The role of macrophages TLR4 signaling
and stem cell transformation to form cancer
stem cells in the pathogenesis of ALD-HCC
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
). 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
). 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
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
). 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
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
). 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
) (Figure 3
). 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
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
). 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
). 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
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
) 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
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
). 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
) 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×)
- Oliva J, Bardag-Gorce F, French BA, et al. Fat10 is
an epigenetic marker for liver preneoplasia in a drugprimed
mouse model of tumorigenesis. Exp Mol Pathol
- Chedid A, Mendenhall CL, Gartside P, et al. Prognostic
factors in alcoholic liver disease. VA Cooperative Study
Group. Am J Gastroenterol 1991;86:210-6.
- Nakanuma Y, Ohta G. Is mallory body formation a
preneoplastic change? A study of 181 cases of liver bearing
hepatocellular carcinoma and 82 cases of cirrhosis. Cancer
- French SW, Oliva J, French BA, et al. Alcohol, nutrition
and liver cancer: role of Toll-like receptor signaling. World
J Gastroenterol 2010;16:1344-8.
- French SW, Nash J, Shitabata P, et al. Pathology of
alcoholic liver disease. VA Cooperative Study Group 119.
Semin Liver Dis 1993;13:154-69.
- French SW, Eidus LB, Freeman J. Nonalcoholic fatty
hepatitis: An important clinical condition. Canadian J
- French BA, Oliva J, Bardag-Gorce F, et al. Mallory-Denk
bodies form when EZH2/H3K27me3 fails to methylate
DNA in the nuclei of human and mice liver cells. Exp Mol
- Abbas T, Dutta A. p21 in cancer: intricate networks and
multiple activities. Nat Rev Cancer 2009;9:400-14.
- Cazzalini O, Scovassi AI, Savio M, et al. Multiple roles
of the cell cycle inhibitor p21(CDKN1A) in the DNA
damage response. Mutat Res 2010;704:12-20.
- Li CH, Tzeng SL, Cheng YW, et al. Chloramphenicolinduced
mitochondrial stress increases p21 expression and
prevents cell apoptosis through a p21-dependent pathway.
J Biol Chem 2005;280:26193-9.
- Li Y, Jenkins CW, Nichols MA, et al. Cell cycle expression
and p53 regulation of the cyclin-dependent kinase
inhibitor p21. Oncogene 1994;9:2261-8.
- Fang JW, Bird GL, Nakamura T, et al. Hepatocyte
proliferation as an indicator of outcome in acute alcoholic
hepatitis. Lancet 1994;343:820-3.
- Crary GS, Albrecht JH. Expression of cyclin-dependent
kinase inhibitor p21 in human liver. Hepatology
- Lunz JG 3rd, Tsuji H, Nozaki I, et al. An inhibitor of
cyclin-dependent kinase, stress-induced p21Waf-1/Cip-1,
mediates hepatocyte mito-inhibition during the evolution
of cirrhosis. Hepatology 2005;41:1262-71.
- Koteish A, Yang S, Lin H, et al. Ethanol induces
redox-sensitive cell-cycle inhibitors and inhibits liver
regeneration after partial hepatectomy. Alcohol Clin Exp Res 2002;26:1710-8.
- Luo Y, Hurwitz J, Massagué J. Cell-cycle inhibition
by independent CDK and PCNA binding domains in
p21Cip1. Nature 1995;375:159-61.
- Morgan DO. Principles of CDK regulation. Nature
- Waga S, Hannon GJ, Beach D, et al. The p21 inhibitor
of cyclin-dependent kinases controls DNA replication by
interaction with PCNA. Nature 1994;369:574-8.
- Macleod KF, Sherry N, Hannon G, et al. p53-
dependent and independent expression of p21 during cell
growth, differentiation, and DNA damage. Genes Dev
- Gray-Bablin J, Rao S, Keyomarsi K. Lovastatin induction
of cyclin-dependent kinase inhibitors in human breast cells
occurs in a cell cycle-independent fashion. Cancer Res
- French SW. Epigenetic effects of alcohol-induced
hepatocellular carcinoma. Alcohol Res Health 2012 (Epub
ahead of print).
- Essers J, Theil AF, Baldeyron C, et al. Nuclear dynamics
of PCNA in DNA replication and repair. Mol Cell Biol
- Shivji KK, Kenny MK, Wood RD. Proliferating cell
nuclear antigen is required for DNA excision repair. Cell
- Hoege C, Pfander B, Moldovan GL, et al. RAD6-
dependent DNA repair is linked to modification of PCNA
by ubiquitin and SUMO. Nature 2002;419:135-41.
- Abbas T, Sivaprasad U, Terai K, et al. PCNA-dependent
regulation of p21 ubiquitylation and degradation via
the CRL4Cdt2 ubiquitin ligase complex. Genes Dev
- Fataccioli V, Andraud E, Gentil M, et al. Effects of chronic
ethanol administration on rat liver proteasome activities:
relationship with oxidative stress. Hepatology 1999;29:14-20.
- Zhang C, Wang J, Lü G, et al. Hepatocyte proliferation/
growth arrest balance in the liver of mice during E.
multilocularis infection: a coordinated 3-stage course.
PLoS One 2012;7:e30127.
- Mitselou A, Karapiperides D, Nesseris I, et al. Altered
expression of cell cycle and apoptotic proteins in human
liver pathologies. Anticancer Res 2010;30:4493-501.
