MDA-MB-468 cells showed comparable morphologic alterations (Fig. S2B). While the observed cell cycle and proliferation arrest upon MTX treatment was rapidly reversible (Fig. S2C, D) upon MTX removal, [U-13C]serine tracing and metabolic flux analysis confirmed that 50 nM MTX did strongly inhibit nucleotide synthesis (Fig. 2b). Of note, nucleotide synthesis was partially restored in MTX-resistant MDA-MB-468 cells (Fig. S2E). Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative proteomics analysis further confirmed the efficacy of the selected MTX dose as mechanistic targets of MTX such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and thymidine kinase (TK1) were upregulated by treatment as expected (Fig. S2F). DHFR expression was also increased in MTX resistant cells, while FolRa expression was slightly decreased (Fig. S2G). Moreover, superoxide dismutase (SOD) was one of the most significantly upregulated proteins in response to treatment (Fig. S2F), which corresponds to prior reports that depict MTX as a potent inducer of oxidative stress30,34,35. We validated these prior findings with our low dose of MTX by measuring a moderate but significant increase of mitochondrial and cytosolic reactive oxidant species (ROS) upon acute MTX treatment and in the MTX resistant cells (Fig. S2H, I, J). In line with increased ROS levels we observed NRF2 stabilization, however, this did not result in activation of the BACH1 axis, as reported earlier for metastatic lung cancer36,37 (Fig. S2K, I). Increased ROS levels upon MTX correspond to earlier reports that highlight the potential of oxidative stress as a driver of cancer cell transformation via epithelial-mesenchymal transition (EMT)38,39,40,41,42,43,44. EMT is also a known general mechanism to escape chemotherapy induced effects.

Fig. 2: Enhanced motility under MTX is supported by a ROS-driven EMT phenotype.
figure 2

a Morphology of MDA-MB-468 cells after 48 h 50 nM MTX. Bright-field images are representative of 3 independent experiments. Scale bars correspond to 60 and 300 μm. b Mass isotopomere distribution (MID) of intracellular ATP upon [U-13C]serine tracer in response to 24 h 50 nM MTX in MDA-MB-468 cells. Mean ± SEM of 5 independent experiments each measured in triplicate wells. c mRNA expression from the indicated target genes in MDA-MB-468 cells upon 50 nM MTX at the indicated time points measured by real-time RT-qPCR. Mean ± SEM (n = 3 (24 h, 48 h), 6 (72 h, 144 h)). No data measured for SLUG expression at 72 h. d Protein levels of vimentin in MDA-MB-468 cells after 240 h 50 nM MTX; β-actin as loading control. Quantification of vimentin signal relative to total protein stain. Mean ± SD (n = 3); unpaired, two-tailed t-test with Welch’s correction. e Migratory potential of MDA-MB-468 cells in response to 10 mM N-acetylcysteine (NAC) and 50 nM MTX as quantification of relative wound density in IncuCyte and respective AUC. Graph shows mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple comparisons test. f mRNA expression from the indicated target genes in MDA-MB-468 cells upon 10 mM NAC and 50 nM MTX at 72 h measured by real-time RT-qPCR. Mean ± SEM (n = 3). Source data are provided as a Source Data file.

In line with previous reports, we found that ZEB1 and ZEB2 expression as well as VIM expression was upregulated in a time-dependent manner in response to acute MTX treatment and in the MTX-resistant cell line (Fig. 2c, d, S2F, M, N). MTX-treatment also induced the expression of the collagenase MMP9 (Fig. S2O), an enzyme which is involved in the degradation of the extracellular matrix during cancer cell invasion45. Treatment of cells with the antioxidant N-acetylcysteine (NAC) reduced ROS levels in MTX-treated MDA-MB-468 cells (Fig. S2P) and resulted in a significant reduction of MTX-induced cell migration (Fig. 2e) and EMT phenotype induction (Fig. 2f). These findings confirm that the chosen low dose of MTX is sufficient to promote EMT in a ROS dependent manner, supporting earlier reports suggesting EMT onset in context of pulmonary fibrosis using high doses of MTX46,47. Such moderate increase in cellular ROS levels after MTX might contribute to a pro-migratory stimulus that subsequently results in the observed sustained migration.

MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis

In addition to the here reported MTX-specific effects on ROS and EMT, we wanted to take advantage of the MTX effect (sustained migration) to identify growth-independent metabolic liabilities that support cell migration.

