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Global Profiling of Metabolic Adaptation to Hypoxic Stress in
Human Glioblastoma Cells
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to
Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE10(1): e0116740. doi:
10.1371/journal.pone.0116740
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Glioblastoma • Common brain tumor • Hypoxia, vascular hyperproliferation & therapy
resistance • Even with surgery, radiation & chemotherapy –
median lifespan is 15 months
http://www.aboutcancer.com/gbm_progression.jpg
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Hypoxia & Tumors
• Hypoxic microenvironment associate with – Invasion – Metastasis – Tumor recurrence – Decreased survival – Resistance to chemoradiotherapy
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Hypoxia & Tumors
• Tumor metabolism changes under hypoxia – Shift from oxidative phosphorylation to anaerobic
glycolysis – Increased synthesis of glycogen, lipids and
phosphorylated lipid metabolites
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Goal of the study
• Use metabolomics to ID the metabolic “Achilles heel” of cancer cells
• Coupled metabolomic and gene profiling to investigate metabolic response to hypoxic stress in human Glioblastoma cells
http://www.med.wisc.edu
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Methods - Cells
Hypoxia (1% O2)
Normoxia (21% O2)
U-87 cells
InVitro2 Hypoxia Work station
6h 24 h 48 h
• Trypsinized, centrifuged, flash frozen
• Media and cells
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Methods - Metabolites 6 samples Each O2 treatment
Non-targetted
UHPLC/MS-MS2 Positive ion mode
UHPLC/MS-MS2 Negative ion mode GC/MS
• Thawed on ice, protein precipitated with methanol, recovery standards added, freeze dried
• 0.1% formic acid • 6.5 mM Ammonium bicarbonate pH 8
• Derivatized under N2 with bistrimethyl-siyl-trifluoroacetamide (MSTFA)
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Methods – mass spectrometry
• UHPLC/MS – Waters Acquity UHPLC with an LTQ mass spec – Electrospray ionization (ESI) with linear ion-trap
(LIT) mass analyzer – Gradient eluted over 11 minutes – Flow rate: 350 ul/min – MS – 900-1000 m/z – MS2 scans – data dependent using dynamic
exclusion
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Methods – mass spectrometry
• GC/MS – 5% phenyldimethyl silicone column – Helium carrier case – Temp ramp 40-300°C over 16 minutes – Analyzed on a Thermo-Finnigan Trace DSQ MS – 50-750 atomic mass unit scan range
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Metabolite Analysis
• Data extraction – peaks ID using Metabolon peak integration software
• Compound ID – compared to LIMS library • Stats – used R, log-transformed, performed
Welch 2-sample T-test, used FDR q-values
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Gene expresssion
• RNA extracted with TRIzol Reagent • 3 samples of each treatment – BeadChip • Data filtered & normalized with BASE2 • Analysis with R, p value < 0.01 • Looked at transcripts differentially expressed
between hypoxic and normoxic
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Results & Discussion
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Fig 1. The metabolic phenotype of hypoxic glioblastoma cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
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Fig 1. The metabolic phenotype of hypoxic glioblastoma cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
PDK3 = pyruvate kinase dehydrogenase enzyme 3
MCT4 - Monocarboxylate transporter 4 solute carrier
GLUT = Glucose transporter, transport glucose over plasma membrane
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Fig 2. Altered glucose shunting in hypoxic GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
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Fig 2. Altered glucose shunting in hypoxic GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
Alternative pathway to glucose – polyol pathway
FUT11 - Fucosyltransferase 11 Implicated in HIF pathway
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Fig 3. Hypoxic effects on the levels of nucleotide cofactors NAD and NADP.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
• Enhanced de novo synthesis of NAD in hypoxic cells
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Fig 4. Hypoxic effects on TCA cycle and glutamine metabolism in GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
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• Less TCA intermediates with prolonged hypoxia
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Fig 5. Hypoxic accumulation of cholesterol precursors in GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
• Conversion to cholesterol requires oxygen
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Fig 6. Effects of hypoxia on glycerolipid metabolism in GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
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Fig 7. Protein and amino acid metabolism in hypoxic GBM cells.
Kucharzewska P, Christianson HC, Belting M (2015) Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells. PLoS ONE 10(1): e0116740. doi:10.1371/journal.pone.0116740 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0116740
• Hypoxia results in accumulation of dipeptides and amino acids with post-translational modifications
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Conclusions
• Tumor samples contain higher levels of enzymes and other compounds associated with hypoxic pathways
• Metabolic studies helpful for therapy – Couple with magnetic resonance & positron
emission tomography
• Important for understanding cancer cell adaptation to microenvironment