Viral Hepatitis-Oncogenic Mechanisms of the Virus

Hans Peter DienesMedical University of Vienna

HCC – The Facts

• Second most frequent cancer related cause of death• Rapid increase in most Western countries; • >90% defined etiology; linked to chronic liver disease and

chronically damaged non-tumorous liver (~90%); epidemiology linked to prevalence of underlying diseases

• Incidence increases with time and duration of chronic liver disease

• Paradigm of viral, metabolic and inflammation induced cancer

HCC: Frequent and significant geographic differences

HCVHCV, NASH

HBV

HBV

Chronic Inflammation and Cancer

• Viral hepatitis – HCC• Steatohepatitis – HCC• Colitis ulcerosa – CRC• PSC – CC• Chronic gastritis (Type B) – Gastric cancer, MALT Lymphoma• Chronic urocystitis – bladder cancer• Chronic pancreatitis – pancreatic cancer

Variable frequency argues for mixed/heterogenous mechanisms

How is Chronic Inflammation linked to Cancer?

• Etiology of inflammation also causes cancer by independent mechanisms

• (Necro-)Inflammation itself is oncogenic– Inflammatory cytokines– Direct cellular response

• Consequences of (necro-) inflammation are oncogenic– Stochastic effects of cellular turnover/repair– Wound healing effects (fibrosis

chronic

liver

damage

chronic viral hepa- titis

genetic

disorders

chronic alcohol abuseNASH

fibrosis

large cell dys- plasia

smallcellproli-feration

dedifferentiation oforiginal hepatocytes?

stem cellactivation?

dys-plasticfoci

dysplasticnodule(low grade)

dysplasticnodule(high grade)

borderline lesions

mal

igna

nttr

ansf

orm

ation

cirrhosis

early

HCC

fullydeve-lopedHCC

tumor

growth

in-vasion

metas- tasis

focu

s fo

rmati

on

size increase

t

Kern et al., Adv Cancer Res, 2002

Does Inflammation cause Liver Cancer?

Liver Fibrosis and HCC

87%

1%6%

3%3%

Stage 4

Stage 3

Stage 2

Stage 1

Stage 0

Yang JD, et al. Clin Gastroenterol Hepatol. 2011;9:64-70.

35-

40-

45-

50-

55-

60-

65-

70-

75-

80-

85+

0.0

50.0

100.0

150.0

200.0

250.0

300.0

Italy 2003 (M)Italy 2003 (F)Hong Kong 2006 (M) Hong Kong 2006 (F) Sweden 2005 (M) Sweden 2005 (F)

HCC increases with age

Incidence and 5-Year Survival of HCC in United States

El-Serag HB. N Engl J Med 2011

HCC has Defined Etiology(Regensburg; Germany; n=374)

Int J Clin Exp Med. 2010; 3(2): 169–179.

Prevalence in general population

Risk estimate of HCC

Current prevalence in HCC cases

Population attributable fraction

HBV 0.5-1% 20-25 10-15% 5-10%HCV 1-2% 20-25 30-60% 20-25%Alcoholic liver disease

10-15% 2-3 20-30% 20-30%

Metabolic syndrome

30-40% 1.5-2.5 20-50% 30-40%

HCC-Pathogenesis

HBV

HCV

Aflatoxins

ASH

NASH

Others

Integration

Trans-Activation

DNA-Modification

Radical Formation

Molecular Signatures

Molecular Epidemiology

Prevention

Etiology Specific Mechanism

Consequences

Diagnostics

Therapy

chromosome losses (%) gains (%)1p 15,4 5,22p 1,4 7,13p 3,9 54p 10,6 65p 1,7 13,66p 1 22,37p 0,9 158p 38 4,69p 14 3,3

10p 2,7 8,311p 5,4 4,312p 6,5 2,413p 0 014p 0 015p 0 016p 16,8 3,417p 32,1 2,918p 4,1 5,519p 6,9 520p 2 14,921p 0 022p 0 0xp 5 11,2yp 5,1 2,3

chromosome losses (%) gains (%)1q 0,6 57,12q 2,9 83q 1,9 8,84q 34,3 1,75q 7,8 11,16q 15 7,97q 3,1 16,88q 1,9 46,69q 11,1 2,9

