Table Of ContentKumaretal.Cell&Bioscience2011,1:5
http://www.cellandbioscience.com/content/1/1/5 Cell & Bioscience
REVIEW Open Access
Molecular carcinogenesis of hepatocellular
carcinoma and intrahepatic cholangiocarcinoma:
one step closer to personalized medicine?
Mia Kumar, Xuelian Zhao, Xin Wei Wang*
Abstract
Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two major forms of primary liver
cancers (PLC), accounting for approximately 90% and 5% respectively. The incidence of each is increasing rapidly in
the western world, however our knowledge of the underlying mechanisms remains limited and the outcome,
dismal. The etiologies of each vary geographically; nevertheless, chronic inflammation has been identified in more
than 80% of the cases and appears to be a key mediator in altering the liver microenvironment, increasing the risk
of carcinogenesis. However, since not all HCC and especially ICC cases have a recognized risk factor, there are
currently two proposed models for liver carcinogenesis. The clonal evolution model demonstrates a multi-step
process of tumor development from precancerous lesions to metastatic carcinoma, arising from the accumulation
of genetic and epigenetic changes in a cell in the setting of chronic inflammation. While the majority of cases do
occur as a consequence of chronic inflammation, most individuals with chronic infection do not develop PLC,
suggesting the involvement of individual genetic and environmental factors. Further, since hepatocytes and
cholangiocytes both have regenerative potential and arise from the same bi-potential progenitor cell, the more
recently proposed cancer stem cell model is gaining its due attention. The integration of these models and the
constant improvement in molecular profiling platforms is enabling a broader understanding of the mechanisms
underlying these two devastating malignancies, perhaps moving us closer to a new world of molecularly-informed
personalized medicine.
Introduction to extrahepatic cholangiocarcinomas which are a form
Primary liver cancer (PLC) is the fifth most common of biliary tract cancer. The incidence of CCA varies with
cancer worldwide and the third most deadly, with regards to the two forms; however, for purposes of this
approximately 600,000 deaths annually. Hepatocellular review we will address ICC, which has the highest inci-
carcinoma (HCC), a primary malignancy of the hepato- dence in eastern Asia, particularly Thailand, with an
cyte, accounts for approximately 85% to 90% of all PLC, increasing risk ratio in the western world [3-5]. The
out of which 80% of HCC cases occur in either sub- remaining PLC subtypes, which account for less than
Saharan Africa or in eastern Asia [1,2]. Cholangiocarci- 5% of cases, are fibrolamellar HCC, hepatoblastoma,
noma (CCA), a malignancy of cholangiocytes in the bili- angiosarcoma, epithelioid hemangioendothelioma and
ary epithelium, is the second most common form and hepatocellular adenoma.
accounts for about 5% to 10% of PLC. CCA is categor- Risk factors that lead to the multistep development of
ized as intrahepatic or extrahepatic according to the HCC are well known and it is established that approxi-
anatomic location of the tumor. Since intrahepatic cho- mately 80% of HCC cases develop in individuals suffer-
langiocarcinomas (ICC) usually present in small biliary ing from chronic hepatitis B or C viral infection (HBV
ducts or ductules they are considered a PLC, compared or HCV), cirrhosis, and also those with a high exposure
to aflatoxin-b1 (AFB). HCC is particularly attributed to
*Correspondence:[email protected] these exposures due to the extensive oxidative stress
LiverCarcinogenesisSection,LaboratoryofHumanCarcinogenesis,Center and release of inflammatory cytokines induced by viral
forCancerResearch,NationalCancerInstitute,Bethesda,Maryland20892,
infection in the setting of liver inflammation. Diabetes,
USA
©2011Kumaretal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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obesity, smoking and alcohol abuse have also been asso- Hepatocellular Carcinoma
ciated with the development of HCC, but with reduced Altered signaling pathways in HCC at the genomic and
frequency [6-8]. Currently, individuals at risk for HCC transcriptomic levels
are routinely screened by ultrasonography and alpha- In a setting of chronic inflammation, the organ microen-
fetoprotein (AFP) levels but most patients are still diag- vironment experiences a variety of molecular changes
nosed with an advanced disease stage and therefore a that, in fact, often stem from the process and conse-
5-year survival for the majority of HCC patients’ remains quence of inflammation. In liver, cytokines and reactive
dismal [9,10]. Due to the high variability in AFP evalua- oxygen and nitrogen species produced by inflammatory
tion, affected by the specificity of the test, ethnic back- cells have been shown to mediate liver damage and
grounds and tumor size, an improvement in screening induce the liver’s regenerative response [22-26]. This
procedures is highly awaited. Furthermore, the impair- predisposes the proliferating cell to a variety of genetic
ment of liver function and the expression of multi-drug changes at the genomic and transcriptional levels.
resistance genes render HCC treatment difficult [11]. Genomic alterations
This review discusses the mechanistic changes that occur Large-scale quantitative comparisons of HCC tumors to
during the development of HCC and the potential targets non-tumors by the use of comparative genomic hybridi-
that are being investigated for new screening techniques, zation (CGH) arrays and loss of heterozygosity (LOH)
with the hope that this will lead to a more personalized has revealed the occurrence of chromosomal and micro-
treatment regimen to improve patient outcome. satellite instability in HCC. The most frequently deleted
Risk factors for ICC, on the other hand, are not so chromosomes arms are 1p, 4q, 6q, 8p, 9p, 13q, 16p, 16q
well established given that approximately 90% of ICC and 17p and regional gains are most often observed in
patients lack a recognized risk factor for the disease 1q, 6p, 8q and 17q [27,28], which, in general corre-
[12]. Furthermore, ICC cases appear to develop in sponds to autosome arms that contain allelic deletions
otherwise healthy livers, with only 10% resulting from identified by LOH: 1p, 1q, 4q, 5q, 6q, 8p, 9p, 13q, 16p,
chronic inflammation [3]. Nevertheless, relatively strong 16q and 17p [27,29,30]. Unrelated to tumor size, indivi-
CCA associations have been established with primary dual HCCs can represent multiple allelic deletions and
sclerosing cholangitis (PSC) [13-15], liver fluke infesta- chromosomal gains and losses, which can accumulate
tions [14,16] and hepatolithiasis [3]. Other possible, but during successive cell proliferation events and results in
not well characterized associations may also exist with a heterogeneous mixture of genomic aberrations [31].
