1 Hepatitis C Virus and Hepatocellular Carcinogenesis May 19, 2009 Stanley M. Lemon, MD Center for Hepatitis Research, Institute for Human Infections and Immunity, and the Department of Microbiology and Immunology, University of Texas Medical Branch Galveston TX University of Texas Medical Branch, Galveston, TX
2 The Spectrum of Hepatitis Viruses p p
3 A “Richter Scale” of Global Mortality Rates Weiss and McMichael, Nat Medicine 10:S70-76, 2004
4 Hepatitis C Virus p 4 C E1 E2 NS2 NS3 NS4B NS5A NS5B 5’ 4A 3’ 7 A Structural Proteins ER Lumen p7 E2 RNA-dependent RNA E1 Serine protease polymerase p7 NS2 NS4B 4A NS3 NS3 NS5A NS5A NS5B NS5B Core RNA helicase Signal peptidase Signal peptidase Signal peptidase Signal peptidase Signal peptide peptidase Signal peptide peptidase g g p p p p p p p p N Nonstructural Proteins t t l P t i Signal peptide peptidase Signal peptide peptidase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS2/3 cysteine proteinase NS3/4A serine protease NS3/4A serine protease Cytoplasm NS3/4A serine protease NS3/4A serine protease
5 The Hepatitis C Virus Life Cycle Polyprotein Processing and E2 E1 Replicase Assembly p7 NS2 NS4B 4A 4A NS3 NS5A NS5B Core Attachment and Entry 5’ RNA 5 5’ Synthesis + 5’ - 3’ 5’ Release Assembly Assembly Translation
6 Natural History of Hepatitis C Necroinflammation Progressive Fibrosis Hepatocellular Steatosis Cirrhosis Carcinoma ALT Anti-HCV Anti-HCV Viremia 0 1 2 3 4 5 10 10 15 15 20 20 25 25 30 30 Months Years
7 Hepatitis C Pathogenesis Virus-Cell Interactions Perturbed intracellular milieu Innate antiviral defenses System Level Interactions Innate immune responses p NK, NKT cells, cyto/chemokines Adaptive immune responses Inflammation Steatosis HSC activation, fibrosis (ROS) Carcinogenesis (DNA damage) Host Consequences Hepatic failure Cryoglobulinemia
8 Hepatitis C Pathogenesis Virus-Cell Interactions Perturbed intracellular milieu Innate antiviral defenses System Level Interactions Innate immune responses p NK, NKT cells, cyto/chemokines Adaptive immune responses Inflammation Steatosis HSC activation, fibrosis (ROS) Carcinogenesis (DNA damage) Host Consequences Hepatic failure Liver cancer Cryoglobulinemia
9 Emergence of HCV-Related Liver Cancer g Japan 1971-1990 000/yr 17 1971-80 C B 15 per 100, B+C 13 1981-90 B 11 11 eaths p C B+C 9 De 7 1978 1978- -79 79 1980- 1980 -81 81 1982- 1982 -83 83 1984- 1984 -85 85 1986 1986- -87 87 1968- 1968 -77 77 Year 9
10
11 How does HCV cause liver cancer? How does HCV cause liver cancer? Any hypothesis must consider: Contribution of chronic inflammation Increased risk with EtOH consumption HCV transgenic mice develop HCC HCV transgenic mice develop HCC Association with cirrhosis Genetically heterogeneous nature of HCC
12 How does HCV cause liver cancer? How does HCV cause liver cancer? Any hypothesis must consider: Contribution of chronic inflammation Increased risk with EtOH consumption HCV transgenic mice develop HCC HCV transgenic mice develop HCC Association with cirrhosis Genetically heterogeneous nature of HCC Other potential causes of HCC: Aflatoxin Hepatitis B virus Helicobacter sp. Hereditary hemachromatosis (30%) Hereditary hemachromatosis (30%) α 1-antitrypsin deficiency
13 Central Role of c-Myc Responsive Genes in Malignant Conversion of Dysplastic Nodules to Early HCC y p y Early Low Grade High Grade High Grade Cirrhotic Cirrhotic Hepatocellular Dysplastic Dysplastic Nodule Carcinoma Nodule Nodule • Accumulating genetic and epigenetic modifications • Increasing c-Myc copy number with 8q duplications Kaposi-Novak et al. Cancer Res , 69:2775-2782, 2009
14 c-Myc and E2F1 Cooperatively Promote Hepatocellular c Myc and E2F1 Cooperatively Promote Hepatocellular Carcinogenesis in Double Transgenic Mice Proliferation Apoptosis CNA PC S. Ladu et al. Gastroenterology 135:1322-1332, 2008
15 Cooperative Actions of c-Myc and E2F1 p y on oliferatio osis Pro Apopto Hepatocellular Proliferation and Survival S. Ladu et al. Gastroenterology 135:1322-1332, 2008
