11 07 Oncology 2006 Hbv & Hcc

Hepatitis B Virus & Hepatoma 謝財源 三軍總醫院 胃腸科

Top Ten Causes of Death in Taiwan

HCC(4500) Lung Ca

Lung Ca HCC(1500)

六 Chronic Hepatitis

& Cirrhosis

七 Pneumonia 八 GN & Chronic Renal Dis. 九 Suicide I-S TR

ER

L

一 二

F

Accident CVA Heart Dis. DM

TA

M

二 三 四 五

S VI CE H O G E N ER AL

PI

一 Malignant Tumor

Hepatitis B Virus Infection

Brandt LJ et al. Clinical Practice of Gastroenterology

Chronic Hepatitis B  HBsAg (+)  Worldwide: 350 M  Taiwan: 3 M (15-20% adult) • Chronic hepatitis:> 80% • Cirrhosis: 2%/yr • HCC: 0.8%/yr

Discovery of Hepatitis Viruses 1965 Blumber

HBsAg

1970 Dane

HBV particle

1973 Feinstone

HAV

1975 Feinstone

Non-A, Non-B hepatitis

1977 Rizzetto

Delta antigen

1989 Choo

HCV clone

HBV Viral Particles (Three forms)

Lee WM, NEJM (97)

The Structure of Hepadnaviral Virions and Subviral Particles

NP40

SDS

Field, Knipe and Howley Fundamental Virology (3rd Ed.)

Schematic Structure of HBV HBsAg

DNA HBcAg

Lee WM, NEJM (97)

Hepadnavirus Family HBV Genome 3.2 kb ORFs S, C, P, X Hosts Humans Chimps

WHV 3.3 kb S, C, P, X Woodchucks

Replica- Liver tion Kidney Pancreas WBC

Liver Kidney Pancreas WBC Other ACS Hepatitis HCC

Diseases ACS Hepatitis Cirrhosis HCC

GSHV 3.3 kb S, C, P, X Ground squirrels Wood-chucks Chipmunks Liver

ACS Hepatitis HCC

DHBV 3.0 kb S, C, P Geese Liver Kidney Pancreas Spleen Other? ACS Hepatitis

HBV Genome

Lee WM, NEJM (97)

HBV Genotypes

A G

A G

E

F F

B,

D D A

B, C

C

Genomic Organization of HBV

Feldman M et al Gastrointestinal and Liver Disease 7th Ed

Viral Proteins of HBV  HBsAg (L-, M- S-)  HBeAg  HBcAg  Polymerase  HBx protein

Replication Cycle of HBV

Golgi

ER/IC S M L (-) -strand DNA (+) -strand DNA

ccc-DNA Core Genomic and protein subgenomic RNAs + preS/S 3.5 kb RNA +X pregenome + precore proteins

P protein

(A)n

Mechanisms of Hepatocarcinogenesis

Farazi and DePinho Nature Reviews Cancer (2006)

Histopathlogical Progression & Molecular Features of HCC

Farazi and DePinho Nature Reviews Cancer (2006)

Chronic Hepatitis B & HCC Epidemiologic:  HCC is common in hepatitis B-endemic area  HCC is infrequent in hepatitis B-uncommon area

Serologic:  HBsAg(+):high risk for developing HCC(human & animals)

Clinical:  Altered & integrated HBV DNA in HCC

HBV infection and HCC

Haubrich, W.S. et. al. Bockus Gastroenterology (5th) (1995).

Natural Course of HBV Infection HBV 0.1% Fulminant hepatitis

5% (adult) 40-80% (newborns)

Minimal liver lesions

Chronic infection Chronic active hepatitis Cirrhosis (20%) Hepatocellular carcinoma (3-5% / year-1; 30-50% / 10year-1 Brechot, C. et al. Seminar in Cancer Biology (2000)

HBV and HCC  HBV carrier vs Non-HBV carrier :30 X  HBV carrier < CHB < Liver Cirrhosis  Compensated LC < Decompensated LC

