Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin
<p>IDH1<sup>R132H</sup> influences intracellular TCA (tricarboxylic acid) cycle metabolite levels: Concentrations of TCA metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and results are shown relatively to the empty vector control for (<b>a</b>) cells transduced with IDH1<sup>R132H</sup>, (<b>b</b>) cells transduced with IDH1<sup>wt</sup>, or (<b>c</b>) empty vector controls cells treated with 1 mM D-2-Hydroxyglutarate. For better overview, Y-values were converted to fractions of control. The control of each cell line was defined as a 1.0 baseline and the Y-values were divided by the baseline. All statistical analyses were performed on untransformed data comparing IDH1<sup>R132H</sup>, IDH1<sup>wt</sup>, or empty vector+ 2-HG to empty vector cells using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc <span class="html-italic">t</span>-test. (* <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01).</p> "> Figure 2
<p>IDH1<sup>R132H</sup> reduces growth and increases radio-sensitivity: (<b>a</b>) Cell viability was determined using a WST-1 based colorimetric assay after 48 h culture in adherent condition. Values were normalized to empty vector cells and means from all experiments performed with different transductions were compared (* <span class="html-italic">p</span> < 0.01; one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc <span class="html-italic">t</span>-test). (<b>b</b>) Quantification of cell numbers counted using CASY<sup>®</sup> TTC 72 h after seeding. (<b>c</b>) Clonogenic survival assays showed that IDH1<sup>R132H</sup> significantly enhanced the capacity of glioblastoma cells to form colonies, but not of the astrocytes. (<b>d</b>) To analyze cell growth under 3-D conditions, U87-MG control and IDH-mutant cells were seeded in liquid overlay and cultured for up to 50 days. Spheroid size and volume were routinely monitored. In the example shown here, 2 × 10<sup>3</sup> cells/well were seeded. Data are expressed as the mean volume ± SD. Representative phase contrast microscopic images of the same spheroids are shown at day 4, 11, and 18 (scale bare = 400 µm). (<b>e</b>) Radiation-dose response curves were derived from clonogenic survival assays. Data are expressed as mean of three biological experiments ± SD with n ≥4 wells per experiment and treatment condition. Dose response curves were fitted using a linear-quadratic model (surviving fraction = exp − (αD + βD<sup>2</sup>)).</p> "> Figure 3
<p>IDH1<sup>R132H</sup> and not 2-HG alone leads to a drop in NADPH and NAD<sup>+</sup> concentrations and sirtuin activity in glioblastoma cells but not in astrocytes: Concentrations of NADPH/t and NAD<sup>+</sup>/t were measured in cell lysates of stably transduced cell lines from three different transductions and in triplicates using the NAD<sup>+</sup>/NADH and NADP<sup>+</sup>/NADPH Quantification Kit (MBL). The activity of NAD<sup>+</sup> dependent sirtuins was measured using the HDAC Fluorimetric Cellular Activity Assay Kit (Enzo Life Science). The values were normalized to the mean value of the empty vector cells and the means of normalized values were compared (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc <span class="html-italic">t</span>-test). (<b>a</b>) Normalized values of NADPH levels (top) and NADPt levels (middle) as well as NADPH/NADPt ratios (bottom) are given. (<b>b</b>) IDH1<sup>R132H</sup> led to a significant drop in NADt (top) and NAD<sup>+</sup> (middle) levels in glioblastoma cells, but not in astrocytes. Sirtuins showed less enzymatic activity in IDH1<sup>R132H</sup> glioblastoma cells compared to the empty vector cells (bottom).</p> "> Figure 4
<p>IDH1-mutation influences the expression of the NAD<sup>+</sup> synthesis enzyme NAMPT: (<b>a</b>) NAD synthesis and salvage pathway (adapted from [<a href="#B27-cancers-11-02028" class="html-bibr">27</a>]). NAD is synthesized de novo from tryptophan or from nicotinic acid, nicotinamide riboside and nicotinamide via a salvage pathway. NAD-Kinase (NADK) generates NADPH from NAD<sup>+</sup> and ATP. NAPRT: nicotinicacid phosphorybosiltransferse, NMRK1: nicotinamide riboside kinase, NAMPT: nicotinaminde phosphoribosyltransferase, QPRT: quinolinic acid phosphoribosyltransferase, 3-HAO: quniolinicacid-synthesis-enzyme 3-hydroxyanthranilate 3,4-dioxygenase. (<b>b</b>) Representative Western Blot showing the expression of NAD-Synthesis enzymes NAMPT, NMRK1, NAPRT, QRPT and 3-HAO in our cell models. GAPDH was applied to determine protein loading. (<b>c</b>) Proteins expression of NAD-Synthesis enzymes in patient-derived cell lines and xenografts (PDX) tissues of IDH1<sup>R132H</sup>-mutant and IDH-wildtype gliomas. To quantify NAMPT protein levels, density per sample was divided by the loading-control (GAPDH) and relative density for that lane is given. (<b>d</b>) Mean values of NAMPT protein expression normalized to GAPDH and the empty vector controls from four Western Blots performed