HBV or HCV infection [17-19] and alcohol consump- The heterogeneity of tumors can help to identify tumor
tion [20,21]. The molecular interactions and genomic origin and due to the sensitivity of CGH and SNP
alterations in cholangiocytes that drive the development arrays, genomic alterations can be used as fingerprints
of CCA are not clear and the absence of specific symp- to identify whether a tumor is a recurrent event or a
toms and diagnostic tests make the disease difficult to second primary tumor [32,33]. The frequent loss of
identify in premalignant stages. Currently, 5-year survi- chromosome regions observed by LOH and SNP arrays
val rate for ICC cases remains below 5% [3], with the has revealed the concomitant loss or mutation of tumor
only hope for improved survival being complete resec- suppressor genes such as TP53 (p53), retinoblastoma
tion of the tumor. This underscores the need for RB1 (Rb) [34,35], CDKN2A (p16INK4A) [29,36] and insu-
increased research in this field and for the development lin-like growth factor-2 receptor IGF-2R [37,38], which
of improved diagnostic criteria and treatment options. are strongly associated with carcinogenetic signaling
The increased utilization of genome-wide association pathways [29,34,39,40]. Gain of function mutations have
studies (GWAS) will be particularly useful to identify also been observed in HCC, for example mutations in
large-scale genetic variants in PLCs, particularly cholan- CTNNBI (b-catenin), which results in the deregulation
giocarcinoma, since so little is currently understood. of similar signaling pathways in HCC [41,42].
As mentioned, in this review we will describe the cur- TP53 gene encodes the p53 protein which plays a
rent understanding in the molecular mechanisms, speci- pivotal role in the DNA-damage response network,
fically the genetic, epigenetic and signaling alterations including cell cycle arrest, apoptosis, DNA repair and
that take place, which give rise to the majority of PLC cellular senescence. Therefore, it is not surprising that
cases observed, separated by HCC and ICC. Further- TP53 loss of function mutations or allelic deletions in
more, it is interesting to note the two models for tumor- chromosome 17p are commonly associated with human
igenesis, a step-wise clonal evolution model and a carcinogenesis [43], and depending on the extent of
cancer stem cell (CSC) model, that have arisen as a con- damage, p53 can either regulate the production of anti-
sequence of our improved understanding in this field. oxidant genes to initiate DNA repair, or induce apopto-
How these models will impact PLC cases in the clinical sis through the activation of pro-oxidant genes[29].
setting will provide for a stimulating discussion. AFB1 is a particular mutagen of TP53, causing G:C
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to T:A transversions at the third base in codon 249 Wnt/b-catenin. This developmental pathway is com-
(converting arginine to serine), and the rate of TP53 monly known for its fundamental role in embryogenesis,
R249S mutation may be accelerated in the presence of which aids the cell in differentiation, proliferation and
viral infection [44,45]. HBV encodes a viral protein, apoptosis. In the absence of Wnt signaling, cytoplasmic
HBx, which can specifically bind to p53 and suppress b-catenin complexes with the tumor suppressors: adeno-
p53-induced apoptosis [46]. Strong associations have matosis polyposis coli (APC) and Axin1, as well as the
been observed between TP53 R249S mutation levels and glycogen synthase kinase-3b (GSK-3b). In this complex,
HCC risk, especially with respect to primary tumor GSK-3b phosphorylates b-catenin, targeting it for ubi-
development and also the interval between surgical quitiniation and subsequent degradation. In the event
resection and recurrence [47,48]. A recent study has that Wnt signaling receptors are engaged, conforma-
linked this p53 hotspot mutation to HCC with aggres- tional changes in the Axin complex cause the release of
sive tumors, poor prognosis and an acquisition of stem b-catenin, which then localizes to the nucleus and acti-
cell-like traits[49], which is not unexpected since a sepa- vates the transcription of Myc, cyclin D1 and COX2
rate study has shown that TP53 mutations have the abil- amongst others [57-59]. In HCC, our studies and a
ity to reprogram terminally differentiated cells into number of other transcriptomic and proteomic studies
pluripotent stem cells[50]. have indicated an increase in Wnt signaling, possibly as
Transcriptomic alterations: the deregulation of signaling a result of an accumulation of Axin1 mutations at sites
pathways in HCC that bind b-catenin and/or CTNNB1 mutations along
Structural genomic mutations and epigenetic changes sites marked for phosphorylation by GSK-3b [60,61]. It
may lead to altered gene expression patterns that signifi- is hypothesized that an increase in signaling from the
cantly affect the signal transduction pathways in HCC Wnt pathway is necessary to maintain “stemness” in
and the variability in pathway expressed may allude to HCC, characterized by cell proliferation and immortal-
the cellular origin of HCC. A selection of the relevant ity, an event that may be representative of CSCs [60,62].