16 HCV-infected Cells within a Regenerative Nodule Imaged by 2PE Microscopy Elastin 10X 40X HCV Core DAPI Y. Liang et al., Submitted, 2009
17 T Transgenic FL-N/35 Mice i FL N/35 Mi NS3 Tg-FLN/35 Non-Tg Lerat et al., Gastroenterology 122: 352-365, 2002
18 Centrilobular Hepatic Steatosis FL-N/35 Lineage, 11-12 months old Normal
19 H Hepatic Tumor in an FL-N/35 HCV Transgenic Mouse ti T i FL N/35 HCV T i M (FL-N/35-171, 13 mo Male) Lerat et al., Gastroenterology 122: 352-365, 2002
20 Hepatic Adenoma and Hepatocellular Carcinomas in HCV Transgenic FL-N/35 Mice in HCV Transgenic FL N/35 Mice Lerat et al., Gastroenterology 122: 352-365, 2002
BrDU Staining for DNA Synthesis FL N/35 Lineage FL-N/35 Lineage Lerat et al., Gastroenterology 122: 352-365, 2002
22 Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis STAT1, gC1qR p53 hnRNP K Bin1 p53 Src kinases DDX3 TRIF Grb2 LT- β R CD81 IPS1/MAVS 2’,5’OAS pRb TNFR-1 PKR p53 PKR DDX5 p 4 C E1 E2 NS2 NS3 NS4B NS5A NS5B 5’ 4A 3’ 7 7 A A miR-122 CD81 Hsp90 FBL2 VAP-A,B PTB eIF3 eIF3 SR B1 SR-B1 VAP-A,B VAP A,B CypA,B CypA,B La La PTB (Claudin-1) TBC1D20 PRK2 PCBP-1,2 La (Occludin) ESR2 hnRNP C PCBP-1,2 L-SIGN HuR DC-SIGN
23 Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis STAT1, gC1qR p53 hnRNP K Bin1 p53 Src kinases Tumor DDX3 TRIF Grb2 Suppressors pp LT- β R CD81 IPS1/MAVS 2’,5’OAS pRb TNFR-1 PKR p53 PKR DDX5 p 4 C E1 E2 NS2 NS3 NS4B NS5A NS5B 5’ 4A 3’ 7 7 A A miR-122 CD81 Hsp90 FBL2 VAP-A,B PTB eIF3 eIF3 SR B1 SR-B1 VAP-A,B VAP A,B CypA,B CypA,B La La PTB (Claudin-1) TBC1D20 PRK2 PCBP-1,2 La (Occludin) ESR2 hnRNP C PCBP-1,2 L-SIGN HuR DC-SIGN
24 Host Factors in HCV Replication Host Factors in HCV Replication and Pathogenesis STAT1, gC1qR p53 hnRNP K Bin1 p53 Src kinases DDX3 TRIF Grb2 LT- β R CD81 IPS1/MAVS 2’,5’OAS pRb TNFR-1 PKR p53 PKR DDX5 p 4 C E1 E2 NS2 NS3 NS4B NS5A NS5B 5’ 4A 3’ 7 7 A A miR-122 CD81 Hsp90 FBL2 VAP-A,B PTB eIF3 eIF3 SR B1 SR-B1 VAP-A,B VAP A,B CypA,B CypA,B La La PTB (Claudin-1) TBC1D20 PRK2 PCBP-1,2 La (Occludin) ESR2 hnRNP C PCBP-1,2 L-SIGN HuR DC-SIGN
25 NS5B Regulation of pRb g p Host genome NS5B NS5B instability instability E6AP Rb UbUbUb… Hepatocellular proliferation p p Modifed from Classon and Harlow, Nature Rev Cancer 2: 910, 2002
26 pRb Abundance is Post-Transcriptionally Down-Regulated in HCV RNA Replicon Cells Down-Regulated in HCV RNA Replicon Cells EMCV IRES Δ C p Neo Neo C C E1 E1 E2 E2 NS2 NS2 NS3 NS3 NS4B NS4B NS5A NS5A NS5B NS5B 5’ 5’ 4A 4A 3’ 3’ Neo N 7 Northern Immunoblot Pulse-chase Munakata et al., PNAS 102:18159-64, 2005
27 pRb Abundance is Reduced by Genotype 2a JFH1 Virus Infection of Huh-7.5 Cells 120 96 72 48 24 0 Hrs � � � � � � � � � � = infection � = harvest 100 100 nt Relative nt Relative bundance bundance 0 48 72 96 120 Hrs 80 80 pRb 60 60 NS5B pRb Ab pRb Ab Percen Percen 40 40 GAPDH 20 20 1 2 3 4 5 0 0 0 0 48 48 72 72 96 96 120 120 Hours Post inoculation Hours Post-inoculation Hours Post inoculation Hours Post-inoculation Munakata et al., PLoS Pathogens, 3(9): e139, 2007
28 NS5B Forms a Complex With pRb plicon) ured) 2-3c (Cu 2-3 (Rep IP: α -pRb IB: α -pRb pRb Rb on) Ig HC Ig HC g g ) -3 (Replico -3c (Cured IP: α -pRb NS5B IB: α -NS5B IB: α -NS5B IB: α -NS5B 2- 2- IP: α -pRb IB: α -Core IB: α -Core Core Core Core IB: α -Core IB: α -Core 1 2 1 2 3 4 3 4 Munakata et al., PNAS 102:18159-64, 2005
29 Cytoplasmic Colocalization of pRb and NS5A (replicase) in Huh 7 5 cells 48 hrs Post infection with JFH1 Virus in Huh-7.5 cells 48 hrs Post-infection with JFH1 Virus DAPI Rb NS5A+Rb+DAPI NS5A Munakata et al., PLoS Pathogens, 3(9): e139, 2007
30 HCV Regulation of pRb in Human Liver pRb Normal liver Chronic Hepatitis C Chronic Hepatitis C Y. Liang
31 Epoxomicin, a Potent Inhibitor of the Proteasome, Restores pRb and PO 4 -pRb Abundance Phospho- nM EPX pRb pRb 0 50 500 EPX 250 nM + + pRb Ubi-pRb 2-3 NS5B GAPDH GAPDH Phospho- pRb pRb pRb NS5B NS5B 2-3C GAPDH GAPDH 1 2 3 1 2 3 4 Munakata et al., PLoS Pathogens, 3(9): e139, 2007
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