172 Anti-Cancer Agents in Medicinal Chemistry, 2010, 10, 172-175
1871-5206/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Liver Cancer Stem Cells as an Important Target in Liver Cancer Therapies
State Key Laboratory for Oncogene and Related Gene, Shanghai Cancer Institute, Shanghai Jiaotong University， 800 Dong Chuan
Road，Wenxuan Medicine Bldg Rm442. Shanghai, 200240. PR. China
Abstract: Hepatic cancer is one of most common cause of cancer-related death. Hepato-epithelial cancers are believed to originate from
the malignant transformation of liver-resident stem/progenitor cells. Liver cancer stem cells have been characterized recently and the
phenotype of liver cancer stem cells has been defined as CD133+ CD44+ cancer cells. Recently, it has been also demonstrated about the
relevance of targeting liver cancer stem cells, due to cancer stem cells are related to cancer metastasis. These advances no doubt to bring
the new strategy in liver cancer treatment and control in this disease. This review describes the current status and progress about cancer
stem cell research in liver and discuss of the implications of these studies in new liver cancer treatment strategies.
Keywords: CD133, CD44, CD90, IL-6, TGF-?.
Hepatocellular carcinoma (HCC) is a primary malignancy can-
cer of the liver. HCC is the fifth most common cancer in the world
and is especially prevalent in Southeast Asia [1, 2]. Recently, there
are emerging evidences to show the existence of liver cancer stem
cells (CSCs) or cancer stem cell-like cells within liver cancer [3-7].
Recent advances showed that the malignant transformation of
liver stem cells into liver CSCs, might be involved in the etiology
and/or pathology of liver cancer [3-7]. In this review, we focus on
the current status in the studies of cancer stem cells in the liver and
their therapeutic implication to develop novel effective therapies in
NORMAL STEM CELLS IN LIVER
In early studies, it was believed that liver lack stem cells or
transit-amplifying cells. However, recent investigations suggested
that normal hepatic stem cells playing a major role in liver devel-
opment and regeneration. Mouse primitive hepatic progenitor cells
have been isolated from mouse liver. These cells express both
CD49f and CD29 (?6 and ?1 integrin subunits), but do not express
hematopoietic cell antigens such as CD45, TER119, and c-Kit.
When these cells are transplanted into the spleen, they migrated to
the recipient liver and differentiated into liver parenchymal cells.
These mouse hepatic progenitor cells can be enriched by fluores-
cence activated cell sorting (FACS) and can form colonies in in
vitro culture . These cells exhibit multilineage differentiation
potential and self-renewing capability. For instance, they differenti-
ate into hepatocytes and cholangiocytes with reconstitution of hepa-
tocyte and bile duct structures, when they are transplanted into re-
cipient animals. Using transplantation assay, it has been suggested
the existence of a bipotent epithelial liver stem cell in nonhuman
primates . More recently, it has been demonstrated that human
hepatic stem cells are located in ductal plates in fetal and neonatal
livers, in pediatric and adult livers with the frequency of liver stem
cells remaining relatively constant throughout life. The phenotype
of these hepatic stem cells is CD133+ ESA+ .
‘Oval cells’ have also been suggested as stem cell in the liver.
These cells appear in the per portal region and then infiltrate along
the bile canaliculi . Oval cell activation has been extensively
studied in the rodent system, where a carcinogen was used to inhibit
hepatocyte replication in response to regenerative stimuli such as a
*Address correspondence to this author at the State Key Laboratory for
Oncogene and Related Gene, Shanghai Cancer Institute, Shanghai Jiaotong
University, 800 Dong Chuan Road, Wenxuan Medicine Bldg. Rm442.,
Shanghai, 200240. PR. China; Tel: 86-21-34206284; Fax: 86-21-3420628;
partial hepatectomy or carbon tetrachloride administration [12, 13].
In severe hepatocellular necrosis, chronic viral hepatitis, alcoholic
and nonalcoholic fatty liver disease, where mature liver cells are
unable to regenerate owing to inhibition or replicative exhaustion,
activation of the potential stem cell compartment leads to formation
of reactive ductules with a high expression of oval cell markers.