To that end, we profiled central carbon metabolism using [U-13C]glutamine and [U-13C]glucose tracing to monitor glycolytic activity and glutamine- or glucose-derived carbon oxidation through the TCA cycle (Fig. 3a, d). Absolute consumption and release (CORE) rates of glutamine and glutamate were sustained in response to MTX treatment (Fig. 3b), while the relative TCA-cycle flux of [U-13C]glutamine was significantly increased in response to MTX (Fig. 3c). Absolute quantification of CORE rates of glucose and lactate also revealed sustained high glycolytic rates upon MTX treatment (Fig. 3e). This came as a surprise, as lactate release rates were previously shown to correlate with cell growth rates48. In MDA-MB-468 cells, the maintained glycolytic rate and the associated lactate release at constant high levels (Fig. 3e) indicate a constant generation of glycolysis-derived ATP even in the presence of reduced energetic demand for anabolic reactions in growth-arresting MTX conditions. Consequently, [U-13C]glucose distribution within the TCA-cycle was sustained by MTX treatment in MDA-MB-468 cells (Fig. 3f) as well as in 4T1 and LN229 cells (Fig. S3A, B). It has previously been shown that MTX inhibits oxygen consumption rates (OCR) in HCT116 cells18. We could replicate this finding and observed a ~30 % reduction of OCR in HCT116 cells upon MTX (Fig, S3C), while OCR in MDA-MB-468, LN229, and 4T1 cells was sustained (Fig. 3g). This indicates cell line specific effects of MTX on OCR. Furthermore, MTX treatment significantly increased the relative flux of glucose to serine despite decreased anabolic demands for nucleotide synthesis (Fig. 3h, S3D). Protein levels of PHGDH, PSPH, and PSAT1 that catalyze the serine de novo synthesis pathway from glucose were unaltered in response to MTX treatment in multiple cell lines (Fig. S3E–H). In agreement with Diehl et al49. who showed that changes in NAD+/NADH ratio can increase PHGDH activity and in consequence serine de novo synthesis rates, we observed an increased NAD+/NADH ratio upon MTX (Fig. 3i). As MTX is well characterized as an inhibitor of DHFR and thus 1C metabolism, it came to our surprise that MTX treatment did also increase the abundance of extracellular formate M + 1 isotopologues derived from [U-13C]glucose through serine and the mitochondrial 1C metabolism (Fig. 3j, S3I).

Fig. 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis.
figure 3

a Interdependence of glycolysis and tricarboxylic acid (TCA) cycle and metabolic 13C label pattern from [U-13C]glutamine. b Absolute consumption and release (CORE) rates of glutamine (Gln) and glutamate (Glut) in the culture medium of MDA-MB-468 cells after 24 h 50 nM MTX. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM (n = 3). c Enrichment of selected isotopologues of Gln, Glut, α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp), and citrate (Cit) upon [U-13C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an independent experiments in triplicate wells; mean ± SEM; unpaired, two-tailed t-test with Welch’s correction (n = 3). dInterdependence of glycolysis and TCA cycle and metabolic 13C label pattern from [U-13C]glucose. e Absolute CORE rates of lactate and glucose in the culture medium of MDA-MB-468 cells after 24 h 50 nM MTX. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM (n = 3). f Enrichment of selcted isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U-13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an individual experiments composed of triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction (n = 3). g Basal cellular respiration upon 24 h 50 nM MTX in MDA-MB-468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Dots represent the mean of an individual experiments composed of six technical replicates; mean ± SEM (n = 3 (LN229), 4 (MDA-MB-468, 4T1)). h Enrichment of M + 3 isotopologue of serine upon [U-13C]glucose upon 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an independent experiments in triplicate wells; mean ± SEM; unpaired, two-tailed t-test with Welch’s correction (n = 3). i Ratio of absolute intracellular NAD+ to NADH in MDA-MB-468 cells upon 24 h 50 nM MTX. Dots represents the mean of an individual experiments composed of triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction (n = 3). j M + 1 isotopologue of extracellular formate in MDA-MB-468 cells from [U-13C]glucose upon 24 h 50 nM MTX. Dots indicate the mean of an independent experiments measured in triplicate wells; mean ± SEM (n = 4). Source data are provided as a Source Data file.

In summary, the metabolic profiling demonstrates that, despite decreased metabolic demand for biomass production, MTX-treated cells sustain high metabolic rates that are comparable to fully proliferating cells. Additionally, and in contrast to general assumptions, cytosolic DHFR inhibition results in increased rates of serine synthesis and downstream mitochondrial formate excretion suggesting an uncoupling of mitochondrial 1C metabolism from cytosolic 1C metabolism.

