Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative and primed pluripotent states

2020 ◽  
Author(s):  
Joshua G. Dierolf ◽  
Andrew J. Watson ◽  
Dean H. Betts
Keyword(s):  
Stem Cells ◽  
Confocal Microscopy ◽  
Cell Fate ◽  
Pyruvate Kinase ◽  
Warburg Effect ◽  
Aerobic Glycolysis ◽  
Embryonic Stem ◽  
Orthogonal Projections ◽  
Cellular Expression ◽  
The Warburg Effect

AbstractMouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg effect. Until recently, metabolism has been regarded as a by-product of cell fate; however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg effect. Both isoforms catalyze the last step of glycolysis generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting, flow cytometry and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm but also accumulate in differential subnuclear regions of mESC, mEpiLCs and mEpiSCs as determined by a quantitative, confocal microscopy colocalization employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 shifts during the transition from mESCs, mEpiLCs and mEpiSCs. We have also authenticated a new method of selecting formative pluripotency cells from naïve and primed populations using the cell surface markers SSEA1 and CD24. Finally, we have comprehensively validated the appropriateness and power of the Pearson’s correlation coefficient and Manders’ overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 assists with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative and primed pluripotency.

scholarly journals TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Feng Gao ◽  
Xiaojun Zhang ◽  
Shuyue Wang ◽  
Lihua Zheng ◽  
Ying Sun ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Warburg Effect ◽  
Kinase Activity ◽  
Aerobic Glycolysis ◽  
Tumor Formation ◽  
Metabolic Reprogramming ◽  
Hepatocyte Proliferation ◽  
Pyruvate Kinase Activity ◽  
Hcc Cells ◽  
The Warburg Effect

AbstractMetabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.

scholarly journals ASO-based PKM Splice-switching Therapy Inhibits Hepatocellular Carcinoma Cell Growth

2020 ◽  
Author(s):  
Wai Kit Ma ◽  
Juergen Scharner ◽  
Ana S. H. Costa ◽  
Hyun Yun Jeong ◽  
Michaela Jackson ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mouse Model ◽  
Pyruvate Kinase ◽  
Antisense Oligonucleotides ◽  
Warburg Effect ◽  
Aerobic Glycolysis ◽  
Pyruvate Kinase Activity ◽  
Potential Therapeutic Target ◽  
Splice Isoform ◽  
The Warburg Effect

AbstractThe M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in the Warburg effect, which is characterized by the preference for aerobic glycolysis for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene, and is a potential therapeutic target. Previously, we developed antisense oligonucleotides (ASOs) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform and induce apoptosis in cultured glioblastoma cells. Here, we explore the potential of ASO-based PKM splice-switching as a targeted therapy for liver cancer. We utilize a lead cEt/DNA ASO, which has a higher potency than MOE modification, to demonstrate that it induces PKM splice-switching and inhibits the growth of cultured hepatocellular-carcinoma (HCC) cells. This PKM isoform switch increases pyruvate-kinase activity and alters glucose metabolism. The lead ASO inhibits tumorigenesis in an orthotopic-xenograft HCC mouse model. Finally, a surrogate mouse-specific ASO induces Pkm splice-switching and inhibits HCC growth, without observable toxicity, in a genetic HCC mouse model.Statement of significanceAntisense oligonucleotides are used to force a change in PKM isoform usage in HCC, reversing the Warburg effect and inhibiting tumorigenesis.

scholarly journals Single-Cell Transcriptome Analysis as a Promising Tool to Study Pluripotent Stem Cell Reprogramming

2021 ◽  
Vol 22 (11) ◽  
pp. 5988
Author(s):  
Hyun Kyu Kim ◽  
Tae Won Ha ◽  
Man Ryul Lee
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Human Cell ◽  
Transcriptome Analysis ◽  
Single Cell Analysis ◽  
Embryonic Stem ◽  
Single Cell Level ◽  
Cell Analysis ◽  
Cell Level

