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Birke Bartosch Team 15 Mechanisms of chronic hepatitis B and C pathogenesis and novel antiviral strategies Cancer Research Center Lyon INSERM 1052 Hepatitis.

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Presentation on theme: "Birke Bartosch Team 15 Mechanisms of chronic hepatitis B and C pathogenesis and novel antiviral strategies Cancer Research Center Lyon INSERM 1052 Hepatitis."— Presentation transcript:

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

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

3 Hepatitis C Virus Pathogenesis Liver cancer Cirrhosis Metabolic Syndrome Obesity, Diabetes Cell cycle control ↓ Cellular proliferation ↑ Metabolic reprogramming Mitochondrial dysfunction ↑ Fibrosis↑ Genomic instability ↑ DNA damage ↑ Steatosis ↑ Insulin resistance ↑ Clinical Pathology of HCV NAFLD Non-alcoholic liver disease NASH Non-alcoholic steatohepatitis ALD Alcoholic liver disease Inflammation ↑ Bartosch et al. J Hepatology, 2009

4 Lipogenesis Glucose Glycolysis ApoB Triglycerides Lipid droplet Specific infectivity  Immune escape HCV infection and hepatic metabolism vLDL Cirrhosis HCC HCV-induced metabolic changes: Role in viral replication? Contribution to pathology? vLDL Steatosis Oxidative stress Inflammation Fibrosis Insulin Resistance Lipo/viro particle assembly

5 Methodology In vitro: Infection of hepatoma cells Huh-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 glucose GLUT PEP lactate pyruvate Glycolysis acetyl CoA TCA cycle mitochondria fatty acids triglycerides Lipogenesis ACC, FAS, SCD TG lipid droplet vLDL synthesis ER MTP, ApoB vLDL cytosol vLDL assembly HCV assembly low density HCV virion Liver carbohydrate metabolism malate OAA Blood Liver parenchyma

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

8 Cell seeding +/- HCV Infection 0 8-hour supernatant harvest 3 5 Glucose / Lactate measurements Method: 8-hour supernatant harvest Glucose utilizationLactate production Day 3 Post-infection Control medium No glutamine No glucose Reduced glucose utilization by HCV infected cells

9 Cell seeding +/- HCV Infection 0 8-hour supernatant harvest 3 5 Glucose / Lactate measurements Method: 8-hour supernatant harvest Reduced glucose utilization by HCV infected cells Day 5 Post-infection Glucose utilizationLactate production Lactate production In the absence of glucose Control medium No glutamine No glucose

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

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

12 Evidence for a glutamine-based glucose-independent metabolism in HCV infected Huh7.5 cells Cell seeding -5-4 Cell seeding 5/14 Method: +/- HCV Infection Day Cell count 0 1 Change medium +/- glc or gln Cell growth analysis after glucose or glutamine deprivation

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

14 glucose GLUT G6P Pentose Phosphate Pathway PEP pyruvate LPK Glycolysis acetyl CoA TCA cycle mitochondria acetyl-CoA  -KG malate OAA lactate MCT lactate glutamine SLC1A5 SLC7A5 glutamine GLS pyruvate ME PC G6PDH fatty acids triglycerides Lipogenesis ACC, FAS, SCD TG lipid droplet vLDL synthesis ER MTP, ApoB vLDL cytosol vLDL assembly HCV assembly low density HCV virion PFKL HCV-induced metabolic reprogramming PKM2 Xu5P -> Nucleoside pool -> RedoxBalance

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

16 glucose GLUT G6P PEP pyruvate LPK Glycolysis acetyl CoA TCA cycle mitochondria acetyl-CoA  -KG malate OAA lactate MCT lactate glutamine SLC1A5 SLC7A5 glutamine GLS pyruvate ME PC G6PDH fatty acids triglycerides Lipogenesis ACC, FAS, SCD TG lipid droplet vLDL synthesis ER MTP, ApoB vLDL cytosol vLDL assembly HCV assembly low density HCV virion PFKL PKM2 Xu5P c Myc a regulator of cell cycle and cell metabolism New concepts in cancer cell metabolism

17 c-MYC expression is increased in HCV infected cells and biopsies Cell seeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 0 RNA extraction 1 RNA extraction 2 RNA extraction 5 RT-qPCR analysis Method: In vitro time course infection (Huh7.5 cells/JFH1 virus) n=4 Time (day)

18 c-MYC expression is increased in HCV infected cells and biopsies Cell seeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 0 RNA extraction 1 RNA extraction 2 RNA extraction 5 RT-qPCR analysis Method: In vitro time course infection (Huh7.5 cells/JFH1 virus) n=4 Time (day) Confirmation in liver biopsies **

19 Cell seeding (Huh7.5) +/- HCV Infection (JFH1) MOI1 0 RNA extraction 1 RNA extraction 2 RNA extraction 5 RT-qPCR analysis Method: Glutamine transportersGlutaminase Currently beeing confirmed in biopsie samples In vitro time course infection (Huh7.5 cells/JFH1 virus) n=4 Time (day) c Myc target genes are induced by HCV

