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Project description:What is known is that methionine-dependency is a feature of some cancers. So far, it was attributed to mutations in genes involved in the methionine de novo or salvage pathways. What is new is that in this work we propose that methionine dependency stems from an altered cellular metabolism. We provide evidence that in U251 glioblastoma cell line, only cancer stem cells -isolated as tumor spheres in non adherent conditions- are methionine dependent and not monolayer cells grown in adherent conditions. Transcriptome wide-sequencing reveals that several genes involved in cytosolic folate cycle are downregulated whereas some transcripts of genes involved in mitochondrial folate cycle are upregulated. Genome wide DNA methylation does not account for these changes in gene expression. Mass spectrometry measurements confirm that tumor spheres display low cytosolic folate cycle unable to produce enough 5-methyltetrahydrofolate to remethylate homocystein to methionine. This decreased 5-methyltetrahydrofolate bioavailability is presumably due to a reprogrammed mitochondrial folate cycle which instead of synthesizing formate, intended to fuel the cytosolic folate cycle, oxidizes the formyl group to CO2 with the attendant reduction of NADP+ to NADPH and release of tetrahydrofolate. The originality of this work resides in that it replaces methionine deprivation as a useful nutritional strategy in cancer growth control since cancer stem cells are much more tumoregenic than their non stem-like counterparts. Second, it reveals that the primary default responsible of the reprogrammation of folate metabolism originates in the mitochondria. Thus, mitochondrial enzymes could be novel and more promising anticancer targets than dihydrofolate reductase (DHFR), the current target of drug therapy linked with folate metabolism.
2019-07-01 | GSE117448 | GEO
Project description:we report that U251 glioblastoma tumor spheres exhibit low cytosolic folate cycle and a reprogrammmed mitochondrial folate cycle that is presumably oriented towards oxidizing the formyl group to CO2 with the production of TetraHydroFolate and release of NADPH instead of synthesizing formate
2019-07-01 | GSE117543 | GEO
Project description:Low cytosolic folate cycling is a hallmark of U251 glioblastoma cells reprogramming towards pluripotency
| PRJNA482425 | ENA
Project description:Low cytosolic folate cycling is a hallmark of U251 glioblastoma cells reprogramming towards pluripotency [methylation]
| PRJNA482423 | ENA
Project description:Low cytosolic folate cycling is a hallmark of U251 glioblastoma cells reprogramming towards pluripotency [RNA-seq]
| PRJNA482432 | ENA
Project description:Methionine dependency of tumor growth, although not well-understood, is detectable by 11C-methionine positron emission tomography and may contribute to the aggressivity of glioblastomas (GBM) and meningiomas. Cytosolic folate cycle is required for methionine synthesis. Its dysregulation may influence cell reprogramming towards pluripotency. We evaluated methionine-dependent growth of monolayer (ML) cells and stem cell-like tumor spheres (TS) derived from 4 GBM (U251, U87, LN299, T98G) and 1 meningioma (IOMM-LEE) cell lines. Our data showed that for all cell lines studied, exogenous methionine is required for TS formation but not for ML cells proliferation. Furthermore, for GBM cell lines, regardless of the addition of folate cycle substrates (folic acid and formate), the level of 3 folate isoforms, 5-methytetrahydrofolate, 5,10-methenyltetrahydrofolate, and 10-formyltetrahydrofolate, were all downregulated in TS relative to ML cells. Unlike GBM cell lines, in IOMM-LEE cells, 5-methyltetrahydrofolate was actually more elevated in TS than ML, and only 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate were downregulated. The functional significance of this variation in folate cycle repression was revealed by the finding that Folic Acid and 5-methyltetrahydrofolate promote the growth of U251 TS but not IOMM-LEE TS. Transcriptome-wide sequencing of U251 cells revealed that DHFR, SHMT1, and MTHFD1 were downregulated in TS vs ML, in concordance with the low activity cytosolic folate cycle observed in U251 TS. In conclusion, we found that a repressed cytosolic folate cycle underlies the methionine dependency of GBM and meningioma cell lines and that 5-methyltetrahydrofolate is a key metabolic switch for glioblastoma TS formation. The finding that folic acid facilitates TS formation, although requiring further validation in diseased human tissues, incites to investigate whether excessive folate intake could promote cancer stem cells formation in GBM patients.
| S-EPMC6687714 | biostudies-literature
Project description:Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
| S-EPMC2906123 | biostudies-literature
Project description:The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Using fibroblasts derived from macroH2A double knockout (dKO) mice, here we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming. Genomic analyses reveal that macroH2A1 and macroH2A2, together with H3K27me3, co-occupy pluripotency genes in wild-type (wt) fibroblasts. In particular, we find macroH2A isoforms to be highly enriched at target genes of the K27me3 demethylase, Utx, which are reactivated early in iPS reprogramming. Finally, while macroH2A dKO-induced pluripotent cells are able to differentiate properly in vitro and in vivo, such differentiated cells retain the ability to return to a stem-like state. Therefore, we propose that macroH2A isoforms provide a redundant silencing layer or terminal differentiation 'lock' at critical pluripotency genes that presents as an epigenetic barrier when differentiated cells are challenged to reprogram.
| S-EPMC4055026 | biostudies-literature
Project description:The ability to induce the reprogramming of somatic mammalian cells to a pluripotent state by the forced expression of specific transcription factors has helped redefine the rules of cell fate and plasticity, as well as open possibilities for disease modeling, drug screening and regenerative medicine. Here, we hypothesized that the non-viral forced expression of the four originally discovered defined factors (OKSM) in adult mice could result in in vivo reprogramming of cells in the transfected tissue in situ. We show that a single hydrodynamic tail-vein (HTV) injection of two plasmids encoding for Oct3/4, Sox2, Klf4 and c-Myc respectively, are highly expressed in the liver tissue of Balb/C adult mice. Hallmark pluripotency markers were upregulated within 24-48 h after injection, followed by down-regulation of all major hepatocellular markers. Generation of transcriptionally reprogrammed cells in vivo was further confirmed by positive staining of liver tissue sections for all major pluripotency markers in Balb/C mice and the Nanog-GFP reporter transgenic strain (TNG-A) with concomitant upregulation of GFP expression in situ. No signs of physiological or anatomical abnormalities or teratoma formation were observed in the liver examined up to 120 days. These findings indicate that virus-free expression of OKSM factors in vivo can transcriptionally reprogram cells in situ rapidly, efficiently and transiently, absent of host tissue damage or teratoma formation.
| S-EPMC3552956 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing GPR123 RNAi we addressed the role of GPR123 for hPSCs. The suppression of GPR123 in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the GPR123 RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the "true" hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency.
| S-EPMC9856511 | biostudies-literature