Mitochondria protect 1C metabolism-dependent serine catabolism upon cytosolic 1C pathway inhibition

Our finding of increased [U-13C]glucose-derived M + 1 formate excretion under MTX (Fig. 3j, S3I), allows the hypothesis that mitochondrial 1C metabolism can function in an autarkic manner when cytosolic 1C metabolism is inactivated (Fig. 4a). To corroborate this hypothesis, we employed [U-13C]serine tracing to monitor serine flux through 1C metabolism. We observed that cytosolic block of 1C metabolism via MTX did not reduce the exchange rates of serine, glycine and formate in MDA-MB-468 or LN229 cells (Fig. 4b, C, S4A, C). In contrast, the formate release rate was significantly increased and the increased fraction of labeled formate proved that most of the released formate is derived from [U-13C]serine and not from other carbon sources that could alternatively generate formate in presence of MTX. Of note, sustained serine consumption and formate overflow via 1C metabolism upon DHFR inhibition could also be confirmed in Plasmax medium50, which is a culture medium closer to human physiology (Fig. S4D). Since cell dry mass composition is constituted by around 60% of proteins51, growth arresting conditions generally result in decreased consumption rates of proteinogenic amino acids. In fact, a general trend for decreased consumption of essential amino acids was observed (Fig. S4b). Hence, sustained serine consumption rates upon growth arrest suggest that spared serine that is otherwise used for anabolic processes such as nucleotide synthesis, is used for alternative metabolic pathways that support cell motility and survival.

Fig. 4: Mitochondria protect 1C metabolism-dependent serine catabolism upon cytosolic 1C pathway inhibition.
figure 4

a Cytosolic and mitochondrial 1C metabolism and the pharmacologic intervention points of MTX. b Absolute consumption and release (CORE) rates of serine (Ser) and glycine (Gly) from MDA-MB-468 cells upon 24 h 50 nM MTX. Dots represent the mean of an individual experiment each measured in triplicate wells; mean ± SEM (n = 6). c Formate release rate upon [U-13C]serine in MDA-MB-468 cells after 24 h 50 nM MTX; mean ± SEM of three independent experiments each measured in triplicate wells; unpaired t-test with Welch’s correction for M + 1 isotopologue (n = 3). d MID of intracellular serine upon [U-13C]serine in MDA-MB-468 cells upon 24 h 50 nM MTX; mean ± SEM of four independent experiments each measured in triplicate wells. e Ratio of absolute intracellular serine and glycine after 24 h 50 nM MTX in MDA-MB-468 cells. Dots represent the mean of an independent experiment in triplicate wells; mean ± SEM (n = 5). f MID of intracellular serine upon [U-13C]serine in SHMT1, MFT, SHMT2, and FPGS-depleted HAP1 cells; mean ± SEM of independent experiments each measured in triplicate wells (n = 8 for CTRL, SHMT1ko, and SHMT2ko; n = 5 for MTX; n = 2 for FPGSko, MFTko and MFT/SHMT1ko). g Ratio of absolute intracellular serine and glycine levels in SHMT1, MFT, SHMT2, and FPGS depleted HAP1 cells. Dots represent the mean of an independent experiment in triplicate wells; mean ± SEM (n = 8 for CTRL and SHMT2ko, n = 7 for SHMT1ko, n = 6 for MTX, n = 2 for FPGSko, MFTko and MFT/SHMT1ko). h Absolute CORE rates of formate from WT and SHMT1- or SHMT2-depleted HAP1 cells upon 24 h 50 nM MTX. Each dot indicates the mean of an individual experiment measured in triplicate wells; Brown-Forsythe and Welch ANOVA test with Dunnetts multiple comparisons test. Mean ± SEM (n = 4 for CTRL, MTX, SHMT2ko, SHMT2ko MTX; n = 3 for SHMT1ko and SHMT1ko MTX) i Absolute CORE rates of formate from WT and MTHFD1L-depleted MDA-MB-468 cells upon 24 h 50 nM MTX. Mean ± SEM (n = 6 for CTRL and MTX; n = 3 for MTHFD1Lko + /- MTX) of an individual experiment measured in triplicate wells; Brown-Forsythe and Welch ANOVA test with Dunnetts multiple comparisons test. j Absolute CORE rates of formate from SCR and MTHFD2 KD MDA-MB-468 cells upon 24 h 50 nM MTX. Dots represent mean of an individual experiment measured in triplicate wells; mean ± SEM; (n = 3) Brown-Forsythe and Welch ANOVA test with Dunnetts multiple comparisons test. k Absolute CORE rates of formate from MDA-MB-468 cells upon 24 h treatment with 2.5 mM Metformin and 50 nM MTX. Dots represent mean of an individual experiment measured in triplicate wells; mean ± SEM (n = 2). Source data are provided as a Source Data file.

