Download Commentary Can antiviral therapy for chronic hepatitis B enhance

Antiviral Therapy 14:139–142
Commentary
Can antiviral therapy for chronic hepatitis B enhance
the progression to hepatocellular carcinoma?
Nadia Warner1 and Stephen Locarnini1*
Victorian Infectious Diseases Reference Laboratories, North Melbourne, Victoria, Australia
1
*Corresponding author: E-mail: [email protected]
See article by Lai et al. on pp. 249–261 of this issue.
Hepatocellular carcinoma (HCC) is the fifth most
common cancer and the third most common cause of
cancer-­related mortality worldwide [1,2]. Approximately 80% of HCC cases have been attributed to
infection with either hepatitis B virus (HBV) or hepatitis C virus (HCV), and approximately half the total
number of HCC cases can be attributed to chronic
hepatitis B (CHB) [2]. The remaining 20% of HCC
cases involve risk factors, including fatty liver disease,
alcohol abuse and metabolic disorders, such as haemochromatosis [3]. The development of HCC in chronic
HBV infection is a multistep process proposed to be
a consequence of the combination of ≥3 mechanisms,
which include ongoing inflammation, liver damage and
regeneration, an increase in chromosomal instability
and a direct effect of the virus or viral proteins [4].
High levels of replicating HBV have been significantly
associated with ongoing liver damage, inflammation,
fibrosis and progression to HCC [5]. Genotype C HBV
has been reported to replicate to higher levels than other
HBV genotypes and can cause more rapid progression to
HCC [6–8]. Infection with hepatitis B e antigen-negative
strains of HBV has also been associated with more rapid
progression to HCC [9,10]. The host immune response
to this higher level of replication might also contribute to
neoplastic transformation [11].
Most HBV-associated HCCs harbour integrated HBV
DNA, which can cause chromosomal instability [12,13];
however, integrated HBV DNA can also be found in nontumourous tissue from CHB patients [14]. Integration of
viral DNA into the host chromosome is not necessary for
HBV replication, but does occur and might allow persistence of the viral genome. Viral integration can lead to the
development of HCC caused by deletion of cellular genes
at the integration site or transposition of viral and cellular genes [15]. HBV DNA can also integrate directly into
genes that regulate cell signalling, proliferation and viability [13]. These integration events can cause the progression to HCC by modifying the activities of genes that are
©2009 International Medical Press 1359-6535 (print) 2040-2058 (online)
Locarnini.indd 139
important in cell growth and differentiation. The ­protein
products of some integrated HBV genes, notably HBx
and truncated large (L) and middle (M) surface proteins,
have also been implicated in the progression to HCC.
HBV proteins that might be involved in the development of HCC, transcribed from either integrated HBV
DNA or the HBV genome, include HBx. HBx is a nonstructural accessory protein of HBV, which transactivates many cellular genes that regulate cell proliferation
[16]. There is also evidence that the level of the HBV
splice protein correlates with the severity of fibrosis,
which is a precursor to the development of HCC [17].
Truncated HBV surface proteins have been implicated
in the progression to HCC as they also possess transactivational activity. The HBV surface gene (pre-S1/preS2/S) encodes three coterminal surface proteins designated L (pre-S1+pre-S2+S genes), M (pre-S2+S genes)
and small (S; S gene). Pre-S2/S genes truncated at the
3′ end have been isolated from integrated HBV DNA
sequences in HCC [18,19]. Subsequent studies demonstrated that the L and M proteins with C-terminal truncations had transcriptional transactivation potential, a
function that is not exhibited by the full-length forms.
This was determined by measuring nuclear factor
(NF)-κB or activator protein (AP)-1 promoter activity
in cells cotransfected with truncated L or M expression
constructs and NF-κB/AP-1 reporter constructs [20].
