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Review Article
Alcoholic liver disease- Hepatocellular carcinoma transformation
1Department of Pathology, Harbor-University of California, Los Angeles, Medical Center, Torrance; 2University of California, Irvine, Department
of Medicine and Veteran Administration Medical Center, Long Beach, California, USA
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Submitted Apr 01, 2012. Accepted for publication Apr 16, 2012.
DOI: 10.3978/j.issn.2078-6891.2012.025 |
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Introduction
In the DDC feeding mouse model where liver cells
proliferate, Mallory-Denk bodies (MDBs) form and later,
after DDC withdrawal, hepatocellular carcinomas (HCCs)
develop (1). Similarly, patients who abuse alcohol develop
alcoholic liver disease (ALD), MDBs form (2) and later,
after alcohol abstinence, the patients develop HCCs (2).
Also MDBs form in many of the HCCs, both in the mouse
model and in ALD. Because of this, it has been suggested
that MDBs are a preneoplastic change formed in balloon
hepatocytes which transform into cancer cells (3-6). But
there may be other links to the preneoplastic process in
ALD-induced HCCs such as the role that macrophages
play in the TLR4 pathway response to LPS (4) or the
transformation of stem cells seen in both cirrhosis and the
associated HCC in ALD (7). In this review the role played
by the following is discussed: (I) cell cycle arrest, (II) TLR
signaling macrophages and stem cell transformation to form
cancer stem cells, (III) ballooned hepatocytes that form
Mallory-Denk bodies as progenitor pre-cancer cells in the
pathogenesis of the ALD/HCC transformation. |
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Cell Arrest
Alcohol-induced cell cycle arrest plays a role in the ALDHCC
transformation. It also plays a major role in alcoholic
hepatitis (AH) as determined in liver biopsies from AH
patients. Our hypothesis is based on the observation that
the expression of both PCNA and cyclin D1 is increased
in almost all of the hepatocytic nuclei in liver biopsies
taken from AH patients. The stain for Ki-67 was positive
in only a very few hepatocytes in the same biopsies. Both
p21 and p27 positive nuclei were very numerous in these
liver biopsies of patients with AH or NASH (7) (Figure 1).
This indicates that p21 and p27 inhibition of the cell cycle
at both the G1/S growth phase and the G2 phase (8,9) was
the reason. Because of the cell cycle arrest, regeneration
of liver cells is impeded and apoptosis, genome instability
and oncogenic effects result (9). P53 dependent and
independent mechanisms of p21 and p27 induction exist.
Stress from liver injury increases the expression of p53
and mitochondrial stress, both increasing p21 expression,
which leads to cell cycle arrest (10,11). It has been reported
that p21, but not Ki-67 expression, is increased in the liver
cell nuclei of patients with AH, but not in NASH (12,13).
This means that the cell cycle progression is arrested and
regeneration of the liver is prevented in AH. A similar
phenomenon occurs in decompensated cirrhosis where
oxidative stress induces p21 up regulation (14-16). Rats fed
ethanol chronically have up regulation of p21 and p27 in
liver cell nuclei and this explains how ethanol inhibited liver
regeneration after partial hepatectomy (15).
Figure 1 Liver biopsy from a patient with alcoholic hepatitis showing (A) an immunostain of numerous p27 positive nuclei (700×); (B)
MDBs also stain positive (arrows) (1,050×)
The increase in PCNA positive nuclei in AH has been
reported previously (12,13). The mechanisms by which
p21 regulates cell cycle progression are complex. Inhibition
of cyclin/CDK kinase activity by p21 induces cell cycle
arrest (17). P21 can directly inhibit PCNA-dependent
DNA replication (16,18). In response to mitogen, p21 is
induced during the G1 phase and plays a role in normal cell
cycle progression (19,20). Activated p53 binds DNA and
activates WAF-1/Cdip-1 encoding for p21, which binds
to the G1-S/CDK2 and S/CDK complexes (molecules
that are important for the G1/S transition) inhibiting their
activation. When p21 (WAF 1) is complexed with CDK2
the cell cannot continue to the next stage of the cell cycle.
PCNA positive nuclei are markedly increased in
hepatocytes in AH (7,21). PCNA is important for
both DNA synthesis and DNA repair (22,23). PCNA
becomes post-translationally modified by ubiquitin (24).