with protein extracts of different transductions are shown. (<b>e</b>,<b>f</b>) NAMPT RNA expression levels were measured using reverse transcription quantitative PCR (RT-qPCR). Gene expression was normalized to the expression of reference genes GAPDH and ARF1 (E-ΔCT) and thereafter to the expression in astrocytes or commercially available RNA from normal brain control tissue (E-ΔΔCT). (<b>g</b>) Box plots showing gene expression of NAMPT in IDH1-wildtype (GBM IV) and IDH1-mutant (AS III and AS II) glioma patients based on The Cancer Genome Atlas (TCGA) RNA-seq normalized read count data. Box limits indicate 25th and 75th percentiles, whiskers extend at most to 1.5 times the interquartile range of the box, dots represent outliers. NAMPT was significantly higher expressed in GBM IV versus AS II and III (Wilcoxon rank sum test: <span class="html-italic">p</span> = 1.456541 × 10<sup>−26</sup> and <span class="html-italic">p</span> = 1.240315 × 10<sup>−36</sup>, respectively). (** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc <span class="html-italic">t</span>-test).</p> "> Figure 5
<p>Proposed hypothesis of different effects of the IDH1<sup>R132H</sup> mutation in glial and tumor cells: We found that the IDH1<sup>R132H</sup> mutation differently affects the redox state of glial cells and tumor cells. Wildtype IDH1 provides essential amounts of NADPH for the cell whereas IDH1<sup>R132H</sup> consumes α-KG and NAPDH, leading to abnormally high concentrations of 2-HG, reduced concentrations of α-KG and downstream TCA cycle metabolites, as well as an imbalance between NADPH and NADP<sup>+</sup> levels. Based on our observations, we hypothesize that in astrocytes, the increased NADPH consumption by IDH1<sup>R132H</sup> can still be compensated for by elevating the total NADP pool via the induction of NADK and the NAD<sup>+</sup> synthesizing enzyme NAMPT. Malignant, proliferating cells, however, cannot compensate for the imbalance of NADPH/NADP<sup>+</sup> due to IDH1<sup>R132H</sup>, leading to decreased NADPH and NAD<sup>+</sup> levels. This could be due to the increased requirement of NAD<sup>+</sup> and/or NADPH in proliferating cells or insufficient upregulation of NAD synthesis pathways, potentially accompanied by additional inhibition of NAMPT expression due to the IDH1<sup>R132H</sup>. Abbreviations: NAD = nicotinamide adenine dinucleotide, NADP = nicotinamide adenine dinucleotide phosphate, NMRK1 = nicotinamide riboside kinase, NAMPT = nicotinaminde phosphoribosyltransferase, QPRT = quinolinic acid phosphoribosyltransferase, NADK = NAD-Kinase.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Stable Transduction of IDH1R132H in Glioblastoma Cells and Immortalized Astrocytes
2.2. IDH1R132H but not 2-HG Treatment Alone Leads to Changes in TCA Cycle Metabolites
2.3. IDH1R132H Inhibits Growth and Enhances Radio-Sensitivity In Vitro
2.4. Intracellular NADPH Levels Significantly Drop in Glioma Cells but not in Astrocytes Upon Transduction with IDH1R132H
2.5. IDH1R132H Leads to a Decrease in NAD+ and the Activity of NAD-Dependent Enzymes in Glioma Cells but not in Astrocytes
2.6. Expression of NAD+ Synthesis Enzymes Varies between Individual Cell Lines
2.7. Different Effect of IDH1R132H on NAMPT-Expression between Glioma Cells and Astrocytes
2.8. Expression of NAD+ Synthesis Enzymes Varies in Patient-Derived IDH1R132H and IDH1wt Glioma Cells
2.9. IDH1R132H Gliomas Show Lower Expression of NAMPT In Vivo
3. Discussion
4. Materials and Methods
4.1. Cells and Cell Culture
4.2. Patient-Derived In Vitro and In Vivo Models
4.3. Plasmid Constructs and Lentivirus Production
4.4. DNA and RNA Extraction
4.5. PCR and Sequencing
4.6. Quantitative Real-Time PCR
4.7. Protein Extraction and Western Blot
4.8. Quantification of TCA Cycle Metabolite Levels Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
4.9. Colorimetric Measurement of Cellular NAD and NADPH Levels
4.10. Fluorimetric Measurement of Sirtuin Activity
4.11. Two-Dimensional Growth Assays
4.12. Three-Dimensional Growth Assay
4.13. Clonogenic Survival
4.14. Statistical Analysis
4.15. TCGA Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Biedermann, J.; Preussler, M.; Conde, M.; Peitzsch, M.; Richter, S.; Wiedemuth, R.; Abou-El-Ardat, K.; Krüger, A.; Meinhardt, M.; Schackert, G.; et al. Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin. Cancers 2019, 11, 2028. https://doi.org/10.3390/cancers11122028
Biedermann J, Preussler M, Conde M, Peitzsch M, Richter S, Wiedemuth R, Abou-El-Ardat K, Krüger A, Meinhardt M, Schackert G, et al. Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin. Cancers. 2019; 11(12):2028. https://doi.org/10.3390/cancers11122028
Chicago/Turabian StyleBiedermann, Julia, Matthias Preussler, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Khalil Abou-El-Ardat, Alexander Krüger, Matthias Meinhardt, Gabriele Schackert, and et al. 2019. "Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin" Cancers 11, no. 12: 2028. https://doi.org/10.3390/cancers11122028