Of clinical significance is the recent observation that
nucleoside/nucleotide analogue (NA) therapy might select
for HBV mutants that encode truncated surface proteins
and therefore could theoretically encourage the progression to HCC. Treatment of CHB with NAs, including
lamivudine and adefovir, inevitably results in the selection
of HBV variants with point mutations in the polymerase
gene that confer NA resistance. HBV has the added complexity of overlapping reading frames, and because the
HBV surface gene completely overlaps the polymerase
gene (Figure 1), point mutations selected by NA therapy
can also result in changes in the surface proteins [21]. In
139
27/3/09 11:53:48
N Warner & S Locarnini
In vitro phenotypical testing has confirmed that
rtA181T confers resistance to most NAs tested, including low levels of resistance to adefovir, lamivudine, tenofovir, entecavir [30,31], telbivudine and emtricitabine,
and high levels of resistance to clevudine and torcitabine [31]. The truncated surface proteins encoded by this
variant are secretion defective and are dependant on wt
surface proteins for secretion, resulting in their intracellular accumulation [25]. The surface protein truncation at sW172* is strikingly similar to those encoded
by HCC integrants, which were subsequently shown to
cause transactivation.
A molecular mechanism for the transactivational
function of truncated proteins has been described, and is
dependent on the cytoplasmic localization of the pre-S2
particular, the point mutation that causes the rtA181T
change in the polymerase also encodes a stop codon
(sW172*) in surface proteins (Figure 1).
Clinically, HBV encoding rtA181T has been selected
during therapy with several NAs and usually occurs
in vivo as a mixed population with wild-type (wt) HBV.
The selection of rtA181T during lamivudine therapy
has been reported by a number of groups [22–25] and
the corresponding mutation has also been reported
in woodchucks infected with woodchuck hepatitis
virus and treated with lamivudine [26]. In addition,
rtA181T has been detected during adefovir [25,27],
telbivudine [28] and more recently, clevudine therapy
[29], at varying frequencies depending on the NA and
duration of treatment.
Figure 1. Polymerase/surface gene overlap
Lamivudine
Adefovir
Telbivudine
Clevudine
A181T
Polymerase
gene
Terminal protein
Surface gene
Reverse transcriptase
BCDE
G F A
Spacer
Pre-S1
PreS2
RNase H
Overlapping
reading frames
S
W172*
L
M
S
}
Surface
proteins
Surface proteins
ER lumen/virion surface
172
Membrane
Cytosol/virion interior
Transactivation and HCC
Treatment with nucleoside/nucleotide analogues can result in the selection of a mutation encoding the A181T mutation in the polymerase (light grey box). The DNA
that encodes the polymerase protein also encodes the surface proteins from another reading frame (dark grey box), and the point mutation that encodes A181T in
the polymerase also encodes W172* in the surface proteins. This results in truncation of the hepatitis B virus large (L), middle (M) and small (S) surface proteins and
loss of the C-terminal hydrophobic region from amino acid 172 (shown as dashed region). Mutations in the overlapping reverse transcriptase and surface genes, and
the corresponding changes to the surface proteins are represented by x. Truncated L and M proteins have transactivational activity and have been implicated in the
development of hepatocellular carcinoma (HCC). ER, endoplasmic reticulum.
140
Locarnini.indd 140
© 2009 International Medical Press
27/3/09 11:53:54
Can therapy for chronic hepatitis B cause HCC?
region (reviewed in [32]). The pre-S2 region of the fulllength M protein is localized inside the endoplasmic
reticulum (ER) lumen and no transactivational activity is displayed. The pre-S2 region of the L protein has
two topologies: approximately half are localized to the
ER lumen and half to the cytosol [33–35]. Hence, fulllength L has some transactivation activity [36]. When
the hydrophobic C-terminal region of the L and M proteins is removed, the pre-S2 region is localized primarily
to the cytoplasmic side of the ER and transactivation
occurs [37]. Specifically, L and M proteins that are truncated anywhere between 22 and 152 amino acids from
the start of the S region have increased transactivational
activity, whereas truncations after amino acid 175 do not
confer transactivational activity [38,39]. It is also possible that during HBV replication these truncated proteins
cause cell stress and damage by their intracellular retention. ER stress induces the unfolded protein response
(UPR), which in hepatocytes can lead to HCC [40,41].