Polyubiquitin-mediated degradation of cell cycle proteins such as p21 is bound to PCNA by the E3 ligase CRL4 (Cdt2
ubiquitination and the 26s proteasome). This promotes
several DNA repair processes when p21 is degraded by
the proteasome. PCNA is then freed for the repair process
of the DNA (25). If the U3 ligase/proteasome digestion
mechanism fails to degrade p21, the cell cycle progression is
arrested. This may turn out to be the mechanism involved in
HCC formation in ALD, since chronic ethanol feeding leads
to inhibition of the 26S proteasome activity in the liver (26).
Chronic infection can also induce p21 levels in the liver
where the balance of the liver cell proliferation/growth arrest
leads to changes in the levels of Gadd 45B, PCNA, cyclin
D1, Gadd 45r, p53 and activated caspase 3 (27).
P21 and p27 are up regulated in cirrhosis and HCCs (28)
and up regulated by deacetylase inhibitors such as vorinostat
(SAHA) used in chemotherapy (29). The implication is that
histone acetyltransferases regulate p21 and p27 expression
such as HADC1 (30). HADC1 is over expressed in the
nuclei of hepatocytes forming Mallory Denk bodies in
alcoholic hepatitis (31). P27 has oncogenic effects (32).
Therefore, p21 and p27 may play important roles in the
pathogenesis of HCCs in ALD patients, probably because
of the DNA damage that develops during cell cycle arrest
caused by p21 and p27 over expression. |
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The role of macrophages TLR4 signaling
and stem cell transformation to form cancer
stem cells in the pathogenesis of ALD-HCC
transformation
Liver cell injury in AH is in part, due to macrophage
generated proinflammatory cytokines and sinusoidal
obstruction. The function of some macrophages (Kupffer
cells) causes injury to hepatocytes by way of innate
immune injury in response to endotoxin. This was found
in rodent models of early alcoholic liver disease and
possibly in AH in humans (33). However, these changes
are increased in response to acute alcohol ingestion. They
are responses that are reversible when ethanol ingestion
is stopped in experimental alcohol fed rodent models.
The question is: What has happened to the macrophages
in chronic alcohol ingestion in humans who have AH?
Plasticity and functional polarization are hallmarks of
different types of macrophages i.e. M1i, M2a, M2b, and
M2c which might be involved in AH.
This differential modulation of the macrophage
chemokine system integrates polarized macrophages
in pathways of resistance to or promotion of immuneregulation,
tissue repair and remodeling (34). The T cell
response to chemokines and cytokines differs when M1 and
M2 macrophages are compared. M1 has a Th1 response
to IFNα and LPS. M2a, b and c give a Th2 response of
immune-regulation, matrix deposition and remodeling. M2a
is a response to IL-4 and 13, M2b is a response to TLR/
IL-1R agonists, and M2c responds to 1L-10 and suppresses
immune responses to tissue remodeling (34,35). The type of
macrophages in the sinusoids determines the inflammatory
process in AH. We have done preliminary studies on the type
of macrophages that occupies the sinusoids in liver biopsies of
AH. We did IHC stains for CD-68 and CD163 to determine
the degree of macrophage infiltrate in the sinusoids in AH
(Figure 2A). We were surprised to find that the sinusoids were
diffusely filled with macrophages (obstructed) all of which
stained heavily for CD163 and not so heavily for CD68. The
CD163 (M2c) plays an immuno-regulation role (34). The
soluble form of CD163 can be measured in the serum to
assess the degree of macrophage activation since CD163 is an
activated macrophage marker (35). To assess the sinusoidal macrophages morphologically, we performed electron
microscopy (Figure 2B, C). The morphology was that of two
types of macrophages. The first type was smaller and filled
with phagocytic bodies (secondary lysosomes). The second
type was much larger and less common and contained
lysosomes and rough ER (Figure 2).
Figure 2 CD-163 positive macrophages fill all the sinusoids in a liver biopsy from an AH patient ×612 (A); EM of the same liver as A showing 2 types of macrophages in the sinusoids, phagocytic on the left (B arrow) and (C) secretory on the right ×1973
The marked increase in the activity of CD163 positive
macrophages involves a cascade of intracellular signals
which lead to the secretion of IL6 and CSF1. CD163
positive macrophages are positive for the CD14 and
CD16 subunits. CD-163 expression is down regulated by
proinflammatory mediators like LPS, IFNg and TNFα.
IL-6 and IL-10 strongly up regulate CD-163 (36). Thus, up
regulation of CD-163 as noted in the livers of AH implies
that the positive staining macrophages are functionally antiinflammatory
(36).
