The loss of SHMT2 mediates 5-fluorouracil chemoresistance in colorectal cancer by upregulating autophagy.

5-Fluorouracil (5-FU)-based chemotherapy is the first-line treatment for colorectal cancer (CRC) but is hampered by chemoresistance. Despite its impact on patient survival, the mechanism underlying chemoresistance against 5-FU remains poorly understood. Here, we identified serine hydroxymethyltransferase-2 (SHMT2) as a critical regulator of 5-FU chemoresistance in CRC. SHMT2 inhibits autophagy by binding cytosolic p53 instead of metabolism. SHMT2 prevents cytosolic p53 degradation by inhibiting the binding of p53 and HDM2. Under 5-FU treatment, SHMT2 depletion promotes autophagy and inhibits apoptosis. Autophagy inhibitors decrease low SHMT2-induced 5-FU resistance in vitro and in vivo. Finally, the lethality of 5-FU treatment to CRC cells was enhanced by treatment with the autophagy inhibitor chloroquine in patient-derived and CRC cell xenograft models. Taken together, our findings indicate that autophagy induced by low SHMT2 levels mediates 5-FU resistance in CRC. These results reveal the SHMT2-p53 interaction as a novel therapeutic target and provide a potential opportunity to reduce chemoresistance.

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

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

Mice deficient in the Shmt2 gene have mitochondrial respiration defects and are embryonic lethal.

Accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for human aging and age-associated mitochondrial respiration defects. However, our previous findings suggested an alternative hypothesis of human aging-that epigenetic changes but not mutations regulate age-associated mitochondrial respiration defects, and that epigenetic downregulation of nuclear-coded genes responsible for mitochondrial translation [e.g., glycine C-acetyltransferase (GCAT), serine hydroxymethyltransferase 2 (SHMT2)] is related to age-associated respiration defects. To examine our hypothesis, here we generated mice deficient in Gcat or Shmt2 and investigated whether they have respiration defects and premature aging phenotypes. Gcat-deficient mice showed no macroscopic abnormalities including premature aging phenotypes for up to 9 months after birth. In contrast, Shmt2-deficient mice showed embryonic lethality after 13.5 days post coitum (dpc), and fibroblasts obtained from 12.5-dpc Shmt2-deficient embryos had respiration defects and retardation of cell growth. Because Shmt2 substantially controls production of N-formylmethionine-tRNA (fMet-tRNA) in mitochondria, its suppression would reduce mitochondrial translation, resulting in expression of the respiration defects in fibroblasts from Shmt2-deficient embryos. These findings support our hypothesis that age-associated respiration defects in fibroblasts of elderly humans are caused not by mtDNA mutations but by epigenetic regulation of nuclear genes including SHMT2.

Mitochondrial translation requires folate-dependent tRNA methylation.

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

Role of cytosolic serine hydroxymethyltransferase in one-carbon metabolism in Neurospora crassa.

Conidiospores of wild type and two mutant strains of Neurospora crassa were grown on [3-13C]serine, [2-13C]glycine, or [13C]formate. Acid extracts of the mycelia were analyzed by 13C NMR for incorporation of the 13C label into choline, serine, and adenine. The goal was to elucidate the function of cytosolic serine hydroxymethyltransferase by comparison of a mutant strain lacking this activity and requiring formate for optimal growth (for mutant strain) to the wild-type strain and the ser3 strain which cannot convert glucose to serine. The results for both the wild-type and ser3 strains showed that the one-carbon adduct of the cytosolic pool of methylenetetrahydrofolate is formed primarily and preferably from C3 of serine. Both organisms could form methylenetetrahydrofolate from formate in the absence of serine and glycine. However, the for mutant strain was restricted in its ability to form methylenetetrahydrofolate from C3 of serine, preferring formate as the one-carbon source. All three strains had an active glycine cleavage complex and mitochondrial serine hydroxymethyltransferase. The formate requirement of the for mutant strain appears to be the result of the inability to form formate in the mitochondria from serine or glycine at a rate sufficient to sustain the biosynthetic pools of methylenetetrahydrofolate and 10-formyltetrahydrofolate in the cytosol. All three strains rapidly accumulated serine from the media to form high intracellular levels of this substrate. However, these strains did not accumulate either glycine or formate from the media at levels that could be detected by 13C NMR.

Psychosis and vulnerability to ECT-induced seizures.

Medical records of patients with major depressive disorders who had received electroconvulsive therapy (ECT) for the first time were studied to test the hypothesis that psychotic patients are more vulnerable to seizures than nonpsychotic patients. This hypothesis was based on studies suggesting a putative purinergic deficiency in psychosis. Results showed that the duration of ECT-induced seizures as a measure of seizure vulnerability was significantly longer in psychotic than in nonpsychotic depressive patients. The association applied for the first ECT as well as for the course of eight ECTs. These findings were still present when covariates such as age, electrical energy applied, dosage of methohexital and succinylcholine, and psychotropic medications such as neuroleptics, benzodiazepines, and tricyclics were included in the statistical analysis. The results are discussed in the context of the role of neurotransmitters such as glutamate, gamma-aminobutyric acid, adenosine, and dopamine on seizure vulnerability and psychosis.