Many markers have been used to identify oval cells, including g-
glutamyl transpeptidase, glutathione-S-transferase, a-foetoprotein
(AFP), neural cell adhesion molecule 1 and chromogranin A. There
is also speculation that oval cells are derived from bone marrow
precursor cells because they express some of the antigens of haema-
topoietic cells such as c-kit, flt-3, Thy-1 and CD34.
LIVER CANCER STEM CELLS
It is a well accepted concept currently that in hematopoietic
system, transformed hematopoietic stem/progenitor cells with an
enhanced or acquired self-renewal capability function as leukemic
stem cells . However, it remains unclear whether disruption of
normal stem cell function directly contributes to cancer initiation in
solid tumor, such as liver cancer. To address this question, a col-
laborative research group in Japan did functional analyses of poly-
comb-group protein Bmi1 and Wnt/?-catenin, the molecules that
are responsible for the self-renewal capability of many types of
stem cells [15-18]. In liver stem/progenitor cells, these Bmi1 and
Wnt gene were conducted in c-Kit-/CD29+/CD49f+/low/CD45-/Ter-
119- hepatic stem/progenitor cells using retrovirus- or lentivirus-
mediated gene transfer. They found that forced expression of Bmi1
(B lymphoma Mo-MLV insertion region 1, Bmi1) and constitu-
tively active ?-catenin mutant similarly promoted the self-renewal
of hepatic stem/progenitor cells. The transplantation of Bmi1 or ?-
catenin-transduced cells clonally expanded from single hepatic
stem/progenitor cells produced tumors . These observations indi-
cate that the enhanced self-renewal level of normal hepatic
stem/progenitor cells serves as an early event in hepatocarcinogene-
Several surface antigens, such as CD90, CD44, CD133 and
ESA (epithelial specific antigen, ESA) have been reported to be
associated as a marker of liver CSCs (Table 1).
CD90+ LIVER CANCER STEM CELLS
Recent investigations suggest that CD90 might also be a poten-
tial marker for liver CSCs . CD45- CD90+ cells were detected in
all the tumor specimens, but not in the normal, cirrhotic, and paral-
lel nontumorous livers. Moreover, CD45- CD90+ cells were detect-
able in 90% of blood samples from liver cancer patients, but none
in normal subjects or patients with cirrhosis. CD90+ cells sorted
from cell lines and CD45- CD90+ cells from the tumor tissues and
Cancer Stem Cells in Liver Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 2 173
blood samples of liver cancer patients generated tumor nodules in
immunodeficient mice .
Table 1. Cancer Stem Cell Marker in Liver Cancer
CD90 Yang et al. 
CD44 Yang et al. 
CD133 Ma et al. 
ESA Schmelzer et al. 
ESA, epithelial specific antigen.
In addition, treatment of CD90+ CSCs with anti-human CD44
antibody caused cell apoptosis in a dose-dependent manner. Identi-
fication of CD45- CD90+ CSCs in both tumor tissues and circula-
tion suggests that CD45- CD90+ could be used as a marker for hu-
man liver cancer and as a target for the diagnosis and therapy of this
CD133+ LIVER CANCER STEM CELLS
CD133 antigen-expressing cells are also found to represent only
a minority of the tumor cell population in human primary HCC
specimens [3, 6]. CD133+ cells isolated from human hepatocellular
carcinoma cell lines possess cancer stem/progenitor cell-like prop-
erties. The CD133 antigen was found to be expressed on the surface
of Huh-7 liver cancer cells line. These CD133+ cells isolated from
Huh-7 liver cancer cell line exhibited a higher in vitro proliferative
potential and lower mRNA expressions of mature hepatocyte mark-
ers (glutamine synthetase and cytochrome P450), than CD133-
population of Huh-7 cells . In vivo study revealed that CD133+
liver cancer cells formed tumors. In contrast, CD133- liver cancer
had a very limited effect in this regard, when either CD133+ or
CD133- cells were subcutaneously injected into severe combined
immune deficency (SCID) mice [3, 6]. This phenomenon regarding
CD133+ cell population in human liver cancer cell line has been
further confirmed by a research group in University of Hong Kong
. They demonstrated the identification and isolation of a popula-
tion of CSCs expressing a CD133 surface phenotype from human
liver cell lines. These CD133+ liver cancer cells exhibited increased
potential for self-renewal, an increased colony-forming efficiency,
higher proliferative potential in vitro, and greater ability to form
tumor in vivo [3,4 6].