10q 11,1 4,111q 10,2 9,412q 2,9 6,913q 26,2 7,414q 11,3 4,115q 5,4 4,616q 35,9 1,817q 3,7 22,218q 10,8 519q 3,8 10,420q 0,9 18,621q 8,8 2,222q 6,4 2,8xq 4,5 15yq 5,6 2,3

Genomic Imbalances in human HCCsGeneral analyses of genomic imbalances (n=719) HBV-specific imbalances (n=386)

HBV + HBV -

Chr. 1

Chr. 4

Moinzadeh et al., (2005) Br J Cancer

DN HCCpoorly differentiatedwell differentiated

1q+ 4q -, 13q -6p+, 8p -, 8q+, 16q -, 17p, 17q+

ArrayCGH in human HCCs

Schlaeger et al., Hepatology 2007

HBV Genom

HBV

Cis-acting mechanisms (Integration)

Trans-acting factors (preS, X)

Necroinflammation

Oncogene activation

Suppressor inactivation

Genome destabilisation

Oncogene activation

Increased replication

Increased mutations

Cytokine induction

Oxidative stress

Boyault et al., 2007 Hepatology

Integrated Subtyping

HBV

• Predominance of etiology-specific mechanisms (cis- and trans) of inflammatory effects

• High carcinogenicity despite low grade inflammation (tolerance/perinatal infection) and absence of cirrhosis

Shlomai A et al., Sem in Cancer Biol. 2014, 26

HBV-Oncogenic Mechanisms

• HBV DNA integrates at random sites into host genome thus inducing great genomic instabilities with loss and gains of functions

• Transgenic mice harbouring HBs and HBx develop HCC• PreS1 contains a promoter and transactivator gene• HBx protein: transactivates promoter of CREB,YAP

-suppresses p53 -activates- RAS,Raf,MAPK,PI3k,Src, wnt-cat, -NFkB,STAT3, miRNAs-26,29a,78-

88,143,602 -inhibits: miRNAs 15a, 16, 122

HBV Vaccination and HCC: Taiwan Experience

• HCC prevention extended from childhood to early adulthood• Failures: incomplete vaccination, maternal HBsAg or HBeAg

HCC in HCV - The Facts

• Prevalence of HCV+ HCC (20-90%)

• Relative Risk of HCC

– Compared to HCV - controls (25 fold)

• Absolute Risk of HCC

– HCC in HCV (1 per 100 at 30 years)

– HCC in HCV-related cirrhosis (3.5 per 100 [1-7])

HCV Cirrhosis and HCC 3% per year

HCV Cirrhosis and HCC 3% per year

Multiple smallfoci of HCCMultiple smallfoci of HCC

HCV Cirrhosis and HCC

HCV genome organization and membrane topology of cleaved viral proteins

Bartenschlager R et al., Nature Reviews 2013, 11, 482-496

Bartenschlager R et al., Nature Reviews 2013, 11, 482-496

The HCV replication cycle

HCV-Host Cell-Interaction

12 24 48 72 h

1

Infe

ctiv

ity/

ml (

log

10)

2

3

4

0

5

6

Lohmann et al., Science (1999); Wakita*, Pietschmann* et al., Nat Med (2005)

Miyanari et al., Nat Cell Biol (2007); Shavinskaya et al., JBC (2007); Appel et al., PLoS Pathog (2008)

Bartenschlager, Lohmann, Longerich

Reiss S, Rebhan I, Backes P, Romero-Brey I, Erfle H, Matula P, Kaderali L, Poenisch M, Blankenburg H, Hiet MS, Longerich T, Diehl S, Ramirez F, Balla T, Rohr K, Kaul A, Bühler S, Pepperkok R, Lengauer T, Albrecht M, Eils R, Schirmacher P, Lohmann V, Bartenschlager R. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe. 2011 Jan 20;9(1):32-45.

