Cancer cells reprogramme one‐carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by ...reversibly converting serine and tetrahydrofolate (THF) into 5,10‐methylene‐THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1.
Database
Structural data are available in the PDB under the accession number 6FL5
We designed a set of three serine hydroxymethyltransferase 1 (SHMT1) mutants to uncouple the oligomeric state of the enzyme from its catalytic activity. We investigated the effects of these mutations on nuclear localization, cell viability, and crosstalk between SHMT1 and SHMT2 in lung cancer cells. In these cell lines, de novo thymidylate synthesis requires SHMT1 to be active regardless of its oligomeric state.
Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one‐carbon ...units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine‐tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo‐enzyme structure, showing that PLP binding triggers a disorder‐to‐order transition accompanied by a large rigid‐body movement of the two cofactor‐binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer‐to‐tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo.
Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous ...studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 μM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types.
Ubiquitin-specific protease 2a (USP2a) is overexpressed in almost half of human prostate cancers and c-Myc is amplified in one third of these tumor types. Transgenic MYC expression drives invasive ...adenocarcinomas in the murine prostate. We show that overexpression of USP2a downregulates a set of microRNAs that collectively increase MYC levels by MDM2 deubiquitination and subsequent p53 inactivation. By establishing MYC as a target of miR-34b/c, we demonstrate that this cluster functions as a tumor suppressor in prostate cancer cells. We identify a distinct mRNA signature that is enriched for MYC-regulated transcripts and transcription factor binding sites in USP2a overexpressing prostate cancer cells. We demonstrate that these genes are associated with an invasive phenotype in human prostate cancer and that the proliferative and invasive properties of USP2a overexpressing cells are MYC-dependent. These results highlight an unrecognized mechanism of MYC regulation in prostate cancer and suggest alternative therapeutic strategies in targeting MYC.
The deubiquitinating enzyme USP2a has previously been shown to be oncogenic, overexpressed in almost half of human prostate adenocarcinomas, and prolongs the half-life of targets such as fatty acid synthase, MDM2, and cyclin D1. Here, we highlight a new mechanism by which USP2a enhances MYC levels through the modulation of specific subsets of microRNAs in prostate cancer, suggesting alternative therapeutic strategies for targeting MYC.
Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one‐carbon units by converting ...serine and tetrahydrofolate (H4PteGlu) to glycine and 5,10‐CH2‐H4PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.
Serine hydroxymethyltransferase (SHMT) has been repeatedly hailed as the missing target for cancer chemotherapy. Here, we identify lometrexol as one of the most potent antifolate inhibitors of human cytosolic SHMT (Kd=2±1 μM) known to date. The results reported represent an initial step toward the development of more potent and effective SHMT inhibitors.
Prostate cancer is one of the most common types of cancer in western country males but the mechanisms involved in the transformation processes have not been clearly elucidated. Alteration in cellular ...metabolism in cancer cells is recognized as a hallmark of malignant transformation, although it is becoming clear that the biological features of metabolic reprogramming not only differ in different cancers, but also among different cells in a type of cancer. Normal prostate epithelial cells have a peculiar and very inefficient energy metabolism as they use glucose to synthesize citrate that is secreted as part of the seminal liquid. During the transformation process, prostate cancer cells modify their energy metabolism from inefficient to highly efficient, often taking advantage of the interaction with other cell types in the tumor microenvironment that are corrupted to produce and secrete metabolic intermediates used by cancer cells in catabolic and anabolic processes. We recapitulate the metabolic transformations occurring in the prostate from the normal cell to the metastasis, highlighting the role of the microenvironment and summarizing what is known on the molecular mechanisms involved in the process.
Melatonin has been known to be a chemopreventive agent since its levels inversely correlate with the risk of developing cancer. We have recently shown that melatonin induces p38-dependent ...phosphorylation of both p53 and histone H2AX. This is associated with a p53-mediated increase in repair of both endogenous and chemotherapy-induced DNA damage. In addition, the inhibition of p38 activities impairs melatonin's capability to induce a p53-dependent DNA damage response and thus its ability to maintain genome integrity. Since melatonin-induced p53 phosphorylation requires an intact p38 phosphorylation cascade and p38 can be activated by G proteins, we supposed that melatonin's activities could be mediated by its G-protein-coupled membrane receptors, MT1 and MT2. Here, we show that the activation of the p53-dependent DNA damage response by melatonin is indeed mediated by MT1 and MT2. As a result, the absence of either receptor impairs melatonin's ability to reduce both cell proliferation and clonogenic potential of cancer cells. In addition, this causes an impairment of the p53-dependent DNA damage response. By providing molecular insight, our findings might have translational impact, suggesting the involvement of melatonin receptors in tumorigenesis.
Background/Aim: The connection between prostate cancer and inflammation has been proposed many years ago, but very little is known about the metabolic adaptations of prostate cells in case of ...infection or inflammation. The aim of this study was to examine the effect of the stimulation of Toll-like receptor 3 (TLR3) on the metabolism of prostate cancer (PCa) cell lines and benign prostate cells. Materials and Methods: Cytofluorimetry, qRT-PCR, western blot and Gas-chromatography/Mass-spectrometry were used. Results: Reprogramming of glucose utilization involving hypoxia-inducible factor 1-alpha (HIF-1α) and the extracellular adenosine axis was observed. TLR3 stimulation synergized with adenosine receptor A2b on PCa cells, and induced a strong production of lactate, exacerbating the Warburg effect. Moreover, stimulation of benign prostate cells with poly I:C reduced lactate secretion, a characteristic typical of the neoplastic transformation. Conclusion: TLR3 stimulation promotes metabolic adaptations likely involved in the mechanisms of disease onset and progression.
Abstract
Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine ...hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5′untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1–UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.
A novel method for bottom-up synthesis of two-dimensional (2D) gadolinium-doped cerium oxide (CGO) nanosheets is developed and demonstrated as an efficient precursor for interdiffusion barrier layer ...deposition in solid oxide fuel cells. The CGO is the standard material used as an interlayer in intermediate-temperature solid oxide fuel cells to avoid undesirable reactions between lanthanum strontium cobalt ferrite (LSCF) cathode and yttria-stabilized zirconia (YSZ) electrolyte materials. Herein, the shape-control of the 2D CGO by a low-cost wet-chemical method allowing for the fabrication of fully dense barrier layer of CGO is reported. The high surface coverage promoted by the 2D CGO nanosheets resulted in a thin (∼ 1 µm) and dense interdiffusion barrier layer sintered at 1150 °C preventing the undesirable reaction between oxide ion conducting phases occurring at higher temperatures. The electrochemical properties of solid oxide fuel cells confirmed the CGO nanosheet as an efficient layer for preventing the formation of resistive phases at the electrolyte/cathode interface.
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•Innovative synthesis method for efficient 2D-layered gadolinium doped-ceria (CGO) electrolytes.•2D CGO nanosheets, with high surface coverage, result in dense layer at mild sintering temperature.•CGO nanosheets prevent resistive phases and enhance solid oxide fuel cell performance.
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