The link between the activated macrophage in
the sinusoids in the liver of patients with AH and the
development of HCC is through chronic activation of
TLR4 in response to a “leaky gut” increase in LPS into the
portal vascular system (4). The link to HCC pathogenesis
was first developed using a model of alcohol-fed NSSA Tg
mice with a diet supplement of LPS. The combination,
over time led to synergistic liver damage and liver tumor
formation due to alcohol-induced endotoxemia (37). In this
mouse model, Nanog, a stem cell/progenitor cell marker,
was up regulated by TLR4 activation. CD133/Nanog
positive cells were found in the mouse liver tumors that
formed (38). These observations supported the concept that
the synergism between alcohol abuse and HCV leads to
liver tumorigenesis through TLR signaling up regulation of
the Nanog expressing stem cells, causing them to transform
into cancer stem cells in HCC formation (TISCs). Nanog
is up regulated by TLR4 activation. CD133/Nanog positive
cells are consequently found in the HCCs of affected Tg
mice (39) (Figure 3, 4, 5). CD133, a marker for cancer stem
cells, is regulated epigenetically by TGFβ (40). In fact there
is compelling evidence that TGFβ signals the expansion of
progenitor liver stem cells, which lead to HCC formation
and stimulate the progression of the HCCs (41-43). It’s
a paradox that the cytostatic, tumor suppressor, TGFβ
becomes a tumor promoter, which stimulates the transition
from stem cells to progenitor cells to cancer stem cells
(39,42,43). Yap1 and Igf2bp3 that are Nanog-dependent genes inhibit TGFβ signaling in TISCs (39). Yap1 and
Igf2bp positive cells are present in the livers of ALD and
associated HCCs (Figures 3, 4, 5). Taken together, TLR4
expression may be a universal proto-oncogene responsible
for the genesis of TLR4-Nanog dependent TISCs (39).
Figure 3 Immunostain (IHC) of liver showing an HCC. A. Shows a positive stained cell for YAP1 (green); B. Same cell stained positive for 1GF2bdr3 (red); and C. tricolor combining A and B (×654)
Figure 4 Liver from a patient with alcoholic liver disease showing alcoholic hepatitis and cirrhosis, immunostained for Oct 3-4 (A green), ubiquitin (B red) and (C tricolor) combining A and B. Note the co localization of Oct 3-4 and ubiquitin in the nucleus (×654)
Figure 5 Liver from a patient with alcoholic liver disease showing cirrhosis and HCC. The photos are of a fibrous septa in the cirrhosis. A. Shows numerous Nanog (green) stem cells; B. One cell staining positive for SOX2 (red) (arrow); C. is tricolor combining A and B (×436)
The role of chronic inflammation of the liver in the
development of liver cancer has long been suspected (44).
Transcription factors such as TLR4, JNK, NFκB, STAT3,
IL-6, IL-1α and EGF receptor are involved in inflammation
associated HCC development (44,45). TLR4 and TLR2
signaling activated by inflammation up regulate NFκB
and JNK cytokine expression. In experimental alcoholic
liver disease TLR4 signaling in mice fed ethanol is
increased through a MyD88 independent pathway (46).
However, in rats fed ethanol by intragastric tube, where
high blood alcohol levels are achieved, TLR4 expression
increased as well as MyD88 protein levels indicating
that the MyD88 signaling pathway was activated (47).
When S-adenosylmethionine was fed with ethanol the up
regulation of TLR signaling was prevented indicating that
the changes in TLR expression were the result of epigenetic
mechanisms. Chronic alcohol feeding also up regulated
CD34, FOS, IRF-1, Jun, TLR1, 2, 3, 6 and 7 and Traf6.
IL-6, IL10 and IFNγ were also up regulated. Both IL-6 and
IL-10 are cytokines that are up regulated by Kupffer cells
(M2) in ALD (48). TL-6 activates STAT3. STAT3 acts as
a proinflammatory signal (34). The activation of the TLR
signaling pathway leads to the up activation of NFκB which
stimulates cytokine expression in chronic liver diseases,
including ALD and this triggers, over time, the formation of
HCC (49). |
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The role of ballooned hepatocytes that form Mallory-Denk bodies (MDB) as progenitor precancer cells
Balloon cell differentiation (BCD) with (MDB) occurs in
chronic hepatitis and cirrhosis due to diverse causes such
as alcoholic hepatitis (5). Their occurrence associated with
HCC is well established (3). In an experimental mouse
model where BCD/MDBs develop in large numbers similar
to alcoholic hepatitis, liver tumors develop many months
after the withdrawal of the carcinogen DCC. This is similar
to the development of the HCCs that develop years after
alcohol abstinence in ALD patients (1). In the mouse
model BCD/MDBs are associated with the development
of preneoplastic changes (48). MDB forming hepatocytes
express the same preneoplastic hepatocyte phenotype in both mice (50) and humans (4). The basic morphology of the
MDB forming BCD is the same in the human liver and the
liver in the mouse model of MDB formation (7) (Figure 6).