Mechanistically, 1C metabolism follows a cycle in which serine can be resynthesized in the cytoplasm and mitochondrion from glycine via serine hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) (Fig. 4a). As the SHMT reaction is highly reversible, the serine labeling pattern from [U-13C]serine is composed of a mix of M + 1, M + 2 and M + 3 isotopologues, which represents the different recombination events with labeled and unlabeled glycine and formate16,21,52. Consequently, M + 1 and M + 2 serine isotopologues are expected to be absent upon complete 1C metabolism inhibition. Intriguingly, cytosolic DHFR inhibition with MTX neither completely abrogated serine M + 1 and M + 2 isotopologues upon [U-13C]serine nor did it significantly affect the serine to glycine level ratio in MDA-MB-468 or LN229 cells (Fig. 4d, e, S4E, F), further supporting our hypothesis of a persistent mitochondrial 1C metabolism upon DHFR inhibition. To mechanistically disentangle cytosolic from mitochondrial 1C metabolism, we analyzed a panel of CRISPR knockouts in HAP1 cells in which either the cytosolic (SHMT1), the mitochondrial (MFT, SHMT2), or both compartments (FPGS, MFT + SHMT1) of 1C metabolism were abrogated53. Here, cytosolic SHMT1 KO did not eliminate intermediary M + 1 and M + 2 serine isotopologues from [U-13C]serine comparable to MTX treatment, whereas mitochondrial or combined cytosolic and mitochondrial inhibition of 1C metabolism upon MFT KO, SHMT2 KO, FPGS KO and MFT + SHMT1 KO resulted in a complete loss of M + 1 and M + 2 serine isotopologues (Fig. 4f). In mutants harboring a full inhibition of 1C metabolism or mitochondrial 1C metabolism alone, we additionally observed an increase in the ratio of serine to glycine levels, whereas MTX treatment and SHMT1 KO did not substantially increase serine to glycine ratio (Fig. 4g).

Of note the protein abundance of mitochondrial serine catabolism enzymes was not increased upon MTX. MTHFD2 abundance even decreased upon MTX, however, this did apparently not affect the rate of serine catabolism (Fig. S4G, H).

To test if knock-out of mitochondrial serine catabolism stops formate overflow, we monitored formate release flux upon MTX in HAP1 cells deficient for SHMT1 or SHMT2. As expected, loss of SHMT2 completely abolished formate overflow, while SHMT1 KO increased formate overflow (Fig. 4h). This experiment was then validated in MDA-MB-468 cells harboring MTHFD1L KO20 or shRNA mediated MTHFD2 knock-down and MTHFD2 KO (Fig. 4i, j, S4I-M). All genetic interventions resulted in a strong suppression of formate overflow during MTX treatment, further suggesting that increased formate overflow upon MTX depends on mitochondrial serine catabolism via the folate pathway (Fig. 4i, j, S4J, M).

As an alternative approach to genetic silencing we used Metformin to target complex 1 of the electron transport chain (ETC). We have previously shown that mitochondrial formate production depends on active ETC21,22. Here, we demonstrate that targeting of complex 1 with Metformin strongly suppresses mitochondrial formate production both at baseline and in presence of MTX, further supporting our finding that formate production in presence of MTX depends on mitochondrial 1C metabolism (Fig. 4k). To proof that Metformin indeed inhibits respiration under the applied conditions, we performed Seahorse experiments to measure the expected drop in oxygen consumption rate (Fig. S4N). We also monitored a reduction in proliferation (Fig. S4O) and we performed [U-13C]glutamine tracing to monitor the expected strong increase in reductive carboxylation (Fig. S4P, Q), a well-known phenomenon upon ETC inhibition54,55.

In summary, these results show that mitochondria provide a protected cellular environment that permits autarkic folate-mediated serine catabolism independent of the cytosolic folate metabolism.