- Dokmanovic M, Clarke C, Marke PA. Histone deacetylase
inhibitors: Overview and perspectives. Mol Cancer Res
- Gui CY, Ngo L, Xu WS, et al. Histone deacetylase
(HDAC) inhibitor activation of p21WAF1 involves
changes in promoter-associated proteins, including
HDAC1. Proc Natl Acad Sci U S A 2004;101:1241-6.
- French SW, Bardag-Gorce F, Li J, et al. Mallory-Denk body
pathogenesis revisited. World J Hepatol 2010;2:295-301.
- Serres MP, Zlotek-Zlotkiewicz E, Concha C, et al.
Cytoplasmic p27 is oncogenic and cooperates with Ras
both in vivo and in vitro. Oncogene 2011;30:2846-58.
- Miller AM, Horiguchi N, Jeong WI, et al. Molecular
mechanisms of alcoholic liver disease: innate immunity and
cytokines. Alcohol Clin Exp Res 2011;35:787-93.
- Mellins ED, Macaubas C, Grom AA. Pathogenesis of
systemic juvenile idiopathic arthritis: some answers, more
questions. Nat Rev Rheumatol 2011;7:416-26.
- Bleesing J, Prada A, Siegel DM, et al. The diagnostic
significance of soluble CD163 and soluble interleukin-2
receptor alpha-chain in macrophage activation syndrome
and untreated new-onset systemic juvenile idiopathic
arthritis. Arthritis Rheum 2007;56:965-71.
- Buechler C, Ritter M, Orsó E, et al. Regulation of
scavenger receptor CD163 expression in human monocytes
and macrophages by pro- and antiinflammatory stimuli. J
Leukoc Biol 2000;67:97-103.
- Machida K, Chen CL, Liu JC, et al. Cancer stem cells
generated by alcohol, diabetes, and hepatitis C virus. J
Gastroenterol Hepatol 2012;27:19-22.
- Morgan TR, Mandayam S, Jamal MM. Alcohol
and hepatocellular carcinoma. Gastroenterology
- Machida K, Tsukamoto H, Mkrtchyan H, et al. Toll-like
receptor 4 mediates synergism between alcohol and HCV
in hepatic oncogenesis involving stem cell marker Nanog.
Proc Natl Acad Sci U S A 2009;106:1548-53.
- You H, Ding W, Rountree CB. Epigenetic regulation of
cancer stem cell marker CD133 by transforming growth
factor-beta. Hepatology 2010;51:1635-44.
- Thenappan A, Li Y, Kitisin K, et al. Role of transforming
growth factor beta signaling and expansion of progenitor
cells in regenerating liver. Hepatology 2010;51:1373-82.
- Dooley S, Weng H, Mertens PR. Hypotheses on the
role of transforming growth factor-beta in the onset
and progression of hepatocellular carcinoma. Dig Dis
- Ikegami T. Transforming growth factor-beta signaling and
liver cancer stem cell. Hepatol Res 2009;39:847-9.
- Berasain C, Castillo J, Perugorria MJ, et al. Inflammation
and liver cancer: new molecular links. Ann N Y Acad Sci
- Maeda S. NF-κB, JNK and TLR signaling pathways
in hepatocarcinogenesis. Gastroenterol Res Pract
- Szabo G, Mandrekar P, Petrasek J, et al. The unfolding
web of innate immune dysregulation in alcoholic liver
injury. Alcohol Clin Exp Res 2011;35:782-6.
- Oliva J, Bardag-Gorce F, Li J, et al. S-adenosylmethionine prevents the up regulation of Toll-like receptor (TLR)
signaling caused by chronic ethanol feeding in rats. Exp
Mol Pathol 2011;90:239-43.
- Tazawa J, Irie T, French SW. Mallory body formation
runs parallel to gamma-glutamyl transferase induction
in hepatocytes of griseofulvin-fed mice. Hepatology
- Luedde T, Schwabe RF. NF-κB in the liver--linking
injury, fibrosis and hepatocellular carcinoma. Nat Rev
Gastroenterol Hepatol 2011;8:108-18.
- Nan L, Bardag-Gorce F, Wu Y, et al. Mallory body
forming cells express the preneoplastic hepatocyte
phenotype. Exp Mol Pathol 2006;80:109-18.
- Margueron R, Li G, Sarma K, et al. Ezh1 and Ezh2
maintain repressive chromatin through different
mechanisms. Mol Cell 2008;32:503-18.
- Katsuma Y, Swierenga SH, Marceau N, et al. Connections
of intermediate filaments with the nuclear lamina and the
cell periphery. Biol Cell 1987;59:193-204.
- Kim S, Wong P, Coulombe PA. A keratin cytoskeletal
protein regulates protein synthesis and epithelial cell
growth. Nature 2006;441:362-5.
- Kachi K, Cadrin M, French SW. Synthesis of Mallory
body, intermediate filament, and microfilament proteins
in liver cell primary cultures. An electron microscopic
autoradiography assay. Lab Invest 1993;68:71-81.
- Canaan A, Yu X, Booth CJ, et al. FAT10/diubiquitinlike
protein-deficient mice exhibit minimal phenotypic
differences. Mol Cell Biol 2006;26:5180-9.
- Lee CG, Ren J, Cheong IS, et al. Expression of the FAT10
gene is highly upregulated in hepatocellular carcinoma
and other gastrointestinal and gynecological cancers.
- Ghodsizadeh A, Taei A, Totonchi M, et al. Generation of
liver disease-specific induced pluripotent stem cells along
with efficient differentiation to functional hepatocyte-like
cells. Stem Cell Rev 2010;6:622-32.
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