signaling pathways altered in HCC is discussed here Myc is a potent oncogene, which appears to be consti-
(Figure 1). tutively up-regulated in many human cancers, represent-
TGF-b is an inflammatory cytokine implicated in an ing a phenomenon of “oncogene addiction.” Though
array of functions such as cell growth, differentiation, about 30% of HCC cases show an up-regulation of Myc
migration, apoptosis, adhesion, survival and immunity because of the Wnt/b-catenin pathway[63], its increased
[51]. IGF-2R, a tumor suppressor gene, promotes the expression in HCC is also attributable to the activation
degradation of mitogen IGF-2 and also the simultaneous of its locus through chromosome amplification [64] One
activation of transforming growth factor-b (TGF-b) sig- possible mechanism by which Myc contributes to hepa-
naling, thereby halting cell proliferation and carcinogen- tocarcinogenesis is through the induction of telomerase,
esis [52]. Inflammation and subsequent genomic which also appears to be active during HCC develop-
mutations in IGF-2R result in IGF-2 over-expression ment[65], thereby bypassing cellular senescence. More-
and a reduction in the inhibitory effects of TGF-b sig- over, the up-regulation of Myc in a variety of tumors
naling, a feature commonly observed early in the devel- has also been associated with deregulated microRNA
opment of HCC [53,54]. Immunohistochemical analysis (miRNA) expression in many human malignancies [66],
of HCC has also revealed a disruption of TGF-b signal- which as discussed in the next section, have a significant
ing coinciding with an increase in the expression of impact on tumorigenesis and progression. On the other
stem cell markers and the activation of interleukin-6 hand, the inactivation of Myc in HCC causes a subpo-
(IL-6). This indicates a link between IL-6, a major stem pulation of cells to differentiate while the rest remain
cell signaling pathway and the disruption of TGF-b sig- dormant, giving rise to a phenotypically diverse tumor
naling, resulting in CSC driven HCC[55]. population and possibly the origin of CSCs [67].
Interestingly, IL-6 activation is a frequent event in PI3K/PTEN/Akt. The activation of the Akt pathway
HCC. Recent studies indicate that gain of function is mediated by either an activated tyrosine kinase recep-
mutations of glycoprotein-130 (gp130), a co-receptor of tor, or more rarely the constitutive activation of PI3K or
IL-6, is associated with a marked activation of IL-6 in the loss of phosphatase and tensin homolog (PTEN).
inflammatory hepatocellular adenomas[56]. Noticeably, PTEN is a tumor suppressor gene and the PTEN pro-
rare gp130 alterations are always accompanied by tein functions as a negative regulator of Akt. The loss of
b-catenin activating mutations in HCC, suggesting that PTEN expression via a loss of heterozygosity in chromo-
these two signaling pathways are converged to contri- some 10q along with an activation of Akt has been
bute to hepatocarcinogenesis. Additional details about reported in 40%-60% of HCC cases [68,69]. Since Akt is
b-catenin involvement in HCC are described below. involved in a number of biological processes, such as
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Figure 1 Signaling Pathways Alteredin Hepatic Cancer Stem Cells.Wnt/b-catenin,PI3K/PTEN/AKT,TGF-b/IGF-2R andIL-6/IL-6R/gp130
signalingpathwayshavebeenshowntobeactivatedinHCC.ActivationoftheWntpathwayresultsinb-cateninaccumulationinthecytosol
andtranslocationintothenucleus,whereb-cateninformstwomajorprotein-DNAcomplexes.1)b-catenin/TCF/LEFisaclassiccomplexofWnt/
b-cateninpathwaythatmediatesWnttargetgenesexpression,e.g.EpCAMandmiR-181;2)EpICD/FHL2/b-catenin/LEF1-DNAcomplexrepresents
across-talkofWnt/b-cateninwithEpCAMsignalingpathway[133].CleavageofEpCAMbyTACEandPS-2releasesEpICDintocytosolwhichin
turntranslocatesintothenuclueswithb-cateninandFHL2,whereEpICD/FHL2/b-cateninformsprotein-DNAcomplexwithLEF1andregulates
EpCAMtargetgenesexpression,e.g.cyclinD1,c-MycanddmiR-181.AKTisactivatedbytwophosphorylationsitessThr308andSer473.
PhosphorylationofThr308ispromotedbyPI3KandsuppressedbyPTEN.ActivatedAKTinducescellsurvivalthroughthesuppressive
phosphorylationofBADandCaspase9,twoapoptosismediatorsinunphophorylatedstatus.AKTalsoactsasacellcycleprogressionregulator
throughactivatingthemTORpathway[134].TwooncogenicpathwaysPI3K/PTEN/AKTandWnt/b-cateninmaybeinterconnectedtopromote
stemnessandcarcinogenesis.LossofIGF-2RimpactscellproliferationbyaccumulatingIGF-2mitogenandactivationofTGF-bsignaling.
cell survival, cell growth, apoptosis and differentiation, miRNAs are a class of small non-coding RNAs that play
its deregulation has been implicated in many human a critical role in biological processes of cell development
cancers. Though the role of Akt in HCC is not con- and differentiation and the deregulated expression of
firmed, its activation is interestingly linked to more miRNAs in HCC has revealed their functional involve-
aggressive tumors in HCC [70] and an activation of b- ment in HCC carcinogenesis and progression [72].