HBeAg and the risk of HCC  In 1991 & 1992: 11,893 men without HCC (30 to 65 y/o) from seven townships in Taiwan)  111 cases of newly diagnosed HCC during 92,359 person-years of follow-up.   The relative risk of HCC: 60.2 X (M/ HBsAg & HBeAg(+)) 9.6 X (M/HBsAg(+) only) 1.0 X (M/HBsAg & HBeAg(-))  CONCLUSIONS:HBeAg (+) is associated with an increased risk of HCC Yang and Chen et al. NEJM (2002)

Yang and Chen et al. NEJM (2002)

Yang and Chen et al. NEJM (2002)

HBeAg(+) and HCC  Case-control studies : a higher HBeAg(+) in HCC cases than matched controls  Cumulative HCC risk from age 30 to 70 years 87% (HBsAg(+) & HBeAg(+)) 12% (HBsAg(+) HBsAg only 1% (HBsAg(-) & HBeAg(-)) You and Chen et al. Ann Med. (2004)

HBV genotype & DNA level and HCC: a prospective study in men  1988 -1992: 4841 (Taiwanese men HBsAg , no HCC.)  HBV viral load: 7.26 X  Genotype C HBV : 5.11 X (vs other HBV genotypes)  HBV genotype and viral load with HCC risk: additive  Genotype C & with high viral load : 26.49 X (vs other HBV genotypes and low viral load )

Yu and Chen et al. J Natl Cancer Inst. (2005)

Risk of HCC and HBV DNA Level & Genotype

Liu CJ et al. Liver Int (2005)

Indirect Effects of Chronic HBV Infection     

Chronic active hepatitis (CAH) Cirrhosis Animal models Transgenic mice Accumulation of viral proteins (HBsAg)

Chronic Active Hepatitis (CAH)  The main driving carcinogenic factors for HCC  Necrosis (liver-cell death)→ Trigger proliferative signals of adjacent cells  Inflammation (Inflammatory response)  Synthesis of cytokines (TNF, IL6 etc)  Stimulating liver-cell proliferation  Emergence of DNA mutations and chromosomal rearrangements

 Fibrosis  Disrupt normal lobular structure (cell-cell & cell-CEM interactions)  Loss of control over cell growth  DNA mutations and chromosomal rearrangements  Genetic events (angiogenesis etc.)

Cirrhosis  HBV-related HCC with cirrhosis (majority)  Fibrosis and regeneration nodules  Histological end point of chronic inflammatory and fibrosis process  Increased liver-cell DNA synthesis  Monoclonal cell expansion in regeneration nodules

Animal Models of HBV Infection  Woodchuchs (WHBV)  Ground squirrels (GHBV)  Duck (DHBV)  CAH → HCC

HBV Transgenic Mice  Pre S1/S2/S HBV envelope protein   

Liver-cell proliferation Liver-cell dysplasia Liver cancer

 HBV-expressing hepatocytes   

Immune response (cytokines) Liver cell regeneration HCC

 HBV-positive transgenic mice  Oxidative DNA damage (related to cytokine synthesis)  Increased sensitivity to chemical carcinogens (HBV infection & Carcinogen)

Accumulation of Viral Proteins in HBV-infected Liver Cell  HBsAg in “ground glass” hepatocytes  Modify detoxification pathway (cyt. p450)  Enhance the metabolism of chemical carcinogen (Viral infection & Carcinogen)

HBsAg in Hepatocyte

Hematoxylin & Eosin staining Immunoperoxidase staining Lee WM, NEJM (97)

The Roles of HBV in Liver-cell Carcinogenesis HBV-induced CAH and cirrhosis Direct effect of HBV  HBV DNA sequences integration  HBV X (HBx)  Pre S2/St  HBV spliced protein (HBSP)

HBV DNA Integration  Occur in most HBsAg positive HCC (~90%)  Monoclonal / oligoclonal proliferation  Multicentric occurrence of tumor nodules (several different integration sites)

 No preferential integration sites (high level in chromosomes 11)

 In non-tumoral, cirrhotic liver cells (with

different restriction profile from HCC) (→ coexistence of different clones)

 In HBV carriers  A progressive clonal expansion of certain HBV infected cells

The Dynamic of HBV Integrants Chr. 1

HBV SCX

FreeHBV DNA Chr. 8

CAH / Cirrhosis or early HCC

HBV HBV S X HBV

HB V

Chr. 7 Chr. 1 Chr. 1 Chr. 8

Advanced HCC Brechot, C. et al. Seminar in Cancer Biology (2000)