CD133+ HCC CSC population might be heterogeneous, consist-
ing of perhaps subsets of cells with differing tumorigenic potential.
Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase
(ALDH), which are most abundant in the liver, are the main en-
zymes involved in ethanol and acetaldehyde metabolism. Analysis
of the expression of several different ALDH isoforms and ALDH
enzymatic activity in liver cell lines suggested that ALDH to be
positively correlated with CD133 expression . The purified
subpopulations found CD133+/ALDH+ cells showed to be signifi-
cantly more tumorigenic than their CD133-/ALDH+ or CD133-
/ALDH- counterparts, both in vitro and in vivo . These observa-
tions suggest the existence of a hierarchical organization in HCC
bearing tumorigenic potential in various subpopulation of CD133+
liver cancer cells . A similar phenomenon was further con-
firmed by a research group in Shanghai Cancer Institute , where
they revealed that CD133+/CD44+ HCC cells were more tumori-
genic than those of CD133+/CD44- cells in vivo. A recent study
suggested that CSC phenotype could also be precisely defined by
co-expression of CD133 and CD44 cell surface markers.
CD133+/CD44+ HCC cells showed stem cell properties, including
extensive proliferation, self-renewal and differentiation into the
bulk of cancer cells. In vivo xenograft experiments revealed that,
actually, the highly tumorigenic capacity of CD133+ cells as previ-
ously described was primarily attributed to CD133+/CD44+ cell
subpopulation, instead of their CD133+/CD44- counterparts .
Those findings suggest that CD133+/CD44+ cells might represent
true cancer stem/progenitor cells in HCC .
TREATMENT RESISTANCE AND NOVEL TARGETED
It has been shown that the cells double-positive for CD133 and
CD44 exhibited preferential expression of some stem cell-
associated genes and were more resistant to chemotherapeutic
agents due to the up-regulation of ATP-binding cassette (ABC)
super-family transporters, including ABCB1, ABCC1 and ABCG2.
ABCB1 gene is also known as MDR1 gene, which plays significant
role in multi-drug resistance. The MDR1 gene encodes a 190kDa
glycoprotein (P-gp) which is involved in multidrug resistance.
ABCG2 gene is also related to drug resistance in tumor cells. So
over-expression of these genes in CD133+/CD44+ liver CSCs obvi-
ously increase drug resistance of liver cancer stem cells, conse-
quently render liver cancer therapy refractory.
TARGETING LIVER CSCS
To examine the sensitivity of CD133+ liver CSCs to che-
motherapeutic agents (doxorubincin and 5-fluorouracil) and the
possible mechanism pathways by which resistance may be regu-
lated, Ma et al.  purified the CD133+ CSCs from HCC cell line
and xenograft. They demonstrated that the treatment of CD133+
HCC cells with an AKT1 inhibitor, specific to the Akt/PKB path-
way, significantly reduced the expression of the survival proteins
that was normally expressed endogenously .
These investigations suggest that CD133+ HCC cells contribute
to chemoresistance through the activation of Akt/PKB and Bcl-2
cell survival response. Consequently, targeting of Akt/PKB signal-
ing pathway in CD133+ HCC CSCs might be a novel therapeutic
strategy for this disease. In addition, recent studied also revealed
that blocking of CD44 signaling by anti-CD44 antibody might be a
potential strategy to eradiate liver CSCs and consequently cure
those patients (Fig. 1) . Altogether, liver CSCs might be an
important target in liver cancer treatment.