catenin signaling in intestinal stem cells, suggesting that For example, in HCC cases, gene expression profiling
the two oncogenic pathways: PI3K/PTEN/Akt and Wnt/ reveals that an up-regulation of miR-181 is associated
b-catenin may be interconnected to promote stemness with increased signaling in Wnt/b-catenin pathways and
and carcinogenesis [71]. conversely, siRNA mediated inhibition of the TGF-b
Aberrant expression of miRNAs in HCC pathway indicates a decreased expression of miR-181
In recent years, the aberrant expression of miRNAs has [73,74]. Moreover, loss of let-7g expression is associated
been implicated in a wide variety of human cancers. with HCC metastasis [75]. miR-26 expression has been
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found to be associated with HCC gender disparity and Since distinct methylation signatures of tumor sup-
silencing of miR-26 in tumor cells is linked to a subtype pressor genes have been observed in high-risk subjects
of HCC with an activated inflammatory pathway and a up to 9 years before clinical diagnosis, DNA methylation
favorable response to interferon therapy [76]. In addi- profiling may provide a unique tool to reliably predict
tion, increased expression of miR-21 has been associated cancer status. Apart from their potential as a diagnostic
with loss of heterozygosity at the PTEN locus, conse- platform though, further understanding of methylation
quently activating the Akt pathway and promoting patterns in HCC may provide them useful in determin-
tumorigenesis [77,78]. Similarly, miRNAs associated ing recurrence and survival, as well [88].
with the cell cycle regulation and apoptosis inhibition in
HCC have also been identified [79]. Transcription profiles and the identification of HCC tumor
A study in our lab has revealed a 20-miRNA-based subtypes
signature that is associated with HCC venous metasta- Recent breakthroughs in technology have provided com-
sis, details of which are expanded upon at the end of prehensive genetic and transcriptomic profiling platforms
this article. This signature provides promise to a future that are successfully used in identifying tumor subtypes
of personalized medicine since it can be used clinically and predicting patient outcome and survival. In addition
to identify patients with early-stage disease or metas- to classifying tumor aggressiveness, high-throughput
tases and can even be used to predict survival and molecular profiling systems are also useful in determining
recurrence [80]. how the tumor will respond to treatment.
Global gene expression analysis of HCC has revealed
Epigenetic modifications may improve the early detection two distinctive subclasses of human HCC that are highly
of HCC cases associated with patient survival [89]. The low survival
In the last decade there has been increasing evidence to subclass was marked by increased expression of cell pro-
support the occurrence of aberrant DNA methylation liferation and antiapoptotic genes, such as PCNA and
patterns in human HCC [27]. Therefore, in addition to PTMA, respectively. Moreover, the poor survival sub-
genetic mechanisms of deletions or mutations, epige- group also expressed a higher number of genes related
netic changes can increase or decrease gene expression to ubiquitination and histone modification, namely
via regulating DNA methylation. DNA methylation in UBE2D1 and HRMT1L2, respectively.
the mammalian genome is found at the cytosine resi- Additional transcriptome analysis of HCC samples has
dues of CpG dinucleotides, often associated with pro- classified HCC into 6 subgroups associated with clinical
moter-related CpG islands. Though methylation is and genetic characteristics, which further identify two
imperative for normal development and differentiation, tumor groups linked to chromosome instability (Groups
aberrant hypomethylation in HCC and many human 1-3) or stability (Groups 4-6) [90]. The first group was
cancers can lead to the expression of oncogenes [81], or, linked to low copy number HBV infection, particularly
similarly, hypermethylation can lead to the silencing of in Africa, increased Axin 1 mutations, absence of TP53
tumor-suppressor genes [82]. In HCC, an increased mutations and possessed an over expression of
expression of DNA methyltransferases (DNMTs), imprinted genes. The second group was linked to high
enzymes which catalyze epigenetic alterations, occurs copy number HBV infection, many regions of LOH, and
early in the development of tumorigenesis. The fre- TP53 and Axin1 gene mutations. These first two groups
quency of aberrant DNA methylation increases from are the only ones that employed the Akt biological path-
precancerous lesions to dysplastic nodules and finally way and appeared most genetically distinct from the
HCC, signifying their important role in tumor progres- remaining subgroups. Groups 5 and 6 were also sub-
sion [83]. For instance, the tumor suppressor genes: RB1 stantially different since they were easily classified based
[84] and CDKN2A [36] have been shown to be hyper- on the abundance of CTNNB1 mutations (near 100%)
methylated in HCC, leading to uncontrolled cell prolif- and the high level of Wnt pathway activation.
eration. Likewise, PTEN promoter methylation has also Recently, our lab identified another 2 HCC subtypes
been reported in HCC, which allows the progression of based on their expression of a hepatic stem cell marker,
the PI3K/PTEN/Akt pathway [85]. epithelial cell adhesion molecule (EpCAM) and EpCAM-
Epigenetic changes in HCC have also been reported at coexpressed genes[91]. EpCAM-positive HCC correlated
the miRNA level. For example, the deregulation of miR- with increased Wnt pathway activation, cytokeratin 19
1 due to hypermethylation in HCC was reversed with and c-Kit, which are all known markers of progenitor
5-azacytidine, resulting in decreased cell proliferation cells. On the other hand, EpCAM-negative HCC
and increased apoptosis [86]. A similar association has resembled gene expression patterns of mature hepato-
been observed with miR-124 and also miR-203, amongst cytes. Further analysis based on AFP levels allowed these
others, in HCC [87]. two subgroups to be further divided into 4 subtypes with
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the ability to predict prognosis. Based on this classifica- of HCC morphology, clinical behavior, and molecular
tion, poor clinical outcome is correlated with AFP profiles [60,94]. Since the liver is an organ with regenera-
expression and good prognosis was only associated with tive capacity, it has bi-potential progenitor cells that can
EpCAM+ AFP- cases. give rise to hepatocytes or chloangiocytes, which could
Insights into tumor response to treatment are being possibly develop into HCC or ICC, respectively [95,96].