Two recent reports, one from Lai and Yeh [42] and the
second from Lai et al. appearing in this issue of ­Antiviral
Therapy (see pp. 249–261), have provided evidence for
involvement of HBV encoding the rtA181T/sW172*
mutation in the pathogenesis of, and progression to
HCC. Analyses of HBV DNA from patients who developed HCC despite lamivudine therapy revealed stop
codon mutations in the envelope gene in seven out of
eight patients compared with the control group, in which
no patients developed HCC. Using expression constructs
encoding the HBV surface proteins, these investigators
demonstrated that surface proteins truncated at amino
acids sL21, sW156 or sW172 (the latter of which equates
to the surface proteins expressed from rtA181T/sW172*)
transactivated the c-myc and SV40 promoters. This was
measured using promoter-luciferase reporter constructs,
and luciferase output was used as a measure of promoter
activity. NIH-3T3 cells transfected with these constructs
were also tumourigenic when injected into nude mice,
whereas the full-length surface proteins were not.
In the studies of Lai and Yeh [42] and Lai et al., HBV
constructs expressing only the surface proteins were used
to compare the effects of truncated surface proteins and
wt surface proteins. However, HBV encoding rtA181T/
sW172* primarily occurs as a mixed population with wt
HBV in vivo [25], and the surface proteins are present
in the context of active viral replication. A study by
Warner and Locarnini [25], using full-length replication­competent HBV expression constructs demonstrated
that when both wt HBV and HBV encoding rtA181T/
sW172* were present in equal amounts (which mimics the situation observed in vivo) there was a dramatic
intracellular retention of the truncated L protein, the
same protein shown by Lai and Yeh [42] and Lai et al.
to be a transactivator. The intracellular accumulation of
the truncated L transactivator protein was even more
Antiviral Therapy 14.2
Locarnini.indd 141
marked in cells transfected with a mixed population
of wt and rtA181T/sW172* than in those transfected
with rtA181T/sW172* alone. Furthermore, intracellular
retention of the wt HBV L protein causes severe liver disease, eventually resulting in neoplasia in transgenic mice
[43]. Taken together, these results suggest that selection
of the rtA181T/sW172* drug-resistant mutant might
substantially increase the risk of development of HCC.
These studies provide compelling data suggestive
of the potential of HBV encoding rtA181T/sW172*
or other surface protein truncations to enhance the
progression to HCC. This might occur via a combination of their transactivation activity and general
evolutionary stress responses, including ER stress
and induction of the UPR, because of their intracellular retention. Further clinical monitoring of patients
for the emergence of these potentially carcinogenic
mutants is required to ensure they are not increasing
in prevalence. As these truncation mutants are defective in secretion, they are invariably detected in conjunction with wt HBV, and might be present at low
levels, hindering their detection.
Hence, although NA therapies significantly decrease
viral load and improve patient survival in the short term
[44], they might select for HBV variants that are potentially oncogenic, negating the overall efficacy of NAs
in preventing hepatocarcinogenesis, the main long-term
goal of antiviral therapy in CHB. Future challenges in
the treatment of CHB involve the development of antiviral therapies that do not select for potentially oncogenic drug-resistant HBV. The development of NAs
that are sufficiently effective as inhibitors of HBV replication such that the risk of drug resistance is negligible
should be considered a primary goal, with a secondary
goal of hastening the development of drugs targeted to
other aspects of the HBV lifecycle.
Disclosure statement
The authors declare no competing interests.
References
1.
2.
3.
4.
5.
6.
Bosch FX, Ribes J, Borras J. Epidemiology of primary liver
cancer. Semin Liver Dis 1999; 19:271–285.
Parkin DM. Global cancer statistics in the year 2000.
Lancet Oncol 2001; 2:533–543.
Thomas MB, Zhu AX. Hepatocellular carcinoma: the need
for progress. J Clin Oncol 2005; 23:2892–2899.
Hino O, Kajino K, Umeda T, Arakawa Y. Understanding
the hypercarcinogenic state in chronic hepatitis: a clue
to the prevention of human hepatocellular carcinoma.
J Gastroenterol 2002; 37:883–887.
Chen CJ, Yang HI, Su J, et al. Risk of hepatocellular
carcinoma across a biological gradient of serum hepatitis B
virus DNA level. JAMA 2006; 295:65–73.
Chan HL, Hui AY, Wong ML, et al. Genotype C hepatitis
B virus infection is associated with an increased risk of
hepatocellular carcinoma. Gut 2004; 53:1494–1498.
