Cisplatin inhibits SIRT3-deacetylation MTHFD2 to disturb cellular redox balance in colorectal cancer cell.

The folate-coupled metabolic enzyme MTHFD2 (the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase) confers redox homeostasis and drives cancer cell proliferation and migration. Here, we show that MTHFD2 is hyperacetylated and lysine 88 is the critical acetylated site. SIRT3, the major deacetylase in mitochondria, is responsible for MTHFD2 deacetylation. Interestingly, chemotherapeutic agent cisplatin inhibits expression of SIRT3 to induce acetylation of MTHFD2 in colorectal cancer cells. Cisplatin-induced acetylated K88 MTHFD2 is sufficient to inhibit its enzymatic activity and downregulate NADPH levels in colorectal cancer cells. Ac-K88-MTHFD2 is significantly decreased in human colorectal cancer samples and is inversely correlated with the upregulated expression of SIRT3. Our findings reveal an unknown regulation axis of cisplatin-SIRT3-MTHFD2 in redox homeostasis and suggest a potential therapeutic strategy for cancer treatments by targeting MTHFD2.

MTHFD2 links RNA methylation to metabolic reprogramming in renal cell carcinoma.

One-carbon metabolism plays a central role in a broad array of metabolic processes required for the survival and growth of tumor cells. However, the molecular basis of how one-carbon metabolism may influence RNA methylation and tumorigenesis remains largely unknown. Here we show MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, contributes to the progression of renal cell carcinoma (RCC) via a novel epitranscriptomic mechanism that involves HIF-2α. We found that expression of MTHFD2 was significantly elevated in human RCC tissues, and MTHFD2 knockdown strongly reduced xenograft tumor growth. Mechanistically, using an unbiased methylated RNA immunoprecipitation sequencing (meRIP-Seq) approach, we found that MTHFD2 plays a critical role in controlling global N6-methyladenosine (m6A) methylation levels, including the m6A methylation of HIF-2α mRNA, which results in enhanced translation of HIF-2α. Enhanced HIF-2α translation, in turn, promotes the aerobic glycolysis, linking one-carbon metabolism to HIF-2α-dependent metabolic reprogramming through RNA methylation. Our findings also suggest that MTHFD2 and HIF-2α form a positive feedforward loop in RCC, promoting metabolic reprograming and tumor growth. Taken together, our results suggest that MTHFD2 links RNA methylation status to the metabolic state of tumor cells in RCC.

Mthfd1 is a modifier of chemically induced intestinal carcinogenesis.

The causal metabolic pathways underlying associations between folate and risk for colorectal cancer (CRC) have yet to be established. Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate and methionine. Methionine is converted to S-adenosylmethionine (AdoMet), the major one-carbon donor for cellular methylation reactions. Impairments in folate metabolism can modify DNA synthesis, genomic stability and gene expression, characteristics associated with tumorigenesis. The Mthfd1 gene product, C1-tetrahydrofolate synthase, is a trifunctional enzyme that generates one-carbon substituted tetrahydrofolate cofactors for one-carbon metabolism. In this study, we use Mthfd1(gt/+) mice, which demonstrate a 50% reduction in C1-tetrahydrofolate synthase, to determine its influence on tumor development in two mouse models of intestinal cancer, crosses between Mthfd1(gt/+) and Apc(min)(/+) mice and azoxymethane (AOM)-induced colon cancer in Mthfd1(gt/+) mice. Mthfd1 hemizygosity did not affect colon tumor incidence, number or load in Apc(min/+) mice. However, Mthfd1 deficiency increased tumor incidence 2.5-fold, tumor number 3.5-fold and tumor load 2-fold in AOM-treated mice. DNA uracil content in the colon was lower in Mthfd1(gt/+) mice, indicating that thymidylate biosynthesis capacity does not play a significant role in AOM-induced colon tumorigenesis. Mthfd1 deficiency-modified cellular methylation potential, as indicated by the AdoMet: S-adenosylhomocysteine ratio and gene expression profiles, suggesting that changes in the transcriptome and/or decreased de novo purine biosynthesis and associated mutability cause cellular transformation in the AOM CRC model. This study emphasizes the impact and complexity of gene-nutrient interactions with respect to the relationships among folate metabolism and colon cancer initiation and progression.

Disruption of the mthfd1 gene reveals a monofunctional 10-formyltetrahydrofolate synthetase in mammalian mitochondria.

The Mthfd1 gene encoding the cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase enzyme (DCS) was inactivated in embryonic stem cells. The null embryonic stem cells were used to generate spontaneously immortalized fibroblast cell lines that exhibit the expected purine auxotrophy. Elimination of these cytoplasmic activities allowed for the accurate assessment of similar activities encoded by other genes in these cells. A low level of 10-formyltetrahydrofolate synthetase was detected and was shown to be localized to mitochondria. However, NADP-dependent methylenetetrahydrofolate dehydrogenase activity was not detected. Northern blot analysis suggests that a recently identified mitochondrial DCS (Prasannan, P., Pike, S., Peng, K., Shane, B., and Appling, D. R. (2003) J. Biol. Chem. 278, 43178-43187) is responsible for the synthetase activity. The lack of NADP-dependent dehydrogenase activity suggests that this RNA may encode a monofunctional synthetase. Moreover, examination of the primary structure of this novel protein revealed mutations in key residues required for dehydrogenase and cyclohydrolase activities. This monofunctional synthetase completes the pathway for the production of formate from formyltetrahydrofolate in the mitochondria in our model of mammalian one-carbon folate metabolism in embryonic and transformed cells.

Mammalian fibroblasts lacking mitochondrial NAD+-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase are glycine auxotrophs.

Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3-10 x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.

Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580.

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA.

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.

Folate and carcinogenesis: an integrated scheme.

Collectively, the evidence from epidemiologic, animal and human studies strongly suggests that folate status modulates the risk of developing cancers in selected tissues, the most notable of which is the colorectum. Folate depletion appears to enhance carcinogenesis whereas folate supplementation above what is presently considered to be the basal requirement appears to convey a protective effect. The means by which this modulation of cancer risk is mediated is not known with certainty, but there are several plausible mechanisms which have been described. Folate plays a major role in the formation of S-adenosylmethionine, the universal methyl donor, as well as in the formation of purine and thymidine synthesis for DNA and RNA. Therefore, most mechanistic studies performed to date have focused on alterations in DNA methylation, disruption of DNA integrity and disruption of DNA repair, all of which have been observed with folate depletion. These aberrations in DNA are believed to enhance carcinogenesis by altering the expression of critical tumor suppressor genes and proto-oncogenes. Recently, the role of a common polymorphism of the methylenetetrahydrofolate reductase gene has been highlighted as well. This review presents those mechanisms which are the most likely candidates to explain folate's effects and it proposes an integrated scheme to explain how these mechanisms might interact.