elucidated and our lab recently identified the impor- Furthermore, cases with a mixed morphology have also
tance of miR-26a and miR-26b in survival and response been identified. Based on the expression of EpCAM, a
to interferon therapy [76]. Compared to paired noncan- substantial number of HCC cases consist of progenitor
cerous tissue, HCC samples had decreased miR-26 cells and their heterogeneous progeny with a capacity to
expression correlating with an increase in nuclear factor self-renew and limitlessly divide [60,91,94]. This relatively
(cid:1)B (NF-(cid:1)B) and IL-6 signaling. Furthermore, clinical new hypothesis is not intended to be contradictory to the
data revealed that those patients with low miR-26 step-wise model, but merely complementary in explaining
expression had a better response to adjuvant interferon the origin of a more comprehensive group of HCC cases
therapy with interferon alpha than those with high miR- and the arising issues in diagnosis and treatment.
26 expression. For example, mature hepatocytes, cholangiocytes or
Taken together, transcriptional profiling platforms are bi-potential progenitor cells that acquire mutations
beginning to provide a vast amount of useful informa- through random genetic or epigenetic events can intro-
tion that can be synthesized to identify HCC subtypes, duce a genetic imbalance in the primary tissue, resulting
patient outcome, and survival and treatment options. in the de-differentiation of mature cells and the loss of
Still, new insights into the potential cellular origin of cell cycle control and/or the ability to continuously self
HCC and its activated molecular pathways are necessary renew. Depending on the extent of genetic alterations,
to improve targeted therapy. the tumor cells may remain benign or develop and
metastasize. Therefore, events initiated by the multistep
Two models of hepatocarcinogenesis may complement carcinogenesis model can also result in heterogeneous
one another tumors with stem cell capability and the potential to be
The longstanding clonal evolution model for HCC more aggressive.
development is a multistep event, which may take 30
years to unfold. As described earlier, the various etiolo- Intrahepatic Cholangiocarcinoma
gical factors, particularly inflammation and viral hepati- AnincreasingglobalincidenceofICC[5]hasrecentlyhas-
tis, appear to contribute significantly to approximately tenedresearchinthisfieldtounderstandthemechanisms
90% of HCC cases by creating phenotypically altered underlyingpathogenesisofthisdreadfuldisease.Reviewing
hepatocytes. The stepwise progression from altered the mechanisms of ICC indicates that similaritiescan be
hepatocytes to dysplastic nodules, or precancerous drawn between ICC and HCC, which may improve the
lesion, occurs as a consequence of chronic inflammation prospectsofthisdiseaseinaclinicalsetting.Particularly,
and genomic alterations, which commonly precede HCC thetumorigenesismodelsproposedforICCdevelopment
[27,39,92]. Therefore, the accumulation of genetic and are remarkably similar to those for HCC. Furthermore,
epigenetic changes, such as the loss of tumor suppressor severalhistopathologicandgeneexpressionprofilingstu-
genes and the gain of an oncogene, gives rise to a mass dies have shown PLC tumors that exhibit a combination
of primary tumor cells that are considered monoclonal of HCC and CCA traits, suggesting an overlap between
in origin[92,93]. Though from a scientific standpoint these tumor types. A subtype of tumors showing com-
this model has vastly improved our understanding of binedcharacteristicsofhepatocellular-cholangiocarcinoma
the various genetic and signaling events underlying (CHC)havebeenreportedandproposedtodevelopfrom
HCC development, it is unfortunate that these findings thebi-potentialliverstemcells[97].Evenmorerecently,a
have not been translated into better treatment options new subtype - cholangiocarcinoma-like HCC (CLHCC)
as liver resection and transplantation still remain the -was discovered and characterized as HCC expressing
best choice and only benefits a small population. CCA-like traits. The heterogeneity observed between all
A more recently proposed stem cell model for HCC 4tumorsubtypescouldbeindicativeoftheircellularori-
tumorigenesis may provide a more personalized approach gins from different developmental stages and may also
to address diagnostic and therapeutic strategies in the represent a novel way to approach targeted therapy in
clinic (Figure 2). This model hypothesizes that HCC CCAandHCC[98].
