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Hepatitis C virus-induced metabolic reprogramming and pathological consequences

10 th Annual Conference of the New Visby Network on. Hepatitis C Riga – February 11th, 2013. Birke Bartosch Team 15 Mechanisms of chronic hepatitis B and C pathogenesis and novel antiviral strategies Cancer Research Center Lyon INSERM 1052.

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Hepatitis C virus-induced metabolic reprogramming and pathological consequences

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  1. 10th Annual Conference of the New Visby Network on Hepatitis C Riga – February 11th, 2013 Birke BartoschTeam 15Mechanisms of chronic hepatitis B and C pathogenesis and novel antiviral strategiesCancer Research Center LyonINSERM 1052 Hepatitis C virus-induced metabolic reprogramming and pathological consequences

  2. Hepatocarcinogenesis – a multistepprocess Risk Factors HCV, HBV Alcohol Aflatoxins Genetic diseases Diabetes, Obesity Metabolic syndrome Chronic inflammation Fibrosis Genetic + Epigenetic Alterations Transformation p53, RB, TGFβ, beta catenin Indirect neoplastic transformation 15 to 40 years 3 to 5% per year Normal liver Chronic Hepatitis Cirrhosis Carcinoma Direct neoplastic transformation

  3. Hepatitis C Virus Pathogenesis Metabolic Syndrome Obesity, Diabetes NAFLD Non-alcoholicliverdisease NASH Non-alcoholicsteatohepatitis ALD Alcoholicliverdisease HCV Insulinresistance ↑ Mitochondrialdysfunction ↑ Steatosis ↑ Metabolicreprogramming Clinical Pathology of HCV Fibrosis↑Genomicinstability ↑ DNA damage ↑ Inflammation ↑ Cirrhosis Liver cancer Cellcycle control ↓Cellular proliferation ↑ Bartosch et al. J Hepatology, 2009

  4. HCV infection and hepatic metabolism • Glucose • Glycolysis Insulin Resistance Oxidativestress Inflammation Fibrosis • Lipogenesis HCV-induced metabolic changes: Role in viral replication? Contribution to pathology? Steatosis • Lipiddroplet • Triglycerides • ApoB • vLDL Cirrhosis HCC assembly • Lipo/viroparticle Specificinfectivity  Immune escape • vLDL

  5. Methodology • In vitro: • Infection of hepatoma cellsHuh-7.5 with HCVcc of genotype 2a (JFH1 strain)   • RTqPCR of enzymes implicated in liver metabolism • Metabolic flux analysis in infected and uninfected control cells • Infection assays in cell lines with altered metabolism • In vivo: • Biopsies from HCV patients at different stages of disease • RTqPCR of enzymes implicated in liver metabolism

  6. Liver carbohydrate metabolism lowdensity HCV virion vLDL Blood Liverparenchyma GLUT cytosol glucose Glycolysis ER vLDL synthesis PEP MTP, ApoB lactate pyruvate vLDL assemblyHCV assembly TG acetyl CoA lipiddroplet OAA fattyacidstriglycerides malate TCA cycle ACC, FAS, SCD Lipogenesis mitochondria

  7. Liver carbohydrate metabolism lowdensity HCV virion vLDL Blood Liverparenchyma GLUT Insulin /IR cytosol glucose IRS 1/2 PI3/Akt Glycolysis ER HGP / vLDL synthesisgenes:PEPCK, G6Pase / MTP, APOB FoxO1 vLDL synthesis Lipogenesis:FAS, ACC SREBP-1c PEP MTP, ApoB Glycolysis, lipogenesis:PK / FAS, ACC, SCD lactate pyruvate ChREBP vLDL assemblyHCV assembly Glucosemetabolism:PK, HK1/2, LDHA, MCT4, GLUT1/3 HIF-1a TG Glucose/glutamine metabolism:PKM2, HK2, SLC745, GLS c-MYC acetyl CoA lipiddroplet OAA nucleus fattyacidstriglycerides malate TCA cycle ACC, FAS, SCD Lipogenesis mitochondria