141
27/3/09 11:53:54
N Warner & S Locarnini
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Ding X, Mizokami M, Yao G, et al. Hepatitis B virus
genotype distribution among chronic hepatitis B virus
carriers in Shanghai, China. Intervirology 2001; 44:43–47.
Orito E, Ichida T, Sakugawa H, et al. Geographic
distribution of hepatitis B virus (HBV) genotype in patients
with chronic HBV infection in Japan. Hepatology 2001;
34:590–594.
Carman WF, Jacyna MR, Hadziyannis S, et al. Mutation
preventing formation of hepatitis B e antigen in patients with
chronic hepatitis B infection. Lancet 1989; 334:588–591.
Akahane Y, Yamanaka T, Suzuki H, et al. Chronic active
hepatitis with hepatitis B virus DNA and antibody against
e antigen in the serum. Disturbed synthesis and secretion of
e antigen from hepatocytes due to a point mutation in the
precore region. Gastroenterology 1990; 99:1113–1119.
Nakamoto Y, Guidotti LG, Kuhlen CV, Fowler P, Chisari FV.
Immune pathogenesis of hepatocellular carcinoma. J Exp
Med 1998; 188:341–350.
Brechot C, Pourcel C, Louise A, Rain B, Tiollais P. Presence
of integrated hepatitis B virus DNA sequences in cellular
DNA of human hepatocellular carcinoma. Nature 1980;
286:533–535.
Paterlini-Brechot P, Saigo K, Murakami Y, et al. Hepatitis
B virus-related insertional mutagenesis occurs frequently
in human liver cancers and recurrently targets human
telomerase gene. Oncogene 2003; 22:3911–3916.
Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S,
Paterlini‑Brechot P. Persistent hepatitis B virus infection
in subjects without hepatitis B surface antigen: clinically
significant or purely “occult”? Hepatology 2001;
34:194–203.
Dandri M, Burda MR, Burkle A, et al. Increase in
de novo HBV DNA integrations in response to oxidative
DNA damage or inhibition of poly(ADP-ribosyl)ation.
Hepatology 2002; 35:217–223.
Bouchard MJ, Schneider RJ. The enigmatic X gene of
hepatitis B virus. J Virol 2004; 78:12725–12734.
Soussan P, Tuveri R, Nalpas B, et al. The expression of
hepatitis B spliced protein (HBSP) encoded by a spliced
hepatitis B virus RNA is associated with viral replication
and liver fibrosis. J Hepatol 2003; 38:343–348.
Caselmann WH, Meyer M, Kekule AS, Lauer U,
Hofschneider PH, Koshy R. A trans-activator function
is generated by integration of hepatitis B virus preS/S
sequences in human hepatocellular carcinoma DNA. Proc
Natl Acad Sci U S A 1990; 87:2970–2974.
Kekule AS, Lauer U, Meyer M, Caselmann WH,
Hofschneider PH, Koshy R. The preS2/S region of
integrated hepatitis B virus DNA encodes a transcriptional
transactivator. Nature 1990; 343:457–461.
Schluter V, Meyer M, Hofschneider PH, Koshy R,
Caselmann WH. Integrated hepatitis B virus X and 3′
truncated preS/S sequences derived from human hepatomas
encode functionally active transactivators. Oncogene 1994;
9:3335–3344.
Locarnini SA. Hepatitis B virus surface antigen and
polymerase gene variants: potential virological and clinical
significance. Hepatology 1998; 27:294–297.
Yeh CT, Chien RN, Chu CM, Liaw YF. Clearance of
the original hepatitis B virus YMDD-motif mutants
with emergence of distinct lamivudine-resistant mutants
during prolonged lamivudine therapy. Hepatology 2000;
31:1318–1326.
Chien RN, Yeh CT, Wang PN, et al. Acute leukaemia in
chronic hepatitis B patients with lamivudine therapy. Int J
Clin Pract 2004; 58:1088–1091.
Yatsuji H, Noguchi C, Hiraga N, et al. Emergence of a
novel lamivudine-resistant hepatitis B virus variant with a
substitution outside the YMDD motif. Antimicrob Agents
Chemother 2006; 50:3867–3874.
Warner N, Locarnini S. The antiviral drug selected hepatitis
B virus rtA181T/sW172* mutant has a dominant negative
secretion defect and alters the typical profile of viral
rebound. Hepatology 2008; 48:88–98.