Figure 6 Liver biopsy stained for H&E (A) ×700 and CAM5.2 (B) ×1,050 for keratin 8 and 18. Balloon cells that formed in alcoholic hepatitis are shown where they have formed MDBs. Note that the balloon cells are devoid of keratin except for the MDBs which stain
intensely. (C) ×1,875 and (D) ×7,500 electron micrographs of an hepatocyte balloon degeneration cell which had formed an MDB (arrow)
The first change that occurs when the balloon cell
degeneration occurs is the disappearance of the keratin
18/8 cytoskeleton and rounding up of the cell. The balloon
cell then differs from the normal polyhedral-shaped cell
of neighboring hepatocytes (5). Electron microscopy of
balloon cells (Figure 6B, C) shows micro-vesicular fat,
reduced numbers of mitochondria, reduced glycogen and
loss of the normal organelle arrangement due to the loss of
the keratin filament structure. The most dramatic change
is in the nucleus, which is large, with euchromatin and
vesicular with a prominent nucleolus. When the balloon cell
nucleus was immunostained for H3K27me3 the fluorescent
intensity was low compared to the surrounding normal
liver cell nuclei as shown by morphometric comparison (7).
Similarly, pEZH2 was increased in the balloon cells that
had formed (7). PEZH2 was increased in the liver when
measured by Western blot. These observations supported
the working hypothesis that the balloon cell change is
due to epigenetic alteration of gene expression where the
nuclear DNA methylation was reduced and gene expression
was up regulated globally (1).
The working hypothesis is that balloon cells are
phenotypically changed due to a failure of the H3K27me3/
EZH2 to repress gene expression (51). The hallmark of
the balloon cell/MDB forming cell is the loss of keratin
intermediate filaments which normally span from the
plasma membrane to the nuclear membrane (52). Keratin
protein regulates protein synthesis and epithelial cell
growth in keratinocytes (53). When MDBs form in the
balloon cells in AH, the bile canaliculi disappear and
organelles become randomly arranged. In an electron
microscopic autoradiography study of synthesis of keratin
filament protein using radio labeled S35 methionine as a marker, we showed that the nascent keratin proteins went
to MDBs preferentially compared to the normally formed
intermediate filaments (54).
Most relevant to the role of the BCD/MDB cells linking
them to the formation of HCCs is the fact that HCCs
often form MDBs in large numbers in humans and in the
mouse model (7). In the mouse model the BCD/MDB cells
(FAT10+cells) have a growth advantage compared to the
normal neighboring cells in response to liver cell injury (1).
They show an increased expression of α-fetoprotein, have
a decreased expression of DNA repair enzyme glycosylase
OGG1, have decreased levels of DNA 5’methyl cytosine,
decreased nuclear levels of DNA methyltransferase enzyme
DNMT36 and have a large increase in the expression of
the mouse form of FAT10 (UBD). Fat10 is over expressed
in human HCCs (1,55,56). The markers for the MDB
associated preneoplastic phenotype, which indicate that the
BCD/MDB cells are preneoplastic; include A2 macroglobin,
gamma glutamyl transpeptidase, GSTmu2, fatty acid synthase,
glypican-3, p38 and AKT, as well as AFP (1). The BCD cell as
well as the MDBs stain positive with an antibody to SOX2
(Figure 7) a marker for hepatic stem cells, suggesting that
these cells are stem cell/progenitor cells which have the
potential to transform into cancer stem cells, which drive
the formation of HCCs (57).
Figure 7 Liver from a patient with alcoholic hepatitis
immunostained for SOX2 (red). Note that numerous MDBs stained positive for SOX2 (arrows) (2,224×)
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Acknowledgements
The authors thank Adriana Flores for typing the
manuscript. Supported by a grant from NIH/NIAAA 6772
and the Morphology Core from grant P50-011999.
Disclosure: The authors declare no conflict of interest.
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References
Cite this article as: French SW, Lee J, Zhong J, Morgan
TR, Buslon V, Lungo W, French BA. Alcoholic liver disease-
Hepatocellular carcinoma transformation. J Gastrointest Oncol
2012;3(3):174-181. DOI: 10.3978/j.issn.2078-6891.2012.025
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