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Zheng et al. Hepatoma Res 2018;4:17 I http://dx.doi.org/10.20517/2394-5079.2018.08 Page 3 of 8

have been found in association with various forms of acute and chronic liver disease, including fulminant
hepatitis (FH), fibrosing cholestatic hepatitis (FCH) and cirrhosis [42-44] . Both pre-S1 and pre-S2 mutants led
to defective secretion of mutant large surface antigens which then accumulated in ER, leading to ground
glass hepatocytes (GGH) formation in chronic HBV infection [45,46] . Under electron microscopy, GGHs were
characterized by an abundance of ER, and overloaded ER made the cytoplasm of GGH become “foggy”
or “glassy”. GGH was recognized as a risk factor for HCC, in particular, type II GGHs that harbor pre-S2
mutations accumulated on the ER of hepatocytes were considered biomarkers of HCC and were helpful
in predicting recurrence and survival in HBV-infected HCC patients . Previous studies reported several
[47]
tumorigenic mutants, including sL95*, sW182*, and sL216*, that did not promote ER stress but rather
activated cell proliferation and transformational abilities; the sW182* mutant was demonstrated to have
potent tumorigenic activity ; MHBst167 mutants have been shown to interact with proteins associated
[48]
with tumor progression/progression in vitro . A recent study reported that a pre-S2 start codon mutation
[49]
of HBV subgenotype B3 affected nuclear factor κB (NF-κB) expression and activation in Huh7 cell lines .
[50]
The frequency of pre-S mutations increased successively in the various stages of chronic hepatitis B (CHB)
infection. A meta-analysis showed that the frequency of pre-S mutants was approximately 10%, 20%, 35%,
and 50% in asymptomatic HBsAg carriers, CHB patients, patients with liver cirrhosis and HCC patients,
respectively . The prevalence of pre-S mutants varied among countries with endemic HBV genotypes with
[39]
a higher prevalence of genotypes B and C . Pre-S deletion mutants detected in serum were also reported
[51]
to increase the risk of post-operative recurrence of HCC . To efficiently detect pre-S deletion mutants in
[15]
serum, Su et al. successfully developed an oligonucleotide pre-S gene chip to detect pre-S deletion mutations
[7]
in sera as a predictive hallmark of HCC. Combined detection of pre-S mutations and other markers of
HBV replication such as HBeAg and viral loads may offer a reliable method for predicting HCC risks in
chronic HBV carriers. Among those mutants, the pre-S2 mutation in particular was found to be significantly
associated with the risk of HCC development [20,31,52-55] . Pre-S2 deletion mutations in sera can be detected in
nearly half of children with HCC , and in tissue samples, pre-S2 deletion mutations can be detected in
[56]
about 80% of pediatric HCC .
[57]

VARIOUS MECHANISMS OF PRE-S2 CONTRIBUTING TO HCC
Pre-S2 transcriptional activator proteins
During the infectious process, HBV DNA integrates into hepatocellular chromosomes and encodes
two transcriptional activators: the HBV X protein and the family of the pre-S2 activator proteins of
HBV, including the LHBs and C-terminally MHBst . The pre-S/S genomic region, when deleted in the
[23]
C-terminus portion (including the viral transmembrane hydrophobic region III of the S domain) produces
C-terminally truncated middle surface protein . HBs transactivators (LHBs and MHBst) function based by
[31]
cytoplasmic orientation of the pre-S2 domain . Unlike full-length MHBs, truncated MHBst is retained in
[58]
the endoplasmic reticulum and is not secreted. Therefore, the pre-S2 region of MHBst can interact with the
cytoplasmic protein in the cytoplasmic region, resulting in transcriptional activation [59,60] .

The discovery of transactivating functions exerted by LHBs and MHBst supports the notion that
transactivation of cellular gene expression could be relevant to hepatocarcinogenesis. Pre-S2 activators
LHBs and MHBst exerted tumor promoter-like functions by activating c-Raf-1/Erk2 signaling in transgenic
mice, leading to enhanced proliferative activity of hepatocytes , Liang et al. found that overexpressing
[58]
[61]
MHBst in hepatoma cells enhanced TNF-related apoptosis-inducing ligand (TRAI)-induced apoptosis. In
addition, a study showed that pre-S2, functioning as a transcriptional activator, promoted the development of
hepatocellular carcinoma by activating oncogenes, including c-myc, human telomerase reverse transcriptase
(hTERT) and forkhead box P3(Foxp3) [18,22,23,62] . Another recent study provided evidence that HBV protein
pre-S2 was responsible for reactivation of two oncogenes, alpha-fetoprotein (AFP) and glypican 3 (GPC3),
in HCC . Other studies reported that pre-S2 increased protein levels of transcriptional co-activators with
[63]