could be derived from progenitor cells or de-differen- Comparable to HCC, ICC most commonly arises in
tiated transformed cells; based on the observation that the setting of chronic inflammation, often within bile
embryonic stem cells (ESC) and CSCs behave similarly. ducts [99] and likely due to liver fluke Opisthorchis
This would be able to explain the heterogeneous nature viverrini infestation [100], PSC[101] or hepatolithiasis
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Figure2CancerStemCellModelforHCCTumorigenesis.ThegenerationofaCSCmodelwillmoreeffectivelybenefittheclinicaltreatment
ofHCCpatients,allowingtherapydirectedatthemostaggressivecells.Sofar,thereisnocompellingdatademonstratingthatHCCfollowsthis
model.TotesttheCSCmodel,atleasttwotermshavetobeaddressed:1)ThevastmajorityofHCCcells,excludingthesmallsubpopulationof
CSCs,lacktumorigeniccapacity;2)TheseCSCpopulationsaredistinguishedbyepigeneticratherthangeneticdifferencesbecausetheCSC
modelarguesthatCSCsundergohierarchicaldifferentiationandtheepigeneticchangesareirreversible.TwoHCCsubgroupswererecently
identifiedbasedontheexpressionofAFPandEpCAM.EpCAM+AFP+HCCsubgroup(HpSC-HCC)hadthefeaturesofhepaticstem/progenitor
cellsandEpCAM-AFP-HCCsubgroup(MH-HCC)featuredasmaturedhepacytes.HpSC-HCCdisplayedtheabilitytoself-renew,differentiateand
alsogeneratehighlyinvasiveHCC.Basedontheseobservations,itisplausiblethatHCCmayrepresentanothersolidcancertypethatfollowsthe
CSCmodelbesidesbreast,brainandcoloncancers.Consistentwiththeclonalevolutionmodel,HCCCSCscanarisefromthemutationof
normalhepaticstem/progenitorcells.ThoughtherehasnotbeenevidenceshowingthatHCCCSCscanarisefromdifferentiatedhepatocytes,
thepossibilitystillexits,asthereareexamplesofthisinhematopoieticmalignancies.Overall,notincontrasttotheclonalevolutionmodel,an
accumulationofmutationsduringthenormaldevelopmentofhepatocytesasaconsequenceofexposuretothevariousriskfactorsofHCC
mightcontributetotheriseofthehepaticCSCs;thereforethetwomodelsdonotcontrastbutcomplementeachother.
[102]. The continuous production of inflammatory cyto- cells to biliary dysplastic lesions and eventually cancer
kines and the induction of inducible nitric oxide [104].
synthase (iNOS) lead to oxidative and nitrosative DNA There is growing evidence supporting a hepatic stem
damage [100], increased cell turnover and inhibition of cell model of cholangiocarcinoma [96,105,106]. Interest-
DNA repair mechanisms [103]. In the stepwise model, ingly, the same bi-phasic progenitor cell can give rise to
an accumulation of inflammatory-mediated genetic and hepatocytes and cholangiocytes, as mentioned earlier,
epigenetic alterations has been proposed to lead to the and each of these cells has longevity and repopulating
successive development of ICC from biliary epithelial potential [107]. Therefore, in the setting of chronic
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inflammation, a tumor could arise from the clonal evo- division kinase (CDK) 4:cyclin D complex and prevents
lution of either mature cholangiocytes which de-differ- the phosphorylation of Rb protein. The CDK4:cyclin D
entiate, or progenitor cells; allowing a combination of complex is also influenced by the p16INK4A inhibitory
the two proposals. Furthermore, depending on the protein[117], which coincidentally is altered by LOH
degree of differentiation achieved before maturation and/or promoter hypermethylation in 25%-83% of
arrest, one can observe a heterogeneous tumor with a resected cholangiocarcinoma specimens [118,119].
range of neoplastic phenotypes [107]. One author K-ras mutations occurring at codon 12 are often
explains that the dysplastic nodules observed during car- observed in ICC, involving either a glycine to aspartic
cinogenesis may be adaptive non-oncogenic responses to acid or a glycine to cystine transition [111,115]. Less fre-
carcinogenic substances, rather than a multistep accu- quent mutations have been observed in codon 13 (sec-
mulation of genomic alterations [105]. ond nucleotide) and codon 61 (third nucleotide) [120].
K-ras mutations corresponding to over expression are
Genetic alterations that manifest in ICC observed early in carcinogenesis, which suggests an
Genomic alterations important role in ICC development. Furthermore, K-ras
In a study aimed at distinguishing chromosomal changes has been implicated in aggressive ICC downstream of
between HCC and ICC, CGH analysis reveals the fre- the biliary tree with increased expression levels in meta-
quency of chromosomal losses in ICC is higher [53]. static lymph nodes [121]; therefore K-ras expression
Short segments of chromosomes 1p [108], 3p, 6q and correlates to poor prognosis [122].
9q [28,108] are commonly deleted in ICC, with a fre- Transcriptomic alterations: Enhanced proliferation signaling
quency of at least 55%, whereas the frequency of such in ICC
events in HCC are usually less than 40% [28]. Com- The inactivation of tumor suppressor genes and the
monly amplified regions in ICC are in segments of 1q, concordant amplification of proto-oncogenes, such as
7q, 7p and 8q [28,108], with an amplification frequency TP53 and K-ras, respectively, play a significant role in
of at least 30%. Losses in regions of 6q and 3p appear to altering the signaling network and promoting tumori-
be highly characteristic of cholangiocarcinoma, but over- genesis. The exact mechanism by which these pathways
all the high frequency of gains and losses appears to are affected is currently unknown, especially since most
carry a poor prognostic value [28]. Further studies to of the mechanisms have only been observed in ICC cell
detect the loss of tumor suppressor genes during a con- lines. Since in vivo studies in animal models poorly cor-
sistent LOH have indicated a high rate of allelic losses relate with clinical outcome, the need to recreate an
at 5q and 17p [109,110]. LOH has been observed in ICC model to better understand this disease is under-
other chromosomal regions, but to a lesser extent, and scored. Despite this lack of conclusive data, several stu-
may represent random error. HCC and ICC share simi- dies have identified a selection of important
lar allelic losses in the 5q and 17p regions, allowing mechanisms contributing to the development of ICC,
some to propose that these two tumors arise from the emphasizing the role of TGF-b, IL-6, STAT-3, COX-2
same CSC and therefore may share similar genetic and b-catenin [99,123]. Because of the similarities
changes during tumorigenesis [105,110]. Persistent already observed in the etiology and structural changes
structural genomic changes in these cells have been between HCC and ICC, and since some pathways most
associated with a variety of mutations, conferring a loss likely overlap, one may try to infer the mechanisms
in tumor suppression and the amplification of oncon- underlying HCC on ICC and compare this with gene
genic pathways. expression arrays to arrive at a more functional under-
TP53 mutations in ICC are common and their fre- standing of molecular pathogenesis in ICC.