Limiting serine availability decreases formate overflow and cancer cell migration

As we observed increased glucose derived serine synthesis and subsequent downstream catabolism of serine via mitochondrial 1C metabolism, we investigated if interfering with serine synthesis is effective to reduce sustained cell migration upon pharmacologic inhibition of cytosolic 1C metabolism. First, to identify an effective inhibitor of serine de novo synthesis, we tested multiple available allosteric and one competitive inhibitor of PHGDH. Indicated by the strong abrogation of labeled serine, we observed that the competitive inhibitor BI-4916 (BI)12 had superior efficacy compared to the allosteric inhibitors WQ-2101 (WQ)11, NCT-502 (NCT)9, and CBR-5884 (CBR)15 (Fig. 5a). Released M + 1 formate from [U-13C]glucose was also effectively inhibited after BI treatment alone or in combination with MTX (Fig. 5b). Additionally, routine screening for metabolic side effects revealed that treatment with the allosteric inhibitors WQ, NCT, and CBR did result in a significant reduction of mitochondrial OCR (Fig. 5c), which was not observed with the competitive inhibitor BI (Fig. 5c). WQ and NCT also negatively affected TCA cycle activity as characterized by a reduction of M + 2 isotopologue abundance of TCA cycle associated metabolites (Fig. 5d). In contrast to the other inhibitors, BI had no adverse effect on TCA cycle activity and proliferation rate and thus emerged as the preferred PHGDH inhibitor in all subsequent experiments (Fig. 5d, S5A–D). Combined treatment with the specific PHGDH inhibitor BI significantly reduced MTX-mediated cell migration (Fig. 5e). This inhibitory effect was further enhanced upon combined PHGDH inhibition and serine and glycine (S/G) starvation (Fig. 5f). While S/G starvation alone resulted only in moderate inhibitory effects on migration of MTX-treated cells, combined treatment with PHGDH inhibition fully blunted cell migration (Fig. 5f). Similar results were observed in 4T1 cells (Fig. S5F). As all MTX conditions resulted in full growth arrest (Fig. S5E), this finding indicates a role for mitochondrial 1C metabolism in cell motility that is independent of serine’s anabolic function to support biosynthetic processes.

Fig. 5: Limiting serine availability decreases formate overflow and cancer cell migration.
figure 5

a MID of intracellular serine in MDA-MB-468 cells upon [U-13C]glucose and 24 h 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15 μM BI-4916, and 50 nM MTX; mean ± SEM (n = 10 for CTRL; n = 3 for WQ, MTX, WQ MTX, BI, BI MTX; n = 2 for NCT; n = 1 for CBR) of independent experiments each measured in triplicate wells. b Formate M + 1 isotopologue exchange rate in MDA-MB-468 cells using [U-13C]glucose and treatment as in (a). Dots indicate independent experiments measured in triplicate wells; mean ± SEM (n = 10 for CTRL; n = 3 for WQ, MTX, WQ MTX, BI, BI MTX; n = 2 for NCT; n = 1 for CBR). c Basal cellular respiration upon 24 h 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 in MDA-MB-468 cells as fold mitochondrial OCR compared to control. Dots represent individual experiments composed of six technical replicates; mean ± SEM (n = 10 for CTRL, n = 7 for WQ, n = 3 for BI, n = 2 for NCT, n = 1 for CBR). d M + 2 isotopologues of selected TCA-metabolites in MDA-MB-468 cells upon [U-13C]glucose and treatment as in (b). Dots indicate independent experiments measured in triplicate wells; mean ± SEM (n = 7 for CTRL, n = 3 for WQ, BI, n = 2 for NCT, n = 1 for CBR). e Migration of MDA-MB-468 cells upon 50 nM MTX, 15 μM BI-4916 and f serine- and glycine starvation and respective AUC; mean ± SEM of independent experiments (n = 5 for (e), n = 4 for (f)); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. g mRNA expression from indicated genes in MDA-MB-468 cells upon 72 h 50 nM MTX, 15 μM BI-4916, and serine- and glycine starvation measured by real-time RT-qPCR. Mean ± SEM (n = 4 for BI, BI + MTX, n = 3 for MTX-SG, MTX-SG + BI, n = 2 for -SG, n = 1 for BI-SG). h Cell death induction in MDA-MB-468 cells upon 48 h 50 nM MTX, 15 μM BI-4916 and serine- and glycine starvation was assessed by flow cytometry and AnnexinV-FITC/PI-staining; mean ± SD of independent experiments (n = 3). Two-way ANOVA with Dunnett’s multiple comparisons test. i Migration of MTX-resistant MDA-MB-468 cells upon 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM of independent experiments (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. j Cytosolic and mitochondrial compartments of 1C metabolism and the pharmacologic intervention points of MTX and inhibitors of PHGDH. Source data are provided as a Source Data file.

While neither BI treatment alone nor S/G starvation alone were sufficient to prevent the induction of the observed EMT-phenotype, combined inhibition and starvation did minimize ZEB1, ZEB2, and VIMupregulation upon DHFR inhibition (Fig. 5g). This indicates that serine represents an underlying requirement for EMT onset upon DHFR inhibition. Importantly, S/G starvation did not further increase cell death after combined MTX and BI treatment (Fig. 5h). Of note, we also found that BI treatment was effective to reduce the migratory capacity of MTX-resistant, pro-migratory MDA-MB-468 cells (Fig. 5i). In summary, using MTX as a tool compound to study cell migration in absence of proliferation revealed that serine is essential to sustain full cancer cell migratory capacity (Fig. 5j).