  8. Reduced glucose utilization by HCV infectedcells 8-hour supernatant harvest 8-hour supernatant harvest Cellseeding +/- HCV Infection Method: Glucose / Lactate measurements -1 0 3 5 Glucose utilization Lactate production Day 3 Post-infection No glucose No glutamine Control medium Control medium

  9. Reduced glucose utilization by HCV infectedcells 8-hour supernatant harvest 8-hour supernatant harvest Cellseeding +/- HCV Infection Method: Glucose / Lactate measurements -1 0 3 5 Glucose utilization Lactate production Lactate production In the absence of glucose Day 5 Post-infection No glucose No glutamine Control medium

  10. Expression of metabolic enzymes in HCV infection RT qPCR of proliferative HCV infected versus uninfected Huh7.5 cells

  11. HCV-induced metabolic reprogramming SLC1A5 SLC7A5 MCT lowdensity HCV virion GLUT vLDL glutamine lactate glucose cytosol Pentose Phosphate Pathway Xu5P G6P Glycolysis ER G6PDH PFKL -> Nucleoside pool -> RedoxBalance vLDL synthesis vLDL assemblyHCV assembly PEP PKM2 lactate LPK MTP, ApoB pyruvate lactate TG PC pyruvate acetyl CoA lipiddroplet OAA ME acetyl-CoA fattyacidstriglycerides malate TCA cycle ACC, FAS, SCD GLS Lipogenesis glutamine mitochondria

  12. Evidence for a glutamine-based glucose-independentmetabolism in HCV infected Huh7.5 cells Cellgrowthanalysisafter glucose or glutamine deprivation Change medium +/- glc or gln Cellseeding Cell count Cell count Cellseeding +/- HCV Infection Cell count Method: -5 -4 -1 0 1 5/14 Day

  13. Evidence for a glutamine-based glucose-independentmetabolism in HCV infected Huh7.5 cells Cellgrowthanalysisafter glucose or glutamine deprivation Change medium +/- glc or gln Cellseeding Cell count Cell count Cellseeding +/- HCV Infection Cell count Method: -5 -4 -1 0 1 5/14 Day -HCV +HCV Cell Count Cell Count N = 4 Time (day) Time (day)

  14. HCV-induced metabolic reprogramming SLC1A5 SLC7A5 MCT lowdensity HCV virion GLUT vLDL glutamine lactate glucose cytosol Pentose Phosphate Pathway Xu5P G6P Glycolysis ER G6PDH PFKL -> Nucleoside pool -> RedoxBalance vLDL synthesis vLDL assemblyHCV assembly PEP PKM2 lactate LPK MTP, ApoB pyruvate TG PC pyruvate acetyl CoA lipiddroplet OAA ME acetyl-CoA fattyacidstriglycerides malate TCA cycle ACC, FAS, SCD GLS Lipogenesis a-KG glutamine mitochondria

  15. Evidence for a glutamine-based glucose-independentmetabolism in HCV infected Huh7.5 cells Cellgrowthanalysisafter glucose or glutamine deprivation Change medium +/- glc or gln Cellseeding Cell count Cell count Cellseeding +/- HCV Infection Cell count Method: -5 -4 -1 0 1 5/14 Day -HCV +HCV Cell Count Cell Count N = 4 Time (day) Time (day) Glutamine is an essential source to the TCA cycle in HCV infectedcells

  16. New concepts in cancer cellmetabolism SLC1A5 SLC7A5 MCT lowdensity HCV virion GLUT vLDL glutamine lactate glucose cytosol Xu5P G6P Glycolysis ER G6PDH PFKL vLDL synthesis c Myc aregulator of cell cycle and cellmetabolism vLDL assemblyHCV assembly PEP PKM2 lactate LPK MTP, ApoB pyruvate TG PC pyruvate acetyl CoA lipiddroplet OAA ME acetyl-CoA fattyacidstriglycerides malate TCA cycle ACC, FAS, SCD GLS Lipogenesis a-KG glutamine mitochondria

  17. c-MYC expression isincreased in HCV infectedcells and biopsies Cellseeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 RNA extraction RNA extraction RNA extraction Method: RT-qPCRanalysis -1 0 1 2 5 Time (day) In vitrotime course infection (Huh7.5 cells/JFH1 virus) n=4