TARGETING OF CYTOKINE PATHWAYS
Aberrant transforming growth factor-? (TGF-?) pathway? has
been suggested to contribute to liver stem cells committing to liver
cancer stem/progenitor cells . Currently, the functional interac-
tions between specific signaling pathways in solid organ "cancer
stem cells," such as those of the liver, still remain elusive. Examina-
tion of human HCC reveals cells that label with stem cell markers
have unexpectedly lost TGF-?? receptor type II (TBRII) and em-
bryonic liver fedrin (ELF) . Elf(+/-) mice spontaneously de-
velop HCC. The expression analysis of these tumors highlighted the
marked activation of the genes involved in the IL-6 signaling path-
way, including IL-6 and Stat3, suggesting that HCC could arise
from an IL-6-driven transformed stem cell with inactivated TGF-?
signaling. These studies suggested a novel functional link between
IL-6, a major stem cell signaling pathway, and the TGF-??signal-
ing pathway in the modulation of mammalian HCC . Me-
thionine adenosyltransferase (MAT) is an essential enzyme required
for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls
during chronic liver injury, and mice lacking Mat1a develop spon-
taneous hepatocellular carcinoma by 18 months. CD133+/CD45-
oval cells isolated from 16-month-old Mat1a (-/-) mice represent a
liver CSC population. To test the response of tumorigenic liver
stem cells to TGF-?, CD133+/CD45- oval cells were isolated from
premalignant 16-month-old Mat1a(-/-) mice by flow cytometry and
expanded as five clone lines derived from a single cell . Al-
though TGF-? inhibited cell growth equally in CD133- and CD133+
cells from each clone line, the CD133+ population demonstrated
significant resistance to TGF-?-induced apoptosis compared with
174 Anti-Cancer Agents in Medicinal Chemistry, 2010, Vol. 10, No. 2 Gang-Ming Zou
CD133- cells . One mechanism of resistance to TGF-?-induced
apoptosis in CD133+ CSCs is an activated mitogen-activated pro-
tein kinase (MAPK)/extracellular signal-regulated kinase (erk)
pathway . A recent investigation revealed normal hepatic stem
cells commit to malignant transformation due to aberrant TGF-?
and activated IL-6 signaling . Therefore, inhibition of IL-6
signaling may be a potential therapeutic strategy in liver cancer
Hypoxia is a general phenomenon in cancer tissue. Hypoxia
may mediate chemorsistance in liver cancer cells and liver CSCs
(alternatively named as tumorigenic hepatic progenitor cells). A
research group in university of Hong Kong recently demonstrated
that Akt/HIF-1?/PDGF-BB antocrine loop is associated with
chemoresistance of this malignant disease, suggesting that the
blockade of Akt /HIF-1?/PDGF-BB antocrine signaling could en-
hance the chemosensitivity of liver CSCs (Fig. 1) .
In malignancy, actively proliferating cells may be effectively
targeted and killed by anti-cancer therapies, but CSCs may survive
and support re-growth of the tumor and metastasis. Thus, new
strategies for the treatment of liver cancer obviously need to target
CSCs. Liver CSCs may secret cytokines to sustain their survival,
proliferation as shown in Fig. (1). Liver CSCs exhibit aberrant
TGF-? and activated IL-6 signaling, therefore anti-IL-6 targets
might be a potential therapeutic strategy in liver cancer treatment.
Another strategy is of blocking CD44 signaling by anti-CD44 anti-
body to eradiate liver CSCs, and consequently improve the therapy
effect on the malignancy. In addition, inhibition of Akt/HIF-
1?/PDGF-BB antocrine signaling in combination with traditional
chemotherapy may enhance the chemosensitivity of liver CSCs and
subsequently reduce metastasis and relapse. Altogether, liver CSCs
now become an important target in treatment of the liver cancer.
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Received: July 11, 2009 Revised: October 30, 2009 Accepted: January 11, 2010