Mitochondrial 1C metabolism supports cell motility and depends on mitochondrial THF availability

As withdrawal of serine can affect different aspects of cell physiology, we aimed to investigate more specifically the role of mitochondrial 1C metabolism in promoting cell migration. Analogue to the interventions presented in Fig. 4i–k, we used Metformin to target the ETC and genetic interventions to target MTHFD2 or MTHFD1L both in MDA-MB-468 and 4T1 cells.

Metformin treatment of MTX exposed (growth arrested) cells reduced the migration of MDA-MB-468 cells (Fig. 6A), suggesting that one function of the ETC is to support cell migration.

Fig. 6: Mitochondrial 1C metabolism supports cell motility and depends on mitochondrial THF availability.
figure 6

a Migration of MDA-MB-468 cells upon 2.5 mM Metformin and 50 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. b Migration of MDA-MB-468 cells upon MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. c Migration of 4T1 cells upon Mthfd2 knockdown and 75 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. d Migration of 4T1 cells upon Mthfd1l knockdown and 75 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. e Migration of MDA-MB-468 cells upon MTHFD1L knockout and 50 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. f ALDH1L2 and MTHFD1L protein expression in MDA-MB-468 and 4T1 cells. g Signal intensity of ALDH1L2 on Western Blot was quantified relative to β-actin as loading control. Mean ± SEM (n = 3); unpaired t-test with Welch’s correction. h MTHFD1L- and ALDH1L2-dependent reactions in mitochondrial 1C metabolism. i Migration of MDA-MB-468 cells upon MTHFD1L knockout, ALDH1L2 knockdown and 50 nM MTX and respective AUC; mean ± SEM of independent experiments (n = 3); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. j Model summarizing the autarkic activity of mitochondrial and cytosolic 1C metabolism and their respective relevance for cell migration and cell proliferation. Source data are provided as a Source Data file.

Next, we investigated the role of mitochondrial serine catabolism in context of migration directly, by monitoring the migration of MTX treated MTHFD2 or MTHFD1L silenced MDA-MB-468 and 4T1 cells. Of note, neither MTHFD2 nor MTHFD1L silencing reduced cell proliferation or sensitivity to the growth-arresting effects of MTX (Fig. S6A–D). Also, cell death rates upon MTX were not affected by MTHFD2or MTHFD1L silencing (Fig. S6E–H). Scratch assay analysis showed that MTHFD2 silencing markedly reduced MTX-mediated cell motility both in MDA-MB-468 and 4T1 cells (Fig. 6b, c). As shRNA used to silence human and murine MTHFD2 were different, we do not expect shRNA specific off-target effects. Nevertheless, we confirmed the shMTHFD2 KD results in MDA-MB-468 cells with MTHFD2 knock-out cells (Fig. S6I).

Mthfd1l silencing of MTX treated 4T1 cells also resulted in decreased migration (Fig. 6d). Somewhat surprisingly, loss of MTHFD1L in MDA-MB-468 cells had no negative effect on the migration of MTX treated cells (Fig. 6e). By following different hypotheses and various experiments, we realized that there is a striking difference in ALDH1L2 expression in 4T1 versus MDA-MB-468 cells, while MTHFD1L expression was comparable between these two cell lines (Fig. 6f, g).

This observation reminded us that in MDA-MB-468 cells, ALDH1L2 can act as an alternative outlet to oxidize formyl-THF to CO2 thereby regenerating mitochondrial THF (in contrast to 4T1). That said, in MTHFD1L deficient cells, the ALDH1L2 status can be decisive whether THF can be regenerated or if THF will be trapped as 10-CHO-THF in the mitochondrion. Loss of free THF will potentially inhibit the autarkic mitochondrial cycle that can otherwise act in presence of MTX (Fig. 6h). In support of this hypothesis, we also measured increased formate release flux in 4T1 cells compared to MDA-MB468 (Fig. S6J), suggesting that in 4T1 cells less 10-CHO-THF is oxidized via Aldh1l2 and instead catabolized through Mthfd1l.

To test this hypothesis, we targeted ALDH1L2 in MTHFD1L KO cells and monitored migration. We found that combinatorial depletion of MTHFD1L and ALDH1L2 in MDA-MB-468 cells resulted at least in a reduction of migration upon MTX compared to single depletion of MTHFD1L alone. This was observed both for shRNA as well as for siRNA mediated knockdown of ALDH1L2 (Fig. 6i, S6K).