quency ranges from 20-80% depending on the geo- Transcriptomic alterations: the recent identification of
graphic region [111,112]. Though the deregulation of miRNAs in ICC
p53 in ICC is similar to HCC, as there is a loss of cell Just as in HCC, the altered expression of miRNAs in
cycle control and a decrease in apoptotic events, the ICC has been reported to contribute to tumor growth.
deregulation in ICC is sometimes more associated with Malignant cholangiocytes appear to be marked by an
an accumulation of inactive wild-type p53 and its inhibi- over-expression of miR-21, miR-141 and miR-200b
tor mdm-2, rather than a loss of function mutation [124]. The increased expression of miR-21 and miR-
[113,114]. This renders the p53 regulatory pathway non- 200b has been linked to increased cellular proliferation,
functional and supports the notion that either TP53 mediated by a down-regulation of PTEN and ZFHX1B
mutations or an alteration to the p53 pathway may be tumor suppressors, respectively [78,124]. In addition, the
critical to the development of ICC [115,116]. The regu- decrease in miR-29b has been linked to the increased
lation of cell cycle entry by p53 involves, amongst expression of MCL-1, an anti-apoptotic protein, result-
others, the p21WAF1/Cip1 protein, which binds to the cell ing in decreased apoptosis [125]. Though the network of
Kumaretal.Cell&Bioscience2011,1:5 Page9of13
http://www.cellandbioscience.com/content/1/1/5
miRNA involvement is far greater in HCC, the study of The exciting identification of miRNA signatures
miRNAs in ICC is likely to lead to novel diagnostic and unique to HCC offers new platforms for cancer diagno-
prognostic methods once their function is confirmed. sis and prognosis. For instance, in addition to the 20-
miRNA-based signature described above that is able to
Future Perspectives differentiate between CSC-like and mature hepatocyte-
Currently, surgery such as liver resection remains the like HCC tumors, there are additional miRNAs that can
best treatment option for early HCC and ICC but tumor help differentiate between benign and malignant tumors,
recurrence is still predominant in about 80% of HCC or between alcohol induced or HCV induced HCC
cases [126] and may be higher in ICC cases [127]. To [130]. Prognostic miRNA markers of HCC also exist,
make matters worse, effective treatment is limited for and in fact they are specific enough to help with deter-
advanced stage carcinoma, which emphasizes the need mining metastasis [80], recurrence [131] and survival
to improve our understanding of primary liver cancers [75,76,80], independently.
and consequently help improve patient diagnosis during Studieshave alsoindicatedthetherapeutic potential of
the early stage. Intervention early in the process will miRNAs, particularly with theobservance that anti-miR-
improve treatment and prognosis, which may be con- 181canreducetumorigenicityinmicewithhepaticCSCs,
ferred by an attempt at personalized medicine. cellsthatareotherwiseresistanttochemotherapywith5-
Individual genetic background has been suggested to Fluorouracil (5-FU) [60]. Along with others, our studies
contribute to HCC risk, given that only a fraction of indicatethatmiR-26functionallyactsasatumorsuppres-
patients with chronic liver disease or PSC actually sor by inhibiting cell proliferation, and a low miR-26
develop HCC or ICC, respectively, even though greater expressionisassociatedwithpoorprognosis[76].miR-26
than 50% of cases occur in the setting of inflammation. expression is significantly down-regulated in HCC, but
For this reason, the identification of genetic susceptibil- gene therapy with the delivery of miR-26 to hepatocytes
ity loci and new biomarkers are essential to improving considerablyblocksMyc-inducedHCC[76,132].Further-
diagnosis and treatment outcome, and GWAS studies in more,studiesinourlabonmiR-26,NF-(cid:1)BandIL-6have
liver cancer are highly necessitated. revealedthepotentialofmiRNAexpressionprofilesinthe
The hypothesized CSC model for the development of stratificationofpatientsforinterferontherapy[76].
HCC and ICC, and the molecular pathways, such as the
Wnt/b-catenin pathway and miR-181, provide valuable Conclusion
information about tumor growth and invasiveness. Since The studies of molecular mechanisms involved in the
recently,theroleofEpCAMinmaintainingastemcellphe- progression to HCC have been investigated at length,
notypeinHCCandICCisbeingelucidatedbutourstudies, and they have helped infer testable hypotheses in ICC.
aswellasothersprovideevidenceforitsroleinthepromo- Advances in molecular profiling studies using DNA-
tion of proliferation, migration and invasion potential in microarray based gene-expression profiling have pro-
cells with activated Wnt/b-catenin signaling [60,91,128]. vided increased awareness about the regulatory networks
Therefore,b-cateninmaybeanoveltargetinthepreven- altered in human HCC and have also provided useful
tionofcarcinogenesis[57],highlightingtheimportanceof gene expression-based signatures that can distinguish
molecularprofilingtocharacterizethepopulationofcells tumor subtypes, assist clinical staging and predict
andtheirdistinctmolecularpathways[60]. patient outcomes. Since molecular profiling is proving
The role of miRNA signatures has recently been eluci- to be an efficient way to gain insight into the molecular
dated through the examination of miRNA expression mechanisms underlying carcinogenesis, these techniques
profiles of samples from two different subtypes in HCC, should also be employed more extensively in ICC so
indicating miRNA profiling can be used to indicate that we can obtain a more inclusive picture of regula-
tumor origin and aggressiveness. In these studies tory elements in pathogenesis.