Cells are the basic units of all organisms and are involved in all vital activities, such as proliferation, differentiation, senescence, and apoptosis. A human body consists of more than 30 trillion cells generated through repeated division and differentiation from a single-cell fertilized egg in a highly organized programmatic fashion. Since the recent formation of the Human Cell Atlas consortium, establishing the Human Cell Atlas at the single-cell level has been an ongoing activity with the goal of understanding the mechanisms underlying diseases and vital cellular activities at the level of the single cell. In particular, transcriptome analysis of embryonic stem cells at the single-cell level is of great importance, as these cells are responsible for determining cell fate. Here, we review single-cell analysis techniques that have been actively used in recent years, introduce the single-cell analysis studies currently in progress in pluripotent stem cells and reprogramming, and forecast future studies.

scholarly journals Nicotinamide promotes pancreatic differentiation through the dual inhibition of CK1 and ROCK kinases in human embryonic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yumeng Zhang ◽  
Jiaqi Xu ◽  
Zhili Ren ◽  
Ya Meng ◽  
Weiwei Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Fate Determination ◽  
Rna Seq ◽  
Pancreatic Cell ◽  
Pancreatic Progenitor ◽  
Pancreatic Progenitors ◽  
Rock Inhibition

Abstract Background Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. Methods We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. Results First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. Conclusions In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.

scholarly journals Valproic acid Suppresses Breast Cancer Cell Growth Through Triggering Pyruvate Kinase M2 Isoform Mediated Warburg Effect

2021 ◽  
Vol 30 ◽  
pp. 096368972110275
Author(s):  
Zhen Li ◽  
Lina Yang ◽  
Shuai Zhang ◽  
Jiaqi Song ◽  
Huanran Sun ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Valproic Acid ◽  
Cancer Therapy ◽  
Pyruvate Kinase ◽  
Histone Deacetylases ◽  
Warburg Effect ◽  
Broad Class ◽  
Pyruvate Kinase M2 ◽  
Breast Cancer Cell Growth ◽  
The Warburg Effect

Energy metabolism programming is a hallmark of cancer, and serves as a potent target of cancer therapy. Valproic acid (VPA), a broad Class I histone deacetylases (HDACs) inhibitor, has been used as a therapeutic agent for cancer. However, the detail mechanism about the potential role of VPA on the Warburg effect in breast cancer remains unclear. In this study, we highlight that VPA significantly attenuates the Warburg effect by decreasing the expression of pyruvate kinase M2 isoform (PKM2), leading to inhibited cell proliferation and reduced colony formation in breast cancer MCF-7 and MDA-MB-231 cells. Mechanistically, Warburg effect suppression triggered by VPA was mediated by inactivation of ERK1/2 phosphorylation through reduced HDAC1 expression, resulting in suppressing breast cancer growth. In summary, we uncover a novel mechanism of VPA in regulating the Warburg effect which is essential for developing the effective approach in breast cancer therapy.

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

scholarly journals The Key Role of the WNT/β-Catenin Pathway in Metabolic Reprogramming in Cancers under Normoxic Conditions

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5557
Author(s):  
Alexandre Vallée ◽  
Yves Lecarpentier ◽  
Jean-Noël Vallée
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Warburg Effect ◽  
Target Genes ◽  
Glucose Transporter ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Metabolic Reprogramming ◽  
Pyruvate Dehydrogenase Kinase ◽  
The Warburg Effect

