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Folates (B9 vitamins) are essential cofactors in one-carbon metabolism. Since C1 transfer reactions are involved in synthesis of nucleic acids, proteins, lipids, and other biomolecules, as well as in epigenetic control, folates are vital for all living organisms. This work presents a complete study of a plant DHFR-TS (dihydrofolate reductase-thymidylate synthase) gene family that implements the penultimate step in folate biosynthesis. We demonstrate that one of the DHFR-TS isoforms (DHFR-TS3) operates as an inhibitor of its two homologs, thus regulating DHFR and TS activities and, as a consequence, folate abundance. In addition, a novel function of folate metabolism in plants is proposed, i.e., maintenance of the redox balance by contributing to NADPH production through the reaction catalyzed by methylenetetrahydrofolate dehydrogenase, thus allowing plants to cope with oxidative stress.

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing. Metabolic pathways are closely intertwined with longevity. Here the authors perform metabolomic profiling of canonical longevity pathways and show that folate and methionine cycle intermediates are changed in common, and further, genetic manipulation of pathway enzymes and supplementation with metabolites indicates that they causally regulate longevity.

Resistance to 5-Fluoruracil (5-FU) has been linked to elevated expression of the main target, thymidylate synthase (TYMS), which catalyses the de novo pathway for production of deoxythymidine monophosphate. The potent oncogenic forkhead box transcription factor, FOXM1 is is regulated by E2F1 which also controls TYMS. This study reveals a significant role of FOXM1 in 5-FU resistance. Overexpression and knock-down studies of FOXM1 in colon cancer cells suggest the importance of FOXM1 in TYMS regulation. ChIP and global ChIP-seq data also confirms that FOXM1 can also potentially regulate other 5-FU targets, such as TYMS, thymidine kinase 1 (TK-1) and thymidine phosphorylase (TYMP). In human colorectal cancer tissue specimens, a strong correlation of FOXM1 and TYMS staining was observed. Elevated FOXM1 and TYMS expression was also observed in acquired 5-FU resistant colon cancer cells (HCT116 5-FU Res). A synergistic effect was observed following treatment of CRC cells with an inhibitor of FOXM1, thiostrepton, in combination with 5-FU. The combination treatment decreased colony formation and migration, and induced cell cycle arrest, DNA damage, and apoptosis in CRC cell lines. In summary, this research demonstrated that FOXM1 plays a pivotal role in 5-FU resistance at least partially through the regulation of TYMS.

Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3' untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.

Thymidylate synthase is the rate-limiting enzyme required for DNA synthesis and overexpression of this enzyme causes resistance to cancer cells. Long treatments with 5-FU cause resistance to Thymidylate synthase targeting drugs. We have also compiled different mechanisms of drug resistance including autophagy and apoptosis, drug detoxification and ABC transporters, drug efflux, signaling pathways (AKT/PI3K, RAS-MAPK, WNT/β catenin, mTOR, NFKB, and Notch1 and FOXM1) and different genes associated with resistance in colorectal cancer. We can overcome 5-FU resistance in cancer cells by regulating thymidylate synthase by natural products ( Coptidis rhizoma) , HDAC inhibitors, mTOR inhibitors, Folate antagonists, and several other drugs which have been used in combination with TS inhibitors. This review is a compilation of different approaches reported for the regulation of thymidylate synthase to overcome resistance in colorectal cancer cells. Graphical abstract

Alteration of O -GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O -GlcNAcylation was reported but not investigated yet. We hypothesize that O -GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G ( O -GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O -GlcNAcylation and, reciprocally, elevation of O -GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O -GlcNAcylation by decreasing O -GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O -GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O -GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.

High expression of nucleotide synthetic enzyme thymidylate synthase (TYMS) is responsible for the resistance to fluorouracil (FU) treatment and worse survival in colorectal cancer (CRC). Herein, we revealed that pleckstrin-2 (PLEK2) cooperated with YTHDF2 to enhance TYMS mRNA stability in CRC via an m 6 A dependent manner . Silencing of PLEK2 led to the degradation of TYMS mRNA that suppressed DNA replication, which activated p53/p21 signaling and consequent inhibition of CRC cell proliferation via the cellular senescence. Additionally, PLEK2 is also required for CRC cell migration, invasion and stemness-like properties. PLEK2 inhibition is sufficient to ameliorate the progression of AOM/DSS-induced CRC . Together, our study identified PLEK2 as a key regulator for the progress of CRC via the regulation of TYMS expression, and demonstrated that PLEK2 is a novel therapeutic target for CRC.

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2 H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

Background: We investigated the predictive value of dihydropyrimidine dehydrogenase (DPD) phenotype, measured as pretreatment serum uracil and dihydrouracil concentrations, for severe as well as fatal fluoropyrimidine-associated toxicity in 550 patients treated previously with fluoropyrimidines during a prospective multicenter study. Methods: Pretreatment serum concentrations of uracil and dihydrouracil were measured using a validated LC-MS/MS method. The primary endpoint of this analysis was global (any) severe fluoropyrimidine-associated toxicity, that is, grade ⩾3 toxicity according to the NCI CTC-AE v3.0, occurring during the first cycle of treatment. The predictive value of uracil and the uracil/dihydrouracil ratio for early severe fluoropyrimidine-associated toxicity were compared. Pharmacogenetic variants in DPYD (c.2846A>T, c.1679T>G, c.1129-5923C>G, and c.1601G>A) and TYMS ( TYMS 5′-UTR VNTR and TYMS 3′-UTR 6-bp ins/del) were measured and tested for associations with severe fluoropyrimidine-associated toxicity to compare predictive value with DPD phenotype. The Benjamini-Hochberg false discovery rate method was used to control for type I errors at level q <0.050 (corresponding to P <0.010). Results: Uracil was superior to the dihydrouracil/uracil ratio as a predictor of severe toxicity. High pretreatment uracil concentrations (>16 ng ml −1 ) were strongly associated with global severe toxicity (OR 5.3, P =0.009), severe gastrointestinal toxicity (OR 33.7, P <0.0001), toxicity-related hospitalisation (OR 16.9, P <0.0001), as well as fatal treatment-related toxicity (OR 44.8, P =0.001). None of the DPYD variants alone, or TYMS variants alone, were associated with severe toxicity. Conclusions: High pretreatment uracil concentration was strongly predictive of severe, including fatal, fluoropyrimidine-associated toxicity, and is a highly promising phenotypic marker to identify patients at risk of severe fluoropyrimidine-associated toxicity.

Cancer cells frequently boost nucleotide metabolism (NM) to support their increased proliferation, but the consequences of elevated NM on tumor de-differentiation are mostly unexplored. Here, we identified a role for thymidylate synthase (TS), a NM enzyme and established drug target, in cancer cell de-differentiation and investigated its clinical significance in breast cancer (BC). In vitro, TS knockdown increased the population of CD24+ differentiated cells, and attenuated migration and sphere-formation. RNA-seq profiling indicated repression of epithelial-to-mesenchymal transition (EMT) signature genes upon TS knockdown, and TS-deficient cells showed an increased ability to invade and metastasize in vivo, consistent with the occurrence of a partial EMT phenotype. Mechanistically, TS enzymatic activity was found essential for maintenance of the EMT/stem-like state by fueling a dihydropyrimidine dehydrogenase—dependent pyrimidine catabolism. In patient tissues, TS levels were found significantly higher in poorly differentiated and in triple negative BC, and strongly correlated with worse prognosis. The present study provides the rationale to study in-depth the role of NM at the crossroads of proliferation and differentiation, and depicts new avenues for the design of novel drug combinations for the treatment of BC.