  18. c-MYC expression isincreased in HCV infectedcells and biopsies Cellseeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 RNA extraction RNA extraction RNA extraction Method: RT-qPCRanalysis -1 0 1 2 5 Confirmation in liver biopsies ** Time (day) In vitrotime course infection (Huh7.5 cells/JFH1 virus) n=4

  19. c Myctargetgenes are induced by HCV Cellseeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 RNA extraction RNA extraction RNA extraction Method: RT-qPCRanalysis -1 0 1 2 5 Time (day) Time (day) Time (day) Glutamine transporters Glutaminase In vitrotime course infection (Huh7.5 cells/JFH1 virus) n=4 Currentlybeeingconfirmed in biopsie samples

  20. c Mycrequired for HCV replication • Establishment of a Huh7.5 cell line silenced for MYC • Strongdecrease of HCV RNA level in shMYCcell line despite a reduction of only 60% in MYC mRNAlevels (*) • MYC seems to be essential for HCV replication  MYC mRNAlevel shCTRL shMYC 48h 24h

  21. c-mycmediates HCV-induced glutamine dependence but not decrease of glucose utilization Glucose utilization Lactate production c-mycinduced HCVinfected

  22. Glycolytic reprogramming in HCV infection SLC1A5 SLC7A5 MCT GLUT glutamine lactate glucose Pentose Phosphate Pathway G6P R5P Xu5P G6PDH PGD TALDO TKT Glycolysis PFKL NADP+ NADPH PEP PKM2 LPK lactate pyruvate PC acetyl CoA pyruvate OAA acetyl-CoA fattyacids ME malate TCA cycle .02- ACC, FAS, SCD Lipogenesis a-KG GLS NAD + NADH, H + glutamine mitochondria Huh7.5 cells Biopsies

  23. Cellular defenceagainstoxidative stress MCT GLUT glutamine lactate glucose Pentose Phosphate Pathway G6P R5P Xu5P G6PDH PGD TALDO TKT Glycolysis mitochondria H2O+ 02 H2O+ 02 PEP NADPH GSH GSH lactate pyruvate CAT GR mGPX cGPX GSSG GSSG NADP+ H2O2 H2O2 MnSOD CuZnSOD TCA cycle glutamine .02- .02- a-KG NAD + NADH, H +

  24. Reactiveoxygenspecies (ROS) production duringHCVcc infection Day 2 p.i. Day 3 p.i. Day 1 p.i. Time (days) Oxidative stress Measurement (H2O2 using DCFDA / O2.- using DHE) Huh7.5 seeding HCVcc infection

  25. Reactiveoxygenspecies (ROS) production duringHCVcc infection Day 2 p.i. Day 3 p.i. Day 1 p.i. Time (days) Oxidative stress Measurement (H2O2 using DCFDA / O2.- using DHE) Huh7.5 seeding HCVcc infection MOI • H2O2 production 3 days p.i. • O2.- production 3 days p.i. • Glutathione turnover during infection n= 3 n= 3 n= 1 ** ** ** MOI

  26. Reactiveoxygenspecies (ROS) production duringHCVcc infection Day 2 p.i. Day 3 p.i. Day 1 p.i. Time (days) Oxidative stress Measurement (H2O2 using DCFDA / O2.- using DHE) Huh7.5 seeding HCVcc infection Increase in O2.-but not in H202levels: Overall no increase in oxidative stress MOI • H2O2 production 3 days p.i. • O2.- production 3 days p.i. • Glutathione turnover during infection n= 3 n= 3 n= 1 ** ** ** MOI

  27. HCV inducesglutathioneperoxidases 1 and 4 mRNA Day 3 p.i. Day 1 p.i. Time (days) seeding HCVcc infection RNA extraction • qPCR analysis at day 3 p.i. n= 12

  28. HCV inducesglutathioneperoxidases 1 and 4 activity Day 3 p.i. Day 1 p.i. Time (days) seeding HCVcc infection RNA extraction tBOOH or CHP • Western-blot Analysis at day 3 p.i. • Activity test at day 3 p.i. Measure the decreaseat 340 nm HCV infection n= 4 n= 3 - + - + NS3 NS3 β-actin β-actin GPx1 GPx4 Densitometric analysis of Western-blots ** Substrate : tBOOH Substrate : CHP