In conclusion, these results reveal a growth-independent role of mitochondrial 1C metabolism for cancer cell motility. Interestingly, the pool of free THF might be relevant for the functionality of the autarkic mitochondrial 1C cycle and ALDH1L2 can serve as an alternative outlet to regenerate THF when MTHFD1L is missing (summarized in Fig. 6J).

Genetic targeting of mitochondrial 1C metabolism reduces metastasis formation in vivo

According to our model, we predict that sole inhibition of mitochondrial 1C metabolism might be sufficient and effective to reduce cancer cell escape from the primary tumor and subsequent metastasis formation. To test this hypothesis, we employed the 4T1 cells with Mthfd1l KD to study the metastatic cascade in orthotopic tumors in vivo.

To evaluate first if Mthfd1l KD cells are more prone to oxidative stress, which could impact metastatic dissemination in vivo56, we measured ROS levels in MTHFD1L silenced MDA-MB-468 and 4T1 cells at baseline and after MTX treatment. At baseline, ROS levels between MTHDF1L silenced and control cells were unchanged. MTX treatment induced ROS levels to comparable extends and 500 µM of H2O2treatment did induce cell death rate comparable to Ctrl cells (Fig. S7A–C). In summary, these experiments do not indicate that loss of MTHFD1L results in increased cellular ROS levels or increased susceptibility to oxidative insults.

We then injected 4T1 cells with or without silenced Mthfd1l into the mammary fat pad of immunocompetent BALB/c mice to form orthotopic tumors (Fig. 7a). Primary tumor growth was monitored over 6 weeks. In concordance with the in vitro data, primary tumor growth was not affected and tumor weight at endpoint was not different compared to Ctrl conditions (Fig. 7b, c). Knock-down of Mthfd1l in the primary tumors was confirmed by Western Blot (Fig. 7d, S7D–F). In line with our hypothesis, the number of macroscopic lung metastases was significantly reduced in mice that were carrying tumors deficient in Mthfd1l (Fig. 7e). While all mice that were injected with Mthfd1l-expressing 4T1 cells exhibited lung metastases, over 50% of mice injected with Mthfd1l KD cells did not show macroscopic metastatic lesions. Of note, expression of EMT related genes in the primary tumor was unchanged between the two groups (Fig. S7G). H&E staining of lungs and quantification of the metastatic area per lung confirmed reduced metastasis in Mthfd1l deficient tumors (Fig. 7f, g).

Fig. 7: Genetic targeting of mitochondrial 1C metabolism reduces metastasis formation in vivo.
figure 7

a 4T1 breast cancer cells transfected with non-targeting control (SCR, n = 9) and 4T1 cells with KD of Mthfd1l (n = 9) were injected into the mammary fat pads of immunocompetent female Balb/c mice. Primary tumor growth was monitored and lung metastasis formation was evaluated at end-point. b Primary tumor size was measured and mean tumor volume for each group was calculated; mean ± SEM (n = 8 (Scr) and 9 (Mthfd1l KD)). c Primary tumor weight was measured at the end-point. Dots indicate individual animals; mean ± SEM (n = 9); unpaired, two-tailed t-test with Welch’s correction. d Primary tumor tissue was analyzed for Mthfd1l protein expression by Western Blot and quantified relative to β-actin as loading control. Dots indicate individual animals; mean ± SEM (n = 7 (Scr) and 8 (Mthfd1l KD)); unpaired, two-tailed t-test with Welch’s correction. Protein lysates from tumors with significantly low Mthfd1l and β-actin expression relative to total protein were judged to be not pure tumor lysate and discarded from analysis (see Figure S7D–F for details). e Macroscopic lung metastases were counted and are depicted as number of metastases per lung. Dots indicate individual animals; mean ± SEM (n = 9); unpaired, two-tailed t-test with Welch’s correction. f Lung tissue was embedded in paraffin and stained with H&E. 2 representative images per group are presented to show microscopic lung metastasis; scale bar corresponds to 100 μm. g Metastatic area per lung was quantified and is expressed as % area of lung tissue. Dots indicate individual animals; mean ± SEM (n = 7 (Scr) and 9 (Mthf1l KD)); unpaired, two-tailed t-test with Welch’s correction. Source data are provided as a Source Data file.

These in vivo findings further strengthen the key relevance of mitochondrial 1C metabolism as a cornerstone to support cancer cell migration independent of primary tumor growth and cancer cell proliferation rates.