The canonical WNT/β-catenin pathway is upregulated in cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Nuclear β-catenin accumulation is associated with cancer. Hypoxic mechanisms lead to the activation of the hypoxia-inducible factor (HIF)-1α, promoting glycolytic and energetic metabolism and angiogenesis. However, HIF-1α is degraded by the HIF prolyl hydroxylase under normoxia, conditions under which the WNT/β-catenin pathway can activate HIF-1α. This review is therefore focused on the interaction between the upregulated WNT/β-catenin pathway and the metabolic processes underlying cancer mechanisms under normoxic conditions. The WNT pathway stimulates the PI3K/Akt pathway, the STAT3 pathway and the transduction of WNT/β-catenin target genes (such as c-Myc) to activate HIF-1α activity in a hypoxia-independent manner. In cancers, stimulation of the WNT/β-catenin pathway induces many glycolytic enzymes, which in turn induce metabolic reprogramming, known as the Warburg effect or aerobic glycolysis, leading to lactate overproduction. The activation of the Wnt/β-catenin pathway induces gene transactivation via WNT target genes, c-Myc and cyclin D1, or via HIF-1α. This in turn encodes aerobic glycolysis enzymes, including glucose transporter, hexokinase 2, pyruvate kinase M2, pyruvate dehydrogenase kinase 1 and lactate dehydrogenase-A, leading to lactate production. The increase in lactate production is associated with modifications to the tumor microenvironment and tumor growth under normoxic conditions. Moreover, increased lactate production is associated with overexpression of VEGF, a key inducer of angiogenesis. Thus, under normoxic conditions, overstimulation of the WNT/β-catenin pathway leads to modifications of the tumor microenvironment and activation of the Warburg effect, autophagy and glutaminolysis, which in turn participate in tumor growth.

scholarly journals Proton export drives the Warburg Effect

2021 ◽  
Author(s):  
Shonagh Russell ◽  
Liping Xu ◽  
Yoonseok Kam ◽  
Dominique Abrahams ◽  
Bryce Ordway ◽  
...  
Keyword(s):  
Warburg Effect ◽  
Cancer Metabolism ◽  
Aerobic Glycolysis ◽  
Lactate Production ◽  
Glycolytic Enzymes ◽  
Hek293 Cells ◽  
Driver Mutations ◽  
The Warburg Effect

Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect”. It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. To test this hypothesis, we stably transfected lowly-glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton exporting systems: either PMA1 (yeast H+-ATPase) or CAIX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Therefore, cancer cells with increased H+ export increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards a Warburg phenotype.

scholarly journals Cell Type-Specific Role of RNA Nuclease SMG6 in Neurogenesis

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3365
Author(s):  
Gabriela Maria Guerra ◽  
Doreen May ◽  
Torsten Kroll ◽  
Philipp Koch ◽  
Marco Groth ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Cortical Neurons ◽  
Embryonic Stem ◽  
Normal Structure ◽  
Mutant Mice ◽  
Cell Type ◽  
Nonsense Mediated Mrna Decay ◽  
A Cell ◽  
Cell Type Specific

SMG6 is an endonuclease, which cleaves mRNAs during nonsense-mediated mRNA decay (NMD), thereby regulating gene expression and controling mRNA quality. SMG6 has been shown as a differentiation license factor of totipotent embryonic stem cells. To investigate whether it controls the differentiation of lineage-specific pluripotent progenitor cells, we inactivated Smg6 in murine embryonic neural stem cells. Nestin-Cre-mediated deletion of Smg6 in mouse neuroprogenitor cells (NPCs) caused perinatal lethality. Mutant mice brains showed normal structure at E14.5 but great reduction of the cortical NPCs and late-born cortical neurons during later stages of neurogenesis (i.e., E18.5). Smg6 inactivation led to dramatic cell death in ganglionic eminence (GE) and a reduction of interneurons at E14.5. Interestingly, neurosphere assays showed self-renewal defects specifically in interneuron progenitors but not in cortical NPCs. RT-qPCR analysis revealed that the interneuron differentiation regulators Dlx1 and Dlx2 were reduced after Smg6 deletion. Intriguingly, when Smg6 was deleted specifically in cortical and hippocampal progenitors, the mutant mice were viable and showed normal size and architecture of the cortex at E18.5. Thus, SMG6 regulates cell fate in a cell type-specific manner and is more important for neuroprogenitors originating from the GE than for progenitors from the cortex.

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