  29. Effects of GPx1 & GPx4 silencingon HCV replication, secretion & infectivity Day 1 p.i. Day 2 p.i. Day 3 p.i. Time (days) n.s. Extraction of : -Intra RNA -extra RNA -supernatant seeding cells/cm2 HCVcc infection & siRNA transfection • Specificinfectivity • intracellular viral RNA • extracellular viral RNA • Efficiency of siRNA n.s. n= 3 n= 3 n=3 • GPx1 silencing: n.s. n.s. n= 3 n=4 n= 4 • GPx4 silencing: *** *** GPx4, but not GPx1, is a pro-viral factor for HCV replication & infectivity

  30. HCV replicationis sensitive to lipidperoxidation Day 1 p.i. Day 2 p.i. Day 3 p.i. Time (days) Extraction of : -Intra RNA -extra RNA -supernatant Huh7.5 seeding HCVcc infection & CHP incubation • Linden A. et al, Toxicol In Vitro 2008 • Cumene hydroperoxide (CHP) incubation induces lipid peroxidation • intracellular viral RNA n= 3 • Huang H. et al, Proc NatlAcadSci USA2007 • α-tocopherol blocks CHP mediated lipid peroxidation Lipidperoxidationinhibits HCV replicationRole of GPx4 ? • Measurement of hydroxyalkenals and MDA in infected vs uninfectedcells • Collaboration with Institut Multidiscplinaire de Biochimie des Lipides • Measurement by GC-MS

  31. NS5A induces GPx4 transcription Day 1 Day 2 Day 3 Time (days) +/- Protein induction with Doxycycline RNA & protein extraction Huh7.5 seeding • Western-blot analysis 3 post induction to check protein expression: • qPCR analysis 3 post induction: 7.5 TA GFP n= 2 Doxycycline (5 µg / mL) - + 7.5 TA Core 7.5 TA NS3/4A 7.5 TA NS5A Core Doxy cycline NS3/4A - + - + - + NS5A β-actin β-actin β-actin

  32. Conclusions low density HCV virion vLDL glucose extracellular cytosol Pentose Phosphate Pathway G6P Glycolysis PEP HCV replication and morphogenesis NS5B pyruvate HCV genome Core E1E2 TCA cycle glutamine .02- .02- NAD + NADH, H + mitochondrion ER III I IV II

  33. Conclusions low density HCV virion vLDL glucose extracellular cytosol Pentose Phosphate Pathway G6P NS5A Glycolysis PEP Constant turn over H2O+ 02 H2O+ 02 NADPH GSH GSH HCV replication and morphogenesis GR GPx4 NS5B GSSG GSSG NADP+ pyruvate HCV genome H2O2 H2O2 Phospholipid peroxidation Core E1E2 TCA cycle glutamine .02- .02- NAD + NADH, H + mitochondrion GPx4 ER III I IV II

  34. Perspectives • Validation in clinicalstudies • NMR flux and GC-MC analysis • What about NASH, chronic HBV, HCC? in vitroclinicalcohorts • « metabolic »  cancer drugs  for treatment of liverdiseases • Understand the role of alterd carbohydrate metabolism and glutaminolysis not only in liverdisease but also in cancer

  35. Acknowledgements Cancer Research Center Lyon Team 15 : U1052 Fabien Zoulim Pierre Levy Charlène Brault David Durantel Romain Parent Maud Michelet Aurélie Salle Kennel Audrey Hepato-Gastroenterology HCL Lyon Fabien Zoulim U823, Grenoble, France Patrice Marche Christian Villiers U886, Lyon, France Michel Ovize U1060, Lyon France Michel Guichardant Hubert Vidal PathologischesInstitut, Köln, Germany MargaretheOdenthal Hans-Michael Steffen Hannah Eischeid Ulrike Koize CRMN, Lyon, France Bénédicte Elena-Herrmann Gilles Rautureau Elodie Jobard Engelhard Institute, Moskow, Russia Alexander Ivanov

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