[60,91,129], the two subtypes are hepatic stem cell like Advances in the specificity and sensitivity of molecular
HCC (HpSC-HCC; EpCAM+AFP+) and mature hepato- profiling platforms including expression analysis and
cyte like HCC (MH-HCC; EpCAM-AFP-), where HpSC- comparative genomics with the additional incorporation
HCC cells are hepatic CSCs with the ability to self- of PLC databases and bioinformatics tools, we are
renew, differentiate and initiate aggressive tumors in approaching a new era for understanding the heteroge-
vivo [60]. Coincidentally, among HpSC-HCC tissues, neity of HCC and ICC tumors. Such integrated plat-
miR-181 is found to be up-regulated and functions in forms are enabling our improved understanding of the
promoting stemness by targeting hepatic transcriptional etiology, tumor microenvironment and the carcinogen-
regulators of differentiation, such as CDX2 and GATA6, esis of these two devastating diseases, which we hope to
and nemo-like kinase (NLK), an inhibitor of Wnt/b- integrate with a personalized approach in improving the
catenin signaling [73]. clinical outcome of these cases.
Kumaretal.Cell&Bioscience2011,1:5 Page10of13
http://www.cellandbioscience.com/content/1/1/5
Abbreviations 16. ParkinDM,SrivatanakulP,KhlatM,ChenvidhyaD,ChotiwanP,InsiripongS,
5-FU:5-Fluorouracil;AFB:aflatoxinB1;AF:alpha-fetoprotein;CCA: L’AbbeKA,WildCP:LivercancerinThailand.I.Acase-controlstudyof
cholangiocarcinoma;CDK:celldivisionkinase;CGH:comparativegenomic cholangiocarcinoma.IntJCancer1991,48:323-328.
hybridization;CSC:cancerstemcells;DNMT:DNAmethyltransferases; 17. KobayashiM,IkedaK,SaitohS,SuzukiF,TsubotaA,SuzukiY,AraseY,
EpCAM:epithelialcelladhesionmolecule;GWAS:genome-wideassociation MurashimaN,ChayamaK,KumadaH:Incidenceofprimary
studies;HBV:hepatitisBvirus;HCC:hepatocellularcarcinoma;HCV:hepatitis cholangiocellularcarcinomaoftheliverinjapanesepatientswith
bvirus;HpSC-HCC:hepaticstemcelllikeHCC;ICC:intrahepatic hepatitisCvirus-relatedcirrhosis.Cancer2000,88:2471-2477.
cholangiocarcinoma;LOH:lossofheterozygosity;MH-HCC:mature 18. SorensenHT,FriisS,OlsenJH,ThulstrupAM,MellemkjaerL,LinetM,
hepatocytelikeHCC;PLC:primarylivercancer;PSC:primarysclerosing TrichopoulosD,VilstrupH,OlsenJ:Riskofliverandothertypesofcancer
cholangitis;SNP:singleneucleotidepolymorphism. inpatientswithcirrhosis:anationwidecohortstudyinDenmark.
Hepatology1998,28:921-925.
Acknowledgements&Funding 19. DonatoF,GelattiU,TaggerA,FavretM,RiberoML,CalleaF,MartelliC,
WewouldliketothankJunfangJiforhercriticalreviewofthismanuscript SavioA,TrevisiP,NardiG:Intrahepaticcholangiocarcinomaandhepatitis
andhelpfulsuggestionsandKarenYarrickforherbibliographicalassistance. CandBvirusinfection,alcoholintake,andhepatolithiasis:acase-
ThisworkwassupportedbytheIntramuralResearchProgramoftheCenter controlstudyinItaly.CancerCausesControl2001,12:959-964.
forCancerResearch,theNationalCancerInstitute(Z01BC010313andZ01 20. BergquistA,GlaumannH,PerssonB,BroomeU:Riskfactorsandclinical
BC010876). presentationofhepatobiliarycarcinomainpatientswithprimary
sclerosingcholangitis:acase-controlstudy.Hepatology1998,27:311-316.
Authors’contributions 21. ChalasaniN,BaluyutA,IsmailA,ZamanA,SoodG,GhalibR,
MRKdraftedthemanuscript,XZdesignedthefiguresandXWWcontributed McCashlandTM,ReddyKR,ZervosX,AnbariMA,etal:Cholangiocarcinoma
hisideasandhelpededitthepaper.Allauthorsparticipatedequallyinthe inpatientswithprimarysclerosingcholangitis:amulticentercase-
designanddevelopmentofthisreviewandallauthorsreadandapprove controlstudy.Hepatology2000,31:7-11.
thefinalmanuscript. 22. HussainSP,HofsethLJ,HarrisCC:Radicalcausesofcancer.NatRevCancer
2003,3:276-285.
Competinginterests 23. BhogalRH,CurbishleySM,WestonCJ,AdamsDH,AffordSC:Reactive
Theauthorsdeclarethattheyhavenocompetinginterests. oxygenspeciesmediatehumanhepatocyteinjuryduringhypoxia/
reoxygenation.LiverTranspl2010,16:1303-1313.
Received:22December2010 Accepted:24January2011 24. MakiA,KonoH,GuptaM,AsakawaM,SuzukiT,MatsudaM,FujiiH,RusynI:
Published:24January2011 Predictivepowerofbiomarkersofoxidativestressandinflammationin
patientswithhepatitisCvirus-associatedhepatocellularcarcinoma.Ann
SurgOncol2007,14:1182-1190.
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