Integrated HBV DNA in HCC KT589 Alph-a 339 bp E(P)

Alph-b 339 bp E

Alph-a 339-29 bp

E

3.2k bp E

1k bp E

E(P)

HBV DNA (899 bp)

910

1080

1234

1374

Enhancer II 1811

(3bp del) Enhancer I

X Gene

1812-1838 (27bp del)

Miyaki, M. et al Int. J. Cancer (2000)

Consequences of HBV DNA Integration  Chromosomal DNA instability  Chromosomal rearrangements  Chromosomal deletions (4q, 16q, 11p, 11q)  Translocations (17/x, 5/9, 17/18)

 Synthesis of X and truncated preS2/S protein  Insertional mutagenesis (cis-activation)

HBV X (HBx)  HBx expression in human HBV-related HCC  Role of HBx in viral life cycle ?  Indispensable for infectively of WHBV  HBx transactivate cellular and viral genes (c-jun, c-fos & c-myc, TNFα , transforming growth factor-β, HBV Ens, HIV-LRT)

 HBx transgenic mice  Susceptibility to chemical carcinogens  Development of c-myc-induced HCC

 HBx acting as a tumor promoter during liver carcinogenesis

Different Targets of HBx Transactivation: NF-kB, AP-1, AP-2, CRE, NF-AT Cytoplasmic (proteasome, mitochondria?): ras/raf/MAPK, Jak1/STAT, c-Jun N-ter kinase, Src family kinase Nuclear: RNA Pol (RPB5, RMP), TATA-binding Cellular partners: CREB/ATF2 RPB5 IkBα P53 UVDDB ERCC3 (DNA repair) Cell senescence factor Proteasome unit hu-Sui1

HBx

Proliferation Concentration Extracellular signals Cell differentiation Apoptosis

Brechot, C. et al. Seminar in Cancer Biology (2000)

Biological Effects of HBx Acute

High intracellular concentration

 Cell-cycle block: G1/S  Apoptosis

Chronic HBV infection

HBx

Low intracellular concentraion HBx mutations

 Abrogation of cellcycle block and apoptosis  Cell-cycle progression ? Brechot, C. et al. Seminar in Cancer Biology (2000)

PreS2/S  Expressed in up to 25% HCCs  Transactivating effect of preS2/S proteins on cellular genes (c-myc and c-fos)  Cytoplasmic location (Indirect transactivating effect)  Mechanisms involved ? (generation of tree-radical oxides)

HBV Spliced Protein (HBSP)   

Spliced HBV transcripts Anti-HBSP (positive in 1/3 HBV carriers) In liver biopsy from HBV carriers (Western blot)  Ectopic expression of HBSP in liver cell lines leads to apoptosis  Regulate liver cell viability & favor viral particle dissemination  Liver-cell apoptosis may also favor viral persistence (high versus low HBV proteins expression)

HBSP, a New HBV Protein

Kremsdorf and Brechot et al. Oncogene (2006)

Insertional Mutagenesis  Frequent in woodchuck HCC (> 50%)  c-myc or N-myc gene  Rare in human HCC (few cases reported)  Retinoic acid receptor B (RARB) gene  Cyclin A2 gene  Sarcoendoplasmic calcium ATPasedependent pump (SERCA1) gene  Carboxypeptidase N and epidermal growth factor receptor gene  Mevalonate kinase gene (PLC/PRF/5 cell line)

Mechanisms involved in HBV-related chronic liver lesions and HCC Chronic inflammation Fibrosis

Immune response

Genetic alterations

Clonal-cell expansion Transformation

Cell-death Cell cycle signalling regulation HCV Core, E2 NS3, NS5A

Angiogenesis Metastasis

Genetic alterations: deletions , amplifications random? cis-activation (HBV) Demethylation HBV •HBx, HBSP •PreS2/St •Integrated genomes

Brechot, C. et al. Seminar in Cancer Biology (2000)

Pathogenesis of Human Hepatocellular Carcinoma

Levrero M Oncogene (2006)

Conclusions  HBV constitutes a major environmental and etiological factor for HCC in humans  HBV can deregulate the proliferation, differentiation and viability of liver cells (by viral proteins by inducing genetic alterations)