20  Establishment of a Huh7.5 cell line silenced for MYC  Strong decrease of HCV RNA level in shMYC cell line despite a reduction of only 60% in MYC mRNA levels (*)  MYC seems to be essential for HCV replication 24h 48h shCTRL shMYC  MYC mRNA level c Myc required for HCV replication

21 c-myc mediates HCV-induced glutamine dependence but not decrease of glucose utilization Glucose utilizationLactate production c-myc induced HCV infected

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

23 mitochondria Cellular defence against oxidative stress.02-.02- H2O2H2O2 MnSOD.02-.02- CuZnSOD mGPX H 2 O + 0 2 CAT cGPX GSH GSSG GSH GSSG GR NADPH NADP + lactateglucose GLUTMCT G6P R5P Xu5P Pentose Phosphate Pathway PEP pyruvatelactate Glycolysis G6PDH PGD TALDO TKT TCA cycle  -KG NAD + NADH, H + H2O2H2O2 glutamine

24 Huh7.5 seeding Oxidative stress Measurement (H 2 O 2 using DCFDA / O 2.- using DHE) HCVcc infection Day 1 p.i. Day 2 p.i. Day 3 p.i. Time (days) Reactive oxygen species (ROS) production during HCVcc infection

25 Huh7.5 seeding Oxidative stress Measurement (H 2 O 2 using DCFDA / O 2.- using DHE) HCVcc infection Day 1 p.i. Day 2 p.i. Day 3 p.i. Time (days) Reactive oxygen species (ROS) production during HCVcc infection Glutathione turnover during infection n= 3 O 2.- production 3 days p.i. MOI n= 1 H 2 O 2 production 3 days p.i. MOI ** n= 3

26 Huh7.5 seeding Oxidative stress Measurement (H 2 O 2 using DCFDA / O 2.- using DHE) HCVcc infection Day 1 p.i. Day 2 p.i. Day 3 p.i. Time (days) Reactive oxygen species (ROS) production during HCVcc infection Glutathione turnover during infection n= 3 O 2.- production 3 days p.i. MOI n= 1 H 2 O 2 production 3 days p.i. MOI ** n= 3 Increase in O 2.- but not in H 2 0 2 levels: Overall no increase in oxidative stress

27 HCV induces glutathione peroxidases 1 and 4 mRNA seeding RNA extraction HCVcc infection Day 1 p.i. Day 3 p.i. Time (days) Enzymeexpressionstructure GPx1strong expressiontetramer GPx2weak expressiontetramer GPx3Plasmatic GPxtetramer GPx4 strong expression and specific of membranes lipid peroxidesmonomer qPCR analysis at day 3 p.i. n= 12

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

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

30 HCV replication is sensitive to lipid peroxidation Huh7.5 seedingHCVcc infection & CHP incubation Time (days) Extraction of : -Intra RNA -extra RNA -supernatant intracellular viral RNA Cumene hydroperoxide (CHP) incubation induces lipid peroxidation  Linden A. et al, Toxicol In Vitro 2008 Measurement of hydroxyalkenals and MDA in infected vs uninfected cells Collaboration with Institut Multidiscplinaire de Biochimie des Lipides Measurement by GC-MS α-tocopherol blocks CHP mediated lipid peroxidation  Huang H. et al, Proc Natl Acad Sci USA 2007 n= 3 Lipid peroxidation inhibits HCV replication Role of GPx4 ? Day 1 p.i. Day 2 p.i. Day 3 p.i.

31 NS5A induces GPx4 transcription Huh7.5 seeding +/- Protein induction with Doxycycline Time (days) RNA & protein extraction - + Core β-actin NS3/4A NS5A Doxy cycline - + Doxycycline (5 µg / mL) 7.5 TA GFP 7.5 TA Core7.5 TA NS3/4A 7.5 TA NS5A qPCR analysis 3 post induction: Western-blot analysis 3 post induction to check protein expression: n= 2 Day 1 Day 2 Day 3

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

33 Conclusions.02-.02- H2O2H2O2.02-.02- H 2 O + 0 2 TCA cycle NAD + NADH, H + H2O2H2O2 IV III II I mitochondrion cytosol extracellular ER E1E2 low density HCV virion vLDL Core HCV genome NS5B HCV replication and morphogenesis GSH GSSG GSH GSSG GR NADPH NADP + glucose G6P Pentose Phosphate Pathway PEP Glycolysis glutamine pyruvate Phospholipid peroxidation Constant turn over GPx4 NS5A

34 Perspectives Validation in clinical studies NMR flux and GC-MC analysis What about NASH, chronic HBV, HCC? in vitro clinical cohorts « metabolic » cancer drugs for treatment of liver diseases Understand the role of alterd carbohydrate metabolism and glutaminolysis not only in liver disease 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 Pathologisches Institut, Köln, Germany Margarethe Odenthal 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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