HCV as the Dominant Risk Factors for HCC in the US

• HBV most frequent in Asians

• HCV most frequent in whites and blacks

• HBV most frequent in Asians

• HCV most frequent in whites and blacks

HCVHCV47%47%

HBVHBV

15%15%

BothBoth

5%5%

NeitherNeither

33%33%

(N=691)(N=691)

HCV

Viral factors Core NS5A

Necroinflammation

Oxidative stress

Metabolic changes (steatosis)

Transactivation (MAPK, AP-1)

Increased replication

Increased mutations

Cytokine induction

Oxidative stress

HCV-Oncogenic Factors

• Transgenic mice with HCV core,E1/2 develop HCC

• HCV core –ROS, suppresses p53, blocks miRNA 122

• NS 3/4A – inhibit ATM (ataxia teleangiectasia suppressor gene)—

reduced DNA repair

• NS 5A– inhibits p53, blocks TGF-β signalling HCV mutants: core-Gln

70 and NS3-Tyr 1082 are linked to HCC development,

more than infection with wild type (El-Shamy, Hepatol. 2013)

Risk Factors for HCC in Chronic HCV

– Older age– Duration of HCV infection– Male sex– Race– Alcoholism– Obesity– Diabetes– HBV co-infection– HIV co-infection– HCV RNA positive, GT1 and GT3

Ashahina et al., Hepatology 2010

HCC and Hepatitis C Treatment

Non-SVR

SVRNon-SVR

SVR

EntzündungRegeneration

Normale Leber

Dysplastische Foci

Dysplastische Knoten

Frühes HCC

InvasionMetastasenv

Virale Replikation Radikalbildung

Transaktivatoren Virale Integration

Tumorsuppressoren

Onkogene

Wachstumsfaktoren

Genomische Instabilität

Intrazelluläre Signalwege

Zellschaden/Zelltod

Immunreaktion

HBV HCV C2 genet. Erkr.

Stepwise Carcinogenesis

Increased Incidence of HCC in Patients Infected with Mutated HCV Strains

El-Shamy et al., Hepatology 2013,58

Animal Models for HCC

• Chimpanzees serve as models for chronic Hep. B and C however they do not develop HCC

• Woodchucks develop HCC upon infection with WHV; the DNA integrates close to c-myc gene, which is different from human disease

• Transgenic mouse models can be used to explore the oncogenicity of viral components; but different from human disease

• Chimeric mouse models are useful because they contain human hepatocytes that can be infected with HBV and HCV

HCC in a Chimeric Mouse Model

• We generated a chimeric mouse model in which transgenic SCID/Bg mice carrying the uPA (urokinase Plasminogen Activator) under the MUP (major urinary protein) received human hepatocytes and were infected after 5 weeks either with HBV or HCV. (Tesfaye, Stift,Dienes,Feinstone,‘ PloS One 2013,14,8-18).

• They developed mild hepatitis but no fibrosis or cirrhosis• After one year 21 % infected with HBV and 26% infected with

HCV developed HCCs. The HCCs originated from the human hepatocytes that were engrafted and showed histopathology identical to human HCC.

• So HBV and HCV infection can induce HCC without significant inflammation.

No significant difference between ages of encraftment, total length of timeencrafted and total length of time infected

Histopathology of Chimeric Mice

GGH inHBVinfected mice

HCC in HBV-infected chimeric mouse

Chimeric Mice

HCC in HCV-infected chimeric mouse

HCC in HCV-GT3-infected chimeric mouse (HRP)

Chimeric Mice II

FIG3_GLY-3hALB_HBV_HCV-3

Staining for Glypican and Albumin in a mouse infectected with HCV

Summary 1

• In human chronic hepatitis B and C HCC develops on the basis of chronic inflammation with fibrosis and mostly cirrhosis due to the increased and uncontrolled liver cell regeneration.

• HBV and HCV have above that direct oncogenic potential• HBV: due to the integration of viral DNA into the host genome

a great chromosomal instability is induced• HBV proteins can function as transactivators: preS1 and HBx

for CREB,YAP,RAS,Raf,wnt b-cat or suppressors: p53,miRNAs: 26,29,78,143,148

Summary 2

• HCV does not integrate its genome into the host but viral components directly act:

• HCV core inceases ROS, blocks p53,promotes ras oncogene• HCV NS3/4 blocks ATM• NS5A inhibits p53&TGF beta signalling• HCV neutralizes miRNA 122.• HCV infection with mutations in core GLN70 and NS3Tyr 1082

carries a higher risk to induce HCC than wild type HCV• Chimeric mice are an excellent model to study HCC