26. Tatti KM, Korba BE, Stang HL, et al. Mutations in the
conserved woodchuck hepatitis virus polymerase FLLA
and YMDD regions conferring resistance to lamivudine.
Antiviral Res 2002; 55:141–150.
27. Lacombe K, Ollivet A, Gozlan J, et al. A novel hepatitis
B virus mutation with resistance to adefovir but not to
tenofovir in an HIV-hepatitis B virus-co-infected patient.
AIDS 2006; 20:2229–2231.
28. Rhee SS. Microbiology Review. US Food and Drug
Administration Centre For Drug Evaluation And Research
2006. (Updated 23 October 2006. Accessed 10 February
2009.) Available from http://www.accessdata.fda.gov/
scripts/cder/drugsatfda/index.cfm
29. Yoo BC, Kim JH, Chung YH, et al. Twenty-four-week
clevudine therapy showed potent and sustained antiviral
activity in HBeAg-positive chronic hepatitis B. Hepatology
2007; 45:1172–1178.
30. Durantel D, Brunelle MN, Gros E, et al. Resistance of
human hepatitis B virus to reverse transcriptase inhibitors:
from genotypic to phenotypic testing. J Clin Virol 2005;
34 Suppl 1:S34–S43.
31. Qi X, Xiong S, Yang H, Miller M, Delaney WEI. In vitro
susceptibility of adefovir-associated hepatitis B virus
polymerase mutations to other antiviral agents. Antivir Ther
2007; 12:355–362.
32. Hildt E, Hofschneider PH. The PreS2 activators of the
hepatitis B virus: activators of tumour promoter pathways.
Recent Results Cancer Res 1998; 154:315–329.
33. Bruss V, Lu X, Thomssen R, Gerlich WH. Post-translational
alterations in transmembrane topology of the hepatitis B
virus large envelope protein. EMBO J 1994; 13:2273–2279.
34. Ostapchuk P, Hearing P, Ganem D. A dramatic shift in the
transmembrane topology of a viral envelope glycoprotein
accompanies hepatitis B viral morphogenesis. EMBO J
1994; 13:1048–1057.
35. Prange R, Streeck RE. Novel transmembrane topology of
the hepatitis B virus envelope proteins. EMBO J 1995;
14:247–256.
36. Hildt E, Saher G, Bruss V, Hofschneider PH. The hepatitis
B virus large surface protein (LHBs) is a transcriptional
activator. Virology 1996; 225:235–239.
37. Hildt E, Urban S, Hofschneider PH. Characterization
of essential domains for the functionality of the MHBst
transcriptional activator and identification of a minimal
MHBst activator. Oncogene 1995; 11:2055–2066.
38. Lauer U, Weiss L, Hofschneider PH, Kekule AS. The
hepatitis B virus pre-S/S(t) transactivator is generated by 3′
truncations within a defined region of the S gene. J Virol
1992; 66:5284–5289.
39. Natoli G, Avantaggiati ML, Balsano C, et al.
Characterization of the hepatitis B virus preS/S region
encoded transcriptional transactivator. Virology 1992;
187:663–670.
40. Li J, Lee AS. Stress induction of GRP78/BiP and its role in
cancer. Curr Mol Med 2006; 6:45–54.
41. Shuda M, Kondoh N, Imazeki N, et al. Activation of the
ATF6, XBP1 and grp78 genes in human hepatocellular
carcinoma: a possible involvement of the ER stress pathway
in hepatocarcinogenesis. J Hepatol 2003; 38:605–614.
42. Lai MW, Yeh CT. The oncogenic potential of hepatitis B
virus rtA181T/surface truncation mutant. Antivir Ther
2008; 13:875–879.
43. Chisari FV, Klopchin K, Moriyama T, et al. Molecular
pathogenesis of hepatocellular carcinoma in hepatitis B
virus transgenic mice. Cell 1989; 59:1145–1156.
44. Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for patients
with chronic hepatitis B and advanced liver disease. N Engl
J Med 2004; 351:1521–1531.
Accepted for publication 23 February 2009
142
Locarnini.indd 142
© 2009 International Medical Press
27/3/09 11:53:55