Discussion

In this study, we uncover a growth-independent function of mitochondrial serine catabolism to drive cancer cell motility. Importantly, by using MTX as a tool compound to study migration independent of proliferation, we observed that upon inhibition of cytosolic 1C metabolism, mitochondria sustain an autarkic 1C metabolism that is sufficient to sustain full migratory capacity.

Such compartmentalization of 1C metabolism during MTX treatment is feasible because release of the 1C unit from THF in the mitochondrial MTHFD1L reaction is, in contrast to cytosolic thymidylate synthetase reaction, not oxidizing THF to DHF. Therefore, the mitochondrial THF pool is sustained to allow SHMT2-dependent mitochondrial serine catabolism, while cytosolic 1C metabolism enzymes are inhibited due to accumulating DHF species. Our finding that ALDH1L2 can serve as an alternative source for THF regeneration in MTHFD1L deficient cells, supports the notion that mitochondria can maintain an independent THF pool that is required for an autarkic mitochondrial 1C metabolism as long as there is THF available that can accept the hydroxymethyl group in the first (SHMT2 dependent) step of serine catabolism.

An autarkic mitochondrial 1C metabolism is further supported by a chemical modification of folate species that alters their transport activities across the mitochondrial membrane. For maximal biologic activity, folate species need to be polyglutamated by folylpolyglutamate synthase (FPGS) and the resulting polyglutamate species were shown to be only poorly transported across the mitochondrial membrane57,58. Consequently, polyglutamated folate species can be chemically trapped within the mitochondria to sustain the mitochondrial 1C cycle. Especially, upon growth arrest when cells can retain their mitochondrial content and don’t need to amplify their content as it is required during proliferation. Additionally, there is evidence that transport activity of the mitochondrial folate transporter is limited to reduced folates59. This indicates a limited transport of MTX and folic acid itself across the mitochondrial membrane. Taken together, such compartmentalization of 1C metabolism and autarkic function of mitochondrial 1C metabolism emerges as a selective advantage in cells upon perturbation of cytosolic 1C metabolism and indicates a function of the mitochondrial 1C metabolism pathway to support cell motility.

While our data demonstrate that mitochondrial serine catabolism is needed to allow full migratory capacity, the specific biochemical output conferring this function still needs to be determined in more detail. Mitochondrial serine catabolism can generate NADH, NADPH, ATP, glycine, CO2, methylene- and formyl-THF and formate. The plasticity of this pathway allows tailoring the catabolism towards current cellular needs38. Therefore, it can be possible that serine is differentially catabolized along the metastatic cascade in a context dependent manner. We have previously shown that exogenous formate promotes cancer cell invasion22 and various cellular stress situations such as energy stress21 or MTX treatment (this study) can increase the rate of formate overflow which results in increased extracellular formate concentrations. It is therefore tempting to speculate that increasing formate concentrations in the tumor microenvironment promote a more invasive phenotype of stressed cancer cells. In more general terms, it could be possible that mitochondrial serine catabolism can selectively promote cell motility in response to extrinsic or intrinsic stress stimuli such as growth inhibition or nutrient deprivation. On the other hand, MDA-MB-468 MTHFD1L KO cells are also formate overflow negative but do not show reduced migration. As ROS levels are neither increased in MDA-MB-468 nor in 4T1 MTHFD1L deficient cells, we do not think that the observed migration effects in this study are ROS dependent. Given previous work22 and the current study, there might also be differences when comparing migration with invasion. Ongoing work in our lab will help clarifying these questions in the future. To do so, it will be of particular importance to further disentangle effects arising from exogenous formate from effects depending on endogenous serine catabolism. Independent of these yet unknown details, we remain with the conclusion that mitochondrial serine catabolism contributes to cancer cell migration and metastasis independent of its known anabolic functions.

Finally, we would like to note that although we mainly use MTX as a tool compound to separate proliferative from migratory processes and to inhibit cytosolic 1C metabolism, our results have also some implications in regard to MTX as an anchor drug in chronic treatment of autoimmune diseases. Intracellular erythrocyte and mean plasma concentration of MTX were reported to be in a comparable, even slightly higher, nM range to our chosen drug concentration60,61. Finally, we also would like to refer to a parallel and independent (back-to-back) publication that provides additional evidence that purine depletion promotes serine de novo synthesis and cell migration62. Interestingly, in this parallel work, the authors provide data that MTX also promotes the metastatic potential of melanoma cells in vivo. Hence, our findings on mitochondrial 1C metabolism might justify future investigations in cancer patients and patients that undergo chronic MTX therapy for an arthritic comorbidity.

Article classification: Biological abstract
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